Accessory molecules 79

OPEN POSTERS Accessory molecules

OP1 OP2 Association of invariant chain (CD74) and HLA-DR on Association with atopy of a single nucleotide the surface of transfected fibroblasts detected by polymorphism in the gene encoding the single molecule fluorescence imaging immune-regulatory protein, PD-1 I. Karakikes, I. E. G. Morrison, N. Fernandez & R. J. Cherry E. S. James, S. J. Davis, W. O. C. M. Cooksony & M. F. Moffatty University of Essex, Department of Biological Sciences, Wivenhoe Park, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Colchester, CO4 3SQ, UK Headington, Oxford, OX3 9DU, UK, yAsthma Genetics, Wellcome Trust Centre for Human, Genetics, Roosevelt Drive, Headington, Oxford, The interaction of the major histocompatibility complex (MHC) class II- OX3 7BN, UK associated invariant chain (Ii) with MHC class II molecules in the ER facilitates correct assembly and export of class II dimers. It is known that a Programmed Death 1 (PD-1), a new member of the CD28 family of co- proportion of Ii appears at the cell surface, known as CD74 antigen. stimulatory molecules, appears to attenuate T-cell activation. Mice lack- Biochemical data suggest that a proportion of the surface Ii is associated ing PD-1 have been shown to develop a variety of autoimmune diseases, with HLA-DR. We have developed a novel technique based on digital suggesting that PD-1 might be involved in the maintenance of peripheral fluorescence imaging for the study of the association of individual CD74 tolerance. In the PD-1 gene, we have identified an SNP consisting of a and HLA-DR molecules on living cells. Co-localization experiments silent C to T mutation in the cytoplasmic domain-encoding exon at show that 25 2% of the CD74 forms complexes with HLA-DR abdimers position þ7714 relative to the initiation codon. The SNP has been on the plasma membrane. The data provide for the first time quantitative genotyped in a panel of 364 subjects in 80 nuclear families from a general evidence for formation of HLA-DR:Ii complexes at the surface of antigen population sample from Western Australia. Significant association to total presenting cells. In agreement with this finding, FRET analysis demon- serum IgE levels P ¼ 0Á002 and the skin test index (the sum of skin test strates close proximity of HLA-DR and CD74 at the cell surface of living responses to house dust mite and grass pollen) P ¼ 0Á009 were seen. human transfected fibroblasts. We demonstrate a physical interaction of Association was also found for serum IgE levels to whole Timothy Grass MHC class II and Ii that could account for the recycling of empty HLA- (Phleum pratense) P ¼ 0Á001. Similar results were seen in a replication DR molecules from the cell surface and/or the delivery of newly synthe- panel of 410 subjects. These data are the first to implicate the involvement sized class II:Ii complexes to processing compartments via the cell of the gene in human immune pathology, suggesting that PD-1 may surface. become an important target for immunotherapy.

Allergy

OP3 mice by discrete classes of chemical allergens are associated with Intracellular cytokine patterns induced in mice by differential frequencies of cells expressing IL-4. chemical contact and respiratory allergens OP4 N. E. Humphreys, R. J. Dearman & I. Kimber The role of antigen presenting cells in Syngenta, CTL, Alderley Park, Macclesfield, SK10 4TJ, UK histamine-induced T helper cell polarization We have demonstrated previously that repeated topical exposure of A. Hogg, B. N. Hudspith, N. B. Rayment & J. Brostoff BALB/c strain mice to the contact allergen 2,4-dinitrochlorobenzene King’s College London, Infection and Immunity Research Group, Division (DNCB) or to the respiratory allergen trimellitic anhydride (TMA) of Life Sciences, 150 Stamford Street, London, SE1 9NN, UK induces, respectively, a selective type 1 or a type 2 cytokine secretion profile in draining lymph node cells (LNC). Using flow cytometry to Type 1 allergic reactions are characterized by the release of mediators, measure intracellular cytokine expression, we have now investigated the including histamine, from mast cells and basophils. It is these mediators relative frequencies of interleukin (IL) 4 and interferon (IFN)-gþ CD4 that induce the characteristic allergic symptoms. However it has been and CD8 cells in the allergen-activated LNC population. The majority of shown that histamine also has a role in regulating the immune response IFN-g was detected within CD8þ cells, with a small subset of CD4þ IFN- that control the events leading to allergic sensitization. Allergic sensitiza- gþ cells; interestingly there were no significant differences between tion is initiated by the development of an aberrant Th2 response against TMA- or DNCB-activated LNC. Quiescent (vehicle-treated) LNC also common environmental antigens. It is known that histamine suppresses had a significant population of CD8þ IFN-gþ cells at approximately half lymphocyte proliferation and Th1 type cytokine production, and this the frequency found in allergen-treated mice. Exposure to TMA induced suppression is more marked in atopic individuals. It has therefore been twice as many CD4þ CD3þ IL-4þ cells, as did treatment with DNCB. A proposed that this is an important factor contributing to the severity and small population of B220þ IL-4þ cells was also observed that was not persistence of allergies. In this study, we show that histamine induces its detectable in populations derived from DNCB- or vehicle-treated mice. observed immunoregulatory effects through modulating antigen present- These data demonstrate that the divergent immune responses induced in ing cell function. Histamine inhibited the uptake of antigen and increased

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 80 Allergy

the level of prostaglandin E2 released by these cells. This led to enhanced degrading tight junction proteins. The possible protective role of the production of the cytokine IL-10 and suppressed IL-12 production. These endogenous pulmonary antiprotease secretory leucoprotease inhibitor histamine treated APC’s, when added to antigen, stimulated T cell- (SLPI), however, has not been explored. We investigated the effect of induced polarization of the responses towards a Th2 phenotype. SLPI on the protease activity of immunopurified Der p1. This was measured by the rate of hydrolysis of the substrate N-benzoyl-Phe-Val- Arg-p-nitroanilide. Immunopurified SLPI and sputum containing SLPI OP5 (gift from Professor Stockley) were incubated with Der p1 in ratios of Pigeon fanciers’ lung: exposure to respiratory 0Á1–3 : 1 mol/mol and protease activity measured. Der p1 and SLPI were pathogens and development of disease then incubated (1 : 1 mol/mol) at 378C with DTT in 10 mM phosphate buffer, pH 7Á4, for 30 min and analyzed by SDS–PAGE and silver R. M. Tailford, A. Todd, J. E. Calvert,y A. Allen,y S. J. Bourkez & stain. Immunopurified SLPI at physiological concentrations (0Á3–3 mm) C. I. Baldwin§ inhibited the protease activity of Der p1 by 72–81%. Sputum containing Public Health Laboratory, Cumberland Infirmary, Carlisle, CA2 7HY, 0Á5–2Á0 mm SLPI inhibited activity by 27–58%. SDS–PAGE showed ySchool of Cell and Molecular Biosciences, Medical School, University of cleavage of SLPI by Der p1 after 30 min incubation. In conclusion, Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, zDepartment of immunopurified SLPI and sputum containing SLPI inhibit the protease Respiratory Medicine, Royal Victoria Infirmary, Newcastle upon Tyne, activity of Der p 1; the SLPI appears to be cleaved during this process. NE1 4LP, §School of Applied Sciences, Northumbria University, Ellison Building, Newcastle upon Tyne, NE1 8ST, UK Pigeon fanciers’ lung (PFL) is caused by hypersensitivity reactions to OP7 inhaled pigeon antigens. Only a small percentage of subjects exposed to HLA associations with sensitization to rats pigeon antigens develop PFL and thus it has been suggested that envir- H. Jeal, A. Draper, M. Jones, J. Harris, K. Welsh,y A. N. Taylor & onmental factors, including respiratory infection, may influence the P. Cullinan immune response and subsequent development of disease. In this study, Department of Occupational and Environmental Medicine, yInterstitial serum samples from 156 pigeon fanciers were tested for IgG antibodies to Lung Disease Unit, 1b Manresa Road, London, SW3 6LR, UK a range of organisms known to cause atypical pneumonia and viral lung diseases, which may predispose to PFL. These included influenza A, HLA class II molecules are involved in presenting allergens to T cells and influenza B, respiratory syncitial virus, adenovirus, cytomegalovirus, are therefore likely candidates for controlling the immune response. We Epstein-Barr virus, Mycoplasma pneumoniae, Chlamydia pneumoniae hypothesized that HLA class II genotype might be associated with and C. psittaci. Individuals with PFL were significantly more likely to sensitization to rats among exposed individuals. 109 cases and 397 have had previous infection with influenza B (P ¼ < 0Á003). This suggests referents were HLA typed for DRB1 and DQB1 loci. HLA-DR7 was that infection with influenza B predisposes the development of PFL. associated with sensitization (OR 1Á99), symptoms (OR 2Á98) and sensi- tization with symptoms (OR 4Á81). HLA-DR3 was protective against sensitization (OR 0Á55). We provide a biologically plausible explanation OP6 for these associations. Amino acid analysis of HLA-DR3 and HLA-DR7 The effect of secretory leucoprotease inhibitor on Der reveals that these molecules differ in their hydrophobicity at certain p1 locations. HLA-DR7 is made up of hydrophobic residues whereas HLA-DR3 is made up of hydrophilic residues at the positions where V. Barlow, N. Sehgal, F. Smillie, A. Custovic & A. Woodcock these two molecules differ. The hydrophobic residues of HLA-DR7 may North-west Lung Centre, Wythenshawe Hospital, Manchester, M23 9LT, bind with greater affinity to Rat n 1, which functions in the transport of UK hydrophobic ligands, while the hydrophilic residues of HLA-DR3 may Der p1 exposure is a major risk factor for mite sensitization, yet the total have a negative effect on binding. In this study, we also investigated dose of inhaled allergen is very low (2 mg/year). Invitro studies suggest the associations between HLA and the response to T-cell epitopes of that Der p1’s proteolytic activity increases its trans-epithelial passage by Rat n 1.

Antigen presentation

OP8 were isolated from Raji cells in peptide loading complexes containing Identification of specific glycoforms of MHC class I TAP, tapasin and ERp57 with and without the lectin-like folding chaper- heavy chain indicates that class I peptide loading is an one, calreticulin (CRT). CRT is a soluble protein that recognizes the

adaptation of the quality control pathway involving terminal glucose of the oligosaccharide precursor Glc1Man9GlcNAc2 calreticulin and ERp57 attached to all nascent glycoproteins in the ER. This work demonstrates that monoglucosylated glycoforms of HC are present in the peptide C. M. Radcliffe, G. Diedrich,y D. J. Harvey, R. A. Dwek, loading complex. The data are consistent with a model in which the P. Cresswelly & P. M. Rudd release of peptide-loaded MHC class I molecules from CRT, induced Department of Biochemistry, The Glycobiology Institute, University of by deglucosylation of the HC N-linked glycan, signals the dissociation of Oxford, South Parks Road, Oxford, OX1 3QU, UK, ySection of the complex, supporting the hypothesis that the class I loading process is Immunology, Howard Hughes Medical Institute, Yale University School of an adaptation of the quality control mechanism involving CRT and Medicine, New Haven, CT 06510, USA ERp57. Glycan analysis was used to probe the sequence of events accompanying the binding of antigenic peptides to MHC class I heavy chains (HC). HC

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Antigen presentation 81

OP9 surface. These MHC II-restricted epitopes are organism specific and in Antigen presentation and the induction of peripheral conjunctionwithco-stimulatorymolecules,primeCD4þ T-cellresponsesas tolerance amongst CD8þ T cells part of the adaptive immune response. DC were produced from murine bone marrow by invitro culture in the presence of GM-CSF and TNF-a. The DC J. M. Fraser & D. J. Morgan were then pulsed with peptides comprising known MHC II-restricted Department of Pathology and Microbiology, School of Medical Sciences, epitopes from the F1 protein of Yersinia pestis (J.H. Robinson, personal University Walk, University of Bristol, Bristol, BS8 1TD, UK communication) and maturation induced. Confirmation of the presence of InsHA mice express, as a transgene, the influenza virus haemagglutinin an MHC II-epitope complex on the DC surfacewas achieved using confocal (HA) on pancreatic islet b cells. Mice demonstrate immunological microscopy todemonstrate theco-localization of epitopeandMHCII on the tolerance to the HA protein, which is associated with a loss from the cell surface and the proliferation of F1-specific T-cell lines after co-incuba- repertoire of CD8þ CTL precursors having high avidity for the dominant tion with antigen-pulsed DC. MHC II-restricted epitopes were extracted by Kd-restricted HA epitope. Such tolerance occurs via a peripheral mechan- acidelutionandmassspectraacquiredon aQuattroIITM tandem quadrupole ism. Following their adoptive transfer into InsHA mice, naive Clone-4 mass spectrometer with an electrospray ionization source. Initial results CD8þ T cells, which are specific for the dominant Kd-restricted HA suggestidentificationofY. pestisspecificMHCII-restrictedepitopesmaybe epitope IYSTVASSL (518–526), proliferate in the pancreatic lymph possible within 6 hr of the addition of the DC maturation signal. nodes. We have demonstrated that proliferation of Clone-4 T cells occurs # Crown Copyright 2002 Dstl due to the cross-presentation of KdHA epitopes by bone marrow-derived antigen presenting cells (APC). In order to determine the tolerogenic potential of several phenotypes of bone marrow-derived APCs, InsHA OP12 mice were injected with KdHA peptide-pulsed population of BM-derived Peptide binding preferences of the HLA-class II APCs and Clone-4 T-cell responses measured. The phenotype of the APC molecule, DR15 for the RhD autoantigen involved in both cross-presentation of HA, and the subsequent induction L. S. Cairns, F. J. Ward, S. J. Urbaniak & R. N. Barker of tolerance amongst KdHA-specific Clone-4 CD8þ T cells were deter- Institute of Medical Sciences, University of Aberdeen, Foresterhill, mined by transferring purified population of putative APCs into chimeric Aberdeen, AB25 2ZD, UK InsHA mice that were deficient in Kd-expressing APCs. The RhD polypeptide is the major alloantigen in haemolytic disease of the newborn and an important autoantigen in human autoimmune haemolytic OP10 anaemia (AIHA). The production of anti-RhD antibodies has previously Alteration in presentation of an immunodominance been shown to be driven by the activation of T-helper cells specific for hierarchy by the MHC class I-associated chaperone epitopes on the Rh proteins. T-helper cells, isolated from AIHA patients tapasin and individuals immunized with RhD protein, proliferate in response to M. Howarth, A. Tolstrupy & T. Elliottz epitopes from a panel of overlapping peptides spanning the RhD MRC Human Immunology Unit, John Radcliffe Hospital, Oxford, sequence. The HLA type of the individual is one factor that determines OX3 9DU, UK, yInoxell, Kogle Alle´ 5, Hoersholm, DK-2970, Denmark, the distribution of the proliferative responses. HLA-DR15 is the most zCancer Sciences Division, Southampton General Hospital, Southampton, predominant allele in both AIHA patients and those alloimmunized SO16 6YD, UK donors, demonstrating a high anti-RhD antibody titre. We examined the binding preferences of DR15 molecules for the panel of RhD peptides. CTL responses focus on a very small number of MHC class I-presented The results obtained were compared to the ability of these peptides to peptides. The most important factor in this immunodominance is the induce proliferative responses in DR15-positive patients. The majority of affinity of the peptide for class I. Our aim was to look at how the peptides previously shown to induce Th-cell proliferation in one or more presentation of an immunodominance hierarchy would be altered in cells AIHA patients were identified as having intermediate or high affinity for lacking chaperones involved in loading of class I with peptides. We DR15. These results are important for identifying potential therapeutic selected derivatives of SIINFEKL that have varying affinities for Kb. This peptides for the immunization of patients. series was expressed as minigenes and their presentation was determined with the class I peptide-specific antibody 25-D1Á16. In tapasin-competent cells, a clear immunodominance hierarchy was obtained with greatest OP13 presentation of the high affinity peptide and correspondingly reduced GP96 as a tool for studying indirect and direct presentation of lower affinity peptide variants. However, in tapasin- xenoantigenic responses deficient cells this hierarchy disappeared and three peptides with varying affinities were presented at the same level. This study demonstrates L. K. Slack, B. Fairburn, M. Muthana, S. Mirza & A. G. Pockley qualitative changes in the peptide repertoire by tapasin, which had not Immunobiology Research Group, Clinical Sciences Centre, Northern been observed by mass spectrometry and shows that peptide presentation General Hospital, Herries Road, Sheffield, S5 7AU, UK at the cell surface is not a simple function of class I binding affinity. Molecular engineering shows promise in controlling hyperacute xenograft rejection, and a better insight into the cellular responses that lead to OP11 xenograft rejection is therefore required. Dendritic cells (DC) internalize The identification of Yersinia pestis class II major the heat shock protein gp96 by receptor-mediated endocytosis and direct histocompatibility restricted epitopes by mass peptides chaperoned thereon (which represent the ‘antigenic fingerprint’ spectrometry of the cells from which it has been isolated) into the intracellular pathway for MHC-restricted presentation to T cells. This study reports a technique G. D. Healey, K. Anderson, D. D. Despeyroux, M. Morton & for assessing T-cell responsiveness to indirectly presented xenoantigens. E. D. Williamson Lewis rat bone marrow was cultured with rat GM-CSF (10 ng/mL) and IL- Dstl, Porton Down, Salisbury, SP4 0JQ, UK 4 (5 ng/mL). On day 7, adherent DCs were harvested and incubated for Dendritic cells (DC) capture and process antigens from invading patho- 24 hr with gp96 purified from C57BL/6 mouse liver (0–100 mg/mL). DCs gens and present them in association with MHC II molecules on their were washed and cultured for up to 72 hr with CFSE-labelled Lewis

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 82 Antigen presentation

splenic T cells. A progressive decline in the fluorescent intensity of CFSE- T cells, TCR-restricted responses are best measured using autologous labelled cells reflects their proliferation, and their phenotype can be presentation assays. HSP-specific T cells are implicated in autoimmune determined by co-staining with anti-CD4, anti-CD8 and anti-cytokine diseases such as RA and have a high precursor frequency among the naive monoclonal antibodies. The precursor frequency of responding cells can T-cell repertoire. We examined newborn responses to these endogenous also be calculated. proteins. KLH, a known neoantigen, was included for comparison. Acknowledgement This study was funded by the National Heart, Lung MDDCs, pulsed prior to LPS maturation, were used in an autologous and Blood Institute, USA (HL 69726). system. Primary and secondary CD4þ responses were investigated, study- ing proliferation and cytokine production. Responses to HSP70 were above an unpulsed mature DC background (S.I. 1Á8 0Á72), equivalent to OP14 KLH (S.I. 2Á02 0Á98). Real-time PCR revealed IL2 equal to background, HSP70 elicits primary responses from newborn CD4 T but significantly higher IFNg when DCs were pulsed with HSP70 or KLH. cells in vitro No IL4 was detected. Levels of cell surface activation markers were higher on cells encountering HSP70, with CD25 and CD45RO expression O. Curran & D. J. Reen increased. Restimulation with HSP70-pulsed DCs confirmed antigen- Children’s Research Centre, Our Lady’s Hospital for Sick Children, specific responses, showing increased proliferation and IFNg release, Crumlin, Dublin 12, Ireland with no IL4 production. In this system, HSP70 elicits responses from Cord blood is a model system for investigating truly naive T-cell newborn CD4 cells to a self-antigen, suggesting a role in autoimmune responses. While mitogen and superantigen stimulation are used to study responses.

Apoptosis

OP15 possible role of mitochondria-dependent mechanisms has not been Making macrophages die: can we switch off equally evaluated in this condition. We have examined mitochondrial FLIP? membrane depolarization by two-colour flow cytometry on enriched thyrocytes using the lipophilic cationic probe, JC-1, which exists as a R. Buchan, R. Dawson, L. Duncan & J. Liversidge green fluorescent monomer at low mitochondrial membrane potential Institute of Medical Sciences, University of Aberdeen, Foresterhill, (DCm) and forms orange aggregates at higher DCm. In basal conditions, Aberdeen, AB25 2ZD, UK 5/7 Graves and 2/6 multi-nodular goitre preparations showed lower ED1þ monocyte-macrophages generate nitric oxide and superoxide but levels of JC-1 aggregate staining. Following pro-inflammatory cytokine show resistance to apoptosis during experimental autoimmune uveitis. An stimulation, a decrease in DCm of at least 10–15% was obtained in understanding of the mechanisms that allow these primary effectors of 3/7 Graves’ versus 1/6 multi-nodular thyroids. DCm changes were photoreceptor damage to remain resistant to apoptosis, and a means to confirmed by a simultaneous decrease in MitoTracker orange staining lower such resistance, may enable better treatment of uveitis. In this study, of active mitochondria. Double immunofluorescence staining of enriched we characterized the expression of FLICE-inhibitory protein (FLIP), a thyrocyte monolayers showed cytochrome c release into the cytoplasm in caspase 8-inhibitory molecule, throughout the disease time course in preparations where MitoTracker orange staining was disrupted or lost. Lewis rats. FLIP was expressed late in the disease process and primarily These data suggest that pro-inflammatory cytokines may affect the within cells in the target retinal tissue. Expression increased significantly mitochondrial integrity and possibly contribute to pro-apoptotic factor from peak disease onwards and declined rapidly as disease subsided, a release. pattern correlating broadly with changes in the ED1þ population. FLIP was expressed in ED1þ macrophages, but was also found in ED1– cells. Additionally, in vitro bone marrow derived macrophages show upregu- OP17 lated levels of FLIP following stimulation, which is suppressed using The effect of Cord blood (CB) sera on lymphocyte therapeutic treatments (L-NAME and TNF-a R-Ig fusion protein) known apoptosis to reduce ocular pathology in EAU, as well as a PKC inhibitor. Together, K. Bogunia-Kubik,y S. Brown, P. Natarajan, J. A. Madrigal & these data suggest a complex regulatory network controlling FLIP expres- S. B. A. Cohenz sion, which may prove a useful therapeutic target in uveitis and other Anthony Nolan Research Institute, Pond Street, London, NW3 2QG, UK, autoimmune inflammatory diseases. yInstitute of Immunology & Experimental Therapeutics, Weigla 12, Wroclaw, 53–114, Poland, zUK Cord Blood Immunology Group, Herts, EN5 4ZD, UK OP16 Our previous results showed that CB sera affect T-cell responsiveness to Effects of pro-inflammatory cytokines on IL-2 by increasing the frequency of IL-2 producing cells (but not the mitochondrial membrane potential in thyroid amount of IL-2 secreted) and CD25 expression on stimulated PBMC. In cells contrast to mitogen or allostimulation, CB sera also enhanced the pro- liferation of adult cells in response to mitogen plus IL-2 when compared to L. J. Hammond, S. Waheed & R. Mirakian the adult sera (P < 0Á05). In the present study, we analysed the influence of Department of Immunology & Surgical Unit, St. Barts and the Royal different sera and IL-2 on lymphocyte apoptosis. Activated adult cells London School of Medicine, 38 Little Britain, London, EC1A 7BE, UK were stained with merocyanine and anti-CD3 and the percentage of the Thyrocytes from severely infiltrated autoimmune thyroid glands die by total lymphocyte population and CD3þ lymphocytes undergoing apop- apoptosis. Death receptor activation appears to be involved but the tosis were determined by flow cytometry. CB sera decreased the frequency

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Apoptosis 83 of apoptotic cells as compared to the adult sera (P < 0Á005). This effect population was becoming apoptotic. It was concluded that CsA is involved was independent of the presence of IL-2 (P < 0Á005). We suggest that the in apoptosis of Molt4 cells during the first 48 hr of drug addition. After this serum that CB cells were derived from, may play a role in anergy time, the same population of leukaemic cells were stopped in the G1 phase induction and not enhance apoptosis. These data may help to explain of the cell cycle with no signs of apoptosis or cell division. the lower incidence of GvHD post CB transplantation. It might be possible that since CB cells have been previously incubated in CB sera (in vivo), they are anergic and therefore unable to initiate or perpetuate OP19 GvHD. T-cell apoptosis as a mechanism of immune privilege R. A. Harry, Z. Walters, J. Greenwood & V. L. Calder Department of Clinical Ophthalmology, Institute of Ophthalmology, OP18 University College London, 11–43 Bath Street, London, EC1V 9EL, Cyclosporin A induces apoptosis in a cell UK cycle-dependent manner The blood–brain barrier (BBB) is known to selectively control the entry of J. Ochando, E. Sala,y J. O’Neill & P. Whiting immune cells to the central nervous system (CNS). Activated T cells, School of Pharmacy and Pharmaceutical Sciences, which cross the BBB, have been shown to be susceptible to apoptosis. This De Montfort University, Leicester, LE1 9BH, yDivision de study, using a rat brain derived EC line (GP8/3Á9) as a model of the BBB, Genetica, Universidad de Alicante, Alicante, E03080, investigates apoptosis of T cells which transmigrate the EC monolayer. A Spain migration assay was established where GP8Á3 and control HEV EC (non- The therapeutic- and side-effects of the fungal-derived immunosuppres- CNS EC) were cultured on 3Á0 mmol transwell inserts. ConA stimulated T sive drug Cyclosporin A (CsA) are still controversial. Several authors have cells were added to EC monolayers for 24–48 hr. Cells were then suggested that CsA inhibit apoptosis while others have claimed the harvested, stained and analysed by FACS and the percentage of live, opposite. In this study, the effects of CsA on cell death were investigated early and late apoptotic cells calculated. T-cell apoptosis as a result of EC within the different stages of the Molt-4 cell cycle using flow cytometry migration was also investigated by TUNEL staining, immunofluorescence techniques. The percentage of mitotic cells decreased significantly after and e/m. Transmigration of T cells across GP8/3Á9 monolayers resulted in the addition of CsA (5m mol) compared to normal controls (P < 0Á05). The significant T-cell apoptosis (56Á3% versus 22Á4% controls; r ¼ 0Á015). No percentage of apoptotic cells in CsA-treated cells was higher than that of significant apoptosis was observed using HEV EC. TUNEL positive cells controls (P < 0Á01), reaching maximum values after 36 hr of drug addi- were detected post migration while e/m investigation showed chromatin tion. After this time, the percentage of apoptotic cells decreased changes characteristic of apoptosis. Results from this study suggest (P < 0Á01), reaching normal values after 72 hr in culture. A decrease in T-cell apoptosis as a mechanism for maintaining immune privilege within the percentage of mitotic cells was positively correlated with that the CNS and support an important role for microvascular EC in this of apoptotic in CsA-treated cells indicating that the G2/M-phase cell process.

Asthma

OP20 IL4 can be detected by ELISPOT in atopic asthmatics compared to normal Use of ELISPOT to demonstrate increased subjects. aeroallergen-specific IL4 producing T cells in asthmatic adults OP21 S. D. Message, P. Pala, P. J. M. Openshaw & S. L. Johnston Antisense IL-4 constructs as possible therapeutic Respiratory Medicine, NHLI, Imperial College, London, W2 1PG, tools for the treatment of asthma UK S. F. Khurrum, L. J. Hammond, P. A. Biro & R. Mirakian Atopic asthma is thought to be associated with excess type 2 cytokine Department of Immunology, QMUL, London, EC1A 7BE, UK production. Conventional methods for detecting type 2 cytokine produc- tion such as intracellular staining do not detect allergen-specific cells and Asthma is a chronic inflammatory disease of increasing prevalence. Th2 have shown increased production of type 1 but not type 2 cytokines in lymphocytes control the inflammatory response through cytokine secretion, asthma. Our aim was to compare allergen specific type 1 and type 2 particularly IL-4, IL-5 and IL-13. IL-4 plays a major role in IgE class responses in peripheral blood by ELISPOT between atopic asthmatic switching and production which is responsible for the allergic symptoms. (AA) and non-atopic normal adults (N). PBMC were obtained from 21 AA Down-regulating IL-4 synthesis might be an effective way of controlling the and 10 N. Atopy was defined by skin prick testing. ELISPOT was used to inflammatory response. Antisense constructs are designed to specifically determine frequencies of cells producing IL4 and IFNg after ex vivo hybridize to a target mRNA and interfere with protein synthesis. We have culture with cat Feld, birch Betv and dust mite Derp allergens. The prepared 58 bp oligonucleotides spanning the 50-end of the mouse IL-4 gene median frequency per 106 of cells producing IL4 in AA was significantly in both sense and antisense orientations and cloned them into an inducible increased to Feld (37 versus 7 P ¼ 0Á01) and Betv (13 versus 5 P ¼ 0Á04) expressionvector.Theseconstructsweretransfectedintoamouselymphoma and non-significantly increased to Derp (73 versus 13 P ¼ 0Á2) relative to cell line, EL-4, with a transfection efficiency of >60%. Ponasterone, an N. No significant differences were observed for IFNg. In cat-allergic AA, inducing agent, was added and then withdrawn and supernatants assayed for there was a significant positive correlation between size of skin prick test IL-4 production. A substantial inhibition (>50%) in the production of IL-4 response and frequency of IL4 producing lymphocytes after culture with was obtainedinthesamplestransfectedwith theantisense constructsandthis Feld (r ¼ 0Á662 P ¼ 0Á01).Increased allergen specific T cells producing inhibition was not observed when the inducing agent was withdrawn.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 84 B cells

B cells

OP22 OP24 B cells as regulators of autoimmune Visual analysis of k5 transcription during B-cell T cells development M. J. McGeachy & S. M. Anderton M. J. D. Parker, L. Erlandsson & I-L. Martensson-Bopp University of Edinburgh, ICAPB, Edinburgh, EH9 3JT, UK The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK The importance of B cells in the T cell-mediated autoimmune demye- B-lymphocyte development occurs in adult bone marrow and is comprised linating diseases, multiple sclerosis in humans and experimental auto- of several distinct cell stages, each characterized by discrete cell surface immune encephalomyelitis (EAE) in mice, is controversial. Recent work markers and by the status of the immunoglobulin heavy (IgH) and light has shown that while B cells are not required for induction of EAE as chain gene rearrangements. One important receptor in B-cell development previously thought, they are crucial for remission of disease. Using the is the precursor-B cell receptor (pre-BCR) composed of the IgH chain MOG35-55-induced model of EAE in C57BL/6 mice, we used bone protein and the surrogate light (SL) chain. The SL chain is composed of l5 marrow chimeras in which chosen defects are targeted specifically to B and VpreB proteins, both of which are only expressed in pre-B cells. We cells. B-cell activation (via CD40) and their IL10 production are abso- have shown that the SL chain as part of the pre-BCR is crucial for lutely required for remission of disease. We have also found that mice that proliferation to occur. Mice lacking functional VpreB or l5 genes show an have recently recovered from EAE are relatively resistant to re-induction, impairment in B-cell development at the pre-BI to large pre-BII cell stage showing a milder course of disease. Furthermore, transfer of lymphoid transition, resulting in a 40-fold reduction of pre-BII and immature B cells confers similar resistance to naive recipients. The ability of various cells. Our hypothesis is that the transcription of the SL chain genes and cell populations to mediate protection is under investigation, with pre- their surface expression as part of the pre-BCR controls proliferation of liminary results indicating the importance of B cells, as expected. the cell. To test this hypothesis, we analysed l5 gene transcription with Ongoing experiments to further define how and where B cells exert their primary transcript RNA FISH. Our results show that the l5 gene is regulatory effects will be presented. expressed as RNA primary transcript at the pre-BI cell stage, while at the pre-BII cell stage, expression has been or is being terminated. At the immature B-cell stage, we detect virtually no transcription of the l5 gene.

OP23 Identification and characterization of an alternative OP25 transcript of mouse B29 The alternative transcript of CD79b is over-expressed in B-CLL and inhibits signalling for apoptosis M. D. Fox, M. S. Cragg & M. J. Glennie Directors Group, Tenovus Research Laboratory, Southampton General M. S. Cragg, H. T. C. Chan, M. D. Fox, A. Tutt, D. Oscier,y Hospital, Southampton, SO16 6YD, UK T. J. Hambliny & M. J. Glennie Tenovus Research Laboratory, Cancer Sciences Division, General B29 (Igb, CD79b) and mb-1 (Iga, CD79a) form the CD79 heterodimer Hospital, Southampton, SO16 6YD, yDepartment of Haematology, Royal which is crucial for surface expression of immunoglobulin (Ig), and B-cell Bournemouth Hospital, Bournemouth, BH7 7DW, UK receptor (BCR) formation. An alternative B29 transcript has been reported in human B cells (hDB29) where exon 3 is deleted, resulting in a truncated The B-cell receptor for antigen (BCR) is composed of surface Ig, and a extracellular domain. Increased expression of hDB29 is observed in non-covalently associated signalling unit, the CD79a/b heterodimer. certain B lymphomas, including CLL. The physiological role of Recently, an alternative transcript of CD79b(DCD79b) has been reported hDB29 remains unclear, although recently we demonstrated that which is up-regulated in B-CLL and may explain the diminished BCR hDB29 appears to inhibit apoptotic signalling through the BCR. We have expression in these cells. Here, we assess the expression of DCD79b in B- now addressed whether alternative transcripts of B29 are present in other CLL and other lymphoid malignancies and investigate its function. High species. RT-PCR of murine B-cell lines demonstrated that all express a relative expression of DCD79b was confirmed in the majority of cases of truncated transcript of B29 (mDB29). Sequence cloning revealed it to be a B-CLL, and found in 6/6 cases of SLVL and hairy cell leukemia. In a range genuine product of alternative splicing and homologous to hDB29. To of Burkitt’s cell lines, expression of DCD79b correlated inversely with the investigate its translational properties, mDB29 was cloned into the pCI- ability of the BCR to signal apoptosis when cross-linked by Ab. Inter- puro expression vector and assessed in in vitro transcription/translation estingly, when Ramos cells with low DCD79b were transfected with this and transient transfection assays. Localization of the mDB29 protein was transcript, they were transformed from being sensitive to anti-Fcm- investigated following cloning of the transcript into a YFP expression induced apoptosis to being resistant. Although DCD79b was expressed vector. WEHI-231 were also transfected with pCI-puro-mDB29 vector to as protein, its over-expression did not reduce the level of cell surface BCR. observe effects on apoptotic signalling through the BCR in murine Finally, we show that the inhibitory activity of DCD79b depended on an B cells. intact leader sequence and a functional ITAM in the cytoplasmic tail.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Cellular immunology 85

Cellular immunology

OP26 OP28 Analysis of T1/T2 responses by intracellular Co-localization of lipopolysaccharide and CD14 staining of IFNc and IL-4 in T-lymphocyte subsets of studied by Single Particle Fluorescence Imaging goats immunized with a killed Ehrlichia ruminantium (SPFI) vaccine R. E. Barber, I. E. G. Morrison, R. J. Cherry & I. Esteves, N. Vachie´ry, C. Sheikboudou, D. Martinez & N. Ferna´ndez P. Totte´ Department of Biological Sciences, University of Essex, Wivenhoe Park, CIRAD EMVT, Guadeloupe, 97170, France Colchester, CO4 3SQ, UK All five animals vaccinated with the obligate intracellular bacterium The membrane bound receptor CD14, a GPI-anchored protein, is thought Ehrlichia ruminantium inactivated by azide (group I) were protected to be the primary receptor for lipopolysaccharide (LPS) in innate against a homologous challenge. In contrast, none of the three animals immune recognition. The absence of a transmembrane component vaccinated with denatured E. ruminantium survived (group II). We implies that other proteins must be involved in signalling the presence observed in group I animals, using flow cytometry-based intracellular of LPS to the intracellular domain. Associations between LPS and these staining of IFNg, a comparable contribution of CD4þ and CD8þ T cells to receptors are being investigated by three-colour digital fluorescence the production of IFNg triggered by invitro stimulation with E. ruminan- microscopy. Individual antibodies labelled by R-phycoerythrin at 1 : 1 tium antigen. In group II, we detected IFNg only in two out of three ratio can be imaged by a back-illuminated CCD camera, and appear animals, and only in CD8. Simultaneous staining of intracellular IFNg and as diffraction-limited spots; oligomers are identified by their higher IL-4 indicated that stimulated immune CD4 T cells produced IFNg in the intensity. LPS can be labelled by the fluorophore Alexa-488; in the absence of detectable IL-4, which is suggestive of a strict Th1 response. presence of serum much of the LPS forms small aggregates. Sub-micro- On the other hand, when the same cells were stimulated with concanavalin scopic autofluorescence can be detected by a third image using red A, Th1, Th2 and Th0 responses were detected. We confirmed by ELISA emission filters. Correlation between the images allows identification that stimulated immune cells also secreted IFNg. IFNg secretion was of autofluorescence, antibodies, LPS aggregates and co-localized anti- entirely dependent on CD4 as shown by addition of anti-MHC class II body and LPS. The high spatial precision of SPFI allows co-localization antibodies. In conclusion, our data strongly suggest that protective between particles to be determined at a molecular level. Receptors immunity induced in goats by the killed E. ruminantium vaccine is co-localized with LPS can be quantified and compared with LPS-free associated with a dominant Th1 response. controls; the implications for signalling of responses to bacterial infection are discussed.

OP27 Characterization of NOD-RIP-CD80 and NOD-RIP-CD86 OP29 mice Maintenance of a CD45RAþ CD4þ T cell pool in humans I. J. Thomas, L. Wen,y S. Guerder,z R. A. Flavell,y C. A. Janeway Jry & F. S. Wong M. V. D. Soares, J. E. Cook,y E. Derrett-Smith, M. Salmon,z Department of Pathology, University of Bristol, Bristol, P. C. Beverleyy & A. N. Akbar BS8 1TD, UK, zCentre d’Immunologie de Marseille-Luminy, Department of Immunology, Royal Free Hospital, Pond Street, London, CNRS-INSERM-Universite´ medicale de Marseille, NW3 2QG, yThe Edward Jenner Institute for Vaccine Research, Berkshire, 13288, France, yYale School of Medicine, New Haven, Compton, RG20 7NN, zMRC Centre for Immune Regulation, University of CT O6520, USA Birmingham, Birmingham, B15 2TT, UK CD80 and CD86 are important co-stimulatory molecules. Studies using The CD45RAþ T-cell pool is under two main constraints. Thymic Rat Insulin Promoter (RIP) CD80 and CD86 transgenic mice, where the involution decreases the number of these cells released to the periphery. transgene is expressed on the pancreatic islet b cells in B6 mice, showed Secondly, antigenic exposure drives CD45RAþ T cells into the ROþ T-cell that RIP-CD80 transgenic mice developed little insulitis or diabetes, pool. However, a CD45RAþ T-cell pool is maintained during ageing. whereas RIP-CD86 mice had islet infiltrating IL-4 and IFN-g expressing Three mechanisms may allow the replenishment of this pool: the presence cells, but did not develop diabetes. The aim of our study was to compare of residual thymic tissue; cytokine driven expansion; and reversion from these RIP-CD80 and RIP-CD86 transgenic mice on the non-obese diabetic ROþ to RAþ. We have previously shown that CD45RAþ T cells from (NOD) genetic background. Greatly accelerated diabetes occurs in the umbilical cord blood proliferate in response to IL-7. We have further NOD-RIP-CD80 mice, from 4 weeks of age compared to 12 weeks for found that similar culture in IL-7, CD4þ CD45RAþ T cells show telomere wild-type NOD mice by the N3 generation. The acceleration increases shortening with ageing, further suggesting that bystander proliferation with each backcross. In contrast, although NOD-RIP-CD86 mice also by cytokines may allow the maintenance of this pool. Reversion develop accelerated diabetes, the onset begins at 8 weeks of age with 50% from CD45ROþ to CD45RAþ has been described in CD8þ T cells. of mice developing diabetes by 14 weeks of age (in two separate founder Heteroduplex analysis of CD4þ CD45RAþ and CD45ROþ T cells from lines). This effect was seen once the mice were backcrossed >12 humans has revealed the presence of clones in both subsets. These clones generations to NOD mice. Thus, diabetes onset is less aggressive in increase in number with age, suggesting that apart from cytokine driven the NOD-RIP-CD86 mice compared with NOD-RIP-CD80 mice. The expansion, reversion may contribute to the maintenance of the CD45RAþ mechanism for this phenomenon is under study. T-cell pool.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 86 Cellular immunology

OP30 Recently, they are emerging as key ‘danger’ signals for the immune Identification of proteins associated with natural killer system and they have been shown to be able to generate protective cell lipid rafts immunity by eliciting T-cell responses. Here, we report using confocal microscopy, flow cytometric and biochemical studies that members of the S. B. Taner & D. M. Davis hsp family, hsp70 and hsp90, are present on the cell surface of unstressed Department of Biological Sciences, Sir Alexander Fleming Building, cells. Furthermore, we show that hsp expression is sensitive to pretreat- Imperial College of Science, Technology and Medicine, London, ment with trypsin and pronase (a compound that hydrolyses cell surface SW7 2AZ, UK proteins including membrane bound receptors). Natural killer (NK) cells provide a crucial first line of defence by their ability to lyse certain virally infected or transformed target cells without the need for prior antigen exposure. Lipid rafts, cholesterol- and sphin- OP32 golipid-rich membrane microdomains, have been implicated in NK cell The anergic T-cell immunological synapse activation, potentially acting as platforms to increase the local concentra- K. Yanagi, A. Verhoef,y M. Dallman & D. Davis tion of molecules involved in signal transduction. Identifying proteins Department of Biological Sciences, Sir Alexander Fleming Building, associated with NK cell lipid rafts should, therefore, provide an insight Imperial College of Science, Technology and Medicine, London, into the mechanisms underlying NK cell activation. Detergent-resistant SW7 2AZ, yDepartment of Allergy and Clinical Immunology, National lipid raft material was isolated from NK cells by sucrose density gradient Heart and Lung Institute, Imperial College School of Medicine, London, centrifugation, confirmed by Western blotting for the raft residing lipid, SW3 6LY, UK GM1 ganglioside. With a view to understanding the role of lipid rafts in assembly of the NK cell immune synapse, we also used Western blotting T-cell anergy is one way by which peripheral tolerance to specific antigens to identify some components of NK cell lipid rafts. can be maintained. However, the molecular mechanisms underlying T-cell anergy are largely unknown. Recently, it has emerged that the supramo- lecular organization of proteins at immune synapses can influence T-cell OP31 responses. Thus, here we set out to determine the organization of proteins Heat shock proteins hsp70 and hsp90 are present on at anergic T-cell immune synapses. Antigen-specific anergy was induced the cell surface of unstressed cells by stimulation of T cells with a high dose of a class II-restricted peptide in the absence of APCs. Anergized or resting T cells were then co-incubated R. Haston, M. Triantafilou & K. Triantafilou with antigen-coated APCs. To compare the ability of anergic and resting T Institute of Biomedical and Biomolecular Sciences, University of cells to bind the peptide-pulsed APCs, the number of cell conjugates made Portsmouth, School of Biological Sciences, King Henry Building, King was assessed by flow cytometry. To compare the organization of proteins Henry I Street, Portsmouth, PO1 2DY, UK at the anergic and resting T-cell synapses, conjugates were fixed, stained Heat shock proteins (hsps) represent a family of proteins whose expres- with mAb and imaged by laser scanning confocal microscopy. These data sion is induced by stress. Hsps are normally localized in the cytoplasm and indicate that anergic T cells are deficient in their ability to construct nucleus, and act as molecular chaperones for antigen presentation. immunological synapses.

Chemokines

OP33 OP34 Interactions between human mycoplasmas and In vitro chemotaxis of ovine eosinophils: cross- eosinophils in vitro reactivityofrecombinantinterleukin-5(IL-5)andeotaxin C. Donohue, C. Jepson & J. B. March L. A. Wildblood, D. A. S. Clark & D. G. Jones Moredun Research Institute, Edinburgh, EH26 0PZ, UK Moredun Research Institute, Edinburgh, EH26 0PZ, UK Mycoplasmas lead an obligate parasitic way of life. They communicate As part of the chemotactic host immune response in acute hypersensitivity with the host cell via their trilaminar membrane, allowing immunomo- conditions and parasitic infections, eosinophils are recruited to sites of dulation of host immune responses, hence enabling evasion or suppression inflammation by locally released and cell-specific chemotactic agents of their host defense mechanisms. Human strains, M. fermentans and such as IL-5 and eotaxin. IL-5 is known to be highly conserved amongst M. hominis are both common components of the normal flora, while mammals and functional cross-reactivity for sheep cells has been demon- M. pneumoniae is associated with asthma. As eosinophils are the major strated in terms of eosinopoiesis and survival. In this study, modified component of the inflammatory infiltrate in allergic disease, the relation- Boyden chambers were used to investigate in vitro chemotaxis of bone ship between these mycoplasmas and their chemotactic activity towards marrow-derived ovine eosinophils, in response to recombinant murine and eosinophils was investigated. A quantitative measure of invitro eosinophil human IL-5 and eotaxin, and the effectof specific antichemokineantibodies migration was calculated using the Boyden chamber assay. M. fermentans on these responses. Both eotaxin as well as IL-5 recombinants evoked a and M. hominis did not cause any significant chemoattraction of ovine significant (P < 0Á01) chemotactic response, which was inhibited in a dose- eosinophils. However, viable whole M. pneumoniae and a specific super- dependent and species cross-reactive manner by chemokine specific anti- natant fraction elicited a significant chemotactic response (P ¼ 0Á01) in a bodies. These results add further support to the highly conserved nature dose-dependent manner. This suggests an eosinophil chemotactic factor is of IL-5 and its biological reactivity. Moreover, for the first time, it has also secreted by M. pneumoniae. Further characterization of this active mole- been shown that eotaxin exhibits functional species cross-reactivity. These cule may enable its therapeutic use in protection against M. pneumoniae findings provide further evidence that the eosinophilic response has been infection. closely preserved during mammalian evolution.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Chemokines 87

OP35 regulates gene expression in certain cell types by targeting intracellular Characterization of the promoter for the CCR1 thiols and proteins. Questions arise regarding the cell and gene selectivity chemokine receptor of NO. In this study we examined the susceptibility of chemokine mRNA expression in human A549 epithelial and HMC-1 mast cells to NO. R. M. Phillips, V. E. L. Stubbs, T. J. Williams, I. Sabroey & Stimulation of A549 cells with IFN-g, TNF and IL-1b induced mRNA for J. E. Pease the chemokines RANTES, IP-10, MCP-1 and IL-8. Exposure of the cells Leukocyte Biology, Biomedical Sciences Division, Imperial College for up to 24 hr with chemical NO donors did not influence the chemokine Faculty of Medicine, Sir Alexander Fleming Building, Exhibition Road, mRNA response. Stimulation of human HMC-1 cells with the intracellular London, SW7 2AZ, yDivision of Genomic Medicine, Royal Hallamshire Ca2þ-mobilising agent, thapsigargin, induced expression of the chemo- Hospital, Sheffield, S10 2JF, UK kines MIP-1a/b, MCP-1, IL-8 and I309. Again, NO did not influence the CCR1 is expressed by neutrophils, basophils and eosinophils. We have chemokine mRNA response. These results contrast with parallel results previously shown that high levels of functionally active CCR1 are exp- showing that NO time-dependently inhibits cytokine mRNA responses in ressed on eosinophils in approximately 15% of individuals. Definition of rat RBL mast cells and human PBMC. We conclude that either chemokine the CCR1 promoter should facilitate our further understanding of the and cytokine expression are differentially regulated by NO, or immune regulation of CCR1 expression by eosinophils. We used 50RACE analysis cell types are phenotypically diverse in NO responsiveness. of CCR1 mRNA to identify the transcription start site (þ1), and analysed a 1Á7-kb region upstream of and including 114 bp of the untranslated first OP38 exon for promoter activity. Using a reporter gene assay, a promoter was Leukocyte expression of CCR1 and CCR3 identifiedina 182/þ114 bp sequence of the CCR1gene. Promoter activity V. E. L. Stubbs, M. R. Henson,y T. J. Williams & I. Sabroey was observed in eosinophilic HL-60 clone 15 cells, HEK 293 cells and Leukocyte Biology, Biomedical Sciences Division, Imperial College monocytic RAW cells. Preliminary data indicates that a region of 1588/ London, London, SW7 2AZ, yDivision of Genomic Medicine, University of 182 bp may enhance this minimal promoter activity in HL-60 clone 15 Sheffield, M Floor, Royal Hallamshire Hospital, Glossop Road, Sheffield, cells. Comparison of promoter regions from individuals expressing high S10 2JF, UK levels of CCR1 on eosinophils has not revealed any polymorphism to date, but analysis of the CCR1 promoter may help elucidate the mechanisms Eosinophils from a subgroup of donors express relatively high levels of the underlying the phenomenon of increased responsiveness to CCL3/MIP-1a. chemokine receptor, CCR1, associated with enhanced responsiveness to CCL3, which may affect the utility of CCR3 antagonists in the treatment of asthma. We investigated expression of CCR1, and the major eosinophil OP36 chemokine receptor CCR3, in 110 normal and atopic donors. Eosinophil Production of CXCR3/CCR5 chemokine ligands in CCR1 expression levels were non-normally distributed, and did not juvenile idiopathic arthritis (JIA) correlate with levels of CCR3. CCR3 expression was observed on D. Pharoah, R. Tatham,y H. Varsani, N. Kleinz & L. Wedderburn eosinophils and basophils at high levels, and monocytes and neutrophils Rheumatology Unit, Institute of Child Health, 30 Guilford Street, at low levels, and showed strong correlation in expression between cell London, WC1N 1EH, yUniversity of Liverpool Medical School, Liverpool, types. In contrast, CCR1 was seen at highest levels on monocytes and zInfectious Disease and Microbiology Unit, ICH, London, UK basophils, and showed poor correlation between cell types. There was no correlation between expression of these receptors on any cell type and a Introduction Juvenile arthritis involves the accumulation of activated, diagnosis of atopy or levels of serum IgE. These data support the existence polarized T cells into the joint. One aspect of polarization is over- of a subgroup of individuals whose eosinophils express high levels of expression of chemokine receptors CCR5 and CXCR3. The production CCR1, and also highlight differences in the control of CCR1 and CCR3 of ligands for these receptors by synovial T cells may serve to propagate expression on selected cell types. the inflammatory response. Acknowledgement Funded by the National Asthma Campaign and the Methods RANTES, MIP-1a and IP-10 production by synovial fluid (SF) MRC. T cells was compared to adult normal and matched JIA peripheral blood (PB) T cells by RT-PCR and by intracellular flow cytometry. Results While no difference was observed with MIP-1a and IP-10, OP39 RANTES mRNA was expressed at higher levels in SF T cells compared The relative contribution of CCR4 and CCR8 to function to PB. RANTES protein was also increased in SF CD8 T cells. In PB, of in vitro polarized human Th2 cells þ RANTES protein was produced by CD8 CD28-perforinþ cells whereas C. Rothwell, B. Waters, C. Walker & S. J. Charlton RANTES production in SF T cells is not restricted to this mature effector Novartis Horsham Research Centre, Horsham, RH12 5AB, UK population. Increased, dysregulated RANTES production by SF T cells provides further evidence on the factors propagating chronic synovial The chemokine receptors CCR4 and CCR8 are expressed on Th2 cells. inflammation in JIA. Here, we compare the functional responses of human Th2 cells after CCR4 and CCR8 ligation by MDC and I-309, respectively. Th2 cells were prepared from CD4þ CD45RAþ PBMCs. Phenotype was confirmed at OP37 second week and function was examined at third week. In chemotaxis Comparative resistance of chemokine mRNA assays, MDC was a potent and efficacious agonist, with a pEC50 of 8Á88, expression by human epithelial and mast cells to nitric whereas I-309 had no effect. Taqman analysis revealed increased CCR4 oxide and CCR8 mRNA levels upon activation with anti-CD3/CD28 over a 48- hr timecourse. CCR4 levels were approximately twofold that of CCR8 at y C. A. Gibney, B. F. Flanagan & J. W. Coleman all time points. Activation did not increase Th2 chemotaxis in response to y Department of Pharmacology, Department of Immunology, University MDC, but increased I-309-induced chemotaxis to 16Á0% of that to MDC, of Liverpool, Liverpool, L69 3GA, UK with a pEC50 of 9Á01. The ability of MDC and I-309 to induce calcium Nitric oxide (NO) is produced at high and sustained levels by inducible transients in Th2 cells was examined using FLIPR. Both MDC and NO synthase in immunity and inflammation. It is a short-lived radical that I-309 increased intracellular calcium with pEC50s of 8Á94 and 9Á64,

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 88 Chemokines

respectively. However, the maximal I-309 response was only 14Á4% of Conclusions On activation with cytokines both HIEC and BEC express that to MDC. In conclusion, CCR4 induces calcium signalling and CXCR3 ligands that promote lymphocyte recruitment. We have demon- chemotaxis of polarized Th2 cells. CCR8-induced calcium responses strated for the first time that CXCR3 ligands can support capture of and chemotaxis were much weaker, consistent with either lower effector lymphocytes from flow. receptor expression levels or diverse functions of the two chemokine receptors. OP41 The role of PI3-kinase(s) in SDF-1-mediated signalling OP40 and chemotaxis Differential expression and functional significance of A. P. Curnock & S. G. Ward adhesion molecules and CXCR3 binding chemokines University of Bath, Bath, BA2 7AY, UK by liver-derived endothelial and epithelial cell populations The CXC chemokine SDF-1 and its receptor CXCR4 play essential roles in development of the immune system and inflammation. We have S. M. Curbishley, P. Lalor & D. H. Adams previously demonstrated that SDF-1 stimulates phosphoinositide 3-kinase Liver Research Laboratories, MRC Centre for Immune Regulation, (PI3K) activity in human T cells and that this activity is required for SDF- University of Birmingham, Edgbaston, Birmingham, B15 2TH, UK 1-mediated chemotaxis. The use of pharmacological inhibitors is unable Introduction The recruitment via endothelium of CXCR3high T cells to assign SDF-1-stimulated accumulation of PtdIns(3,4,5)P3 and chemo- results in immune damage in liver diseases such as primary biliary tactic response to a particular PI3K isoform. To determine the relative roles cirrhosis and allograft rejection and the retention of these effector of different PI3K isoforms in SDF-1 signalling, Jurkat clones were estab- lymphocytes via interactions with biliary epithelium drives chronic lished that stably express dominant-negative constructs of class 1A and 1B inflammation and tissue damage in these diseases. We have investigated PI3Ks, under the control of the Tet-Off inducible gene system. Our results the role of liver-derived CXCR3 ligands in the recruitment of lymphocytes indicatethatexpressionofkinasedeadPI3KgorDp85(unabletobindp110a, via hepatic endothelium. b or d) inhibits SDF-1-mediated PI3K activation (as indicated by the ab- Results Human intrahepatic endothelial (HIEC) cells and biliary epithe- rogation of PKB phosphorylation and activity), but had no effect on SDF-1- lial cells (BEC) secreted IP-10, ITAC and HuMig in response to IFN-g and mediated ERK1/2 phosphorylation. Expression of kinase dead PI3Kg levels of chemokines were increased by adding TNF-a, TNF-b or IL-1. partially inhibited SDF-1-mediated chemotaxis (60–80% inhibition) and, Cytokine-stimulated HIEC were able to support the adhesion of surprisingly, Dp85 also inhibited chemotaxis (40–50% inhibition). The CXCR3high lymphocytes in a flow-based adhesion assay. The addition residual chemotactic activity in these dominant-negative PI3-K clones of a function blocking anti-CXCR3 mAb reduced the levels of adhesion could be further inhibited by PI3K inhibitors. These studies indicate crucial recorded. roles for multiple PI3-kinase isoforms in the co-ordination of chemotaxis.

Complement

OP42 OP43 A monoclonal antibody recognizing SCR 1–2 of CD55 Complement-mediated lysis by anti-CD20 mAb blocks Daf activity giving enhanced C3b/c deposition correlates with segregation into lipid rafts on cells M. S. Cragg, S. M. Morgan, H. T. C. Chan, B. P. Morgan,y R. Bradley, J. Morgan, I. Spendlove & L. G. Durrant A. V. Filatov,z P. W. M. Johnson,§ R. R. French & M. J. Glennie CR UK Department of Clinical Oncology, Nottingham University City Tenovus Research Laboratory, Cancer Sciences Division, General Hospital, Nottingham, NG5 1PB, UK Hospital, Southampton, SO16 6YD, yUniversity of Wales, Department Medical Biochemistry, Cardiff, CF14 4XN, UK, zInstitute of Immunology, CD55 (decay accelerating factor) is one of a number of membrane- Kashirskoye Shosse 24–2, Moscow, 115478, Russia, §Medical Oncology, bound complement regulatory proteins that is widely expressed in most Cancer Sciences Division, General Hospital, Southampton, SO16 6YD, UK cells including endothelial cells and leukocytes: CD55kocytes. CD55 functions to accelerate the decay of both complement activation path- Despite the success of anti-CD20 mAb in the clinic, there remains ways, specifically C4b2a or C3bBb and corresponding C5 convertases, uncertainty about its mechanism of action. In vitro studies revealed that thus giving rise to bystander protection from complement-mediated anti-CD20 mAb, with one exception (B1), are unusually effective at attack. CD55 comprises of four short consensus repeat domains recruiting human complement (C0). This activity is important invivo, (SCR). Classical pathway regulatory activity has been located in since C0 depletion using CVF markedly reduced the efficacy of two anti- SCR 2–3 and that for the alternative pathway in SCR 2–4. SCR 3 CD20 mAb (1F5 and Rituximab) against lymphoma xenografts, but did mutations have shown to disrupt regulatory activity for both pathways. not affect the potency of B1. Differences in C0 recruitment could not be This inhibition can also be achieved using antibodies to SCR 3. Using a explained by the level of mAb binding or isotype, but did correlate with CD55 overexpressing cell line we have demonstrated that monoclonal redistribution of CD20 into membrane rafts. Membrane fractionation antibodies to SCR 3 (1C6, 1H4 and BRIC 216) are able to block CD55 confirmed that B1, unlike 1F5 and Rituximab, was unable to translocate function, enhancing C3b/c deposition. Furthermore, a monoclonal anti- CD20 into lipid rafts. In addition, we were able to drive B1 into a 0 body (791T/36) recognizing SCR 1–2 is superior at blocking CD55 detergent-insoluble fraction of the cell with an F(ab )2 anti-Ig Ab, a activity than SCR 3-directed antibodies, resulting in greater C3b/c treatment which also conferred the ability to activate lytic C0. Thus, we deposition. This provides a novel therapeutic approach to inhibiting have shown that an important mAb effector function appears to be CD55 function. controlled by movement of the target molecule into membrane rafts.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Cytokines 89

Cytokines

OP44 OP46 The role of IL-18 in CD8 T cell-mediated immune Alterations in the hepatic cytokine environment regulation in vitro induced by malignancy M. Salagianni, M. J. Thomas, A. Noble & A. M. Kelly, L. Golden-Mason, G. McEntee,y O. Traynor,y D. M. Kemeny D. Doherty,z J. E. Hegartyy &C.O’Farrelly GKT School of Medicine, Department of Immunology, Education & Research Centre, yNational Liver Unit, St. Vincents 123 Coldharbour Lane, London, SE5 9NU, UK Hospital, Dublin 4, zNational University of Ireland, Maynooth, Kildare, Ireland CD8 T cells promote Th1 and inhibit Th2 responses invivo via dendritic cell (DC)-IL-12. As IL-12 is not the only DC cytokine A defect in the intrahepatic cytokine network may contribute to the that induces CD4 T cell IFNg, we investigated the role of IL-18. CD4 development of hepatic malignancy. Normal (n ¼ 11) and tumour-bearing T cells from C57BL/6 mice were cultured with anti-CD3/CD28. (n ¼ 12) liver was snap frozen and powdered. IL-2, -10, -12, -18, TNF-a Addition of IL-18 and IL-12 up-regulated CD4 T cell IFNg synergisti- and IFN-g levels were quantified by ELISA. IFN-g inducing cytokines, cally. IL-12-induced Th1 cells responded to IL-18 by proliferation and IL-12 and IL-18 were increased in tumour-bearing liver (4Á18 versus IFNg secretion. Induction of Th1 cells was significantly impaired in CD4 2Á99 ng/100 mg protein, P < 0Á05; 236Á6 versus 112Á4 ng, P < 0Á005, T cells from both IL-12 and IL-18 knockout mice, while CD8 T cell respectively). In contrast, TNF-a, involved in liver regeneration as well differentiation into Tc1 cells was IL-12- and IL-18-independent. IL-18 as IFN-g production, was significantly reduced (0Á19 versus 0 ng, had no significant effect on functionally committed CD4 Th1/Th2 or CD8 P < 0Á01). However, levels of IFN-g were significantly raised (13Á57 Tc1/Tc2 cells. Cognate induction of IL-12 and IL-18 from DC by CD8 versus 4Á82 ng). There was no associated rise in IL-2 levels. IL-10 levels and CD4 T cells was also investigated invitro. Bone marrow DC (BM- were also raised in tumour-bearing liver (12Á74 versus 2Á31 ng, DC) were generated from C57BL/6 mice cultured with GM-CSF and IL-4 P < 0Á0001). The IFN-g:IL-10 ratio was significantly reduced in for 9 days. Mature DC, expressing high levels of both MHC class II and tumour-bearing liver (1Á37 versus 4Á35, P < 0Á05), reflecting the greater CD11c (80%), were tested in a mixed lymphocyte reaction (MLR) with IL-10 levels in these samples, which may suppress local antitumour CD8 T cells from BALB/c mice. The data show that DC-CD8 T cell responses. The lack of a TNF-a response in tumour-bearing liver suggests interaction in the MLR induces DC-IL-12p40 and IL-18 cytokine secre- a defect in inflammatory antitumour responses and in tissue repair. Failure tion. These data suggest that the ability of CD8 T cells to regulate Th1/Th2 to up-regulate IL-2 may result in insufficient expansion and activation of immune responses is by the enhancement of IL-12/IL-18 production by tumour-specific cytotoxic T cells. These results provide a rationale for the DC. use of IL-2 in the treatment of hepatic malignancy.

OP47 IL-13 and IL-13Arg110Gln demonstrate similar activity OP45 in an in vitro model of cytokine-induced eotaxin release Cytokine response of ovine c/d T cells to mitogenic from human lung fibroblasts stimulation S. Parveen, I. Hunt, R. Schmitt,y M. Zurini,y J. F. Zuber,y M. S. Rocchi, K. McKay, G. Entrican, S. Wattegedera & H. Sparrer,y C. Walker & E. Campbell D. J. McKeevery Novartis Pharma, Horsham, RH12 5AB, UK, yNovartis Pharma, Basle, Moredun Research Institute, Edinburgh, EH26 0PZ, CH 4002, Switzerland yUniversity of Edinburgh, Edinburgh, EH25 9RG, UK IL-13 is implicated in the pathogenesis of asthma and induces a number of Although g/d T cells constitute a major T-cell subset in ruminants, their inflammatory changes. To assess the activity of recombinant hIL-13 function is poorly defined. To date, there are no reports on the ability of proteins, we have established an invitro model of eotaxin generation ovine g/d T cells to produce cytokines in response to different stimuli. We from primary human lung fibroblasts. IL-13 is usually expressed with an have investigated the expression of IFN-g and IL-4 in g/d T cells exposed Arg at position 110, but a polymorphism results in the substitution of to mitogenic stimuli by intracellular flow cytometry. We have chosen to R110Q in a region of the protein thought to bind to IL-13Ra1. This variant test the effect of mitogens in first instance, since antigenic specificities and has been associated with disease parameters in some asthmatic popula- restriction parameters of g/d T cells are not sufficiently defined to explore tions, but its functional significance is not clear. Both IL-13 and IL- antigenic responses. Chinese hamster ovary (CHO) cells transfected with 13R110Q induced comparable time- and dose-dependent induction of cDNAs encoding for IFN-g or IL-4 were used as positive control. Analysis eotaxin (EC50 1Á6 0Á6 ng/ml, 4Á0 0Á5 ng/ml respectively, at 24 hr), of ovine PBMCs exposed in vitro for 6 hr to a variety of mitogens (ConA, despite the slightly lower affinity with which IL-13R110Q bound to

PHA, LPS or PMA/ionomycin) revealed that cytokine production sIL-13Ra1/Fc chimera (IL-13 Kd ¼ 4Á9 0Á5nM, IL-13 R110Q ¼ occurred predominantly in the g/d TCR negative lymphocyte population. 14Á1 0Á9nM by BIAcore). We assessed affinity of the proteins for the This may be due to differential requirement for this cell subset when sIL-13Ra2/Fc chimera. IL-13 and IL-13R110Q bound with comparable compared with other T cells. We plan to investigate ovine g/d T-cell Kd ¼ 161 26 pM and 158 28 pM respectively. No differences were requirement for cytokine production further, both invitro and invivo, observed in the ability of this chimera to inhibit IL-13R110Q or IL- specifically in lesions associated with intracellular pathogens of sheep 13-induced eotaxin generation. In conclusion, no clear functional differ- (e.g. mycobacteria, chlamydia). ences were observed between IL-13 and IL-13R110Q in this assay system.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 90 Cytokines

OP48 age-related disease, then there may be an age-related reduction in Interleukin-6-gene C/G174 polymorphism in octo/non- homozygotes, who produce higher levels of IL-6. This hypothesis was agenarians in the BELFAST study: reciprocal effects on tested in competent, octo/non-agenarians from the Belfast elderly long- IL-6, sIL-6R and IL-10 itudinal free-living ageing study (BELFAST), and found a frequency reduction in GG homozygotes of 10%. CC homozygotes had higher serum I. M. Rea, O. Ross,y M. A. Armstrong,z S. E. McNerlan, IL-6 versus GG (P ¼ 0Á055), with reciprocal changes in anti-inflammatory B. Loughrey, D. H. Alexander,§ M. Currany & D. Middletony IL-10 (P ¼ 0Á05). There was spontaneous production of Il-6 and IL-10 Department of Geriatric Medicine, Whitla Medical Building, 97 Lisburn from mononuclear cell monolayers in aged subjects. In the BELFAST, Road, Queens University of Belfast, Belfast, BT9 7BL, yNorthern Ireland there is a reduction in frequency of GG homozygotes in octo/non- Histocompatability and Immunogenetics Centre, Belfast City Hospital, agenarians, higher IL-6 associated with CC homozygotes and reciprocal Belfast, BT9, zImmunology and Microbiology Department, Royal Trust changes for anti-inflammatory IL-10. Group Hospitals, Queens University of Belfast, Belfast, BT12, §Haematology Laboratory, Belfast City Hospital, Belfast, BT9, UK This study measured the frequency of GG homozygotes of 174C/GIL-6 polymorphism, arguing that if IL-6 tracks with functional disability and

Dendritic cells

OP49 by these two forms of the same protein, the internalization pathways Dendritic cell (DC) accumulation in draining lymph utilized by DC for the uptake of LF have been explored. Immature murine nodes induced by chemical contact and respiratory bone marrow-derived DC were pulsed for 30 min with equivalent con- allergens centrations of nLF or rLF (0Á1–1000 mg/mL) at 378 and 48C, and uptake visualized by flow cytometry using an anti-human LF antibody. At all K. Clelland, M. Cumberbatch, R. J. Dearman & I. Kimber concentrations tested, rLF was internalized more efficiently than nLF, Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, with DC exhibiting 2–5-fold higher mean fluorescence intensities, despite SK10 4TJ, UK the anti-LF antibody recognizing both LF species equally when tested by It has been reported previously that preferential activation of T helper ELISA. Pretreatment of DC with mannose resulted in greater inhibition of (Th)1 and Th2 cells is observed following repeated topical exposure of rLF uptake (80%) than that observed for nLF (45%). These data suggest BALB/c strain mice to chemical contact and respiratory allergens, and that that nLF and rLF are handled differently by the mannose-sensitive uptake only those chemicals that provoke respiratory allergy in humans induce pathway(s) of DC; observations which may in turn impact upon the IgE antibody responses in these mice. To explore the contribution of DC to outcome of the immune response. such responses, BALB/c strain mice have been exposed topically to the contact allergen dinitrochlorobenzene (DNCB) or the respiratory allergen trimellitic anhydride (TMA), at doses shown previously to be of similar OP51 immunogenicity. Excision of draining lymph nodes (DLN) 24 hr follow- Chemical allergen-induced changes in cytokine ing exposure revealed increases in cellularity and CD11cþ/Iaþ DC expression in human blood-derived dendritic cells number that were both significantly higher for DNCB compared with (DC) TMA. These observations reflect the delayed kinetics of LC migration C. Sellick, C. J. Betts, R. J. Dearman & I. Kimber reported previously for TMA. DLN cellularity and DC accumulation Syngenta CTL, Alderley Park, Cheshire, Macclesfiled, SK10 4TJ, continued to increase for both allergens 48 and 72 hr later, with similar UK values being obtained for DNCB- and TMA-treated mice. No differences in phenotypic characteristics of DC were observed. It is proposed that Langerhans cells (LC) play pivotal role in cutaneous immune responses, these early kinetic differences in DC responses to chemical allergens may including skin sensitization. Analyses of these cells have been facilitated influence the quality of immune response induced. by methods for the generation of DC with an LC-like phenotype from peripheral blood mononuclear cell precursors. CD14þ precursors have been isolated by negative selection and cultured for 5 days with inter- OP50 leukin IL-4, granulocyte/macrophage-colony stimulating factor and trans- Influence of glycosylation status on forming growth factor-b. In 21 of 23 separate isolations, the cell the uptake of native and recombinant population acquired an LC-like phenotype with high expression of lactoferrin by bone marrow-derived dendritic CD1a and MHC class II (57–96% and 88–99% positive cells, respec- cells tively), and low or absent expression of CD14 and CD86 (<3%) as determined by flow cytometry. On day 5, cells were stimulated with M. Cumberbatch, R. J. Dearman & I. Kimber 0Á5mM of the potent skin sensitizer 2,4-dinitrofluorobenzene or vehicle Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, alone (0Á01% dimethyl sulphoxide) for between 1 and 24 hr, cells har- SK10 4TJ, UK vested and RNA prepared for cytokine analysis by RT-PCR. Despite the Human recombinant (r) and native (n) lactoferrin (LF) exhibit identical relatively consistent cellular phenotype, allergen-induced up-regulation in amino acid sequences and 3-D X-ray crystallographic structures. How- expression of IL-1b, IL-6, IL-12 and IL-18 was observed in approximately ever, these proteins differ with respect to glycosylation status and their 50% of donors. These data reveal interdonor variation in responsiveness to ability to provoke IgG antibody responses in BALB/c strain mice. To allergen which may be related to the level of constitutive cytokine mRNA explore the mechanisms responsible for the different responses stimulated expression.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Dendritic cells 91

OP52 dependent upon 25-hydroxyvitamin-D31a-hydroxylase (1aOHase), a Respiratory tract dendritic cells (RTDCs) prime T-cell mitochondrial enzyme that catalyses the conversion of inactive 25-hydro- responses:implicationsforintranasaltolerancetherapy xyvitamin-D3 (25(OH)D3) to the active 1a25D3. Using a human monocyte- in experimental autoimmune uveoretinitis (EAU) derived DC (moDC) model, we have examined the relationship between DC VDR expression and the impact of exposure to 1a25D . We show that C. Calder, N. Taylor,y J. Liversidgey & A. Dick 3 moDCs are able to synthesize 1a25D invitro as a consequence ofincreased Division of Ophthalmology, Medical Sciences, University of Bristol, 3 1aOHase expression. Consistent with this finding is the observation that the University Walk, Bristol, BS8 1TD, yDepartment of Ophthalmology, development of moDCs is inhibited at physiological concentrations of the Medical School, Foresterhill, Aberdeen, AB25 2ZD, UK inactive metabolite 25(OH)D3,aswellasby1a25D3. In contrast to Suppression of EAU can be achieved by a single intranasal application of 1aOHase, VDR expression is down-regulated as monocytes differentiate autoantigen. One proposed mechanism is that the RTDC modulates T-cell and accordingly, the inhibitory effect of 1a25D3 is lost in more mature responses, generating either a Th2 or T-regulatory phenotype within the moDCs.Inconclusion,differentialregulationofendogenous1a25D3 ligand local drainage lymph node. We have assessed the response of RTDC to and VDR appear to be important regulators of DC biology and represent maturation signals and antigen priming, and whether retinal antigen potential targets for the manipulation of DC function. pulsed RTDC prime T-cell responses in vivo. After isolation from murine lung, RTDC phenotype and cytokine profiles were determined in response to IL-4, TNFa, LPS and/or peptide. Antibody production was assessed OP55 following naive or primed RTDC transfer. RTDC have an immature Distinct responses of human dendritic cells to type I phenotype (iRTDC) displaying low levels of coaccessory molecules IFN inducers and subtypes of type I IFN and IL-12 production. Antigen-primed RTDC elicited both Th1 and J. Walker, M. Ashton, H. Kirkbride, D. Flower, P. Borrow & D. Tough Th2 antibody responses invivo. When pulsed with peptide that is known The Edward Jenner Institute For Vaccine Research, Compton, Newbury, to induce EAU, mRTDC are capable of driving a Th1 response. Results RG20 7NN, UK imply that tolerance is not dependent on a specific RTDC behaviour but likely to be as a result of microenvironment of drainage lymph nodes as Dendritic cells (DCs) undergo maturation in response to direct (pathogen- well as route of antigen delivery. associated) and indirect (host-derived) signs of infection. Production of type I IFN is one aspect of the host response to infection that has been linked to DCs, as these cells both produce and respond to type I IFN. OP53 Furthermore, DCs have been shown to be important targets for the invivo The importance of IL-9 during antigen presentation – is adjuvant activity of type I IFN. We have compared the response of human IL-9 a potent type 2 adjuvant? monocyte-derived DCs to three different subtypes of type I IFN (a(2a), M. D. Leech & R K. Grencis a(2b) and b) and to two inducers of type I IFN expression: poly IC (a 3.239 Stopford Building, School of Biological Sciences, Oxford Road, synthetic dsRNA) and RNA from virus infected cells. Preliminary experi- Manchester, M13 9PT, UK ments examining phenotypic maturation and secretion of cytokines and chemokines by DCs have revealed that the three subtypes of type I IFN do Resistance to the intestinal nematode Trichinella spiralis is characterized not stimulate equivalent responses in DCs. In addition, it was evident that by the production of a strong Th2 immune response. In particular, IL-9 has poly IC was a much stronger maturation factor for DCs than type I IFN been demonstrated to be critical in the expulsion of T. spiralis from the alone, indicating that the stimulatory effects of poly IC were not solely small intestine. However, how these protective immune responses are mediated through induction of type I IFN. We are currently investigating initiated by dendritic cells is unknown. Previous work has demonstrated gene expression in DCs treated with the various stimuli using cDNA that adoptive transfer of antigen pulsed dendritic cells (DCs) can accel- microarrays to analyse the expression profiles for over 18 000 genes. erate expulsion of T. spiralis from the intestine and therefore enhances protective immunity. Interestingly, if these DCs have the ability to produce IL-9, expulsion is further enhanced. More recent observations have OP56 demonstrated that adoptively transferred IL-9tg DCs home towards the Dendritic cell maturation enhances CD8þ T-cell spleen and liver where they have the ability to prime a strong Th2 immune responses to exogenous antigen and reveals a response. Moreover, in vitro, these DCs enhance proliferation of naive WT proteasome independent mechanism of MHC class I CD4 T cells in a type 2-dependent manner during antigen presentation and loading appear, themselves, to uptake foreign proteins at a greater rate then their WT controls. Therefore, it can be concluded that IL-9 is not only critical in N. C. Robson, H. Beacock-Sharp, A. M. Donachie & A. M. I. Mowat the expulsion of T. spiralis from the intestine, but also plays an important Department of Immunology and Bacteriology, Western Infirmary, role in initiating Th2 immune response. Furthermore, IL-9 does act as a Glasgow, G11 6NT, UK potent type 2 adjuvant. Immune stimulating complexes (ISCOMS) are vaccine adjuvants that induce strong CTL activity invivo. However, the APC responsible for the OP54 induction of this response has not been characterized. We have found that Differential regulation of vitamin D receptor and ligand resting bone marrow DC pulsed with OVA ISCOMS efficiently prime in human monocyte-derived dendritic cells naive CD8þ T cells through a mechanism that is TAP dependent, but independent of CD40 ligation and CD4þ T cell help. LPS-induced L. M. Freeman, M. Hewison, S. V.Hughes, K. N. Evans, A. Eliopoulos, maturation of DC markedly enhances their ability to prime CD8þ T cells P. A. H. Moss & R. Chakraverty through a mechanism which is also independent of CD4þ T cell help, but Institute for Cancer Studies, University of Birmingham, Edgbaston, is dependent on CD40 ligation. DC maturation also revealed a TAP- Birmingham, B15 2TT, UK independent mechanism of CD8þ T-cell priming. We suggest that DC þ 1a25-dihydroxyvitamin D3 (1a25D3) inhibits the differentiation and may be the principal APC responsible for the priming of CD8 T cells maturation of DCs, an effect dependent upon binding to the nuclear vitamin by ISCOMS invivo and that targeting these vectors to activated DC may

D receptor (VDR). Physiological control of 1a25D3 levels is critically enhance theirpresentationvia a novel pathway of class I antigen processing.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 92 Dendritic cells

OP57 generalized immune activation. We previously showed that circulating Necrosis and dendritic cells in atopy dendritic cells, which have the ability to initiate immune responses, are activated (increased surface CD86) in patients with pre-eclampsia com- H. Kandil, F. M. Gotch & M. A. A. Ibrahimy pared to non-pregnant and healthy pregnant controls. In this study, we Immunology, Chelsea and Westminster Hospital, Imperial College of tested the hypothesis that dendritic cells can be activated by injured Science, Technology and Medicine, London, SW10 9NH, yImmunology, trophoblast. We established an invitro model of interaction between King’s College London, The Rayne Instutite, 123 Coldharbour Lane, dendritic cells and injured choriocarcinoma cells JAr (as a surrogate London, SE5 9NU, UK for syncytiotrophoblast). JAr cells were killed by either repeated cycles of IL-12 secretion by activated dendritic cells (DC) is an important con- freezing and thawing or by heat-shock. Dendritic cells up-regulated CD80 tributing factor in atopy. Studies have shown that DC can be activated by and CD86 and secreted IL-12p40 in response to JAr cells injured by both inflammatory cytokines and necrotic cells. This study aimed at comparing methods. This is consistent with the possibility that injured placental cells DC responses to cellular injury and pro-inflammatory cytokines (TNF-a/ are a potential cause of immune activation in pre-eclampsia and the IL-1b) in atopic and non-atopic individuals. Using an in vitro culture process may play a role in the pathogenesis of this disease. system, monocyte-derived DC were stimulated with necrotic cells and with TNF-a/IL-1b. Up-regulation of co-stimulatory molecules (CD80 and CD86) on DC was measured by immunoflurescence, whereas IL-12 OP60 secretion in culture supernatants was measured by ELISA. Results showed Innate immune responses to Schistosoma mansoni that atopic subjects secreted less IL-12 in response to both TNF-a/IL-1b vaccine in the murine skin and cellular death compared with non-atopic subjects. The up-regulation S. Kumkate, K. Hogg & A. Mountford of co-stimulatory molecules in response to TNF-a/IL-1b was not sig- Department of Biology (Area 5) University of York, York YO10 5YW, UK nificantly different between the two groups. However, in response to cellular injury, only non-atopic subjects up-regulated co-stimulatory Intense inflammatory responses were elicited in mice following vaccina- molecules. These findings indicate that DC from atopic individuals tion with radiation-attenuated cercariae of Schistosoma mansoni as judged respond aberrantly to necrosis and pro-inflammatory cytokines which by thickening of the skin (pinnae). As larvae traverse the epidermis and may contribute to the Th2 bias of their immune response. dermis, they provoke the formation of localized cellular foci which were still evident 8 days postvaccination. By immunohistochemistry, we found that these foci comprised of cells positive for 7/4, CD11b and CD11c, OP58 indicating the presence of neutrophils, macrophages and dendritic cells, Distinct roles for IL-4, IL-12, and CD40 during T-cell respectively. The role of Langerhans cells (LCs) has been investigated response induction by dendritic cells using an mAb against the exclusive intracellular marker Langerin. As anticipated, LCs were abundant in the epidermis of naive mice. However, A. S. MacDonald, A. D. Strawy & E. J. Pearcey while LCs were still present in the epidermis following vaccination ICAPB, Ashworth Laboratories, University of Edinburgh, King’s (day 4), significant numbers of LCs were also observed in the dermis Buildings, West Mains Road, Edinburgh, EH9 3JT, UK, yDepartment of and the cellular exudates after invitro culture of split pinnae. In addition, Pathobiology, University of Pennsylvania, 3800 Spruce Street, we tracked the migration of LCs onwards to the skin draining lymph Philadelphia, PA 19104, USA nodes. Langerin positive cells have been detected only in the T-cell area of Comparison of immune response induction by antigen-pulsed wild-type lymph node where their number has increased after vaccination. The (WT) or gene-deficient (KO) murine bone marrow-derived dendritic cells migration of LCs is likely to be directed by various cytokines since the (DC) transferred into WT or KO recipients has revealed a number of secretion of IL-1b, IL-6, IL-10 and IL-12 from cultured pinnae were also interesting features of T-cell polarization by DC. We have found that DC detected. IL-4 production is not necessary for Th2 induction. More surprisingly, we have also found that DC IL-12 production is not required for Th1 induction. However, neither Th2 nor Th1 response development occurs OP61 in the absence of recipient cells that are able to produce IL-4 or IL-12, Molecules released by schistosomes stimulate the respectively, suggesting that the important source of these cytokines innate immune system during T-cell response induction and development is not the initiating S. J. Jenkins & A. P. Mountford DC. A similar experimental approach, where WT or CD40-deficient DC Biology, University of York, York, YO10 5DD, UK were transferred into WT recipients, unexpectedly revealed that DC CD40 expression is integral for Th2 but not Th1 response induction. These data Schistosomes infect their hosts via the epidermis, and undergo rapid help delineate the source and importance of IL-4 and IL-12, and the role of developmental change as they migrate through the dermis, vasculature or CD40, during the induction of polarized T-cell responses by DC. lymphatics. During this period they are likely to come into contact with numerous innate accessory cells, such as dendritic cells (DC) and macrophages (Mf). We have previously shown that the molecules OP59 released by S. cercariae (0–3 hRAP) stimulate IL-12 and IL-6 production Dendritic cell interaction with trophoblast in by murine Mf, whereas there is little response to other larval stage pre-eclampsia molecules. It is thought that development of acquired T helper (Th) cell- mediated immunity is potentially guided by the innate immune response, V. Bachy, H. Kandil, D. Williamsy & M. Ibrahim via the action of the cytokines that are released, and through the signals Department of Immunology, Rayne Institute, King’s College, 123 received from activated APC. We now show that 0–3 hRAP drives bone Coldharbour Lane, London, SE5 9NU, yDepartment of Obstetrics and marrow-derived DC maturation, as determined by up-regulation of MHC Gynecology, Chelsea and Westminster Hospital, Imperial College School II, CD40 and CD86 surface expression. Moreover, 0–3 hRAP stimulates of Medicine, 369 Fulham Road, London, SW10 9NH, UK DC to produce increased levels of IL-12 and IL-6. Further to this, we Pre-eclampsia is a non-microbial disease unique to human pregnancy demonstrate that DC stimulated with 0–3 hRAP have an increased capa- characterized by extensive placental and endothelial injury and a state of city to prime Th-cells from OVA-TCR transgenic mice. Moreover, these

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Dendritic cells 93

Th cells produce increased levels of IL-4 and IL-5. We are presently We show that reduced numbers of DC produced in cultures of neonatal investigating the influence of 0–3 hRAP-stimulated DC on Th1/Th2 cells are not due solely to reduced numbers of precursors. Increasing the development after in vivo transfer into OVA-TCR transgenic mice. time in culture results in the development of comparable numbers of DC in neonatal and adult cell cultures, although DC generated from neonatal cells remain phenotypically distinct from those produced from adult cells. OP62 The inclusion of adult bone marrow derived stromal cells or cells of the Circulating blood dendritic cells are activated in atopy adult stromal cell line ST2 also result in an increased production of DC from neonatal precursors. These results suggest that the reduced number H. Kandil & M. A. A. Ibrahimy of DC in neonatal mice result from a lack of signals being delivered to Immunology, Chelsea and Westminster Hospital, Imperial College of precursors from stromal cells and not to a reduced number of precursors in Science, Technology and Medicine, London, SW10 9NH, yImmunology, the neonate. King’s College London, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK Studies have shown that dendritic cells are involved in immune deviation OP64 underlying atopic status. We therefore studied the state of activation Murine dendritic cells activated with excretory/ (CD86) and maturation (CD83) of peripheral blood dendritic cells in secretory products of Nippostrongylus brasiliensis atopic and non-atopic subjects. Peripheral blood dendritic cells were drive Th2 responses identified and quantified using a no-wash lysis method for whole blood A. Balic, Y. Harcus & R. M. Maizels staining using flow-count flourosphere beads. Results showed that there is ICAPB, Ashworth Laboratories, King’s Buildings, University of no significant difference in the absolute count of circulating blood Edinburgh, Edinburgh, EH9 3JT, UK dendritic cells between atopic and non-atopic subjects. Similarly, the number of CD83 positive dendritic cells was not different between the two Nippostrongylus brasiliensis is a rodent gastro-intestinal nematode para- groups. However, dendritic cells expressing CD86 were significantly site, which induces strong CD4þ T-cell dependent Th2 type responses in increased in atopic subjects compared to controls. These data suggest its host. Th2 immune response development is so pronounced in the that circulating dendritic cells are activated in atopy. Although it is not environment of N. brasiliensis infection that responses to coincident known whether the activation of dendritic cells is a consequence of the bystander antigens are also driven in this direction. We have shown that allergic phenotype or its origin, it could be speculated that it reflects an the Th2-driving effect can be achieved with a set of soluble proteins intrinsic aberrance of these cells. Our data may emphasize dendritic cells secreted from adult N. brasiliensis. Subcutaneous footpad injection with a as a feasible target for immunotherapy. single dose of N. brasiliensis excretory/secretory (NES) antigen induces CD4þ T-cell dependent IL-4 production by peripheral lymph node cells. Currently, we have focused on the effect of NES on the development and OP63 function of murine dendritic cells (DC). This has been preformed by Dendritic cell (DC) generation in cultures of neonatal analysing the effect of NES on DC antigen presentation function, both murine spleen and bone marrow invitro and in vivo and by examining the influence of NES on dendritic cell cytokine and chemokine production. We have shown that cells from C. Allen & S. Marshall-Clarke mice immunized with NES-pulsed DC showed antigen specific prolif- Department of Human Anatomy and Cell Biology, Sherrington Buildings, erative responses and IL-4 in response to antigen stimulation, but no IFNg. Ashton Street, Liverpool, L69 3GE, UK Conversely, cells from mice immunized with boiled NES, while still The immune systems of neonatal rodents and humans are immature and exhibiting antigen specific proliferation, do not produce significant anti- their immune responses are generally weaker than those of adults. We gen-specific IL-4. Furthermore, DC pulsed with NES, not BNES, show an have reported that neonatal mice possess fewer splenic and bone marrow activated phenotype and produced IL-12 p40. These results indicate that DC, expressing lower levels of MHC II than DC from adults. Our results NES-pulsed DC are selective inducers of Th2 lymphocyte differentiation indicate that culture of neonatal spleen or bone marrow cells with GM- and in this particular case, Th2 lymphocyte differentiation is not depen- CSF generates fewer CD11cþMHC IIþ DC than cultures of adult marrow. dent on a non-activated DC phenotype as has been reported previously.

Immunomodulation

OP65 daily doses of recombinant human growth hormone (rhGH) for 12 weeks. Enhanced T-cell maturation, differentiation and At week 12, a significant increase in effector CD8 T cells assessed by function in HIV-1-infected individuals after growth CD45RA and CCR7 expression, and increases in naive CD4 and CD8 T hormone and highly active antiretroviral therapy cells were observed. In addition, we observed an increase in HIV-1 antigen-specific CD4 and CD8 T cell responses. Randomization into A. Pires, J. Pido-Lopez, G. Moyle,y A. Spentzou, B. Gazzard,y placebo, alternate day or twice weekly, was instigated at week 12. The F. Gotch & N. Imami phenotype and function of the virus-specific effector CD8 T cells seen at Department of Immunology, Imperial College, London, yDepartment of week 12 was maintained at week 24, regardless of randomization, despite HIV/GU Medicine, Chelsea & Westminster Hospital, 369 Fulham Road, the disappearance of HIV-1-specific CD4 T cell responses in all patients. London, SW10 9NH, UK In conclusion, rhGH and HAART reverse the defects exerted on Good correlates of immunity during HIV-1 infection are focused on the immune system by HIV-1, in a dose-dependent manner. This combi- strong HIV-specific helper and cytotoxic T cell responses. We assessed nation represents a valuable immunotherapeutic agent in chronic HIV-1 12 HIV-1þ individuals treated with HAART 4 years, who received infection.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 94 Immunomodulation

OP66 development of pristane-induced arthritis. This protection is associated Tick salivary gland extract inhibits B cell activation with the production of Th2-type cytokines (IL-4, IL-5 and IL-10) in response to stimulation of T cells in vitro with peptide 261–271 or hsp65. S. Hannier, J. Liversidge,y J. M. Sternberg & A. S. Bowman These results have been extended to show that T cells from mice Zoology Building, Tillydrone Avenue, yDepartment of Opthalmology, subcutaneously immunized with peptide 261–271 from hsp65 induce a Medical School, Aberdeen, AB24 2TZ, UK regulatory T cell population that are CD4þ , CD25þ, CTLA-4þ and Tick saliva contains immunosuppressive factors such as Salp15 which was secrete high levels of IL-10 (approximately 1000 pg) but low or virtually recently shown to inhibit CD4þ T cell activation of the host (Immunity undetectable levels of IFNg. Surprisingly, T cells from mice intranasally 2002; 16: 849–859). In this study, we examined the immunomodulation of immunized with peptide 261–271 produce high levels of IFNg and low naive murine B cells by the saliva and salivary gland extract (SGE) of levels of IL-4 and IL-10 in response to stimulation with peptide 261–271. Ixodes ricinus ticks. We found that saliva-specific factors reduced IL-10 Thus, i.n. immunization induces a systemic Th1 inflammatory immune production by lipopolysaccharide (LPS)-stimulated splenocytes more response that exacerbates disease whilst subcutaneous immunization efficiently than IL-10 production by concanavalin A (ConA)-stimulated provides protection from pristane-induced arthritis by enhancing regula- splenocytes. SGE from weight-graded ticks suggested that factors mod- tory T-cell activation. ulating ConA-stimulated splenocytes are, at least in part, different from those modulating LPS-stimulated splenocytes. Flow cytometric analysis determined that SGE mainly inhibited B (B220þ) cell IL-10 production. OP69 Moreover, SGE reduced the early activation marker CD69 expression on Effects of preincubation with nitric oxide on IL-2 B cells in presence of ConA or LPS. Flow cytometric analysis of annexin production by human PBMC V and via probeTM staining demonstrated that SGE did not increase cell S. E. Macphail, B. M. Brooks, B. F. Flanagan,y C. G. Boothz & death in activated B-cell subpopulations and, indeed, SGE slightly, but J. W. Coleman significantly, decreased apoptosis in these lymphocytes. By employing Department of Pharmacology, University of Liverpool, Liverpool, assays with isolated B cells, we further showed that SGE has a direct effect L69 3GE, yDepartment of Immunology, University of Liverpool, on B cells and inhibits LPS-induced B-cell proliferation. Finally, we Liverpool, L69 3GA, zRespiratory and Inflammation Research Area, showed that PKCd mRNA was reduced after 24-hr addition of SGE in AstraZeneca, Mereside, Alderley Park, Macclesfield, SK10 4TG, UK isolated B cells. Taken together, our results indicate that immunomodu- lators in tick saliva induce hyporesponsiveness to mitogen in B cells. Nitric oxide (NO) is a naturally occurring radical with known immune regulatory effects including inhibition of T-cell proliferation. There is controversy over: (1) whether NO targets early signalling events or is OP67 cytostatic/late acting and (2) whether NO inhibits cytokine production. To Enhancement of bovine DTH responses to defined focus on early regulatory events, we prestimulated human PBMC with the mycobacterial antigens using a synthetic lipopetide NO donors S-nitroso-glutathione (GSNO) or S-nitroso-N-acetyl-penicil- A. O. Whelan, D. Clifford, R. G. Hewinson & M. Vordermeier lamine (SNAP) or control non-NO releasing compounds (GSH and NAP) Veterinary Laboratories Agency, Woodham Lane, Addlestone, KT15 3NB, for up to 48 hr and then studied subsequent PHA-induced proliferation UK (measured by 3H-thymidine uptake), release of IL-2 (by ELISA) and expression of IL-2 mRNA (by RNase protection assay). Preincubation The incidence of bovine tuberculosis is currently on the increase in the with the NO donors but not control compounds time-dependently inhib- UK. The identification and subsequent removal of infected cattle is based ited PHA-induced cell proliferation, release of IL-2 and expression of IL-2 on the detection of a delayed-type hypersensitivity (DTH) response to mRNA. The inhibitory effect of NO was not accompanied by changes in bovine tuberculin purified protein derivative (PPD). PPD is a crude extract CD25 expression on CD3þ T cells, changes in the apoptosis marker of Mycobacterium bovis, the causative agent of bovine TB. To overcome annexin V, nor changes in expression of the housekeeping gene GAPDH. limitationsofPPDinsituationswhereitsspecificitywouldbecompromised, In conclusion, prestimulation with NO inhibits PHA-induced PBMC for example in BCG-based cattle vaccination strategies, recent studies have proliferation by a non-toxic mechanism that is associated with decreased identifiedseveral defined M. bovis candidate antigens. Todate, these studies expression of IL-2 but not CD25. haveusedinvitroblood-basedassaystodeterminethediagnosticpotentialof such antigens. In the current study, we investigated the DTH inducing potential ofdefined M. bovis antigens.Interestingly,weobserved thatwhilst OP70 a protein cocktail comprising of the antigens ESAT-6, MPB64 and MPB83 Preferential activation of Th2 lymphocytes on stimulated strong invitro cell-mediated responses in cattle experimentally stimulation with recombinant soluble peptide: infectedwithM. bovis,itfailedtostimulateDTHinthesameanimals.During I–A complexes associated with pristane-induced subsequent investigations to identify factors capable of enhancing bovine arthritis DTH to defined mycobacterial antigens, we identified that a synthetic lipopetide demonstrated such invivo immunomodulatory properties. L. Zhang, C. Hall, I. Karadimitris & S. J. Thompson King’s College London, Division of Life Sciences, Franklin-Wilkins Building, 150 Stamford Street, London, OP68 SE1 9NN, UK Induction of regulatory T cells with stress protein (HSP60) epitopes: relevance to pristane-induced Protection from pristane-induced arthritis in CBA mice can be afforded by arthritis immunization with the mycobacterial 60 kDa heat shock protein or the immunodominant T-cell epitope (aa261–271). This protection is mediated C. Hall, I. Karadimitris & S. J. Thompson by the production of high levels of Th2-type cytokines. In this study, King’s College London, Division of Life Sciences, Franklin-Wilkins recombinant hsp peptide (261–71):I–Ak complexes were cloned and Building, 150 Stamford Street, London, SE1 9NN, UK expressed in the baculovirus/insect cell system and used in studies to Previous studies have shown that the i.p. immunization of CBA mice with examine the activation requirements of primed T cells and T-cell lines the immunodominant peptide 261–271 from hsp65 protects from the associated with protection from pristane-induced arthritis. Accordingly,

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Immunomodulation 95 antigen-specific T cells were stimulated in culture with peptide:I–A OP73 complexes in the presence or absence of costimulation provided by Protection against herpes simplex virus type 1 anti-CD28. The results clearly show that these complexes preferentially infection of the eye is associated with modulation of activate Th2 lymphocytes as assessed by the detection of IL-4 but not the immune response IFNg in the culture supernates. The ability of these peptide:I–A complexes C. M. Richards, R. Case, T. J. Hill & N. A. Williams to afford or enhance protection from disease is currently under investiga- University of Bristol, Department Pathology & Microbiology, School of tion. Medical Sciences, University Walk, Bristol, BS8 1TD, UK Acknowledgement This work is supported by the Medical Research Council, UK. Herpes simplex virus type 1 (HSV-1) is a mucosal pathogen which becomes latent in nerve ganglia following primary infection. In the case of the eye, subsequent recurrent infections may result in the potentially OP71 blinding herpetic stromal keratitis. This disease has been shown to be, at Modulation of B lymphocyte signalling by the B subunit least in part, a consequence of immunopathological responses in the eye. of Escherichia coli heat-labile enterotoxin In particular, the influx of large numbers of neutrophils to the cornea is followed by increased levels of CD4þ T cells. Following i.n. immuniza- H. K. Bone, S. Eckoldt & N. A. Williams tion with HSV-1 glycoproteins in the presence of recombinant Escherichia University of Bristol, Department Pathology and Microbiology, School of coli heat-labile enterotoxin B subunit as adjuvant, the incidence and Medical Sciences, University Walk, Bristol, BS8 1TD, UK severity of ocular disease upon infection with HSV-1 was significantly The non-toxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) reduced. In this present study, immunohistochemical analysis carried out is a potent mucosal adjuvant and immunomodulator, capable of blocking on immunized mice following ocular infection with HSV-1 indicated that autoimmune disease. These effects are linked with its ability to modulate protection from disease is associated with a reduction in the influx of lymphocyte populations, a feature that is dependent on binding to inflammatory cells to the eye. Further, analysis of viral expression ubiquitously expressed cell surface receptors. Here, we demonstrate that following ocular infection with HSV-1 virus incorporating the b-galacto- EtxB can trigger up-regulated expression of class II MHC and CD25 on sidase gene showed a reduction in viral replication which may also be purified populations of B lymphocytes, suggesting that EtxB can directly associated with reduced immunopathological damage. activate biochemical signalling pathways in these cells. The nature of the intracellular signalling events was investigated. EtxB induced the activa- tion of Erk in a process that was dependent on MEK, PI3-K and PKC. OP74 While PI3-K was critical for EtxB-induced up-regulation of both class II Inhibition of endogenous nitric oxide leads to a MHC and CD25, inhibition of MEK only partially abrogated up-regula- reduction in antigen-mediated mast cell degranulation tion of MHC class II expression and did not affect CD25 expression. in vivo These findings suggest that addition pathways downstream of PI3-K are E. J. Swindle & J. W. Coleman involved. A role for PKC in these processes was suggested by the findings Department of Pharmacology, University of Liverpool, Liverpool, that inhibitors of PKC completely blocked EtxB-mediated CD25 up- L69 3GE, UK regulation. Thus, EtxB triggers multiple signalling pathways in B cells that regulate expression of key cell surface molecules. Nitric oxide (NO) has been shown to inhibit many aspects of mast cell- mediated inflammation in vivo. We have investigated what effect endo- genous NO, produced by resident macrophages, has on mast cell activa- OP72 tion in the peritoneal cavity of rodents. Twenty-four hours prior to NO Modification of murine nephrotoxic nephritis using synthase, inhibitor administration OVA-sensitized BN rats were injected targeted T cells with 3H-serotonin (25 mCi, i.p.) which is selectively incorporated into mast cell granules. L-NMMA (12 mM in 0Á25 ml, i.p.) was then adminis- D. J. Clarke, W. P. Pearce, D. M. MacCallum, L. Bowie, D. C. Kluth, tered for 0Á5 or 2 hr prior to a 15-min OVA (1 mg/ml, i.p.) challenge, after A. J. Rees & R. N. Barker which peritoneal fluid and plasma were collected and 3H activity assessed. Department of Medicine and Therapeutics, University of Aberdeen, Initial experiments revealed 99% of total 3H-serotonin administered was Foresterhill, Aberdeen, AB25 2ZD, UK fully excreted within 24 hr and the remaining 3H activity was isolated to Goodpasture’s disease is a severe nephritis caused by autoimmunity the resident mast cells. In addition, no increase in inflammatory cells was against the NC1 domain of the a3 chain of type IV collagen, a3(IV)NC1. observed during the 48 hr time–course. L-NMMA (12 mM in 0Á25 ml, i.p.) Murine T lymphocytes have been generated that proliferate in response to pretreatment for 0Á5 or 2 hr significantly reduced the OVA-specific 3H- synthetic 15-mer peptides derived from a3(IV)NC1. Peptide-specificTh serotoninactivityintheperitoneal fluid by 44 and48%,respectively,butthis clones were transfected with recombinant adenovirus coding for rat IL-4, was not mirrored in the plasma. Furthermore, there was no increase in 3H TGFb and GFP. Coxsackie adenovirus receptor (CAR) expression was activity in unfractionated cell lysates. In conclusion, endogenous NO has an increased on the T-cell surface by prior transformation with a CARþ enhancing effect on antigen-mediated mast cell degranulation invivo. retrovirus. The effect of these cells upon inflammation was then ascer- tained by introducing them to murine models of nephritis. T cells specific to a naturally processed peptide p3 localized to glomeruli more efficiently OP75 than those specific to a non-naturally processed peptide p20. Localization Aspirin-treated DC as potential tools for tolerance was increased a further fivefold during renal inflammation, as scored by induction albuminuria, macrophage infiltration and glomerular hypercellularity. p3- C. Jago, C. Germain, R. Lechler & G. Lombardi specific T cells may have a negative effect upon inflammation. We aim to Department of Immunology, Hammersmith Hospitalk, Imperial College direct virus-engineered antigen-specific Th clones and freshly isolated Faculty of Medicine, Du Cane Road, London, W12 0NN, UK splenic T cells to the inflammatory focus of murine models of nephritis, investigating the effects of secreted cytokine upon cell localization and The non-steroidal anti-inflammatory drug (NSAID) aspirin has been disease. shown to arrest dendritic cell (DC) maturation at an immature stage.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 96 Immunomodulation

We investigated aspirin treatment of DC to determine whether matura- capacity, as measured by 3H-thymidine incorporation of allogeneic CD4þ tional arrest was an indicator of tolerogenic potential. Human DC were T cell in an MLR. Stimulation of allogeneic CD4þ T cells by aspirin-DC derived from the adherent fraction of PBMC by culture with GM-CSF and was shown to induce a regulatory activity in responder T cells. This was IL-4. An amount of 2Á5mM aspirin was added from day 2 of DC culture, demonstrated by the ability of these cells to inhibit a subsequent MLR and on day 7, aspirin-DC were compared to immature untreated DC. comprising allogeneic DC and autologous T cells. The mechanisms of Aspirin treatment was shown to significantly inhibit DC allostimulatory action of aspirin-DC-derived regulatory T cells are under investigation.

Immunotherapy

OP76 OP78 OX40R agonists: new insights into M3, the mouse gamma herpes virus encoded biological function and use in tumour broad-spectrum chemokine binding protein, can immunotherapy inhibit site directed migration of neutrophils and macrophages in vivo A. McIlgorm, E. Taylor, C. Kitchener, S. Hill, E. Choolun, A. Weinbergy & C. Entwisle H. A. Arnold & C. J. Bunce 310 Cambridge Science Park, Cambridge, CB4 0WG, UK, yEarle A Xenova Research Ltd, 310 Cambridge Science Park, Cambridge, Chiles Research Institute, Providence Portland Medical Center, Portland, CB4 0WG, UK OR 97213, USA M3 is a 44 000-MW protein that binds with high affinity to a broad- Cancer immunotherapy has a controversial history and although occa- spectrum of mouse and human chemokines. At Xenova, we have been sional successes are reported, few therapies are accepted as best practice exploring the anti-inflammatory properties of this protein with a view to by clinicians. Significant problems are associated with clinical evaluation establishing it as a therapeutic for human disease. Neutrophils and of immunotherapeutic protocols. Although it is now accepted that immune macrophages are considered to play an important role in diseases such responses can be induced against specific antigens, it is still debatable if as rheumatoid arthritis and atherosclerosis and it is well understood that these responses are sufficient to mediate clinical benefit. OX40 receptor chemokines play a key role in the migration of these cell types into sites of (OX40R, CD134) and its ligand, OX40L, belong to a family of molecules, inflammation. Using an established mouse model of sterile peritonitis, we which deliver costimulatory signals and provide a new class of immune have investigated the effect of M3 on the infiltration of these cell types adjuvants with broad application. The OX40R is expressed on activated T in vivo. Consistent with published reports we found a sequential migration cells. Cross linking of the OX40R with an antibody agonist or the native of neutrophils followed by macrophages after injection of 1–3% sterile ligand (OX40L) expressed as a fusion protein, leads to increased antibody brewers thioglycollate (SBT) into the peritoneum. Neutrophils peaked at responses, cytokine secretion, expansion of antigen-specific T cells, 4 hr and macrophages at 3–5 days. A single i.p. injection of M3 had a dose- prolonged T-cell survival and in syngeneic murine tumour models, dependent inhibitory effect on neutrophil migration into the peritoneum. enhanced tumour-cell killing and tumour regression. In this report, we A similar inhibitory effect was seen with more frequent injections of M3 describe the construction and characterization of a human OX40L-fusion on SBT-induced macrophage infiltration. These results suggest that M3 protein and present data to support the future use of this protein in cancer acts on multiple chemokines invivo and promote the use of M3 as a novel immunotherapy trials. therapeutic for inflammation.

OP79 Treatment of collagen-induced arthritis (CIA) by OP77 induced nasal tolerance to type II collagen Cloning and characterization of a CCR9/IL-7 fusion L. Marinova, C. J. Derry, N. S. Ali, D. H. Davies, J. J. Murphy & protein N. A. Staines S. M. Henson & R. Aspinall Division of Life Sciences, King’s College, 150 Stamford St, London, Department of Immunology, Imperial College of Science, SE1 9NN, UK Technology and Medicine, Chelsea and Westminster Hospital, The therapeutic effects of inducing nasal tolerance to type II collagen 369 Fulham Road, London, SW10 9NH, UK (CII) was explored in CIA. This study was based on our previous report The use of therapeutic agents for improving immunity is often approached that suppression of CIA by nasal administration of CII, before disease in a haphazard manner introducing the therapeutic agent into the host at induction, resulted in down-regulation of invitro recall Th1 and Th2 dose levels, which will reach the target organ in insufficient quantity to responses. To determine the effect of nasal tolerance during the induction have an effect. An alternative approach, and one which should reduce phase of arthritis, but before the onset of clinical disease, mice were dosage levels and treatment frequency would be to create a fusion protein nasally dosed with either CII or BSA, as a control protein, on days 14–18 between the therapeutic agent and a chemokine receptor whose ligand is after collagen/CFA immunization. Arthritis developed 32–40 days after organ specific. We report here the generation of a fusion protein between immunization in control mice. Nasal tolerance had a protective effect on the receptor region of CCR9 and IL-7. We have characterized this pCCR9/ the clinical arthritis and this effect was dose dependent. From the three IL-7 fusion protein in terms of its molecular weight, chemokine binding doses (5, 20 & 80 mg of CII) examined, the treatment with a cumulative characteristics and IL-7 activity, in addition to determining its ability to dose of 80 mg CII offered the best protection against disease and arthritis target IL-7 to the thymus invivo. developed 19–23 days later compared to control mice. At day 40 after

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Immunotherapy 97 immunization, only 12% of tolerant mice had first signs of disease, while invivo, including IgE-facilitated antigen presentation to T cells. We in the control group 92% of mice had arthritis. By analogy with earlier hypothesized that following grass pollen immunotherapy, IgG antibodies studies in prevention of CIA by nasal tolerance, we anticipate that active disrupt IgE-dependent processes including allergen processing by antigen immune suppression would account to the therapeutic effect. presenting cells. To test this hypothesis, we developed a flow cytometric Acknowledgement Supported by the ARC. assay based on detection of IgE-allergen interdependent binding to the low affinity IgE receptor on B cells. The blocking effects of sera collected from 35 patients participating in a 2 year double-blind controlled trial of OP80 grass pollen immunotherapy were examined in this system. In all, 18 Development of novel reagents to regulate patients that received active therapy, there was induction of an activity that autoimmune disease models inhibited IgE-allergen interdependent binding to B cells (P ¼ 0Á0004, versus placebo subjects) as well as subsequent allergen presentation to T D. Voulgaraki, S. Simmonds, M. H. Brown & A. N. Barclay cells. This activity was allergen-specific, independent of seasonal influ- Sir William Dunn School of Pathology, University of Oxford, South Parks ences and copurified with IgG. We conclude that allergen-specific IgG Road, Oxford, OX1 3RE, UK antibodies induced by immunotherapy can disrupt formation of IgE– Experimental autoimmune encephalomyelitis (EAE) is a disease model allergen complexes that bind to antigen presenting cells and facilitate for multiple sclerosis (MS), a chronic, inflammatory disease of the central allergen presentation. nervous system (CNS), in which invasion by monocytes and T lympho- cytes leads to demyelination and axonal injury. Lymphocyte cell surface proteins OX40 and OX2 are involved in immune responses and can be OP82 targeted to manipulate autoimmune disease. OX40 belongs to the tumour Indentification of CD4þ CD25þ IL-10þ cells from the necrosis factor receptor (TNFR) superfamily and is expressed on T cells. peripheral blood of patients receiving grass pollen Its ligand OX40L belongs to the TNF superfamily and is found on immunotherapy dendritic cells. OX2 and its receptor belong to the Immunoglobulin J. N. Francis, V. A. Carr, F. Puggioni, S. J. Till & S. R. Durham superfamily (IgSF). OX2 is widely distributed whereas OX2R expression Upper Respiratory Medicine, National Heart and Lung Institute, Imperial is restricted to cells of myeloid lineage. In these studies, we have targeted College London, 1b Manresa Road, London, SW3 6LR, UK the OX40–OX40L and OX2–OX2R interactions in murine models of EAE. The first approach used monoclonal antibodies (mAbs) against The mechanisms of specific allergen immunotherapy (SIT) currently OX2R and OX40L. A second involved recombinant proteins designed to remain unclear. Some immunotherapy strategies have suggested that block the OX2–OX2R interaction. These were designed to be multivalent SIT-induced IL-10 production is a factor in controlling allergic disease. to increase avidity. The binding ability of these reagents and their effect on Here, we aimed to analyse allergen-induced proliferation and cytokine disease models will be discussed. production from patients receiving grass pollen SIT (n ¼ 12) and compare the results with those from atopic (n ¼ 11) and normal controls (n ¼ 11). During the grass pollen season, freshly isolated PBMCs were stimulated OP81 with 0, 2 and 20 mg/mL of Phleum pratense for 6 days and then tested for Inhibition of IgE-allergen interdependent binding to proliferation and production of cytokines by ELISA and intracellular B cells by IgG antibodies following grass pollen cytokine staining. Whilst all groups demonstrated strong proliferative immunotherapy responses to the allergen, both atopic and SIT subjects produced greater amounts of IL-4, but less IFNg, compared to normal controls (P < 0Á01). P. A. Wachholz, S. M. Walker, N. Kristensen,y S. J. Till & Significantly, more IL-10 was observed in cultures derived from SIT S. R. Durham patients (P < 0Á01) compared to atopics or normals. Analysis of these NHLI, Upper Respiratory Medicine, London, SW3 6LR, UK, cells by flow cytometry reveals that all IL-10þ cells are also CD4þ yALK-Abello, Horsholm, 2970, Denmark CD25þ. These results demonstrate that regulatory IL-10þ CD4þ CD25þ Allergen-specific IgG antibodies are markedly increased following immu- cells are present in the peripheral blood after successful grass pollen notherapy and may be involved in blocking IgE-dependent processes immunotherapy.

Innate immunity

OP83 28 000-MW portion (HSP70359610) containing the 18 000-MW peptide- Stimulation of maturation of DC and adjuvant function binding region (aa 359–540). The data suggest that stimulation of human by the peptide binding fragment of HSP70 monocyte-derived dendritic cells with HSP70 is mediated by the C- terminal HSP70 which elicits maturation of DC, as demonstrated Y. Wang, C. Kelly, M. Singh, E. McGowan, L. Bergmeier & T. Lehner 359610 by up-regulation of CD83, CCR7, CD86, CD80 and HLA class II, and Department of Immunobiology, 3rd floor, New Guy’s House, Guy’s production of the cytokine IL-12 and CC-chemokine RANTES. The Campus, London Bridge, London SE1 9RT, UK N-terminal, ATPase portion (HSP701358) failed to induce expression Microbial heat shock protein 70 stimulates innate immunity and induces of these markers or to stimulate any of these cytokines or chemokines. maturation of dendritic cells. It consists of two functionally distinct Immunization with peptide-bound HSP70359610 in mice induced higher domains: the N-terminal ATPase fragment and the C-terminal peptide serum IgG2a and IgG3 antibodies than the native HSP70-bound peptide. binding domain. In an attempt to identify the stimulating domains This study suggests that the C-terminal, peptide-binding portion of HSP70 of HSP70, we generated two major fragments of HSP70; the N- is responsible for stimulating TH1 polarizing cytokines, CC chemokines terminal 44 000-MW ATPase portion (HSP701358) and the C-terminal and the adjuvant function.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 98 Innate immunity

OP84 OP86 The role of human HSP60 in the immune response Selective depletion of Va24þ Vb11þ NKT cells in livers of oral epithelial cells of patients with hepatic malignancy

O. Pleguezuelos, J. M. Thomason & J. J. Taylor T. Kenna, L. G. Mason, S. A. Porcelli,y Y. Koezuka,z J. E. Hegarty, Faculty of Medical Sciences, School of Dental Sciences, C. O’Farrelly & D. G. Doherty§ University of Newcastle upon Tyne, Framlington Place, ERC, St. Vincent’s Hospital, Dublin 4, Ireland, yAlbert Einstein Newcastle upon Tyne NE2 4BW, UK College of Medicine, New York 10461, USA, zKirin Brewery, Gunma, Tokyo 150-8011, Japan, §Department of Biology, NUI Maynooth, Co Kildare, Ireland Extracellular heat shock proteins (HSPs) act as endogenous ligands for Toll-like receptors (TLR) and induce the expression of proinflammatory Murine NKT cells that express the invariant Va14Ja281 TCR accumulate cytokines in leucocytes, thereby amplifying immune responses. Sig- preferentially in the liver where they constitute up to 30% of hepatic nificantly, high levels of human HSP60 and homologues from path- T cells. Mouse models of cancer have shown that these NKT cells have ogenic bacteria (e.g. Actinobacillus actinomycetemcomitans) have been potent antitumour effector functions. Here, we have enumerated and detected in the sera and inflamed gingival tissues of periodontitis characterized their human counterparts, Va24þ NKT cells, from freshly patients. We aimed to investigate the hypothesis that human HSP60 isolated normal and tumour-bearing human liver. In contrast to the mouse, can stimulate the cells of the oral epithelium to provide a source of human Va24þ NKT cells were found in small numbers in healthy liver immunoregulatory cytokines. Addition of 0Á1 mg/ml of recombinant (0Á75% of CD3þ cells) and blood (0Á61%). Most hepatic Va24þ NKT cells human HSP60 (rhHSP60)-stimulated cell division in the oral keratinocyte expressed the NK markers CD56 and CD161 and showed a striking Th1 cell line HOK-16B and this was associated with an up-regulation of cytokine bias. The majority (64%) of hepatic, but not peripheral, the IL-1b, TNF-a and IL-8 genes as determined by RT-PCR. The Va24þ NKT cells expressed the Vb11þ chain, associated with CD1d TLR2 and accessory protein MyD88 genes were up-regulated after the restriction and these invariant NKT cells accounted for 0Á5% of hepatic addition of rhHSP60 but there was no significant expression of the TLR4 T cells and 0Á02% of peripheral T cells. The proportion of gene in HOK-16B cells. We conclude that the oral keratinocyte cell line Va24þ Vb11sþ cells were significantly reduced in tumour-bearing com- HOK-16B responds to endogenous HSP60 by changes in cytokine gene pared with healthy liver (0Á2% versus 0Á6%, P < 0Á05). These data indicate expression and expression of TLR2 and MyD88. This may be an that hepatic NKT cell repertoires are distinct in mice and humans. important local immunoregulatory pathway in periodontal health and Depletions of specific subpopulations of hepatic NKT cells may underlie disease. susceptibility to metastatic liver disease.

OP87 Campylobacter induced proinflammatory signals from human monocytes OP85 M. A. Jones, S. Totemeyer,y C. Bryanty & P. A. Barrow Identification of a microtubule binding linker protein Institute for Animal Health, Compton, RG20 7NN, UK, yCentre for interacting with the IFN-inducible 47 000-MW Veterinary Sciences, University of Cambridge, Cambridge CB3 0ES, UK GTPase IIGP Campylobacter is known to cause inflammatory enteritis; during the F. Kaiser, S. H. E. Kaufmann & J. Zerrahn course of disease, it can penetrate the epithelial barrier and may interact Max-Planck-Institute for Infection Biology, Campus Charite´ Mitte, with lymphocytes. We studied the interaction of Campylobacter with the Schumannstr. 21/22, Berlin 10117, Germany human monocytic cell line, THP-1. We show that Campylobacter can stimulate a range of inflammatory cytokines and that this stimulation is not Intracellularly mediated immunity is an essential component of the dependent on the cytolethal-distending toxin. Campylobacter infection of innate immune response against microbial and viral pathogens. These THP-1 cells can stimulate the degradation of I-kBa and trigger transloca- non-adaptive defence mechanisms are not active by default, but tion of functional NF-kB. Additionally, we have shown differential IL-1 rather upon recognition of pathogen-specific components or cellular induction which would indicate that an NF-kB-independent stimulation stimulation by interferons. Members of the IFN-inducible 47 000-MW may also be occurring. The extent of pro-inflammatory cytokine stimula- GTPase protein family, comprising to date IGTP, GTPI, LRG-47, tion suggests that monocytes could significantly contribute to inflamma- TGTP, IRG-47 and IIGP, contribute significantly to immunity against tion and disease pathology. intracellular pathogens. Mice deficient for individual members of the 47 000-MW GTPases reveal differential susceptibility to Toxoplasma gondii or Listeria monocytogenes, suggesting that the effector mechan- OP88 isms exerted by individual cognates are distinct. The localization of IGTP Salmonella enterica serovar Typhimurium F98 and IIGP to intracellular membranes invoked the notion that these modulation of NO production is not host cell-dependent GTPases could participate in membrane-dependent transport or proces- and is inv/spa dependent sing pathways. We report on the identification of an IIGP-interacting M. A. Jones, N. Foster, K. Page, S. Hulme & P. A. Barrow protein that belongs to a recently described family of microtubules and Institute for Animal Health, Compton RG20 7NN, UK organelles binding linker proteins. The significance of this finding for IIGP function and its potential relevance for intracellular immunity will be We present in vitro data, which characterize the initial response of discussed. Salmonella enterica serovar typhimurium to murine and avian phagocytic

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Innate immunity 99 cells. Under specific circumstances, S. typhimurium fails to induce high strains. However, it is not apparent in infections with other Salmonella levels of nitric oxide (NO) and this lack of induction is accentuated by the serovars. The effect is exhibited with both murine and avian cell addition of IFNg. This modulation of NO production by S. typhimurium is lines, suggesting that this modulation of NO is not a key factor in SPI-1 secretion dependent and is exhibited by a variety of S. typhimurium host-specificity.

Macrophages

OP89 OP91 The glycosylation of mannose receptor and its binding IFN-c and TNF-a act synergistically on macrophages properties to induce bacterial clearance and restore APC-associated marker expression following Y. Su, L. Martinez-Pomares & P. M. Rudd Yersinia pestis infection Biochemistry Department, The Glycobiology Institute, The University of Oxford, Oxford OX1 3JP, UK R. A. Lukaszewski, D. G. C. Rees & M. G. Hartley DSTL Biomedical Systems, Building 7a, Porton Down, Salisbury SP4 0JQ, Mannose receptor (MR) is a macrophage glycoprotein, which binds to UK both terminal mannosylated and sulphated carbohydrates. These different properties allow MR to carry out multiple functions in the immune and Yersinia pestis is a highly virulent bacterial pathogen capable of killing its haematopoietic systems. Different glycosylations of the MR itself might host within 2–6 days post-infection. It is predominantly an extracellular affect the protein function. In this study, MR from spleen, liver, lung pathogen of rodents transmitted to humans via fleas to cause bubonic tissues were purified using 5D3 mAb affinity column. N-glycans attached plague. Previous in vivo studies have demonstrated an intracellular phase to MR were analysed by HPLC, MS and lectins assays. The results within the macrophages of mammals shortly after infection. It is likely indicated that the glycans attached to spleen MR mostly contained a2,6 that this early macrophage–plague interaction plays a crucial role in linked terminal sialic acids and less a2,3 sialylation. MR isolated from determining the outcome of infection. Y. pestis induces down-regulation of lung had more a2,3 than a2,6 sialylated configurations. Also terminal the macrophage inflammatory response, preventing clearance of intra- mannose and Gal a1,4 GlcNAc were found in lung MR. No glycans were cellular bacteria. Treatment of J774 macrophages with either IFN-g or detected on the MR from liver, which suggests the liver MR might have TNF-a had no effect on bacterial clearance. Co-administration with both unusual glycan structures. MR from different sources showed different IFN-g and TNF-a had a synergistic effect on the macrophage inflamma- binding properties to mannosylated and sulphated ligands. The affinity of tory response with a reduction of intracellular numbers by approx. 3 logs spleen MR to mannan is 4 folds, and 12 folds to that of liver and lung MR, after 24 hr post-infection. This synergistic effect also prevented the respectively. Also spleen MR binds to Lutropin with nine times affinity to down-regulation of CD40, CD54 and CD80, with high levels of exp- liver and lung MR do. Considering only one MR gene form, these different ression of these costimulatory molecules throughout infection. These binding properties are probably related to its different glycosylations observations indicate possible targets for therapeutic intervention as well found here. as surrogate markers of protection induced by other treatment candidates.

# Crown Copyright 2002 Dstl OP90 Multi-component analysis of bovine macrophages from OP92 breeds differing in resistance to disease FIZZ1 is a novel macrophage gene in chronic Th2-mediated inflammation K. McGuire, G. Makins & E. J. Glass Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK M. G. Nair, P. Loke,y D. Cochrane & J. E. Allen University of Edinburgh, ICAPB, Kings Buildings, West Mains Road, Bovine macrophages are the principal host cell for the protozoan parasite Edinburgh EH9 3JT, UK, yUniversity of California, Berkeley, Theileria annulata. Infection can result in high levels of mortality in CA 94704, USA susceptible cattle (e.g. Holsteins), while Sahiwals (indigenous to endemic areas) develop relatively mild symptoms. Previous work suggests that the ‘Alternatively-activated’ macrophages (AAM) are found in Th2-mediated control of cytokine responses by macrophages is key to the observed inflammatory settings such as nematode infection and allergic pulmonary difference in pathogenesis. RT-PCR analysis has revealed phenotypic and inflammation. We have used murine macrophages elicited by nematode functional differences in the response of Holstein and Sahiwal macro- infection (NeMf) as a source of in vivo-derived AAM. NeMf have a phages to in vitro activation. The expression dynamics of several genes novel IL-4 dependent phenotype characterized by two striking features (1) differ. Infected cell lines generated for both breeds also exhibit phenotypic the ability to reversibly suppress the proliferation of T cells and (2) the differences. Furthermore, a distinct population of macrophages has been over representation of FIZZ1, a gene product not previously associated identified in Sahiwals. To date these studies have not elucidated the with macrophage function. FIZZ1 is a small secreted cysteine-rich protein mechanism involved in macrophage regulation of disease pathogenesis. recently identified as an abundantly expressed protein in the broncho- We are now undertaking a more global approach to investigate the alveolar lavage fluid of a mouse asthmatic model. FIZZ1 has been shown relationship between bovine macrophages and T. annulata infection. to inhibit the action of nerve growth factor but its function in nematode We have generated a cDNA library from unactivated, activated and infection is unknown. We have shown by real-time PCR that FIZZ1 is up- infected Holstein and Sahiwal macrophages. This library will be used regulated in three distinct nematode infection models. In vitro, FIZZ1 is to develop a bovine macrophage microarray at the ARK-Genomics unit responsive to IL-4/IL-13 and is predominantly expressed in antigen (http://www.ark-genomics.org) at the Roslin Institute. The microarray presenting cells activated in Th2 cytokine settings. We are currently will be made available to interested groups. determining the role of FIZZ1 in Th2-mediated chronic inflammation

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 100 Macrophages

by studying further the regulation of its expression and by functional they have been stimulated by necrotic cell uptake. Here we show that assays with the recombinant protein. BMDM are able to suppress proliferative responses from splenic T cells. We first investigated the role of macrophage derived nitric oxide in the co- cultures. In some experiments an inverse correlation was seen between OP93 NO production and splenocyte proliferation, but only partial recovery Mechanisms involved in macrophage suppression of resulted from the use of NO blockers. Furthermore, BMDM from iNOS ko splenocyte proliferation mice were found to be no less suppressive than wild type (B6) macro- phages. However the BMDM produced high levels of TGFb and TGF-b K. S. K. Hill, A. J. Rees & R. N. Barker blocking antibody reversed the suppression. Suppression of T-cell pro- Department of Medicine and Therapeutics, University of Aberdeen, liferation by activated macrophages has been recorded in other systems, Foresterhill, Aberdeen AB25 2ZD, UK and the mechanism described has been lymphoblast IFN-g stimulation of We have previously demonstrated that murine bone marrow derived macrophage NO. Here, by contrast, the predominant suppressive effect macrophages (BMDM) are ineffective antigen presenting cells unless appears to be elicited via secreted TGF-b.

Mucosal immunology

OP94 Tonsillar mononuclear cells were isolated and cultured in RPMI medium BMP signalling pathway components are up-regulated with the addition of CCS and LTB. Cell culture supernatants were in epithelial cells in chronic inflammatory lung collected and tested by immunoassay for CbpA-specific IgA and IgG, diseases total IgA and IgG antibodies, and IFNg, IL-2 and IL-4. Preliminary data have shown that LTB co-culture with pneumococcal CCS induced a dose- E. Lewis, B. Mahon & S. O’Dea dependent enhancement of CbpA-specific IgG and IgA responses, Epithelial Immunobiology Laboratory, Biology Department, NUI although no difference was found in the levels of total IgG and IgA Maynooth, Maynooth, Co. Kildare, Ireland when compared to controls. There was also an increase in the concentra- Bone morphogenetic proteins (BMPs) are a highly conserved subgroup of tions of IFNg, but not IL-2 and IL-4 in tonsillar cell culture supernatants the TGF-b superfamily and have been shown to have important roles in after co-culture with LTB. The results suggest that LTB may be able to morphogenesis, organogenesis, proliferation, differentiation and apopto- enhance mucosal immune responses to CbpA in the upper respiratory tract sis. BMP-4 is believed to be an important communicator in mesodermal in children and IFNg may be an important mediator in this immune and epithelial interactions and is essential during early development. enhancement. During lung development, relatively high levels of BMP-4 at the tips of growing buds are required to maintain distal airway phenotypes whereas reduced BMP-4 levels lead to increased numbers of proximal cell types in OP96 peripheral regions. However, the role of BMP-4 in mature lungs is Further evidence for extrathymic T-cell development in unknown. We used immunofluorescence to identify BMP signalling the human intestine pathway components in normal lung tissue and in chronic inflammatory A. M. Williams, S. Turner,y A. Phillips, T. Brooklyn,y D. Carroll,y lung disease tissues. The BMP receptor, BMPRII, localized to the cell P. Bland & C. S. J. Probert membranes of bronchiolar and alveolar epithelial cells in normal lungs. Laboratory 13, URCN, University Division of Medicine, University of Increased immunostaining was evident in disease sections, particularly in Bristol, Old Medicine Building, Bristol Royal Infirmary, Marlborough areas exhibiting altered architecture. The intracellular signalling mole- Street, Bristol BS2 8HW, UK, yBristol Royal Infirmary, Marlborough cules, Smad 5 and 8, were also up-regulated in disease sections, parti- Street, Bristol BS2 8HW, UK cularly in the alveolar epithelium. We conclude that BMP signalling may play a role in the pathology of chronic inflammatory lung disease. Transcripts of genes associated with TCR rearrangement have been found in the intestine of infants. Here, we provide further evidence for extra- thymic development. Lymphocytes were isolated from resected normal OP95 intestinal tissue from children aged 6 days15 years. For each sample, Enhancement of pneumococcal protein antibody IELs and LPLs were triple-stained for FACS analysis. For children responses by LTB in tonsillar lymphocytes <18 months, RAG gene transcripts were sought by PCR. CD4þ CD8þ (DP) T-cells were found in the small and large intestine of subjects Q. Zhang, K. Arnaoutakis & A. Finn <18 months, particularly amongst LPLs. Immature T cells exhibiting Education Centre, Institute of Child Health, Upper Maudlin Street, Bristol CD2, but not CD3, were evident among IELs and LPLs. Furthermore, BS2 8AE, UK CD3–CD7þ CD2þ cells were found; these were likely to be immature T Choline-binding protein A (CbpA) may be a good pneumococcal protein cells. CD3þ DP cells are seen throughout infancy. These cells may be a vaccine candidate against carriage. E. coli heat-labile toxin subunit B subset of gd cells or immature ab T cells undergoing intestinal develop- (LTB) enhances mucosal antibody responses in animal studies. We have ment. RAG 1 and 2 (which are expressed during such rearrangement) were tested the immune responses to CbpA in human tonsillar lymphocytes in often expressed <18 months. The T-cell repertoire of these immature T- response to stimulation with a concentrated culture supernatant (CCS) of cells is likely to be influenced by luminal antigens and thus may determine Pneumococcus (containing secreted CbpA) in combination with LTB. susceptibility to intestinal diseases in later life.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Mucosal immunology 101

OP97 OP98 IP-10 and ITAC stimulation of CXCR3 Escherichia coli heat-labile enterotoxin B subunit signalling pathways in subepithelial intestinal (EtxB) breaks oral tolerance associated with a myofibroblasts predominantly Th2 response

A. Kouroumalis, G. Koliosy & S. G. Ward A. Plant, R. Williams & N. A. Williams Department of Pharmacy and Pharmacology, University of Bath, Department of Pathology and Microbiology, University of Bristol, Claverton Down, Bath BA2 7AY, UK, yDepartment of Gastroenterology, University Walk, Bristol BS8 1TD, UK Faculty of Medicine, University of Crete, Heraklion 71110, Greece Encountering antigen at mucosal surfaces in the absence of adjuvant Wound healing is a complex response involving recruitment of inflam- usually leads to a state of systemic non-responsiveness. EtxB is a potent matory cells and deposition of extracellular matrix. The pivotal role mucosal adjuvant inducing strong immune responses to co-administered played by myofibroblasts in this process has been recognized in recent antigen. Here, we demonstrate that feeding OVA/EtxB to DO11.10 years. Activation of the chemokine system is also reported in the presence chimaeric mice abrogates tolerance to OVA/CFA induced by feeding of inflammation and fibrosis, two processes that are part of the wound OVA alone. In contrast to unfed animals, oral OVA/EtxB resulted in a healing response. Several studies show that intestinal myofibroblasts predominantly Th2 serum antibody profile (high IgG1:IgG2a ratio). may express chemokines and be targets of the action of chemokines. In vitro studies showed that the presence of EtxB restored proliferative The production of IFN-g-inducible chemokines, IP-10 and ITAC, by responses in spleen, MLN and PP cells upon restimulation, associated human intestinal epithelium and the expression of their cognate with mainly Th2 cytokine production. Consistent with this, CFSE- receptor CXCR3 by intestinal myofibroblasts suggest that interactions labelling experiments showed that OVA or OVA/EtxB treatment resulted between these cells can play a role in modulating physiologic and in DO11.10 T-cell division, and in increased proportion of CD4þKJ1.26þ pathologic mucosal inflammation. Here, we show, using intestinal myofi- memory cells (CD45RBlo). However, EtxB was required for prolonged broblasts, that CXCR3 ligation with ITAC and IP-10 leads to transient proliferation, which occurred primarily in the CD25– population. CTLA-4 MEK and PI3K-dependent phosphorylation of ERK. These chemokines expression increased in CD4þKJ1.26þ cells recovered from the MLN also stimulate a rapid and transient phosphorylation of the PI3K effectors, of mice fed OVA/EtxB but not OVA alone. Hence, EtxB acts as an PKB and GSK-3. Ongoing work will further define the signalling oral adjuvant, by maintaining Ag-specific proliferative responses both pathways and the role each plays in events such as chemotaxis and invitro and in vivo and inducing a predominantly Th2 type immune proliferation. response.

Structural immunology

OP99 OP100 Complete N- and O-glycan analysis of human secretory Structural analysis of leucocyte surface proteins with IgA reveals substantial differences in glycosylation a high content of cysteine residues – CD200, CD200R between the J chain, secretory component and IgA and CD6 heavy chains D. Hatherley, N. Hassan, M. H. Brown & A. N. Barclay L. Royle, D. J. Harvey, M. R. Wormald, R. A. Dwek & P. M. Rudd Sir William Dunn School of Pathology, Oxford OX1 3RE, UK Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK Cysteine residues play a key role in maintaining the folding of the Secretory IgA (SIgA) consists of two monomeric IgA units and two extracellular domains of leucocyte surface molecules. Many of these additional polypeptide chains, the J chain and Secretory Component (SC). proteins contain the immunoglobulin fold characterized by a beta The H, L and J chains are synthesized and assembled into dimeric IgA in sandwich with a disulphide between the two sheets but some domains plasma cells, whereas SC is added during transport across the mucosal such as in CD200 and CD200R contain additional disulphides that may epithelia. The N- and O-glycans of the individual peptides from human be important for maintaining rigidity/stability. Other domains such as colostrum SIgA were identified using a sensitive sequencing strategy in the cysteine rich scavenger receptor domain found in CD6 contain a which the released glycans were fluorescently labeled and analysed by a high proportion of disulphide bridges. In order to determine the struc- combination of HPLC, exoglycosidase digestions, MALDI-MS and LC- ture of these, constructs have been transfected into a mutant CHO line MS/MS. SIgA1 has several sites capable of binding to pathogens, which that enables the proteins to be expressed with abnormal carbohydrate can block their attachment and subsequent infection of mucosal surfaces. side chains that can easily be cleaved by endoglycosidase H. It is In addition to four Fab sites that can bind to specific antigen epitopes, there important to reduce the carbohydrate heterogeneity and amount for are two O-glycan regions (with up to 20 glycans per region), and a SC structural analysis as it makes up around 50% by weight of these region (with seven N-glycans), all of which present complex fucosylated glycoproteins. Human CD200 and CD200R have been purified and and sialylated glycan epitopes capable of interaction with bacterial deglycosylated in sufficient quantity and purity for crystallographic adhesins. In contrast, the N-glycans on the H chain are mainly short studies. The ligand-binding domain of human CD6 has also been non-galactosylated structures, whilst the J chain has a single sialylated expressed. Progress in the production of these three proteins and their N-glycan. analysis will be described.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 102 T cells

T cells

OP101 OP103 The role of costimulatory molecules in T-cell activation Peripheral tolerance of islet a cell specific CD8þ and tolerance T cells K. Hochweller & S. M. Anderton B. J. Raveney & D. J. Morgan University of Edinburgh, ICAPB, Edinburgh EH9 3JT, UK Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK Although the precise signals controlling the decision between T-cell activation and tolerance are not well-defined, differential expression of InsHA mice, express the influenza virus hemagglutinin (HA) on pancrea- costimulatory molecules on APC is likely to be of crucial importance. tic islet a cells. Self-tolerance to HA is associated with the functional loss Administration of soluble ovalbumin peptide (p. 323–339) is a standard of peripheral CD8þ CTL precursors with high avidity for HA, leaving procedure to establish T-cell tolerance to ovalbumin. In order to examine only low avidity CTL. Following adoptive transfer, Clone-4 (CL4) CD8þ the molecular basis of tolerance induction, we administered OVA323-339 T cells, expressing a TcR with high avidity for KdHA epitope proliferated intraperitoneally in the presence or absence of agonistic aCD40 or aOX40 in pancreatic lymph nodes (PLN). However, activation was abortive, as antibodies. Our results suggest that both antibodies can prevent tolerance CL4 T cells were eliminated from the peripheral T-cell pool without in naive C57BL/6 mice, as well as in naive CD40L–/– mice. aCD40 ligates mounting an autoimmune response to islet a cells. In contrast, exposure to CD40 on APC, probably leading to the up-regulation of costimulatory KdHA epitopes following influenza virus infection of conventional reci- molecules necessary for T-cell priming, while aOX40 binds OX40 on T pients resulted in the productive activation of CL4 cells and the formation cells and directly provides priming signals, which prevent the induction of of KdHA-specific CTL. Comparing phenotypes of activated CL4 cells, T-cell tolerance. To further investigate whether a lack of CD40 on APC showed that unlike productively activated CL4, CL4 cells responding HA may have a role in the induction of tolerance, we intravenously transferred as a self-antigen did not differentiate into terminally divided CD69– OVA323-339-loaded splenic dendritic cells (spDC) from either wild-type CD25þ CTL. Previous studies have reported that abortively activated or CD40 deficient mice into naive C57BL/6 mice. Preliminary results CL4 cells die by apoptosis in the PLN of InsHA recipients. Our studies show that CD40-deficient spDC do not prime in vivo. Experiments to show that following activation by cross-presented HA in PLN, CL4 cells establish whether T cells, which have been in contact with CD40-deficient migrate to the lamina propria (LP) of the small intestine of InsHA spDC, are rendered tolerant will be presented. recipients. The LP contains only late stage divided CL4 cells that are not detected in any other lymphoid compartment.

OP102 Assessing sensitivity to estimate antigen-specific OP104 frequencies of CD4þ T cells Human T cells acquire HLA class II and costimulatory molecules from allogeneic S. Stevenson, M. P. Hernandez-Fuentes, O. Barroso-Herrera, D. Diaz antigen-presenting cells & R. I. Lechler Department of Immunology, ICSM, Hammersmith Hospital, Du Cane D. S. Game, N. J. Rogers & R. I. Lechler Road, London W12 0NN, UK Department of Immunology, Imperial College, Hammersmith Hospital, London W12 0NN, UK Our aim was to assess the sensitivity, specificity, labour-intensity and cost of different invitro assays to enumerate antigen specific CD4þ T cells In murine models it has been shown that T cells can acquire MHC and during the course of an immune response. The IL-2-producing precursor costimulatory molecules. To date there is no published data on acquisition frequency of antigen specific CD4þ T cells was measured by ELISA on by human T cells. We co-cultured human CD4þ T cells with M1 (human LDA designed experiments and ELISpot. Similarly, we compared the fibroblast) cells transfected with HLA-DR1 and human CD80, CD86 or proliferating precursor frequencies by 3H-Thymidine uptake on LDA and CD40. Flow cytometry demonstrated significantly increased levels of CFSE cell tracking. Known numbers of CD4þ T cells from DO11.10 mice HLA-DR1 and the appropriate costimulatory molecule on the surface of were mixed at different ratios with CD4þ T cells from BALB/c mice. the T cells at different time points. There are four main reasons why this is CD4þ T cells were stimulated with cells transfected with the appropriate acquisition and not up-regulation: first, the T cells display the molecules at restriction molecule (H2-Ad) and mouse CD86 that had been pulsed their cell surface too quickly for up-regulation to have occurred (less than with OVA peptide. In this system the measured frequency should 4 hr); secondly, the number of T cells that displayed the costimulatory correspond to the input number of DO11.10 T cells allowing the accuracy molecules after co-culture makes cognate interaction unlikely (75% of these methods to be compared. Frequency calculation by ELISpot became positive for CD80); thirdly, pretreatment of the T cells with analysis proved to be the most accurate method overall and is the cyclohexamide did not affect expression of the costimulatory molecules least labour-intensive. CFSE cell tracking was superior to LDA to post-co-culture whereas up-regulation of CD69 was prevented; finally, measure proliferative precursor frequencies and is the cheapest assay. when M1 cells cotransfected with HLA-DR1 and porcine CD86 were Inter-assay variability was similar for ELISpot and CFSE. The informa- placed in coculture with human CD4þ T cells, 60% of the T-cells-bound tion thus obtained will shape the design of in vitro assays for clinical antisera to porcine CD86 but not antihuman CD86. The functional applications. consequences of these findings are under investigation.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 T cells 103

OP105 micevaccinated s.c. with F.tularensis LVS.C57BL/6 J mice showed higher Distinct effects of CD86-mediated costimulation on bacterial counts in the spleen and liver during the first week of infection, resting versus activated human CD4 T cells compared to BALB/c mice. However, later in the infection bacterial loads were similar in both mouse strains. In order to establish whether the N. Rogers, I. Jackson, N. Camara, G. Lombardi & R. Lechler differences in the course of the infection could be related to differences Department of Immunology, Imperial College, London W12 0NN, UK in T-cell immunity, the proliferative response of CD4þ cells to heat-killed CD80 and CD86 appear to be the most important costimulatory molecules LVS was analysed in both mouse strains. We found that CD4þ T-cells from in the initiation of T-cell immunity. Whether the biological significance of both mouse strains proliferated and released IL2 and IFNg in response to the two isoforms resides in their different patterns/kinetics of expression LVS, but no differences were found between BALB/c and C57BL/6 J mice. or in their function is unclear. We have addressed this, using HLA-DR1 The results suggest that differences in susceptibility to F.tularensis LVS in transfectants coexpressing CD80, CD86, or both, as APC for naive, BALB/c and C57BL/6 J occur in the early stages of the infection and do not memory and activated human CD4 cells. Both CD80 and CD86 efficiently correlate with the intensity of long-term memory CD4þ T-cell responses. costimulate alloresponses by whole peripheral blood CD4 cells, however, CD86 is inferior in costimulating alloresponses by memory T cells, and unable to costimulate three human CD4 T-cell clones. CD80/CD86 double OP109 APC stimulated lower responses than cells expressing CD80 alone. That ICOS–B7RP1 interactions are important for the clonal CD86 is actively inhibitory is evidenced by the increased response to the expansion and B-cell helper function responses of double CD80/CD86 APC when anti-CD86 antibody is present. These naive, Th1 and Th2 T cells effects are accompanied by phenotypic changes; CD80-mediated costi- K. M. Smith, P. Webb, J. M. Brewer, J. C. Gutierrez-Ramos,y mulation is accompanied by sustained CD28 and minimal CTLA4 A. J. Coyley & P. Garside expression on the CD4 T cells, while in response to CD86-expressing Department of Immunology, University of Glasgow, Western Infirmary, cells CD28 expression is reduced and high levels of CTLA4 induced. Glasgow G75 0PH, UK, yMillenium Pharmaceuticals Inc., Boston, MA These data indicate that CD86 mediates distinct signals in previously 02139, USA activated T cells, and suggest that CTLA4 ligation may dominate the outcome of CD86-mediated costimulation of activated CD4 T cells. Understanding the roles of costimulatory molecules in T-cell activation and B–T-cell interactions is crucial for effective vaccine design. Although OP106 signal 1, cognate recognition of MHC-peptide by the TcR, and signal 2, Measurement of telomere length in CMV-specific CD4 T binding of CD28 to CD80/CD86 have been defined, the recent avalanche cells by three-colour fluorescence in situ hybridization of costimulatory molecules may represent a potential third signal. ICOS is (FISH) CD28 family member expressed on T cells rapidly after activation which L. L. Belaramani, J. M. Fletcher, M. Salmony & A. N. Akbar augments both Th1 and Th2 responses. ICOS interactions may be important in B–T-cell co-operation, as germinal centre formation is Department of Immunology, Royal Free and University College Medical –/– School, London NW3 2PF, UK, yThe MRC Centre for Immune Regulation, impaired in ICOS mice. Using the adoptive transfer of tg B- and T invivo Birmingham University Medical School, Birmingham B15 2TT, UK cells, we can track immune responses . Using this technique we have assessed ICOS–B7RP1 interactions in the clonal expansion, migra- In order to maintain immunity, virus-specific cells need to persist through- tion and helper function of naive, TH1 and TH2 T cells. In vivo blockade out life. One of the constraints upon the maintenance of a memory T-cell of ICOS resulted in decreased primary B- and T-cell responses. Further- pool may be telomere erosion as a consequence of repeated cell division. more, Th1 and Th2 cells require ICOS signalling to achieve maximal Thus, during persistent viral infections, repeated stimulation of virus- clonal expansion and to support fulminant B-cell responses. specific T cells might lead to the loss of replicative capacity in old age. Our previous work has shown that telomere shortening occurs in EBV- specific CD8 cells, however, the impact of persistent viral infection on OP110 telomere length in CD4 cells has not yet been investigated due to the lack Ontogeny of human NK receptor-positive T cells of CD4 tetramers. In this study, CMV-specific CD4 cells were identified S. Cookson & D. Reen by flow cytometry as IFN-g-producing cells following stimulation with Children’s Research Centre, Our Lady’s Hospital for Sick Children, CMV lysate. Development of a new three-colour flow FISH technique has Crumlin, Dublin 12, Ireland now allowed for the simultaneous detection of antigen-specific CD4 cells as well as telomere length. The results have shown that CMV-specific CD4 NK receptor-positive (NKRþ) T cells differ phenotypically and function- cells have very short telomeres compared to the total CD4 population. ally from NK and T cells. NKRþ T cells occur less frequently in human Loss of replicative capacity through telomere erosion may contribute to umbilical cord, compared to adult blood. We have examined the frequency the loss of virus specific immunity during ageing. and ontogeny of these cells in cord blood. Mononuclear cells were isolated and CD56-positive cells were depleted using microbeads. Following OP108 culture with IL-15, cells were harvested and stained for CD3, CD56 Role of T cells in the immune response of mice to and other cell surface markers, in addition to intracellular perforin, IL-2, Francisella tularensis live vaccine strain (LVS) IL-4 and IFN-g. Lymphocyte populations were analysed by three-colour flow cytometry. Both NKRþ T cells (CD3þCD56þ) and NK cells J. J. Campos-Perez, S. Ugrinovic, K. F. Griffiny & P. Mastroeni (CD3–CD56þ) could be generated from CD56-depleted populations dur- Center for Veterinary Science, University of Cambridge, Madingley ing the first week of culture. Whilst the CD56þ T cells expressed a Road, Cambridge CB3 0ES, UK, yDSTL, Porton Down, Salisbury, predominantly na¨ıve phenotype (CD45RAþCD62LþCD31þCD27þ), the Wiltshire SP4 0JQ, UK majority of cells were CD8þ, and expressed a gdTCR and perforin at a F.tularensis LVS is a live attenuated protective vaccine against tularemia higher frequency than CD56– T cells. Furthermore, the majority of CD56þ in mice and humans. We found that BALB/c mice are more resistant than T cells could produce IL-2 and IFN-g, but not IL-4. These results suggest C57BL/6 J mice to s.c. infection. The reasons for these differences are that IL-15 may act to up-regulate CD56 on particular T cells, and that unclear. We studied the course of the infection in BALB/c and C57BL/6 J CD56 acquisition may be associated with effector function. The ability of

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 104 T cells

newborn T cells to acquire CD56 may therefore provide the neonate with a influence of EtxB on the immune response to an unrelated antigen. Here cytotoxic cell population that can be rapidly expanded and mobilised we show, using DO11.10 chimaeric mice, that intranasal (i.n.) EtxB can following infection. also inhibit immune responses to non-self antigens when administered prior to antigen priming in the periphery. In vitro proliferation and cytokine production were reduced upon re-stimulation of spleen and OP111 draining lymph node cells from EtxB treated mice compared to those Intranasal administration of Escherichia coli heat- receiving PBS. Phenotypic analysis of lymphocytes from such mice labile enterotoxin B subunit (EtxB) prior to revealed clear differences in the distribution of cell types within the immunisation induces CD4þCD25þ regulatory T-cell CD4þKJ1.26þ population with a reduction in the presence of Th1 cells. development Further experiments showed that CD4þCD25þ, but not CD4þCD25– cells, from EtxB treated mice inhibited proliferation and cytokine produc- A. Plant, R. Williams & N. A. Williams tion by primed lymph node cells in coculture. Altered cytokine profiles Department of Pathology and Microbiology, University of Bristol, were also seen when CD4þCD25þ cells from PBS and EtxB treated mice University Walk, Bristol BS8 1TD, UK were stimulated under non-antigen specific conditions. These data suggest EtxB is a potent immune modulator capable of preventing or treating EtxB may prevent autoimmune disease by favouring the expansion of a collagen induced arthritis in DBA/1 mice. We have investigated the regulatory T-cell subset.

T-cell receptors

OP112 chain peptide (B15-23) restricted by H-2Kd and cause diabetes. We have Isolation and characterization of recombinant TCRs for generated three founder lines of transgenic mice that express the TCRVb6 a murine autoantigen from this clone. Mice from all the lines show that the ratio of CD8:CD4 T cells is 50% greater than that found in transgene negative mice. Although L. Bowie, W. J. Harris,y C. R. Shen,z C. J. Elsonz & R. N. Barker T cells do respond to islets, the mice develop less diabetes than their Department of Medicine and Therapeutics, IMS, Forester Hill, Aberdeen transgene negative littermates and disease onset is delayed. We have AB25 2ZD, UK, yDepartment of Molecular and Cell Biology, Foresterhill, examined mice for selection of T cells that recognize a Kd-B15-23 Aberdeen AB25 2ZD, UK, zDepartment of Pathology and Microbiology, tetramer. Overall, there is little difference in the percentage of CD8 University of Bristol, Bristol BS8 1TD, UK thymocytes or peripheral CD8 T cells that bind to the tetramer in the We aim to measure, for the first time, the affinity with which autoreactive transgene positive mice compared with transgene negative littermates. In T cells bind to their self-peptide/MHC ligands. NZB mice will be used, as addition, we have examined the Va–Ja join in mature CD8þ TCR high they spontaneously develop autoimmune haemolytic anaemia. We have thymocytes that express Va18 and these have not revealed limited amino previously shown that Band 3, the red blood cell anion channel protein, is acid sequences. Thus, presence of the TCRb chain does not necessarily the major target for pathogenic autoantibodies and helper T cells. Further increase the selection of the TCRa chain from which the T cell was work identified aa861–874 as containing a dominant naturally processed derived. helper epitope within this antigen. T-cell hybridomas specific for aa861– 874 were generated from NZB mice, and the a and b variable chain genes were isolated and sequenced using ligation-anchored PCR and RACE OP114 techniques. Analysis of the genes isolated showed that both products were EBV as a model to compare self- and allo-restricted murine variable TCR chains, and the TCR b chain belongs to the Vb5Á2 T cells specific for the same MHC-peptide target subfamily. We will use these chains to produce soluble ab TCR hetero- A. Tranter, A. Hislop, A. Rickinson & N. Steven dimers, which we can then use for analysis of ligand binding using surface Cruk Institute for Cancer Studies, University of Birmingham, Birmingham plasmon resonance. These experiments will show whether pathogenic T B15 2TT, UK cells in a spontaneous autoimmune disease express TCRs with high or low affinity for self-peptide/MHC ligands. The T-cell repertoire contains cells that also recognize peptides presented on nonself MHC alleles. A viral peptide model was used to compare the functional properties of self- and allo-restricted clones that recognize the OP113 same MHC-peptide target. The clones were generated from HLA-A2þ TCR-b chain transgenic mice derived from a CD8 T-cell and A2– donors by stimulation with peptide-coated HLA-A2þ cells, clone labelling with HLA-A2-peptide tetramer and immunomagnetic sorting. Staining intensity with the tetramer was used as a measure of TCR–MHC- Z. J. Li, M. Augustine,y C. A. Janeway Jr,y L. Wenz & F. S. Wong peptide affinity. Functional avidity was determined in killing assays by Department of Pathology, University of Bristol, Bristol, BS8 1TD, UK, peptide titration on HLA-A2þ cells. For all but two HLA-A2– clones, ySection of Immunobiology, Yale School of Medicine, New Haven, CT peptide-specific lysis of HLA-A2þ targets was concordant with tetramer 06520, USA, zSection of Endocrinology, Yale School of Medicine, New labelling. Half maximal lysis was seen at 107M for self-restricted and Haven, CT 06520, USA 106 M for most allo-restricted clones. Both clone sets labelled similarly The CD8 T-cell clone (G9C8) from non-obese diabetic (NOD) mice bears with tetramer. Killing assays with substituted peptides indicated that the T-cell receptor (TCR) Va18/Vb6. The T cells recognize an insulin B peptide residues 6–8 were critical for recognition by both allo- and

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 T-cell receptors 105 self-restricted clones. Interestingly, the allo-restricted clone with the The structural properties that underlie the functional variation within self- highest functional avidity, 108 M stained with tetramer at low intensity. and allo-restricted clones is now being explored.

Adhesion molecules

OP115 combined immunodeficient (SCID) mice, it is possible to target human Identification of homing peptides specific for human MVE determinants, using a peptide phage display library. In this model, synovial microvascular endothelium by in vivo phage peptide phage which home specifically to synovial grafts in preference to display selection other human (skin graft) or mouse tissues have been identified, and are shown to bind in vivo to synovial graft MVE independently from phage L. Lewis, C. Buckley,y M. Blades, G. Panayi, A. Georgez & component and degree of human/murine graft vascularization. Sequence C. Pitzalis analysis of synovial homing phage peptide reveals enrichment in specific Department of Rheumatology, GKT School of Medicine, 5th Floor consensus motifs. One such sequence maintains synovial homing speci- Thomas Guy House, Guy’s Hospital, London SE1 9RT, UK, yDepartment ficity both when expressed by a single phage clone and as a free synthetic of Rheumatology, MRC Centre for Immune Regulation, University of peptide. In addition, the free peptide competes and inhibits in vivo Birmingham, Birmingham B15 2TT, UK, zDepartment of Immunology, the binding of the original peptide phage to the cognate synovial MVE Division of Medicine, Faculty of Medicine, Imperial College, ligand. Hammersmith Hospital, Du Cane Road, London W12 ONN, UK The microvascular endothelium (MVE) represents an important therapeutic target in inflammation. By grafting human tissues into severe

Antibodies

OP116 OP117 A combination of phage antibodies and cDNA libraries Development of antibody–porphyrin conjugates for identifying novel tumour markers for use in photodynamic therapy K. A. Smith, L. A. Madden, K. P. Topping, J. R. T. Monson & L. A. Madden, O. J. Clarke, H. Savoie, R. W. Boyle & J. Greenman J. Greenman Postgraduate School of Medicine, University of Hull, Cottingham Road, Departments of Surgery and Chemistry, University of Hull, Hull Hull HU6 7RX, UK HU6 7RX, UK Tumour cell-specific antibodies are ideal reagents for the diagnosis and A previously described method was used for conjugating porphyrins to treatment of cancer. Phage antibody technology offers a tool for gen- antibodies, to allow targeted delivery. A hydroxylated porphyrin iso- erating such potentially useful antibodies. A panel of antibodies specific thiocyanate (PITC) was linked to an anti-epithelial cell adhesion mole- for different colorectal cancer cell lines was isolated from the Nissim cule (EpCAM) antibody via the lysine residues. An irrelevant control phage display library after panning against Colo320 and LoVo cells. antibody was similarly conjugated. Polyacrylamide gel electrophoresis Three of these clones (CAF-1, LAG-3 and LBA-1) were selected for verified successful conjugation, as labeled antibodies were visualized by further study on the basis of high target-molecule expression on the cell UV. FACS analysis showed no change in the level of antibody binding to lines as well as relatively efficient expression of soluble scFv fragments. the EpCAM positive Colo320 cell line after conjugation at PITC:anti- Western Blotting of Colo320 cell lysates with CAF-1, LAG-3 and LBA- body loading ratios (LR) varying from 20 : 1 to 2Á5 : 1. Non-specific 1 scFv fragments showed that CAF-1 bound to proteins of 13, 30 and binding of the irrelevant bioconjugate was not seen. Cytotoxicity assays 80 MW, whereas LAG-3 and LBA-1 demonstrated no binding. This were carried out on Colo320 cells after irradiation with non-thermal red suggests that the epitope recognized by the latter two antibodies is either light (>600 nm). The LD90 concentration varied from 8Á2 106 to 6 conformational or of non-peptide origin. We have used a Colo320 cDNA 9Á5 10 M for LRs of 2Á5 : 1 and 20 : 1, respectively. The control phage library as a way of identifying the molecules recognized by the conjugate killed <5% of cells under the same conditions at a LR of antibodies. This methodology is simple and rapid, and provides infor- 10 : 1. The results show that porphyrins can be conjugated to antibodies mation to assess the usefulness of newly generated phage antibodies. using a simple method that does not affect antibody binding. The low This approach can now be applied to antibodies and cDNA libraries LD90 concentration of the bioconjugates after irradiation compared with generated from fresh tumours for use in identifying novel disease the minimal inherent cytotoxicity mean that these novel PDT reagents markers. are potential anti-tumour drugs.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 106 Autoimmunity

Autoimmunity

OP118 OP120 Anticardiolipin antibody and an ti-nuclear antibody in Intranasal gene therapy in a murine model of women with recurrent pregnancy loss rheumatoid arthritis O. Safa, A. S. Jahromi & S. Zarey A. M. Woods, P. Hobson, A. Barnes, S. J. Thompson, G. S. Panayiy Department of Immunology, and yDepartment of Statistics, Faculty of & L. S. Klavinskis Medicine, Hormozgan Univercity of Medical Sciences, Bandar Abbas, Immunobiology, 3rd Floor, New Guys House, Guys Hospital, London SE1 791496153, Iran 9RT, UK, yAcademic Department of Rheumatology, 5th Floor, Thomas Guy House, Guy’s Hospital, London SE1 9RT, UK Foetal loss and recurrent abortion are responsible for significant emotional distress for couples desiring children. Regarding the etiologic importance, Rheumatoid arthritis is a progressive autoimmune disease affecting this study has been carried out to determine the prevalence of ACLA and approximately 1% of the population. It is extremely debilitating and ANA in women with recurrent miscarriage. This descriptive study was significantly lowers quality of life. Recently, an effective anti-TNF-a carried out in Bandar Abbas University of Medical Sciences, during therapy has become available. However, it is expensive to produce and 2000–2001. One hundred and nine women with definite diagnosis of administer. Recent studies have shown that intra-articular adenoviral recurrent abortion or foetal loss as patients and 220 matched women with mediated IL-10 expression lead to a significant reduction in disease at least two successful deliveries and negative history of miscarriage as course and severity. However, this route is problematic in terms of controls were studied. The sera of the two groups were tested by ELISA patient compliance. We intend to circumvent these problems by the method for determining ACLA and ANA. There was a significant intranasal delivery of plasmid DNA encoding interleukin-10 using a difference between prevalence of ACLA and ANA in the patients and murine model of rheumatoid arthritis (CIA). Silent mutations introduced the controls. The results of this study suggest that autoantibodies play a into the murine IL-10 cDNA sequence (mut-IL10) allow the construct to significant role in recurrent miscarriage. Further studies are needed to be tracked in vivo. Mut-IL-10 cDNA was cloned into a commercial explore the role of autoantibodies in abortion. expression vector and transfected into HEK293 cells. Sandwich ELISA and Western blot confirmed IL-10 secretion. Inhibition of lymphocyte proliferation and down-regulation of MHC class II expression demon- OP119 strated biological activity. Ongoing studies with this construct are Autoreactive T cells in Goodpasture’s disease evaluating its potential to prevent the induction of and ameliorate recognize less efficiently presented epitopes in the established CIA. Goodpasture antigen W. Saweirs,y L. Cairns, R. Barker, A. J. Rees & OP121 R. G. Phelpsy Identification of nuclear spliceosomal epitopes Department of Medicine and Therapeutics, University of Aberdeen, targeted by NOD mouse antibodies following NaI Aberdeen AB25 2ZD, UK, yDepartment Clinical and Surgical Sciences, exposure in vivo University of Edinburgh, Edinburgh EH8 9JZ, UK C. Thompson, S. Verma, D. P. Krummel,y K. Nagaiy & Goodpasture’s disease is an HLA DR15-associated nephritis defined by A. Cooke autoantibodies to the glomerular basement membrane and a pattern of University of Cambridge, Department of Pathology, Tennis Court Road, injury associated with CD4 T cells. The autoantibodies are pathogenic and Cambridge CB2 3QP, UK, yStructural Studies Division, MRC bind to the first non-collagenous domain of the a3 chain of type i.v. Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, collagen (GA), but the specificities of relevant T cells are less clear. UK Patients’ PBMC include T cells specific for GA, but not the homologous a5 chain. Here, we have compared the fine GA specificity and cytokine Patients with autoimmune diseases can produce antibodies specific for phenotype of peripheral blood T cells from six patients and five healthy nuclear antigens. The mechanism for this process is not understood. DR15-bearing individuals. PBMC were incubated with overapping pep- However, specific autoimmune diseases can be characterized by the tides spanning the sequence of GA. Proliferation and cytokine production patterns of immunohistochemical staining by anti-nuclear antibodies were assayed at 5 days. PBMC from patients (all DR15) proliferated in (ANA). The non-obese diabetic (NOD) mouse develops a range of response to significantly more GA-peptides than did those from control autoimmune pathologies and spontaneously develops autoantibodies DR15-positive donors (P ¼ 0Á004). The proliferative responses of patients specific for nuclear antigens. Sodium iodide (NaI) exposure has pre- but not controls were highly focused (P ¼ 0Á0002) on two peptides, 8 and viously been shown to increase autoimmune pathology in the thyroid of 14. PBMC from patients with acute disease were stimulated by GA NOD mice. Here, NOD mice were provided with NaI to assess its effect on peptides to produce interferon-g with little IL10 and no IL4, but PBMC the development of ANA. We have shown that high dietary NaI increases from two of the patients collected 6–8 weeks later proliferated less and the incidence of ANA shown immunohistochemically to bind to HEp-2 produced more IL10. Notably, the peptides that most frequently stimu- cells with a uniform nuclear speckled pattern identical to that given by lated GD patients’ T cells were distinct from the GA peptides previously anti-spliceosomal antibodies. The spliceosome is a nucleoprotein com- shown to be the major peptides naturally processed from GA in B cells and plex involved in splicing of nascent mRNA in the nucleus. The spliceo- presented bound to DR15. Therefore, induction of Goodpasture’s disease somal proteins U1A and the complex of Sm proteins EFG were shown to is associated with breakdown of self-tolerance to less abundantly pre- be specifically targeted by the NOD autoantibodies found to be largely sented GA peptides. IgG1 and IgG2b isotypes.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Autoimmunity 107

OP122 architecture in the BB rat model. Control (BBc) and diabetic prone TCR antagonism by altered peptide ligands of a (BBdp) rats were fed diets that either promote or protect against diabetes. diabetogenic CD8 T-cell clone BBdp rats on a normal diet developed significant enteropathy soon after weaning and it remained constant thereafter with significant increases in L. G. P. Marquesini, L. Weny & F. S. Wong crypt length and mitotic activity compared with BBc rats. The enteropathy Department of Pathology & Microbiology, University of Bristol, Bristol was not influenced by a low antigenic diet, nor by thymectomy, both of BS8 1TD, UK, ySection of Endocrinology, Yale School of Medicine, New which prevent diabetes. We conclude that enteropathy and diabetes are Haven, CT 06520, USA due to different mechanisms, but BBdp rats are predisposed to develop Introduction Our studies further characterize a CD8 T-cell clone (G9C8) enteropathy which may be necessary for the development of autoreactivity from the islet infiltrate of 7-week-old female Non Obese Diabetic (NOD) in the pancreas. mice. This clone is restricted by H-2Kd, produces interferon-g and is cytotoxic to islet b cells invitro and invivo. The cells recognize the insulin B chain amino acids 15–23 (B15–23) whose sequence is LYLVCGERG. Our aim was to identify insulin B15–23-altered peptide ligands (APL) that OP125 antagonize the G9C8-TCR. We focused on positions six and eight of the Mechanisms of T-cell death in the avidity model of peptide, previously suggested to be important TCR contact sites. These peripheral T-cell tuning two positions in the native peptide were systematically substituted with K. Ryan, D. McCue & S. Anderton the remaining 19 (mutant) amino acids at each position. We tested the 38 51 ICAPB, Ashworth Laboratories, Kings Buildings, West Mains Road, mutant peptides in Cr release cytotoxicity assays and for IFN-g in Edinburgh EH9 3JT, UK ELISA assays to look for agonist and antagonist activity. Results (1) None of the APL show agonist activity; (2) antagonist In a process known as tuning, a population of peripheral T cells maintains responses were seen with all the peptides; (3) most APL completely adefined sensitivity to antigen by adjusting its reactivity to variations in inhibited the cytotoxic response, whereas IFN-g production was only antigenic strength. Proposed mechanisms include either the adaptation of partially inhibited. We conclude that positions six and eight of the insulin biochemical pathways of individual cells (biochemical tuning), or the B chain peptide are very important residues for TCR stimulation and no selective expansion of T cells bearing TCRs with appropriate affinity for substitutions are tolerated at these positions. the cognate APC (avidity model). Support for the avidity model of peripheral tuning comes from studies of the T-cell response to the encephalitogenic epitope of myelin basic protein (MBPAc1–9) in context OP123 with IAu. An altered peptide (4Y) characterized as a superagonist by B cells as regulators of autoimmunity invitro T-cell proliferation was unable to induce autoimmune disease M. J. McGeachy & S. M. Anderton invivo due to the deletion of high affinity T cells by the superagonist. invitro University of Edinburgh, ICAPB, Edinburgh EH9 3JT, UK Here, we extend those findings by establishing an system to measure T-cell death in response to antigenic stimulation. The 4Y super- The importance of B cells in the T-cell-mediated autoimmune demyeli- agonist peptide clearly induces more T-cell death than the wild type nating diseases, multiple sclerosis in humans and experimental autoim- peptide, and we are currently investigating the mechanisms by which this mune encephalomyelitis (EAE) in mice, is controversial. Recent work has death occurs. Early indications are that the Fas/FasL pathway and antigen shown that while B cells are not required for induction of EAE, as dose play important roles in determining whether death or expansion previously thought, they are crucial for remission of disease. Using the follows antigenic stimulation. MOG35–55-induced model of EAE in C57BL/6 mice, we made bone marrow chimaeras in which chosen defects are targeted specifically to B cells. B-cell activation (via CD40) and their IL10 production were absolutely required for remission of disease. We have also found that OP126 mice which have recently recovered from EAE are relatively resistant to A role for B cells in peptide-induced T-cell re-induction, showing a milder course of disease. Furthermore, transfer of tolerance? lymphoid cells confers similar resistance to na¨ıve recipients. The ability of C. S. Sweenie & S. M. Anderton various cell populations to mediate protection is under investigation, with Edinburgh University, ICAPB, King’s Buildings, West Mains Road, preliminary results indicating the importance of B cells, as expected. Edinburgh EH9 3JT, UK Ongoing experiments to further define how and where B cells exert their regulatory effects will be presented. Administration of peptides in soluble form is a powerful means of inducing antigen-specific T-cell tolerance. The precise nature of tolerance induction (in terms of antigen presentation) remains poorly defined, OP124 however, with conflicting reports that B cells are or are not required. Enteropathy and diet are unrelated risk factors in the Here, we show that B-cell deficient mice are susceptible to peptide- pathogenesis of type 1 diabetes induced tolerance. However, this is dependent on which lymphoid organs are used as a source of T cells to be tested. B-cell sufficient mice appear S. Graham, F. Scott,y W. Malaisse,z J. Rozing§ & A. McI Mowat ‘fully’ tolerant, with no antigen-specific recall response in lymph node or Department of Immunology, University of Glasgow, Glasgow G11 6NT, spleen. In contrast, mice lacking B cells appear tolerant in the lymph UK, yOttowa Hospital Research, Institute, University of Ottowa, Ottawa node, but not in the spleen. We assessed the impact of this ‘split’ K1H 8L6, Canada, zLaboratory of Experimental Medicine, Brussels Free tolerance in B-cell deficient mice on the ability on control autoimmunity. University, Brussels B-1070, Belgium, §Deparment of Histology and Even though these mice harboured antigen-reactive splenic T cells, Cell Biology, University of Groningen, Oostersingel 9713 EZ, The soluble peptide was fully able to prevent onset of autoimmune encepha- Netherlands lomyelitis. These findings implicate the peripheral lymph nodes (rather Diet may play a role in the pathogenesis of Type 1 diabetes. Here, than spleen) as the critical site for the initial priming of encephalitogenic we investigated the association between diet, diabetes and intestinal T cells.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 108 Autoimmunity

OP127 arthritis. To investigate a possible regulatory mechanism in humans we CD4þ T cells specific for the stress protein BiP have characterized the T-cell response to human hsp 60. Responses were modulates the development of collagen-induced shown by all 12 healthy individuals tested and CD4þ T-cell clones were arthritis generated from a single individual. Despite responding to the recombinant hsp 60 preparation, the clones were found to be specific for either groEL R. Brownlie, V. Corrigall,y M. Bodman-Smith,y N. Staines, (E. coli hsp 60) or another unidentified bacterial contaminant. The groEL G. Panayiy & S. Thompson reactive clones recognized an epitope spanning amino acid residues 9–23. Division of Life Sciences, Franklin Wilkin’s Building, 150 Stamford A homologous peptide from human hsp 60 was unable to stimulate the Street, London SE1 9NN, UK, yDepartment of Rheumatology, Guy’s clones. Using alanine-scanning peptides of groEL a number of essential Hospital, London SE1 9RT, UK residues were found to be different to the human suggesting that cross- The stress protein BiP has recently been described as an autoantigen in reactivity is unlikely. This study shows that if presented with hsp 60 human rheumatoid arthritis. Studies using HLA-DR1þ/þ-transgenic mice containing low amounts of groEL (<0Á2 mg/ml in our study) or other showed that BiP administered intravenously before onset of CIA could bacterial protein, responses are preferentially generated to the bacterial indeed protect mice from disease. Recent studies have focused on the less component. This finding casts doubt on the extent of the repertoire for self invasive, subcutaneous route of antigen administration. Results show that hsp 60 and is important because most human studies into the response to T cells purified from the spleen and lymph nodes of mice immunized with human hsp 60 have been performed using recombinant hsp 60. 200 mg of BiP in saline produce considerably raised levels of Th-2 cytokines, IL-4 (600 pg/ml), IL-5 (700 pg/ml) and IL-10 (500 pg/ ml), as compared to saline immunized controls upon in vitro stimulation OP130 with BiP. This production of regulatory cytokines is remarkably long lived A study of IgA antibodies to a T-cell epitope of a-gliadin (up to 7 weeks) considering it is administered in the absence of additional in Coeliac disease adjuvants. Current studies are evaluating this immunomodulating poten- E. Saxby, B. Ferry, R. Anderson,y P. Kelleher,z S. Misbah & tial for prevention and immunotherapy of CIA in DBA-1 mice. H. Chapel Acknowledgement This work is funded by the Wellcome Trust. Department of Immunology, Churchill Hospital, Oxford OX3 7LJ, UK, yWalter and Eliza Hall Institute, Melbourne 3050, Australia, zDepartment of Immunology, St Mary’s Hospital, London W2 1PG, UK OP128 Murine autoreactive T cells to the Goodpasture In coeliac disease patients the dominant DQ2-a-1gliadin peptide recog- autoantigen nized by CD4 T cells is contained within peptide sequence 57–73 (p57– 73) of a-gliadin. To determine whether coeliacs also recognized a B-cell J. V. Marley, J. Lamb & A. N. Turner epitope within this area an ELISA detecting serum IgA antibodies to p57– Centre for Inflammation Research, Hugh Robson Building, George 73 was developed. IgA antibodies to p57–73 were found in 37/80 (46%) Square, University of Edinburgh, Edinburgh EH8 9XD, UK endomysial antibody positive patients, 29 of whom were known coeliacs. Autoimmunity to the NC1 domain of the a3 chain of type i.v. collagen Assay specificity and sensitivity for coeliac disease was 98 and 57%, (a3(i.v.)NC1), the Goodpasture antigen, is the cause of spontaneous respectively. To determine the importance of the amino acid at position 65 human anti-glomerular basement membrane (anti-GBM) disease. The for IgA binding, glutamine was substituted with four amino acids and the generation of murine autoreactive T cells to the Goodpasture antigen was altered peptides assayed for IgA binding. The amino acid at position 65 is assessed by invivo challenge of 129 mice with a3(i.v.)NC1 (enriched from not important for IgA binding, but is crucial for T-cell recognition of p57– bovine testes) followed by in vitro recall using a panel of peptides to 73. To further elucidate the B-cell epitope, nine biotinylated octapeptides, murine a3(i.v.)NC1. Peptides that induced T-cell proliferation and/or each frame shifted one amino acid to span the whole p57–73 sequence cytokine secretion were then used to challenge mice. Peptide 112–126 were examined for binding. No discrete sequence within p57–73 was (ma3p112–126) was able to specifically induce proliferation of autoreac- identified as the antibody epitope inferring a short common motif or a tive T cells that secreted IL-2, IL-3, TNFa and IFNg. When ma3p112–126 polyclonal antibody response to this region. was used in a tolerizing protocol (nasal insufflation on 3 consecutive days), 7 days prior to re-challenge with the same peptide in CFA, antigen specific T cells became anergic. There was a marked decrease in pro- OP131 liferation and IL-3 and TNFa secretion, and no IFNg secretion in recall Bioinformatics analysis of HERV-K10 identifies responses to ma3p112–126, compared to mice tolerized with an unrelated autoimmune epitopes in a gag open reading frame peptide. The identification of peptides that generate autoreactive T cells P. Nelson, H. D. Ejtehadi, S. R. Jones,y A. J. Astley, gives us a remarkable opportunity to determine under what circumstances A. Y. H. Alosaimi & D. Roden tolerance to an autoantigen can be broken, and how it may be re- Division of Biomedical Sciences, School of Applied Sciences, University established. of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1SB, UK, yMolecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK OP129 E. coli hsp 60 specific T-cell clones recognize a Human endogenous retroviruses (HERVs) constitute 1% of the human conserved epitope but fail to cross react with self genome and have been implicated in certain autoimmune diseases and hsp 60 cancers. It is assumed that these viruses are activated and mediate their effects through a variety of mechanisms including molecular mimicry, R. C. Duggleby, M. S. Lillicrap & J. S. H. Gaston superantigen motifs and modulation of appropriate genes. Clearly for Department of Medicine, Box 157, Addenbrookes Hospital, Cambridge molecular mimicry to be important, there must be a region of an CB2 2QQ, UK endogenous virus that shares homology with host protein. In this study, Evidence in rodent models of inflammatory arthritis suggests that T cells we have used bioinformatic tools to investigate potential antigenic sites of specific for self heat shock protein 60 (hsp 60) are able to ameliorate HERV-K10 and regions of homology with host (human) proteins to

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Autoimmunity 109 identify potential cross-reactive epitopes. We highlight two key which prevent determinant spreading and protect mice from the devel- regions within the gag open reading frame of HERV-K10 with sequence opment of PIA. homology to myelin basic protein and collagen. These observations may be of significance to the process of molecular mimicry that could act through the aberrant production of truncated or full-length protein products. OP134 Major T-cell epitopes in the Goodpasture antigen are destroyed by endosomal proteases OP132 J. Zou, A. N. Turner & R. G. Phelps The potential role of human endogenous Department of Clinical and Surgical Sciences, University of Edinburgh, retrovirus K10 in pathogenesis of rheumatoid Edinburgh EH8 9JZ, UK arthritis Antigen processing constrains the range of antigen-derived peptides H. D. Ejtehadi, H. A. Ali,y E. Serhan,y S. Bowmanz & visible to T cells. One mechanism depends on the specificity of endosomal P. N. Nelson proteases. Thus, T-cell epitopes in foreign antigens such as ovalbumin are School of Applied Sciences, University of Wolverhampton, cut out of intact antigen during in vitro digestion with endosomal proteases Wolverhampton WV1 1SB, UK, yDepartment of Rheumatology, such as cathepsin D, presumably making them available to bind class II. New Cross Hospital, Wolverhampton WV10 0QP, UK, zDepartment Here, we have used MALDI mass spectrometry to investigate the gen- of Rheumatology, University of Birmingham, Birmingham B9 5SJ, eration of the major T-cell epitopes in the Goodpasture autoantigen UK (a3(i.v.)NC1) during in vitro digestion with human cathepsin E and D Human endogenous retroviruses (HERVs) are vertically transmitted and and AEP. Both major T-cell epitopes in a3(i.v.)NC1 (VCNFASRNDYand constitute 1% of the human genome. Because of the structural similarities ISLWKGFSFI) were cleaved across the putative HLA DR-binding motif of HERVs to known exogenous retroviruses, they have been suggested as (Nfe´as and Sle´wk) at the earliest time points examined (5 min) during potential etiological agents of certain autoimmune disorders. We devel- cathepsin E and D digestion of intact a3(i.v.)NC1, and the asparagine- oped a multiplex RT-PCR system for detection and semiquantification of containing epitope was cleaved by AEP as expected (Vcne´fasrne´dy). HERV-K10 mRNA level in peripheral blood mononuclear cells from Relative rates of cutting were assessed with overlapping synthetic pep- osteoarthritis (OA), rheumatoid arthritis (RA) and healthy individuals. tides spanning a3(i.v.)NC1. Peptides containing the T-cell epitopes were Patients’ sera were tested for any antibody reactivity to a synthetic peptide among only 5 of 22 >90% digested at 5 min with cathepsin D. Oxidation designed from the most hydrophilic region of HERV-K10 gag region. The of intramolecular disulfide bonds dramatically influenced the activity of ELISA results showed higher antibody reactivities to the peptide in all three enzymes at several scissile bonds, including those in the healthy individuals as compare to RA patients. Despite of low antibody Vcnfasrndy. Thus, major T-cell epitopes in a3(i.v.)NC1 are exquisitely reactivity to HERV-K10 gag peptide in RA patients, the result of RT-PCR sensitive to prominent endosomal proteases, the opposite of what has been showed an enhanced mRNA expression of HERV-K10 gag region in RA observed for exogenous antigens, which might cause less efficient proces- compared to OA and healthy individuals, which could be contribute to the sing under typical conditions. pathogenesis of RA.

OP133 OP135 Regulatory T cells prevent determinant spreading DNA vaccines encoding collagen Cb11 fragment within the 65 MW heat shock protein: relationship to or the immunodominant 260–270 T-cell determinant pristane-induced arthritis modulate collagen-induced arthritis (CIA) in DBA-1 mice S. J. Thompson, I. Karadimitris, C. Hall, L. K. Siew,y J. T. Beechy & C. J. Elsony M. A. Khan, C. O’Shea, N. S. Ali, L. Marinova-Mutafchieva, King’s College London, Division of Life Sciences, Franklin-Wilkins J. J. Murphy, N. A. Staines, W. Caparros-Wanderley & Building, 150 Stamford Street, London SE1 9NN, UK, yDepartment D. H. Davies Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, Division of Life Sciences, Franklin Wilkins Building, King’s College, UK 150 Stamford St, London SE1 9NN, UK T-cell responses to the 65 MW heat shock protein are implicated in both DNA vaccines offer unprecedented opportunities for the development the development of and protection from (by hsp 65 preimmunization) novel immunotherapies. In comparison with infectious disease, relatively pristane-induced arthritis (PIA). Epitope mapping studies have shown that little is known about the efficacy of DNA vaccines in autoimmune disease T cells from naive animals respond to the hsp 65 epitope (aa 291–306). systems. In this study we have produced and characterized vaccine based Animals protected from the development of PIA, by hsp 65 immunization, on the pcDNA3Á1 plasmid vector (Invitrogen). These expressed the respond additionally to aa 261–71. By contrast, animals with PIA do not cyanogen bromide fragment 11 (Cb11) of bovine type II collagen, and exhibit such restricted T-cell responses but can respond to many epitopes the immunodominant CD4 T-cell epitope of Cb11, 260–270. These (determinant spreading) throughout the sequence of hsp 65 (e.g. aa 302– antigens were chosen on the basis of their ability to induce tolerance 314). In addition, peptide 261–71 can protect mice from the development when delivered via mucosal (nasal) routes. Pilot studies with the gene of PIA whereas the other two peptides cannot. T cells from mice vaccines indicate that both significantly reduced the severity of CIA when immunized with the non-protective peptide epitopes secrete high levels introduced i.m. in both prophylactic and therapeutic regimens. Studies are of IFNg and TNFa but low or undetectable levels of IL-10. By contrast, underway to determine the cytokine profiles in the different treatment immunization with peptide 261–71 activates a population of regulatory groups. cells characterized phenotypically as CD4þ, CD25þ, IL-10 secreting Acknowledgement Supported by an ARC Programme Grant.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 110 Bacteriology

Bacteriology

OP136 OP137 Immunopathology of a Chlamydophila abortus The inflammatory response in mice following infection in a pregnant mouse model Burkholderia mallei infection K. Kerr, M. Livingstone, S. W. Maley, G. Entrican, D. McKeever,y C. A. Rowland, K. F. Griffin, M. S. Lever & R. A. Lukaszewski D. Buxton & D. Longbottom Biomedical Sciences, Dstl, Porton Down, Salisbury SP4 0JQ, UK Moredun Research Institute, Edinburgh EH26 OPZ, UK, yDepartment of Burkholderia mallei is a Gram-negative bacterium that is primarily a Veterinary Clinical Studies, University of Edinburgh, Edinburgh pathogen of solipeds including horses and donkeys. It is also infectious in EH9 1QH, UK humans causing the disease Glanders. Glanders presents in either a Chlamydophila abortus is an obligate intracellular bacterium that targets septicaemic acute form or a chronic infection characterized by abscess the placenta, causing tissue damage, inflammation and abortion. Rumi- formation particularly in the liver and spleen. We investigated the host nants are the principal hosts, although humans are susceptible to infection. immune response to B. mallei as little is known about the pathogenesis of We have developed a mouse model to study the pathogenesis of chla- infection to this organism. BALB/c mice were challenged with a high dose mydial abortion. Infection was monitored by recovery of the organism and of B. mallei (106 organisms) and temperatures and symptoms were by immunohistochemical labelling for chlamydial antigen in tissue sec- monitored for 36 days post-infection. Bacteria were cultured from the tions. Pregnant mice were infected at mid-gestation (day 11) with the S26/ spleens of infected mice and cellular activation was investigated using 3 strain of C. abortus to determine the timing and progression of infection flow cytometry. Variable levels of infection were detected. Approximately of the maternal–foetal interface and to define which other organs may be half the mice developed splenic abscesses and this correlated with targeted. Infected cells were found at the maternal–foetal interface, symptom severity. A severe inflammatory response was identified in mice specifically the giant cells and the trophoblast cells within the labyrinth, which developed splenic abscesses – this was characterized by high on days 3 and 5 post-infection (p.i.). Chlamydial inclusions were scattered bacterial loads, an influx of neutrophils and macrophages into the spleen throughout the trophoblastic labyrinth of the placenta between days 3 and and increased expression of the inflammatory marker CD54 on these cells. 7 p.i., which immediately preceded abortion. Infection was accompanied Up-regulation of CD69, CD25 and CD54 on T cells was also observed. by a maternal mononuclear inflammatory cellular infiltrate. The role of # Crown Copyright 2002 Dstl this inflammatory response in disease pathogenesis is under investigation.

Cell signalling

OP138 TNFa-induced gene regulation in bronchial epithelial cells involving Epidermal growth factor receptor-regulated tumour the EGFR. necrosis factor alpha gene expression in bronchial epithelial cells OP139 L. M. Hamilton, I. Kimber,y R. J. Dearman,y S. T. Holgate, Using laser scanning cytometry to track T-cell S. J. Wilson, & D. E. Davies signalling events in vivo The Brooke Laboratory, Divison of Infection, Inflammation and Repair, C. L. Adams, A. M. Y. Morton, A. M. C. L. Mowat, M. M. Harnett & y SGH, Southampton SO16 6YD, UK, Syngenta Central Toxicology P. Garside Laboratory, Alderley Park, Cheshire SK10 4TJ, UK Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, UK Tumour necrosis factor alpha (TNFa) is a cytokine important in regulation of inflammation in asthma. We have demonstrated that TNFa causes The signalling events that occur within the immunological synapse during interleukin 8 (IL-8) release by H292 and primary bronchial epithelial initial antigen recognition can probably determine whether a T cell will cells, and this release is blocked by addition of the selective epidermal become tolerized or primed and what type of effector function it will growth factor receptor (EGFR) inhibitor, tyrphostin AG1478. This sug- display. It is also important to assess these events in the physiological gests that transactivation of the EGFR is involved in IL-8 release mediated context of the specialized anatomical niches in which they occur in vivo. by TNFa. To identify other TNFa-inducible genes that are regulated by Using a laser scanning cytometer (LSC), we have quantified and localized the EGFR, we used microarrays comprising 12 500 cDNAs encoding the activation of signalling molecules during the induction of T-cell genes of known function. H292 bronchial epithelial cells were stimulated tolerance and priming in vitro and in vivo. Antigen-specific TcR tg T- for 6 hr with TNFa without or with AG1478. From the genes up-regulated cells were primed or tolerized in vitro using immobilized anti-CD3 in the by TNFa, we identified a subset where the increased expression was presence or absence of appropriate costimulation. Laser scanning cyto- suppressed by AG1478. These included IL-8 and macrophage inflamma- metry revealed cell cycle arrest and increased apoptosis within the anergic tory protein 2 (MIP2), as well as genes involved in proliferation, differ- T-cell population. Furthermore, there were marked differences in the entiation and lipid metabolism, some of which are already known to be cellular localization and quantity of phosphorylated ERK1/2 between regulated by EGFR. These data highlight a novel mechanism for anergic and primed T cells. We are currently elucidating the location and

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Cell signalling 111 activation state of these signalling molecules within adoptively transferred LPS. Using stably transfected RAW 264.7 macrophage cell-lines expres- antigen-specific TcR tg T cells during the induction of tolerance and sing decreased and increased lipocortin 1 protein, we have shown pre- priming in an intact animal. viously that lipocortin 1 specifically regulates the ERK signal transduction pathway. Lipocortin 1 modulates upstream components of the ERK pathway including protein tyrosine phosphorylation and MEK and associ- OP140 ates with the adaptor protein Grb 2 (Alldridge et al. J. Biol. Chem. 1999; In vivo applications of the laser scanning cytometer in 274: 37620–37628). The NH2 terminal domain of annexin proteins, which immunology varies in both length and sequence, determines their diverse biological A. M. Morton, C. L. Adams, K. M. Smith, A. M. C. I. Mowat, J. M. activities (Crompton et al. Cell 1998; 55: 1–3). Within this domain of Brewer, M. M. Harnett & P. Garside lipocortin-1 the motif LENQEQEYVQAV is thought to contribute to its Department of Immunology and Bacteriology, Western Infirmary, specific functions. In this study we have prepared and characterized two University of Glasgow, Glasgow G11 6NT, UK mutant forms of lipocortin 1 and performed transient transfection studies in order to determine the precise site of lipocortin 1 action. Current flow cytometric technology allows quantitative assessment of surface and intracellularly expressed molecules on isolated cells. How- ever, the need to disrupt tissues prevents correlation of this expression OP143 with anatomical location. In contrast, immunohistochemistry in conjunc- Integration of first and second signal in T-cell tion with conventional or confocal microscopy allow localization of activation staining, but little in the way of quantification. The recently described y laser scanning cytometer (LSC) allows a combination of both approaches S. H. More´, S. Davis & T. Harder as it can apply flow cytometric laser technology to intact tissue. Using the Sir William Dunn School of Pathology, University of Oxford, South Parks y LSC we have begun to determine the level of expression of a variety of T- Road, Oxford OX1 3RE, UK, Nuffield Department of Clinical Medicine, cell-expressed costimulatory and signalling molecules and are relating University of Oxford, Oxford OX3 9DU, UK this to the anatomical location of T cells in different compartments within The activation of T lymphocytes is governed by the interaction of specific lymphoid organs. Such approaches will allow us to relate differences in the receptor/ligand pairs on T-cell- and antigen-presenting cell, respectively. expression of such molecules to the function of T cells in different sites. The engagement of the T-cell receptor by peptide/MHC complexes on the APC and the interaction of CD80/CD86 with their costimulatory or OP141 inhibitory receptors CD28 or CTLA-4 on the T cell are among the most Effect of killed Mycobacterium vaccae on ICOS important. It is still unclear how combinations of these signals are expression in a murine model integrated by the T lymphocyte to mount a distinctive response, such as differentiation, proliferation, anergy, or cell death. To address that y y V. C . Adams, J. R. F. Hunt, L. Rosa Brunet & G. A. W. Rook question, peptide/MHC complexes, in the presence or absence of costi- Department of Medical Microbiology, UCL-Windeyer Institute, 46 mulatory molecules, are coupled to magnetic beads and used for activation y Cleveland Street, London W1T 4JF, UK, SRPharma, 103 New Oxford of naive and effector T cells from TCR transgenic mice. Functional Street, London WC1A 1DD, UK parameters of T-cell activation are compared with the induction of Inducible costimulatory molecule (ICOS) is a member of the CD28 signalling pathways. superfamily and plays a pivotal role in effector T-cell responses. The ICOS pathway is believed to be critical for activation of Th2 effector responses. In its absence, or in the presence of ICOS antagonists, Th1 OP144 rather than Th2 responses develop. Recently, however, evidence has TNFa stimulates class II P13K in airway epithelial suggested a role for ICOS in the development of Th1 responses. We cells and others have shown that injection of a killed Mycobacterium vaccae K. M. Patel, P. Whittaker,y M. L. Watson, & S. G. Ward suspension in a murine airway allergy model induces regulatory T cells University of Bath, Bath BA2 7AY, UK, yNovartis Horsham Research and reduces Th2-mediated pathology independent of Th1 activity. We Centre, Horsham RH12 5AB, UK investigated whether M. vaccae injection affects the expression of ICOS in naive BALB/c mice during a 3-week period. Control mice, which had TNFa can up-regulate adhesion molecules and facilitate the subsequent been injected with saline, showed no significant alteration in ICOS transmigration of inflammatory leucocytes into the airway wall. TNFa has expression in the spleen using real-time PCR. In contrast, following previously been shown to activate the phosphoinositide 3-kinase (PI3K)- M. vaccae injection, ICOS expression was dramatically reduced on day dependent signalling pathway, which has been implicated in the regulation 7 and remained low until the end of the study. In a murine allergy model, of the cyclooxygenase-2 (COX-2) which in turn plays a key role in we expect that ICOS expression will be reduced in the lungs of M. vaccae- inflammatory responses. We report that the human lung epithelial cell line treated compared to that of saline-treated mice. Data on the expression of A549 expresses members of the class I (e.g. p85 regulatory subunit) and ICOS and its ligand will be presented. class II (C2a and C2b) PI3K subfamilies. TNFa (100 ng/ml) markedly increased phosphorylation of protein kinase B (PKB) after 20–30 min, indicating increased PI3K activity. This correlated with increased COX-2 OP142 protein expression at 6 hr. These effects also correlated with activation of Lipocortin 1-mediated regulation of lipopolysaccharide class II PI3Ks rather than class I PI3Ks, as shown by in vitro lipid kinase induced signal transduction pathway assays. We have also shown that whilst class II PI3K isoforms are inhibited by wortmannin (100–500 nM), they are unaffected by L. C. Alldridge, C. E. Bryant & S. Totemeyer LY294002 (1–30 mM). Thus, wortmannin may be a more appropriate Department Clinical Veterinary Medicine, Madingley Road, Cambridge inhibitor to utilize in order to identify the role of Class II PI3K enzymes CB2 OES, UK in TNFasignalling in airway epithelial cells. These data also suggest Lipocortin 1 (annexin-1) is a calcium- and phospholipid-binding protein, class II PI3Ks as potential therapeutic targets for inflammatory airway which modulates anti-inflammatory responses including those induced by diseases.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 112 Cell signalling

OP145 transmembrane adaptor LAT (linker for activation of T cells) is an Co-operative assembly of multimolecular TCR/LAT essential organizer of these assemblies, which contain the enzymes signalling complexes in Jurkat T-cell raft membrane PLCg, PI3 kinase, Vav, and the adaptor/scaffolding proteins Grb-2, Gads, microdomains and SLP-76. Our aim is to study the structure and the mechanism of assembly of these complexes and how these relate to the induction of L. C. S. Hartgroves, T. Zech, J. Lin,y A. Weissy & T. Harder signalling events. Using imaging and biochemical analysis of immunoi- Sir William Dunn School of Pathology, University of Oxford, South Parks solated TCR signalling plasma membrane domains, we follow the for- Road, Oxford OX1 3RE, UK, yHoward Hughes Medical Institute, mation of signalling complexes within membrane subdomains. We University of California, San Francisco, USA perform this analysis employing LAT-deficient JCaM2 T leukaemic cells TCR-elicited signals are transduced through multimolecular signalling reconstituted with LAT mutated in specific tyrosine-based docking sites complexes in raft plasma membrane domains of T lymphocytes. The for adaptors and PLCg.

Clinical immunology

OP146 biotinylated and bound to streptavidin coated microtitre plates. Anti- Altered production of HLA-DR mRNA correlates S. typhi IgG levels are expressed as arbitrary units with reference to a with surface expression, and can be regulated by standard curve. Specificity was assessed by inhibition studies using GM-CSF salmonella antigen. Reproducibility was determined at the upper and lower ranges of IgG antibody levels: Averaged interassay CVs were S. E. Perry, S. M. Mostafa,y R. Wenstone,y N. Grattony & <7Á4% (n ¼ 7) for sera with low levels and <1Á6% (n ¼ 7) for sera with P. J. McLaughlin high levels of anti-S. typhi IgG. Thus far, we have developed the ELISA Department of Immunology, University of Liverpool, Liverpool L69 3GA, based on normal adult responses, both spontaneous IgG anti-S. typhi levels UK, yICU, Royal Liverpool University Hospital, Liverpool L7 8XP, and post-S. typhi vaccination levels. We report here spontaneous IgG UK levels from a group of normal adults and children. Preliminary data show HLA-DR expression in septic patients is often down-regulated. A study that anti-S. typhi IgG values were between 70 and 94% higher for post was undertaken to ascertain if this down-regulation of surface expressed vaccination subjects (n ¼ 8) compared to paired pre-vaccination samples HLA-DR was controlled at the mRNA level. The effect of GM-CSF on (P < 0Á0006). The aim for this assay is to provide a valid alternative HLA-DR mRNA and protein expression on the promyelocytic HL-60 cell capsular polysaccharide antigen for test immunisations. line was also investigated. Monocyte surface expression of HLA-DR was measured by flow cytometry on PBMC. Primers were designed comple- mentary to a nonpolymorphic region of the alpha chain of HLA-DR and LD-PCR was used to measure HLA-DR mRNA. The HL-60 cell line OP148 expressing HLA-DR was cultured with GM-CSF and both surface expres- Invasive microbial populations in inflammatory bowel sion and mRNA levels of HLA-DR were measured as above. On PBMC of disease the septic patients, a down-regulated surface expression of HLA-DR G. Shah, N. B. Rayment, M. Mylonaki,y B. N. Hudspith, correlated with a reduced mRNA production. When the surface expression D. S. Ramptony & J. Brostoff increased, the mRNA production also increased. When the HL-60 cell line King’s College London, Infection and Immunity Research Group, was cultured with GM-CSF the surface expression and the mRNA Division of Life Sciences, 150 Stamford Street, London SE1 9NN, UK, production of HLA-DR was up-regulated. The results show that the yAcademic Department of Adult & Paediatric Gastroenterology, Barts & altered surface expression of HLA-DR in septic patients may be con- London School of Medicine & Dentistry, Turner Street, London E1 2ND, trolled at the mRNA level, and this expression and production of HLA-DR UK can be altered by GM-CSF in HL-60 cells. The pathogenesis of inflammatory bowel disease (IBD) is unknown. However, some abnormalities maybe related to disturbances in the normal immune process. One area of growing interest is the involvement of OP147 intestinal microbial flora in these diseases. Recent data suggest that Development of an anti-Salmonella typhi colonic bacterial flora, particularly those apposed to the mucosal surface, ELISA: potential use as a polysaccharide may play a role in modulating the mucosal immune response. Using test immunogen in suspected immunodeficiency samples obtained at routine colonoscopy we have studied patients with diseases ulcerative colitis, Crohn’s disease as well as normal controls. FISH probes were employed to identify four potentially pathogenic organisms (E. coli, B. L. Ferry, Z. Sherrell, N. Groome,y P. Stephens, H. M. Chapel & bacteroides, sulphate reducing bacteria and clostridia) and well as two S. Misbah probiotic bacteria (bifidobacteria and lactobacillus). Our preliminary data Department of Clinical Immunology, Oxford Radcliffe Hospitals, Oxford have identified substantial differences in mucosa-associated counts of the OX3 7LJ, UK, yDepartment of Cell and Molecular Biology, Oxford different bacterial species from patients with active disease compared to Brookes University, Oxford OX3 7LJ, UK those seen in normal controls and that some of those bacteria found within We have developed a reproducible ELISA to measure IgG responses to the lamina propria appear to colocalize with cells of the inflammatory Salmonella typhi in human sera. Purified S. typhi polysaccharide was infiltrate.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Clinical immunology 113

OP148a anti-CD20 antibody rituximab. Levels of anti-microbial antibodies, B-cell depletion in patients with rheumatoid arthritis: IgA, M and G rheumatoid factors and antibodies to CCP were measured. serial studies of immunological parameters C-reactive protein (CRP) was used as a serological indicator of inflam- mation. 17/22 patients showed marked clinical benefit lasting up to G. Cambridge, M. J. Leandro, J. C. W. Edwards, 33 months. The time to relapse after B cell return was often long (0– M. R. Ehrenstein, M. Saldeny & A. D. B. Websterz 17 months) but was closely correlated with rises in one or more auto- Centre for Rheumatology, UCL, Arthur Stanley House, 40-50 Tottenham antibodies (AAs). Serum immunoglobulin levels rarely fell below the Street, London W1T 4NJ, UK, yEurodiagnostica, Arnhem, NE, The lower limit of normal but a selective effect on AA levels was seen. The Netherlands, zCentre, Royal Free Hospital, London NW3, UK kinetics of autoantibody fall paralleled CRP levels. In contrast, anti- We have investigated the relationship between clinical disease, B-cell microbial antibodies did not differ from baseline. These findings suggest return and serology in 22 patients with rheumatoid arthritis (RA) follow- that clinical benefit and relapse following B cell depletion is related more ing B-cell depletion. This was achieved using therapy based on the closely to circulating AAs than to the physical presence of B lymphocytes.

Comparative immunology

OP149 24 weeks, there was a negative relationship between AGP and daily weight C-reactive protein signals through Syk in THP-1 cells gain (24 weeks, regression coefficient ¼0Á40 0Á11, P < 0Á01) and daily and induces TNF-a and IL-6 expression in monocytes foodintake(24 weeks,regressioncoefficient ¼1Á07 0Á35,P < 0Á01).At 24 weeks age only, haptoglobin was negatively associated with both daily K. J. Woollard & H. R. Griffiths weight gain (regression coefficient ¼66Á7 31Á3, P < 0Á05) and FCR Molecular Pharmacology Research Group, PSRI, Aston University, (regression coefficient ¼24Á5 9Á53, P < 0Á05). These results demon- Birmingham B4 7ET, UK strate an association between productivity and acute phase protein levels. Introduction Recent evidence shows that the major CRP receptor on Subclinical infection could be one cause for this association. leukocytes is the FcgRII receptor; however, the CRP-mediated signal transduction through FcgRII has not been fully explored. OP151 Methods THP-1 cells were treated with reCRP (0–100 mg/ml) or pre- CD8 positive lymphocyte subsets of the pig can be cipitated IgG for 0–30 min at 378 and examined for Syk protein phos- defined by perforin expression phorylation by Western blotting. Whole blood was collected and treated with reCRP (0–100 mg/ml) or LPS (1 mg/ml) with or without the secretion M. S. Denyer, T. E. Wileman & H.-H. Takamatsu inhibitor brefeldin A for 0–24 hr at 378 and (a) plasma was taken off for Institute for Animal Health Pirbright Laboratory, Pirbright, Woking TNF-a and IL-6 ELISA and (b) the brefeldin A-treated blood was fixed GU24 0NF, UK and lysed. Intracellular TNF-a and IL-6 levels were assessed by corre- The lymphocyte subsets of the pig are phenotypically unique and complex, sponding Ab and analysed by flow cytometry and (c) TNF-a and IL- but their functions and role in immunity are not well defined. Here, we 6 CD14þ mRNA expression analysed by RT-PCR. studied the expression of intracellular perforin in combination with cell Results CRP phosphorylated Syk at 10–100 mg/ml after 10 min incuba- surface markers to define cytotoxic lymphocytes in pigs. The results show tion. A dose-dependent expression of TNF-a and a significant increase in most perforin positive cells to be small dense lymphocytes expressing both IL-6 secretion was seen after 16 hr incubation with 10–100 mg/ml of CRP. CD2 and CD8. They expressed CD8aa or CD8ab, but not CD4 nor markers Both TNF-a and IL-6 mRNA expression and intracellular levels were seen forgdTcells.Inyoungpigs,themajority(upto80%)ofperforinpositivecells after 4 hr stimulation. were CD3– and the proportion of CD3þ perforin positive T cells increased Conclusion CRP is able to phosphorylate the non-receptor tyrosine with the age of pigs. The perforin positive CD3– subset was homogeneous kinase Syk and activate monocytes inducing TNF-a and IL-6 expression, and defined as CD2þCD3–CD4–CD5–CD6–CD8aaCD16þMIL4þ and is greatly promoting the inflammatory response. most likely NK cells. CD3þ perforinpositive T cells can be divided into four subsets. These are: CD2þCD3þCD4–CD5þCD6þCD8aaCD16–MIL4–, CD2þCD3þCD4–CD5þCD6þCD8abCD16–MIL4–, CD2þCD3þCD4– OP150 CD5–CD6–CD8aaCD16þMIL4þ and CD2þCD3þCD4–CD5–CD6–CD8 Acute phase proteins are associated with productivity abCD16–MIL4–. The third (and possibly fourth) subset(s) possesses non- in large white pigs specific cytotoxicity and possible porcine NKT cells. M. Clapperton, S. C. Bishop & E. J. Glass Roslin Institute (Edinburgh),Roslin, Midlothian, Edinburgh EH259PS,UK OP152 In pigs, infection often causes the release of acute phase proteins. Age-related changes to immune parameters in Additionally, productivity is associated with health status. We tested Labrador retriever dogs the association between productivity and acute phase proteins in 128 D. G. Blount, P. R. Heatony & D. I. Pritchard apparently healthy large white pigs (62 males; 66 females) at ages 18 and Department of Pharmaceutical Sciences, University of Nottingham, 24 weeks, using multiple regression analysis. The productivity traits were University Park, Nottingham NG7 2RD, yWALTHAM Centre for Pet daily weight gain, daily food intake and food conversion ratio (FCR) and Nutrition, Waltham on the Wolds, Leicestershire LE14 4RT, UK acute phase proteins were haptoglobin and alpha-1 acid glycoprotein (AGP). Mean AGP was 435 221 mg/ml at age 18 weeks and 261 Small volume blood samples were taken from 79 Labrador retriever dogs 110 mg/ml at age 24 weeks. Mean haptoglobin was 1Á16 1Á05 mg/ml at which were grouped into four age categories (1Á1–3Á1, 3Á9–5Á4, 6Á2–7Á7 age 18 weeks and 1Á25 0Á85 mg/ml at age 24 weeks. At ages 18 and and 8Á5–12Á8 years) in order to assess age-related changes to specific

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 114 Comparative immunology

immune parameters. All dogs were fed commercially available complete and cytokine production from dendritic cells. In this study, we have diets throughout the study. Leukocyte subset analysis, proliferative capa- explored the immunostimulatory effects of CpG DNA in the rainbow city and apoptotic resistance were selected as indicators of immune status. trout. A vigorous proliferative response is seen by head kidney cells in Absolute numbers of leukocytes, lymphocytes and monocytes signifi- response to CpG motifs and Escherichia coli DNA but not GpC or calf cantly decreased with increasing age. Relative percentages of lymphocytes thymus DNA. The optimal proliferation-inducing sequences (AACGGC and CD4 cells significantly decreased, and relative percentages of granu- and AACGAG) were derived by screening a panel of oligodeoxynucleo- locytes and CD8 cells significantly increased with age. The CD4:CD8 tides (ODNs). B cell-depleted head kidney cells reduced the proliferative ratio showed a significant age-related decrease. Whole blood proliferative response to CpG DNA, implying that B cells are one of the subpopulations response to phytohemagglutinin and Concanavalin A was significantly that respond to CpG ODN in fish leukocytes. In contrast, spleen cells were decreased with increasing age, although this was attributed to a decrease consistently non-responsive to CpG ODNs. Proliferative responses were in responding cell number rather than decreased cell function. There was a also seen by both spleen and head kidney cells in response to medium significant age-related increase in resistance to 2-deoxy-D-ribose-induced conditioned by CpG-stimulated spleen or head kidney cells. In this case, apoptosis, but no change to levels of spontaneous apoptosis. the proliferative response was not reduced by B-cell depletion. These data suggest that non-B cell populations proliferate in response to cytokines released by CpG-stimulated spleen or head kidney cells. OP153 CpG oligodeoxynucleotides stimulate rainbow trout (Oncorhynchus mykiss) leucocytes to produce cytokines that induce proliferative responses in spleen and head kidney cells K. S. Shen, C. Hogstrand & D. H. Davies Division of Life Sciences, Franklin-Wilkins Building, King’s College London, 150 Stamford St, London SE1 8WA, UK Unmethylated cytosine–guanine motif containing DNA (CpG DNA) is a powerful mitogen of mammalian B lymphocytes and induces maturation

Gene therapy

OP154 endothelium is an attractive approach to treat cardiovascular diseases and Use of bispecific immunoliposomes to target a poorly vascular related transplant rejection. We herein showed that VCAM-1 and endocytosing receptor with a highly endocytosing ICAM-1 were not very good targets to mediate gene transfer, but they are receptor implicated in the pathogenesis of many diseases. In order to improve gene- targeted delivery efficiency based anti-VCAM-1 and anti-ICAM-1 immu- P. H. Tan, M. Manunta & A. J. T. George noliposomes, we produced bispecific immunoliposomes to target VCAM- Department of Immunology, Faculty of Medicine, Imperial College, 1 or ICAM-1 and Tf receptors using anti-VCAM-1 or ICAM-1 mono- Hammersmith Hospital, Du Cane Road, London W12 ONN, UK clonal antibody (mAb) and anti-Tf receptor mAb, respectively. We We have previously reported that targeting at transferrin (Tf) receptors demonstrated that bispecific immunoliposomes can improve the transfec- using liposome-based delivery system is an efficient method of delivering tion efficiency of anti-ICAM-1 immunoliposomes. The approach used is genes to corneal and vascular endothelium in vitro and ex vivo. However, novel and utilizing a highly endocytosing receptor to improve the the ubiquitious expression of Tf receptors somehow restricts its usage as a endocytosis rate of a poorly endocytosing receptor with a significant target-specific vector. Targeting at adhesion molecules in an activated disease-related expression.

HIV

OP155 with expression of NK cell activation markers (CD69, KIR (KIR2DL- Correlation of cytotoxic function with the expression p70)) and Kar (KIR2DS-p50); cytotoxic molecular levels (cytoplasmic of natural killer (NK) cell markers (CD69, killer perforin) and cytokine production (cytoplasmic IFNg). This analysis of inhibitory receptors (KIR2DL-p70/KIR2DS-p50) and NK cytotoxic activity and expression of cell surface and cytoplasmic cytoplasmic perforin and IFNc) in HIV patients activation and effector markers has shown that HIV patients have reduced NK cell numbers and cytotoxic activity and that this was more evident M. Shamji & D. C. Henderson with falling CD4 T cell levels. There was no shift in the balance between Department of Immunology, Chelsea & Westminster Hospital, 369 the CD56 effector and regulator subpopulations (CD56 dim and CD56 Fulham Road, London SW10 9NH, UK bright cells, respectively). The percentages of NK cells positive for the In HIV patients, an attempt was made to correlate invitro cytotoxicity expression of the surface membrane activation markers, CD69, KIR and (Cr51 release) with CD56 expression – CD56 dim and CD56 bright, along KAR, and of the cytoplasmic effector molecules, IFNg and perforin, were

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 HIV 115 inversely proportional to NK-cytotoxic activity. The most consistent using samples from HIV patients grouped according to CD4 counts association was between the reduction in perforin levels and cytotoxicity. (n ¼ 100, Group A: CD4 < 200 cells/ml; n ¼ 100, Group B: CD4 ¼ 200– 400 cells/ml and n ¼ 100, Group C: CD4 > 400 cells/ml) and samples from healthy volunteers. This study shows that the NK cell count and the NK OP156 percentage in blood were reduced in HIV patients with the lowest NK cell Quantification of activated NK cells and expression of counts found in patients with low CD4 cell counts (<200 cells/ml). The CD57 and CD158b in healthy individuals and HIV numbers of NK cells expressing activation markers (CD57 and CD158b), infected patients like the total NK cell count, were reduced in HIV patients and that the lowest counts were found in patients with low CD4 cell counts M. Shamji & D. C. Henderson (<200 cells/ml). However, the percentages of activated cells within the Department of Immunology, Chelsea & Westminster Hospital, 369 NK cell population were higher in the three groups of HIV patients than in Fulham Road, London SW10 9NH, UK the controls. The numbers of CD56þ NK cells expressing CD57 and CD158b in peripheral blood from HIV patients were quantified by flow cytometry

Immunity to infection

OP157 we could show that this programme identified ca. 73% of the empirically Mouse model characterization for anthrax vaccine defined peptides from nine M. bovis antigens recognized by bovine T development cells. Validating this observation, we showed that 4/5 peptides from the mycobacterial antigen Rv3019c that were predicted to contain HLA-DR- H. C. Flick-Smith, E. L. Waters, N. Walker, J. Miller & restricted epitopes were recognized by T cells from M. bovis-infected E. D. Williamson cattle. Dstl, Porton Down, Salisbury SP4 0JQ, UK

A new vaccine for anthrax, composed of recombinant Protective Antigen OP159 (rPA), one of the toxin components of the causative agent of anthrax, A role for eotaxin in protective immunity against the Bacillus anthracis, is being developed for clinical trial. In order to parasite, Brugia malayi evaluate the immunogenicity and effectiveness of this vaccine against y challenge, a suitable animal model is required. From published literature, J. E. Simons, M. E. Rothenberg & R. A. Lawrence the A/J strain of mouse was selected as a potential model, and its immune School of Biological Sciences, 3.239 Stopford Bldg, University of y response to immunization with the vaccine characterized by assessment of Manchester, Oxford Road, Manchester M13 9PT, UK, Division of Allergy rPA-specific antibody production post-immunization, and protection and Immunology, Department of Pediatrics, Children’s Hospital Medical against challenge. Studies were conducted to determine the time required Center, Cincinnati, OH 45229, USA post-immunization to develop a protective immune response, the mini- Brugia malayi causes the chronic human disease, lymphatic filariasis. mum protective dose of vaccine and to assess the long-term immune Delineating the mechanisms of protective immunity has proved elusive. response to immunization. From the results of these studies, it was Previous studies in filarial systems have demonstrated a vital role for IL-5 possible to select a suitable dosing regimen for future assays and further but the function of eotaxin has yet to be investigated. IL-5 and eotaxin co- elucidate the role played by PA in the prevention of anthrax infection. operate to promote eosinophilia. IL-5 is essential for eosinophil expansion # Crown Copyright 2002 Dstl in the bone marrow and mobilization into the blood, while both eotaxin and IL-5 act synergistically in eosinophil recruitment to the tissues. Implantation of adult nematodes into the peritoneal cavity (pc) of OP158 eotaxin-deficient mice ( –/– ) resulted in significant retention of micro- Recognition of T-cell determinants from mycobacterial filariae (Mf) into the pc compared with WT. This was associated with a antigens across mammalian species dramatic reduction in eosinophilic infiltration into the pc. In a different –/– M. Vordermeier, A. O. Whelan & R. G. Hewinson model of infection, eotaxin mice infected with Mf iv displayed a Veterinary Laboratories Agency, Department of Bacterial Diseases, New pronounced blood eosinophilia and cleared Mf more rapidly than WT. Haw, Addlestone KT15 3NB, UK Furthermore, IL-5 levels were significantly higher in the absence of eotaxin. Our data suggest that tissue eosinopenia increases Mf survival The incidence of tuberculosis in cattle is rising in the GB. A scientific while blood eosinophilia correlates with Mf clearance. report concluded that an effective vaccination strategy of cattle will have the best long-term prospects of controlling this disease in GB. Diagnostic OP160 reagents discriminating between vaccinated and infected cattle will also Evolution of systemic Salmonella infections in relation be required. Synthetic peptides are useful diagnostic reagents to detect to bacterial localization bovine TB in cattle. In addition, they have also been used to identify novel M. Sheppard, D. J. Maskell & P. Mastroeni target antigens for diagnostic or vaccination. In this study, we used the Bacterial Infection Group, Centre for Veterinary Science, Department of M. bovis protein ESAT-6 as a model antigen to describe peptides contain- Clinical Veterinary Medicine, University of Cambridge, Madingley Road, ing T-cell epitopes that were frequently recognized across mammalian Cambridge CB3 OES, UK species either relevant as target species of tuberculosis (humans, cattle), or as small animal models of tuberculosis (mice, guinea pigs). In addition, by Using multicolour fluorescence microscopy techniques, we have inves- employing a virtual matrices-based human prediction program (ProPred), tigated, in the mouse typhoid model, the dynamic evolution of systemic

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 116 Immunity to infection

Salmonella infections in relation to bacterial localisation in the tissues. We OP163 have performed concurrent infections with S. typhimurium isogenic deri- Changes in the cellular composition of vatives that express either the O4 or O9 lipopolysaccharide surface mesenteric lymph nodes following Trichuris antigens and that can be individually visualized using appropriate com- muris infection in the absence of the chemokine binations of fluorochrome-labelled antibodies. We have found that bac- CCL2 terial growth in the tissues is paralleled by an increase in the size and M. L. deSchoolmeester, M. C. Little, B. J. Rollinsy & number of infected cells/foci. Low numbers of the O4 and O9 S. typhi- K. J. Else murium derivatives were found in the infected cells throughout the 3.239 Stopford Building, Oxford Road, University of Manchester, infection and the two bacterial strains always segregated to different Manchester, M13 9PT, UK, yHarvard Medical School, Boston, cells and infectious foci. Taken together, the results show that the MA 02115, USA evolution of the local pathogenesis in a Salmonella infection is the result of clonal events. T. muris is a large intestinal dwelling nematode against which a Th2 immune response is required for worm expulsion. Chemokines are chemotactic cytokines which may also be immunomodulatory in parasite OP161 infection. C57BL6 mice deficient in the gene for CCL2 (MCP-1) are Regulatory pathways of cytokine production and unable to expel T. muris before day 35 post-infection (p.i.) whereas wild- responsiveness in epithelial cells infected with type (WT) animals expel between day 21 and 35 p.i., before the larvae Chlamydiae moult to become fecund adults. We have presented data previously showing that the susceptibility of CCL2 knockout mice is ultimately V. Magdalenic,,y S. Wattegedera,y S. E. M. Howie & due to the lack of a Th2 immune response. As CCL2 is a major G. Entricany chemoattractant for macrophages in vivo, we investigated whether there Department of Pathology, Medical School, Teviot Place, Edinburgh, EH8 are any discrepancies in the mesenteric lymph node (MLN) cell composi- 9AG, UK, yMoredun Research Institute, Pentlands Science Park, tion in naive and infected animals. Flow cytometry was used to assess the Edinburgh, EH26 0PZ, UK proportion of macrophages, dendritic cells, CD4þ, CD8þ and B220þ cells Chlamydiae are Gram-negative intracellular pathogens that can cause a in the MLN. Interestingly, the composition of MLN of CCL2-deficient number of acute and chronic inflammatory diseases in a variety of hosts. mice differ from that of WT, containing relatively fewer CD4þ cells and The host inflammatory immune response is important for control of more B cells. However, macrophages are rare cells in the MLN, and infection, but can also contribute to disease pathogenesis. IFN-g is a consequently differences may only be seen at the site of inflammation, key cytokine in control of chlamydial growth and it does so by induction namely the large intestine. of indoleamine-2,3-dioxygenase (IDO), thereby depriving the organism of tryptophan. We have found that some epithelial and trophoblast cells are refractory to IFN-g treatment and fail to control the growth of Chlamydia. To elucidate this, we have investigated the role of suppressors of cytokine signalling (SOCS) in regulation of cytokine responsiveness in chlamydial OP164 infection. We have detected SOCS-3 mRNA in a variety of cell lines in Can measles-specific CD8þ T cells be both infected and uninfected cells. IFN-g signalling can still occur in cells detected in immune donors and if so, to that express SOCS mRNA as measured by expression of IDO mRNA. which epitopes? However, this does not correlate with control of chlamydial growth, y y suggesting a complex relationship between the inflammatory and reg- K. R. Newton, J. R. Stephenson, M. Steward, D. Goldblatt & ulatory pathways in cells exposed to IFN-g. L. R. Wedderburn Rheumatology Unit, Infection and Immunity CW6, Institute of Child Health, UCL, 30 Guilford Street, London WC1N 1EH, UK, yDepartment of OP162 Infectious and Tropical Diseases, London School of Hygiene and Tropical Characterization of the murine immune response to Medicine, Keppel Street, London WC1E 7HT, UK anthrax vaccine immunization Measles (MV) is a highly communicable viral disease. In the UK it is E. L. Waters, H. C. Flick-Smith, M. Green & E. D. Williamson currently vaccinated against in the MMR with a live attenuated strain. Dstl Chemical and Biological Sciences, Porton Down, Salisbury SP4 0JQ, However little is known about recognition of MV by the host immune UK system. Five peptides were selected from four measles proteins (M, C, H and N) predicted to bind in the HLA-A0201 binding groove. Preliminary In the development and testing of new vaccines based on protective studies in seropositive donors using ELIspot to show antigen specific IFNg antigen (PA), it is vital to identify a murine model to demonstrate production showed little specific response, raising the issue of efficiency protective efficacy against pulmonary anthrax infection. In this investiga- of antigen presentation using exogenous peptide. In mice models adeno- tion, A/J and TO mice were immunized with formulated rPA vaccine, via viral vectors containing MV protein cDNA have been used to study the intramuscular route. Half of the mice were administered a subsequent immune responses to these proteins and protection against MV infection. booster vaccination 21 days post-prime immunization, followed by a third We plan to use these constructs to infect human APC in order to allow the immunizing dose on day 41 of the study. Sampling was undertaken pre- measles proteins to be processed and presented in a way that more closely and post-booster vaccinations to assess the immune response for single mimics that of natural MV infection. In this way, we will raise CTL and two dose regimes and the effect of a third dose on immune status. IgG lines specific for these proteins and use these to map the memory and subclass data will be presented. T-cell response to measles. Data resulting from this approach will be # Crown Copyright 2002 Dstl presented.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Immunity to infection 117

OP165 but reinfection rates are very high. Thus, development of successful CD45RO expression and evaluation of Pneumovax immunization programmes is important, and understanding the immune vaccination effect mechanisms underlying chronic infections a prerequisite for developing Z. Huo, J. Miles & P. Riches effective control strategies. To further our understanding of immunity to Department of Biochemistry & Immunology, St George’s Hospital infection, we have been using the intestinal nematode parasite Trichuris Medical School, Cranmer Terrance, London SW17 0RE, UK muris in the mouse to study differential gene expression during chronic intestinal helminth infection. Mesenteric lymph nodes were harvested The protective mechanism of Pneumovax (polysaccharide vaccine) is from infected and uninfected susceptible AKR mice at D60 post-infection. believed to induce specific antibody which can promote phagocytosis Total RNA was extracted, cDNA fluorescently labelled and hybridized on against bacteria. This vaccine is a thymus-independent type 2 (TI2) to compugen glass oligo arrays (UK-HGMP Resource Centre). Arrays antigen. TI2 responses are unable to generate memory. CD45RO is a were read on an Axon scanner, and further analysed using MaxD software. marker for the maturation of nucleated hematopoietic cells. The CD45RO Among the differentially expressed genes, secretory leucocyte protease is present at low density early in the T-cell maturation cycle. Upon inhibitor, the chemokines Scyb9, Scyb10, Scyb11 and interferon g were activation by PHA, naive T cells first acquire CD45RO and then lose found to be up-regulated during chronic infection whereas expression of CD45RA. When these activated T cells are rechallenged, the cells that retinol binding protein was down-regulated. exhibit a secondary response are primarily CD45ROþ, leading to the concept that CD45ROþ cells are a primed population of memory T cells. We hypothesized that if Pneumovax has an effect on the expression of CD45RO, vaccinated individuals might make a good phagocyosis to the OP168 later bacteria challenge. Blood samples were incubated with and without Unconventional T-cell populations in the respiratory Pneumovax for 24 hr, then followed by challenge with Pneumococcus. tract have a non-redundant role in the early immune Results showed that a strong correlation exists between phagocytosis response to B. pertussis against Pneumococcus and the increase of CD45RO after incubation of Pneumovax in these samples. It is concluded that exposure to Pneumovax O. Zachariadis, J. P. Cassidy & B. P. Mahony invitro increases CD45RO and this results in an increased immune Department of Veterinary Pathology, UCD, Belfield, Dublin, Ireland, response to subsequent challenge with Pneumococcus. yMucosal Immunology Laboratory, Institute of Immunology, NUI Maynooth, Co. Kildare, Ireland

OP166 Early interactions between host and pathogen shape the subsequent Contribution of IL-9 to intestinal muscle function and adaptive response. gd T and NKT cells play an important role in early host protection in nematode infection differs defence. We observed previously that pathology did not correlate to according to parasite involved bacterial load during early phase of murine infection by Bordetella –/– W. I. Khan, M. Richard,y H. Akiho, P. A. Blennehasset, pertussis. Wild-type and gdTCR mice were challenged with B. pertus- sis J. Van-Snicky & S. M. Collins , and autopsies performed post-infection. Standard microbiological and histopathological examination of airway sections demonstrated that Department of Medicine, McMaster University, Room 3 N5C, Health –/– Science Centre, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, pathological damage occurs earlier and more extensively in gdTCR Canada, yLudwig Institute for Cancer Research, Brussels Branch, than in the wild-type mice, despite significantly reduced bacterial load. Brussels B-1200, Belgium We extended this using 3-colour flow cytometry. Neutrophil migration to the airways of infected gdTCR–/– mice was altered in terms of magnitude, Interleukin-9 (IL-9) is a T helper 2 cytokine and has pleiotropic activities kinetics and location, correlating with increased tissue damage. Initial on various cells. Studies in IL-9 transgenic mice have suggested a role for observations suggest that this difference is compensated at later time- this cytokine in worm expulsion in nematode infection. However, the points by NKT cell responses. We conclude that gdT cells control the precise effector by which IL-9 mediates the protection remains to be trafficking of early effector cells in the airways mediating both the determined. In this study, by neutralization of IL-9 in mice, we studied the reduction in bacterial load and the degree of pathology role of this cytokine in muscle function and in worm expulsion, using two different species of nematodes, Trichuris muris and Trichinella spiralis. Immuno-neutralization of IL-9 in T. muris infection significantly attenu- OP169 ated colonic muscle hyper contractility and inhibited worm expulsion. Increased susceptibility of C3- and FcRc This attenuated worm expulsion was not accompanied by changes in mast chain-deficient mice to Salmonella typhimurium cell protease-1 or in goblet cells. In contrast, neutralization of IL-9 had no infection significant effect on muscle contractility or on worm expulsion in T. spiralis infection. These results suggest that IL-9 contributes to intest- N. I. V. Me´nager & P. Mastroeni inal muscle function and to host-protective immunity and that its con- Centre for Veterinary Science, University of Cambridge, Madingley Road, tribution may differ depending on the type of nematode infection. Cambridge CB3 0ES, UK Salmonella infections represent a serious health hazard worldwide and a major concern for the food industry. Mice infected with Salmonella OP167 typhimurium are commonly used as a model to study S. typhi infections Changes in the gene expression profiles of mesenteric in humans (typhoid fever). We are currently studying the role of comple- lymph node cells during chronic intestinal infection ment and antibody binding to FcRg in host resistance to Salmonella.We found that infection of C3–/– C57BL/6 mice with S. typhimurium results in R. Datta, A. Bashein, A. Brass & K. J. Else an increase in bacterial numbers in the spleen and liver and in a lower 3.239 Stopford Building, School of Biological Sciences, University of survival rate as compared to control C57BL/6 mice. Passive transfer of Manchester, Oxford Road, M13 9PT, Manchester, UK Salmonella-immune serum in C57BL/6 mice prior to Salmonella infec- Gastrointestinal helminths are amongst the most prevalent infections of tion reduced the bacterial load in the tissues. A similar serum-dependent man and his domestic animals. Infections are controlled with antihelmintics increase in host resistance was seen in C3–/– mice. The effect of serum

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 118 Immunity to infection

transfer was less pronounced in FcRg–/– mice. These findings indicate a whether the parasite is expelled from the gut or if infection reaches different role for C3 and FcRg receptors in immunity to Salmonella. patency. The E, J and S isolates of the parasite differ in immunogenic and immunomodulatory characteristics within the host. Preliminary data suggests that there is differential antigen recognition of the three isolates, OP170 with the more readily expelled J isolate eliciting significantly less IL-12. Does SAP influence host immune responses to Evidence will be presented showing the differential expression between tissue-dwelling nematode infection? the three isolates of putative immunomodulatory genes. J. O. P. Cheung & R. A. Lawrence 3.239 Stopford Building, University of Manchester, Oxford Road, OP173 Manchester M13 9PT, UK Identification of epitopes of Mycobacterium Signalling lymphocyte activation molecule (SLAM)-associated protein tuberculosis 16 000-MW protein recognized by (SAP) is an adaptor protein that plays an important role in the develop- HLA-A0201 CD8 T lymphocytes ment of Th1 and Th2 cell responses. SAP regulates SLAM signalling in T- N. R. Caccamo, F. Dieli, C. Di Sano,y S. Meraviglia, J. Ivanyi,z cell activation and differentiation. Recent studies demonstrated that SAP- A. M. Krensky,§ G. Sireci & A. Salerno deficient mice have an impaired Th2 immune response to Leishmania Department of Biopathology, University of Palermo, Palermo 90134, major infection characterized by low IL-4 and IL-13 production and Italy, yISMEDA, National Research Council, Palermo 90134, Italy, higher resistance to infection. We studied whether SAP influences the T- zDepartment of Oral Medicine & Pathology, Guy’s Hospital, London SE1 cell responses to tissue-dwelling nematode infection. SAP–/– BALB/c and 9RT, UK, §Department of Pediatrics, University School of Medicine, C57Bl/6 mice were implanted with Brugia malayi adult worms i.p. Lower Stanford, CA 94305, USA levels of IgG1 were observed in all SAP–/– mice compared to wild-type mice and this decrease was more significant in the BALB/c strain. CD8 T cells can make an important contribution to protection against However, the Th1-driven isotype IgG2a was low in both SAP–/– strains, tuberculosis, but the antigenic determinants recognized in the context of although there was some increase in IgG2b and IgG3 in SAP–/– C57Bl/6 MHC class I molecules remain ill defined. The aim of this study has been mice. Microfilarial survival was unaffected by the absence of SAP in both to identify nonamer peptide epitopes derived from the acr/16 000-MW mouse strains. In contrast, a higher female adult worm recovery was antigen, which carry a binding motif for HLA-A0201. Two immunogenic obtained in SAP–/– BALB/c compared to wild-type mice. Thus, it appears peptides (p21–29 and p120–128) were identified based on their ability to that a deficiency in SAP in BALB/c mice leads to an impaired develop- elicit cytotoxic CD8 T cells from juvenile patients, recovering from tuber- ment of Th2 cell response with a possible effect on adult parasite clearance culosis. Epitope-specific recognition was demonstrated by the lysis of both upon B. malayi infection. M. tuberculosis-infected and peptide-pulsed macrophages, release of cyto- toxic granules, as well as IFN-g and TNF-a production. CD8 T-cell responses to p21–29 and p120–128 was detected ex vivo in freshly isolated OP171 PBMC from TB patients, but not in healthy PPD negative controls. Our data QTL mapping of resistance and susceptibility in suggest that these antigenic peptides can play a critical role in effective T. muris-infected mice immunity against mycobacterial infection and tuberculosis. J. Hankinson, J. L. Pennock, S. Eyre & R. K. Grencis School of Biological Sciences, 3.239 Stopford Building, University of OP174 Manchester, Oxford Road, Manchester M13 9PT, UK Chronic HCV infection: the role of the hepatic cytokine T. muris is a parasitic nematode which causes characteristic pathology in environment the large intestine of mice. Chronic infection with T. muris has been L. M. Golden-Mason, A. M. Kelly, O. Traynor,y G. McEntee,y studied in two strains; AKR, and BALB/c. AKR are known to be J. E. Hegartyy &C.O’Farrelly susceptible to infection, while BALB/c display a more resistant pheno- Education & Research Centre, St. Vincent’s University Hospital, Elm type. Expulsion is associated with a type 2 immune response and Park, Dublin 4, Ireland, yNational Liver Transplant Unit, St. Vincent’s chronicity with a type 1. Immunological manipulations such as mono- University Hospital, Elm Park, Dublin 4, Ireland clonal antibody treatment and KO mice have identified a number of cytokines important in resistance and susceptibility. In order to identify Our aim was to determine the effect of chronic HCV infection on the

novel genes, we have taken an F2 population from an AKR/Balb/C cross cytokine profile of the liver. HCV-infected liver (HCVL, n ¼ 11) and and have designed a microsatelite marker set with 20 cm spacing across cirrhotic control tissue (CCL, n ¼ 20) was obtained at the time of the genome for QTL analysis using worm counts and antibody levels to transplantation for end-stage disease. Normal controls (NL) consisted determine phenotype. Here, we present preliminary data highlighting of donor liver (n ¼ 12). IFN-g, IL-10, IL-12, IL-18 and IL-2 were important chromosomal loci which correlate with resistance and quantified using ELISAs. A 17-fold increase in IFN-g was observed susceptibility. for HCVL (mean 84.67 ng/100 mg protein) compared to NL (4.83 ng, P < 0.001), while only a threefold increase was observed for CCL. Up- regulation of IFN-g in HCVL appears to be mediated by IL-18 rather than OP172 IL-12, as IL-18 was significantly increased (246.15 ng versus 108.17 ng, Immunogenic and immunomodulatory characteristics P < 0.001) and IL-12 levels were normal. In contrast, \-12 was signifi- of the three isolates of Trichuris muris cantly increased in CCL (P < 0.01). IL-2 was up-regulated in CCL (23.45 ng; normal: 7.21 ng, P < 0.001), but not in HCVL. IL-10 was also C. E. Johnston, D. Tuckwell, K. Matthews & K. J. Else significantly increased in HCVL (4.60 ng) compared to NL (2.31 ng, School of Biological Sciences, Stopford Building, University of P < 0.05); however, the IFNg:IL-10 ratio (18.91 : 1) was higher than Manchester, Oxford Road, Manchester M13 9PT, UK normal (4.35 : 1, P < 0.0001). In ccl, the IFNg:IL-10 ratio was reversed Trichuris muris is a caecal-dwelling parasitic nematode of mice. A (1 : 1). Failure to up-regulate IL-2 may contribute to chronicity in HCV combination of host genetics and parasite-derived factors determines infection.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Immunity to infection 119

OP175 immune effector mechanism responsible for expulsion still remains to be Macrophage accumulation in the large intestine of determined. It is clear that expulsion of intestinal nematodes can involve mice infected with Trichuris muris is reduced in the the interplay between CD4þ T cells and the gut epithelium. T. muris absence of CCL2 (MCP-1) presents a unique situation in which the immune/epithelial interactions can be investigated, as it resides fully or partially embedded within host M. C. Little, M. L. Deschoolmeester, B. J. Rollinsy & K. J. Else enterocytes. The intestinal epithelium is under constant renewal. Inves- School of Biological Sciences, University of Manchester, Manchester, tigations into epithelial cell proliferation kinetics during infection of M13 9PT, UK, yHarvard Medical School, Boston, MA 02115, USA resistant (Balb/c) and susceptible (AKR) mouse strains have been carried T. muris is a large intestine-dwelling nematode. The expulsion of worms out to investigate the hypothesis that an elevation in the rate of epithelial by resistant strains of mice requires a Th2 type response. Chemokines are cell turnover is capable of mediating worm expulsion. Our study has chemotactic cytokines which may also be immunomodulatory in parasite highlighted that the rate of cell turnover in the caecum of Balb/c mice is infection. C57BL6 mice deficient in the gene for CCL2 fail to expel elevated when compared with AKR mice. This suggests that an up- T. muris whereas wild-type (WT) mice expel between day 21 and 35 post- regulation of epithelial cell turnover can act to displace worms from their infection (p.i.), before the larvae moult to become fecund adults. We have optimal niche, so contributing to worm expulsion. It is proposed that this shown previously that the susceptibility of CCL2 knockout (KO) mice is elevation in rate is mediated by the generation of Th2 cytokines by CD4þ due to the lack of a Th2 immune response. As CCL2 is a major T cells in the intestine. chemoattractant for macrophages and T-helper cells, we investigated the accumulation of these cells in the large intestine of WT and KO mice using immunohistochemical techniques. The number of f4/80þ macro- OP178 phages and CD4þ T-helper cells in the large intestine peaked at day 21 p.i. Role of B cells in the development of protective T-cell More than 95% of the leucocytes were in the lamina propria. Interestingly, response in Salmonella infection there were significantly fewer intestinal macrophages in CCL2 KO mice S. Ugrinovic & P. Mastroeni compared to their WT controls, in contrast to T-helper cells, where no such Centre for Veterinary Science, University of Cambridge, Madingley Road, difference was found. The trafficking of macrophages to the large intestine Cambridge CB3 OES, UK may play a key role in the expulsion of T. muris. Infection of mice with Salmonella typhimurium results in a systemic disease similar to human typhoid fever caused by S. typhi. It has recently OP176 been shown that B cells are essential for the establishment of T cell- NKT cells promote antigen-specific T-cell responses dependent acquired immunity to Salmonella. In the present study, we but are not essential for protection against liver stage analysed the role of B cells in protection against S. typhimurium using B malaria in mice after vaccination cell-deficient Igh-6–/– mice. Development of T-cell responses in Igh-6–/– was impaired in the early stages of a primary infection. This impairment S. Korten, R. Anderson, C. M. Hannan, S. Gadola, V. Cerundolo, persisted throughout the course of the disease. The frequency and the M. Taniguchiy & A. V. S. Hill ability of T cells to produce Th1 (IFNg) cytokines was reduced threefold Institute of Molecular Medicine, Oxford OX3 9DU, UK, yRIKEN RCAI in Igh-6–/– as compared to control mice. Thus, B cells seem to be and Chiba University, Chuoku Chiba 260-8670, Japan important at the early stages of the development of T-cell responses in IFN-g producing antigen-specific CD8þ T cells protect against liver-stage Salmonella infection. We further examined possible functions of B cells in malaria and can be induced by heterologous prime-boost vaccination. S. typhimurium infection. B cells from immunized and unimmunized CD1d-restricted NKT cells also protect and enhance specific immunity. animals were able to up-regulate costimulatory molecules upon invitro We investigated the effect of vaccination with a recombinant fowlpox virus stimulation with S. typhimurium, and they were capable of presenting and modified vaccinia virus Ankara encoding the circumsporozoite protein soluble Salmonella extracts to Salmonella-specific CD4þ T-cell lines. We of Plasmodium berghei and subsequent sporozoite challenge on liver NKT are currently investigating other B-cell functions that might be important andCD8þ T-cell responsesinwild-type (WT)andNKT-cell knockout(KO) in the protective immune response to Salmonella typhimurium. BALB/c mice using ELISPOT assay and FACS analysis. Immunization reduced total liver NKT cell numbers while inducing CD8þ T cells. More ofthe remaining NKT cells producedIL-4 or IFN-g thanbeforevaccination. OP179 InKOmice,vaccinationinducedfewerCD8þ Tcellsinthelivercomparedto Characterization of the mucosal and systemic WT mice, but they were boosted sufficiently after challenge resulting in response to long-term Helicobacter pylori infection in similar protection rates in the absence of NKT cells. We conclude that the Mongolian gerbil vaccination increases the cytokine production by NKT cells, which pro- A. H. T. Jeremy, M. A. Aboshkiwa, M. F. Dixon,y P. A. Robinson & motes thegeneration ofantigen-specific Tcells. However, NKTcells are not J. E. Crabtree essential for protection against liver-stage malaria after vaccination. Molecular Medicine Unit, Clinical Sciences Building, St. James’s Hospital, Leeds LS9 7TF, UK, yDepartment of Pathology, The General Infirmary, Leeds LS1 3EX, UK OP177 Immune-mediated control of intestinal epithelial cell Chronic H. pylori infection in the Mongolian gerbil (MG) has been turnover during infection with Trichuris muris: a new demonstrated to result in gastric cancer. This study investigated the role mechanism of worm expulsion? of the inflammatory and immune response in this animal model. MGs were orally infected three times with H. pylori SS1 strain or a more L. J. Cliffe & R. K. Grencis virulent strain 42GX. Infected MGs (n ¼ 17) and controls (n ¼ 18) were 3.239 Immunology, Stopford Building, Oxford Road, Manchester M13 sacrificed at 36 and 62 weeks. The mucosal cytokine (IFNg, IL-12p40 and 9PT, UK IL-10) response was examined by RT-PCR and an ELISA for gerbil IgG It is well established that the development of a Th2 immune response is anti-H. pylori was used to measure the systemic humoral response. All necessary for the expulsion of T. muris from the intestine, although the infected MGs had chronic gastritis with more severe pathology observed

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 120 Immunity to infection

in 62-week infected animals. H. pylori infection was associated with depletions suggest these were of memory phenotype. The majority of high increased (P < 0.01) gastric mucosal IFNg and IL-12p40 transcripts, PorA responses were detected in teenagers but low responses to the PorA- but there was no increase in IL-10 transcripts. Infected MGs also exhibited negative OMVs imply alternative antigens were also involved. No corre- an increase (P < 0.05) in anti-H. pylori IgG. Long-term chronic infection lation was observed between mucosal responses and SBA. of the gastric mucosa in the MG elicits both a Th1 gastric mucosal response and a specific IgG systemic response. The absence of IL-10 transcript up-regulation in H. pylori-infected gerbils may be a contribu- OP181 tory factor to the severe pathology. Phenotypic analysis of Epstein-Barr virus-specific CD4þ T cells in primary and persistent infection E. Amyes & M. F. C. Callan OP180 Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 Mucosal cellular immunity to Neisseria meningitidis 9DS, UK serogroup B During the course of an antigen-specific immune response invivo, the V. Davenport, R. S. Heyderman, R. E. Horton, T. Guthrie, transformation from a naive to memory population of T cells involves R. Borrowy & N. A. Williams transition through several phenotypic and functional states. This process Department of Pathology and Microbiology, University of Bristol, Bristol has been well studied in the context of the CD8þ T-cell response to BS8 1TD, yPublic Health Laboratory, Manchester M20 2LR, UK Epstein–Barr virus (EBV). Here, we have investigated the development of Natural immunity to Neisseria meningitidis serogroup B (Men-B) is the CD4þ T-cell response to EBV. We find that primary infection with associated with colonization of the nasopharynx. An understanding of EBV may stimulate a CD4þ T-cell response comprising 2% circulating the mechanisms behind its acquisition will benefit future vaccine devel- CD4þ T cells. These CD4þ T cells express CD38, CD45RO and have opment. We have therefore looked for T cell-mediated immunity to Men- down-regulated CD45RA. Most cells are CD27þ and CD28þ, although B in tonsils of adults and children and compared our findings with subpopulations of cells have lost expression of these molecules. In healthy systemic immunity. Tonsil mononuclear cell (MNC) suspensions were seropositives, we also find that as many as 2% circulating CD4þ T cells generated and cultured in a standard thymidine proliferation assay with may be EBV specific. Although this ‘memory’ population of cells does not meningococcal outer membrane vesicles (OMVs) from strain H44/76, express CD38, the phenotype of the cells is otherwise similar to that found isogenic porA derivatives: TR52 (p1.5,2), TR4 (p1.7,4) and TR10 in the primary response, suggesting that differentiation of the CD4þ T (p1.5,10) and a PorA deficient OMV (B:15:-). Serum bactericidal activity cells occurs predominantly during early infection. Comparing EBV- (SBA) was measured by standard assay. Mucosal MNC from tonsils specific with CMV-specific CD4þ T cells shows that these populations proliferated in response to recall antigens, primary antigens and menin- of cells tend to lie in different differentiation compartments. Finally, we gococcal OMVs invitro. LPS and CD19 depletions demonstrated find that the pathway of CD4þ T-cell differentiation differs from that of that proliferation was T-cell mediated, while CD45RO and CD45RA CD8þ T-cell differentiation.

Immunodeficiency

OP182 OP183 Mice deficient in XLP gene sh2d1a exhibit The UK multicentre study of subcutaneous increased susceptibility to MHV-68 and immunoglobulin G (SCIG) in primary antibody hypo-gammaglobulinemia deficiency (PAD)

L. Yin, U. Al-Alem, J. Liang, W. M. Tong, M. Badiali, J. Sumegi,y C. H. Dash, H. C. Gooiy & T. M. Lynch on behalf of BPL’s SCIG Z. Q. Wang & G. Romeo investigators International Agency for Research on Cancer, 150, cours Albert-Thomas, Medical Department, BioProducts Laboratory, Elstree WD6 3BX, UK, Lyon 69008, France, yDepartment of Pathology and Microbiology, yDepartment of Clinical Immunology and Allergy, St. James’s University of Nebraska Medical Center, 600th South, 42nd Street, Omaha, University Hospital, Leeds L59 7TF, UK NE 68198, USA Patients with PAD require IgG therapy, usually given IV. Rapid sub- X-linked lymphoproliferative disease (XLP) is an inherited immunode- cutaneous (s.c.) infusion, by syringe driver, is an equally effective ficiency characterized by immune dysregulation and uncontrolled lym- alternative (co-operative UK/Swedish study). The UK multicentre study phoproliferation on exposure to Epstein–Barr virus. This condition is of BPL’s 16% IgG solution has recruited 50 patients. Experience is 47 caused by mutations of the SH2D1A gene. We have generated a sh2d1a- patient-years; this report is of 21 patients for 6 m. The s.c. dose was deficient mouse by gene targeting and have investigated the clinical 100 mg/kg/week to maintain levels of IgG. Clinical effectiveness was phenotypes of the XLP disease in this strain. After murine gammaher- measured by: no. of infections; days on antibiotics; days off work/school. pesvirus (MHV) 68 infection, proliferation of CD8þ cells was exacerbated No difference for infections in study and pre-study periods (P > 0Á1); in sh2d1a-deficient mice compared to normal mice. A more disseminated antibiotic use was low: median proportion of time on antibiotics ¼ 7%; lymphocyte infiltration in various organs was observed in mutant mice, <2% of time spent away from work or school. Infusions were well- suggesting a higher susceptibility to MHV-68. The sh2d1a-deficient mice tolerated: 9/21 patients reported transient local reactions; no systemic had lower basal IgG1, IgG2a, IgG2b, IgG3 and IgE productions than their reactions. Patients reported 31 other adverse drug reactions: none wild-type littermates. The difference became even more evident after serious; one severe (asthma); the rest mild. 19/21 patients transferred MHV-68 infection. This phenotype reproduces therefore one of the main to home therapy after training. The period on home therapy ¼ 7Á6 patient- clinical manifestations of XLP, hypogammaglobulinemia. years. In conclusion, rapid s.c. infusion of BPL’s 16% IgG is

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Immunodeficiency 121 efficacious and well-tolerated. It enables replacement IgG therapy in greater freedom. These findings on s.c. IgG are in agreement with other PAD patients with poor venous access and allows home therapy with its reports.

Inflammation

OP184 apoptotic neutrophils in a cation-deplete environment, although not to as IL-10-expressing macrophages reduce inflammation in great an extent as the cation-replete system suggesting that the CD44 effect experimental glomerulonephritis has both cation-dependent and -independent components. To explore the H. M. Wilson, A. J. Rees & D. C. Kluth cation independent component further, a variety of antibodies and ligands Department of Medicine and Therapeutics, IMS Building, University of were used to try and inhibit CD44 augmentation of phagocytosis. Inhibition Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK of this effect was observed with a blocking CD32 antibody which suggests that FcgRII may play a role in uptake of apoptotic neutrophils by CD44- Macrophages are pivotal in control of inflammation and modifying their stimulated macrophages. However, pretreatment of macrophages with the function provides a way to study their impact on injury. We have CD32antibodyhadnoeffectonphagocyticuptake,suggestingthatCD32on transfected primary cultures of macrophages to express IL-10 and deter- the macrophage is not responsible for this effect. We are currently testing mined their effect on renal inflammation. Transfected macrophages were whether tethering of neutrophils via FcRgII to CD44 mAb bound to the injected into the left renal artery of rats, 6 hr after the induction of macrophage accounts for the cation-independent augmentation of phago- nephrotoxic nephritis (NTN). Ad-IL-10 transfected cells localized cytosis that we observe. to inflamed glomeruli and produced IL-10 invivo. IL-10-expressing macrophages produced a marked reduction in albuminuria compared to unmodified disease or injection of Ad-null transfected macrophages OP186 (albuminuria mg/day 7: IL-10 308 63 versus Unmodified disease Blockade of TIM-3 inhibits neutrophilic pulmonary 730 85, P < 0.01). These effects could not be mimicked by systemic inflammation injection of IL-10 expressing macrophages. The injection of IL-10- y y y expressing BMDM resulted in a reduction in glomerular macrophages S. J. McMillan, J. C. Gutierrez Ramos, A. J. Coyle & C. M. Lloyd and in their state of activation, with reduced numbers of ED3 and class II- Department of Leucocyte Biology, SAF Building, Imperial College, y expressing macrophages. This was seen in both the injected and non- London SW7 2AZ, UK, Inflammation Division, Millennium injected contralateral kidney, but not when IL-10-expressing cells were Pharmaceuticals Inc., Cambridge, MA 02139, USA injected systemically. These studies show that syngeneic macrophages TIM-3 is a surface molecule preferentially expressed on Th1 cells in can be modified by gene transfer and cause reduction in glomerular comparison to Th2 cells. Blockade of TIM-3 in vivo has been shown to inflammation invivo. enhance the symptoms of EAE, a Th1 model. The aim of this study was to investigate the effects of blockade of TIM-3 in antigen-induced airway OP185 inflammation. A model of allergen-driven eosinophilic inflammation and a Characterization of the molecular mechanism of model of neutrophilic inflammation were utilized. Administration of anti- CD44-augmented phagocytosis of apoptotic TIM-3 antibody at the same time as OVA sensitization during eosinophilic neutrophils by macrophages inflammationreduced BAL macrophageand OVA-specific IgE levels.Anti- TIM-3 antibody had no effect on eosinophil recruitment, airway function, S. Vivers, S. Hart & I. Dransfield levels of total IgE or Th2 cytokines. In contrast, treatment with anti-TIM-3 Respiratory Medicine, Medical School, Edinburgh University, Teviot antibody in neutrophilic inflammation-reduced neutrophil recruitment to Place, Edinburgh EH8 9AG, UK the BAL. Moreover, total leucocyte recruitment to the lung was reduced CD44 has been shown to up-regulate phagocytosis of apoptotic neutro- and numbers of CD4 and TIM-3-positive cells were decreased. Levels of phils by human macrophages. We aim to characterize the molecular MCP-1 were also reduced. In conclusion, modulation of TIM-3 has a mechanisms by which this augmentation occurs. We have demonstrated beneficial effect on a model of pulmonary neutrophil accumulation but that CD44 augmentation of macrophage phagocytosis of apoptotic neu- notinamodelofTh2-inducedeosinophilicinflammation.Furtherstudiesare trophils was prolonged. Interestingly, CD44-augmented phagocytosis of ongoing to identify the mechanisms involved in these responses.

Molecular immunology

OP187 leukocytes. It is of central importance to viability in mice, as the null Characterization of a novel isoform of CD147 with an mutant is mostly lethal, and any survivors are sterile. CD147 has been extra Ig-like domain implicated in stimulation of matrix metalloproteinase production, trans- plantation, adhesion, and retina development. We have previously shown S. M. Hanna, P. Kirk, O. Holt, M. J. Puklavec, M. H. Brown & that the rat homologue associates laterally with monocarboxylate trans- A. N. Barclay port molecules, are responsible for maintaining intracellular lactate levels. Sir William Dunn School of Pathology, University of Oxford, South Parks Monoclonal antibodies (mAb) recognizing CD147 did not affect lactate Road, Oxford OX1 3RE, UK transport, and the current model is that CD147 acts to direct the position of CD147 is an integral membrane glycoprotein containing two immuno- the transport molecules so that they line up when cells are in close globulin (Ig) domains that is present on most cells including activated proximity, creating a metabolic synapse. In mice and humans, evidence

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 122 Molecular immunology

from ESTs suggest that there is the potential for a third, membrane distal involved in the immune response. The vaccinia glycoprotein, A38L, is the Ig domain to be expressed (termed d0). A related protein, neuroplastin, viral homologue of CD47. The natural ligand for CD47 is signal reg- exists in 2 and 3 domain forms, and the membrane distal third domain has ulatory protein (SIRP)a. There are three structural subgroups within the been shown to bind homophilically. Here, we present the sequence of SIRP family: a, b and b2, found in humans and cattle, but only a in mouse CD147 d0, mAbs generated against it, and assess its ability to rodents. SIRPa is believed to be an immune inhibitory regulatory protein interact homophilically. whose signal is transduced upon binding to CD47. SIRPb and b2 have no intrinsic signalling ability; however, SIRPb is known to send an activatory immune signal through its association with a DAP-12 homodimer. OP188 SIRPb’s ligand has not been identified. Although the signalling capabil- Interaction of the human SIRP family members with the ities of SIRPb2 are not known, here, we show that it can bind to CD47 CD47 viral homologue A38L although with lower affinity. Data will also be presented on the interaction between A38L and members of the SIRP family in humans. F. G. Cochrane, G. P. Brooke, M. H. Brown & A. N. Barclay Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK Evasion of the immune system is essential for a productive viral infection. One way viruses accomplish this is by mimicking host encoded proteins

Neuroimmunology

OP189 LPS-induced inhibition of long-term potentiation (LTP) in the rat hippo- Dopamine receptor expression on human T- and campus. Pretreatment with PS liposomes reversed LPS-induced increases B-lymphocytes, monocytes, neutrophils, eosinophils in concentration of IL-1b and stimulation of stress-activated protein and NK cells: a flow cytometric study kinases in the hippocampus. In addition, hippocampal activation of microglia, which was observed after LPS administration, was inhibited F. McKennna, P. J. McLaughlin, B. J. Lewis, G. C. Sibbring, by pretreatment with PS liposomes. Moreover, we observed a concomitant J. A. Cummerson, D. Bowen-Jones & R. J. Mootsy increase in the hippocampal concentration of anti-inflammatory cytokines Department of Immunology, University of Liverpool, Duncan Building, IL-10 and IL-4. These data suggest that treatment with PS liposomes Daulby Street, Liverpool L69 3GA, yUniversity of Liverpool, Academic confers a protective effect in the brain by suppressing certain potentially Rheumatology Unit, University of Liverpool University Hospital, Aintree, detrimental effects of LPS, in particular the impairment of synaptic Longmoor Lane, Liverpool L9 7AL, UK function, and that this protection may be related to an enhanced release The expression of dopamine receptors D1–D5 were studied on different of anti-inflammatory cytokines. human leukocyte populations. Flow cytometric techniques were used, identifying dopamine receptors with subtype-specific antibodies. Of the D1-like receptor family (D1 and D5), only D5 was detected and of the D2- OP191 like receptor family (D2, D3 and D4), all dopamine receptors were Multiple sclerosis: a study on Ngf and its receptors detected. T lymphocytes and monocytes had low expression of dopamine M. Alam & I. Hutchinson receptors, whereas neutrophils and eosinophils had moderate expression. Immunology Res. Group, 3.239 Stopford Building, School of Biological B cells and NK cells had higher and more consistent expression. Dopa- Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, mine receptors D3 and D5 were found in most individuals whereas D2 and UK D4 had more variable expression. D1 was never found. The study also indicates how the immune and central nervous systems may commu- Multiple sclerosis (MS), an autoimmune disease, is caused when myelin nicate, and has implications for the role of these neuroimmune interac- and oligodendrocytes are selectively destroyed by activated T cells, tions in immune and inflammatory diseases. macrophages and CNS resident microglia. We are interested to know what triggers the activated T cells to cross the protective blood brian barrier initiating the destruction. Nerve growth factor (NGF) is most OP190 important as a neuroimmuno-regulatory cytokine in the CNS. NGF leads Evidence of an anti-inflammatory effect of apoptotic to survival or death of cells depending on its binding to the high affinity mimetics on LPS-induced inhibition of long-term trkA receptor or the low-affinity p75 receptor. We have taken a candidate potentiation in the hippocampus gene approach to study if the NGF, p75 and trkA cause susceptibility to MS. By PCR-SSCP, DNA sequencing, ARMS-PCR, tissue culture, Y. M. Nolan, D. S. D. Martin, A. E. Bolton,y V. A. Campbell & ELISA and flow cytometry, we want to assess the significance of NGF M. A. Lynch and its receptors in MS patients. We have analysed the expression levels of Department of Physiology, Trinity College, Dublin, Dublin 2, Ireland, p75 and trkA in the peripheral blood of normal and MS people. We have yVasogen Inc., Toronto, Ontario, Canada identified a substitution of T by C at 198 position of the NGF gene Systemic administration of lipopolysaccharide (LPS) induces activation and also a novel polymorphism in the promoter of trkA. Functionality of of macrophages resulting in release of interleukin-1b (IL-1b) and activa- these polymorphisms is being studied. Vitamin D may be an important tion of T cells which infiltrate the brain to stimulate activation of environmental factor in multiple sclerosis. By gel-shift assay, we are microglia. We report that i.m. administration of liposomes bearing assessing the binding of the VDR transcription factor to the NGF 198 phosphatidylserine (PS), i.e. apoptotic mimetics, significantly abrogated alleles.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Parasitology 123

Parasitology

OP192 dependent upon type 2 responses. In addition, IFN-g, IL-12 and IL-18 Sero-epidemiology of toxoplasmosis in pregnant have been shown to be important factors in promoting chronic infection. women in Bandar Abbas, Iran, 1999–2000 Expression of a number of genes which promote type 1/IFN-g responses (T-Bet, Socs-1, Hlx and Wsx-1) or type 2 responses (CIITA) have been O. Safa, A. S. Jahromi & S. Zarey analysed by real-time PCR. The role the parasite may play in promoting its Department of Immunology, yDepartment of Statstics, Faculty of own survival was also investigated by cloning the major somatic and Medicine, Hormozgan University of Medical Sciences, Bandar Abbas excretory/secretory antigen of T. muris, the 43 000 MWantigen, which has 7914964153, Iran previously been shown to have some homology to IFN-g. Recombinant Basedonserologicalevidences,toxoplasmosisisawidespreaddisease.With 43 000 MW was cloned in to pQE-30 (Qiagen) using BamH1 and HindIII regard to congenital toxoplasmosis, social and economic importance, and expressed in E. coli M15 (pREP4). Expressed protein was detected screening and treatment during pregnancy prompted us to do this study. using a penta-his antibody to detect the his-tagged protein. Immune In this cross sectional and descriptive study, the prevalence of toxoplasma responses to recombinant 43 000 MW (T2) were analysed in BALB/c antibodies IgM, IgG in 418 pregnant women was determined by ELISA mice. method. The results showed that the general prevalence of the positive cases of IgG and IgM were 34Á2 and 7Á9%, respectively, there was no relationship between positive cases and age. But, there was a relationship between the OP195 positive cases and keeping cat at home (P < 0Á001), and the way of meat Role of IL-10 in the suppression of DTH induced by the consumption (P < 0Á002). Moreover, 57Á9% of pregnant women were sero- helminth product ABF negative and had predisposition to acute toxoplasmosis. Therefore, con- A. Boitelle, H. E. Scales, M. W. Kennedy,y P. Garsidez & sidering this fact, trying to omit risk factors reduces rate of toxoplasmosis. C. E. Lawrence Department of Immunology, Strathclyde University, Glasgow G4 0NR, yIBLS, Glasgow University, Glasgow G12 8QQ, zDepartment of OP193 Immunology, Glasgow University, Glasgow G11 6NT, UK In vitro production of chemoattractant activity for ovine eosinophils by parasitic and free living nematodes Helminth infections have a potent immunomodulatory effect on the host immune system, inducing decreased responses to non-related antigens. A. G. Cameron, D. A. S. Clark, L. A. Wildblood & D. G. Jones We have investigated the role of IL-10 in the immunomodulation of Moredun Research Institute, Edinburgh EH26 0PZ, UK heterologous responses mediated by an extract (ABF) of the gastrointest- Gastro-intestinal nematode parasites of ruminants are the causal agents of inal helminth Ascaris suum using a sensitive adoptive transfer technique. many diseases of global economic importance to livestock farming. Mice were adoptively transferred with CFSE-stained OVA-specific TCR Despite research identifying eosinophils as the predominant immune transgenic (tg) lymphocytes, and immunized with OVA in FCA ABF. cells associated with helminth infection, their role in immune response Mice were injected i.p. with anti-IL-10R antibody or control isotype remains ill defined. Using modified Boyden chambers, the chemotaxis of before immunization. Tg TCR was detected by an monoclonal antibody ovine bone marrow derived-eosinophils to five species of parasitic nema- (KJ1-26), allowing us to monitor the invivo behaviour of the OVA-specific todes of livestock (Haemonchus contortus, Teladorsagia circumcincta, T cells. ABF inhibition of OVA-specific DTH was associated with a Ostertagia ostertagii, Trichostrongylus colubriformis and Trichostrongylus reduced percentage and number of OVA-specific T lymphocytes in the vitrinus) and a species of non-parasitic nematode (Caenorhabditis elegans), draining lymph nodes. Moreover, OVA-specific T cells had decreased was investigated invitro. Live infective stage larvae (L3) of all five parasitic rounds of cell division when mice were co-immunized with ABF. Injec- species elicited significant (P < 0Á05) and dose-dependent chemotactic tion with anti-IL-10R antibody partly abrogated the inhibition of DTH by responses. Excretory/secretory product collected from T. circumcincta L3 ABF and ameliorated the effect of ABF on the percentage, number and larvae also induced significant (P < 0Á05) eosinophil chemotaxis. In con- cell division of OVA-specific T lymphocytes in draining lymph nodes. trast, no eosinophil chemotaxis was induced either by a whole C. elegans homogenate, or by a mixed population of live C. elegans. The finding that onlytheparasiticnematodespeciesproducedchemoattractantactivityraises OP196 theintriguingpossibilitythattherecruitmentofeosinophilsinvivomaybeof C-reactive protein induces transformation of benefit to the parasite rather than the host. Leishmania in a lipophosphoglycan-dependent manner M. W. Mbuchi & J. G. Raynes London School of Hygiene and Tropical Medicine, Keppel Street, London OP194 WC1E 7HT, UK Chronic helminth infection: the roles played by both the host and the parasite Leishmania parasites are the causative agents of a wide spectrum of diseases, their digenetic life cycle involves cyclic transformation between A. J. Bancroft, J. L. Pennock, J. Hankinson & R. K. Grencis amastigote to promastigote. It has been shown that C-reactive protein School of Biological Sciences, 3Á239 Stopford Building, University of (CRP), a major acute phase protein, binds specifically to the Lipopho- Manchester, Oxford Road, Manchester M13 9PT, UK sphoglycan(LPG) of the infective metacyclic promastigote of Leishmania It is well known that chronic Trichuris muris infection is associated in the donovani. In these studies, we demonstrate that CRP induces trans- host with a type 1 response and conversely expulsion of the parasite is formation of metacyclic promastigotes to an amastigote-like forms in a

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 124 Parasitology

temperature and pH independent manner. This process is LPG dependent vaccine. Despite evidence from animal models and invitro studies,

as an LPG mutant of L. donovani expressing a truncated form of LPG that epidemiological studies of the association between anti-MSP-119 anti- does not bind CRP also fails to transform. Furthermore, binding and bodies and immunity to malaria have given conflicting results. We have transformation were demonstrated in a L. mexicana LPG1 knock out when tested the fine specificity of antibodies in sera from children from Gambia the LPG1 gene was restored. However, CRP-induced transformation did and Uganda for their ability to compete with monoclonal antibodies not confer increase in infectivity or survival of L. donovani in macro- (MAbs) that are: (a) able to inhibit merozoite invasion; or (b) able to phages. Macrophages stimulated with LPS, LTA and SAC produced block these inhibitory antibodies without effect on merozoite invasion.

different amounts of TNF and IL-10 in the presence of L. donovani Sera were also tested for recognition of recombinant MSP-119 proteins compared to macrophages with either L. donovani or stimuli alone, containing mutations which selectively affected binding of the different suggesting synergistic effect with Toll-like receptors. Further experiments MAbs. Competition with some, but not all, MAbs correlated strongly with are currently in progress to characterize this observation. measures of protection, providing support for the strategy of constructing ‘designer’ vaccines to elicit specific responses.

OP197 Fine specificity of serum antibodies to P. falciparum

PfMSP-119 predicts protection from clinical malaria and high density parasitaemia B. D. Okech, P. H. Corran,z J. Todd, A. Joynson-Hicks, C. Uthaipibull,§ T. G. Egwang,y A. A. Holder§ & E M. Riley Immunology Unit, Department Infectious & Tropical Diseases, LSHTM, Keppel Street, London WC1E 7HT, UK, yUK Medical Biotech Laboratories, Kampala, Uganda, zNIBSC, South Mimms, Herts EN6 3QG, UK, §NIMR, The Ridgeway, London NW7 1AA, UK The C-terminus of the Plasmodium falciparum major merozoite surface

protein, MSP-119, is a leading candidate antigen for inclusion in a malaria

Reproductive immunology

OP198 OP199 Placental inflammation and cytokine Lack of apoptotic decidual leucocytes in hydatidiform production in response to Chlamydophila abortus mole: no evidence for Fas-Fas ligand system mediated infection maternal T-cell tolerance in molar pregnancy S. Wattegedera, M. Livingstone, D. Longbottom, I. E. Anderson, S. Pongcharoen, J. N. Bulmery & R. F. Searlez D. Buxton & G. Entrican Department of Microbiology and Immunology, Newcastle University, Moredun Research Institute, Pentlands Science Park, Bush Loan, Newcastle upon Tyne, NE2 4HH, yDepartment of Pathology, Royal Edinburgh EH26 0PZ, UK Victoria Infirmary, Newcastle upon Tyne NE1 4LP, zAnatomy and Clinical Skills Centre, Newcastle University, Newcastle upon Tyne NE2 4HH, UK Chlamydophila abortus mediates an inflammatory host immune response and maternal IFN-g production is associated with the control of the Complete hydatidiform moles are totally paternally derived, representing pathogen. However, type-1 immune responses can be detrimental during a complete allograft that might be expected to provoke maternal immune pregnancy and IFN-g can induce parturition. Peripheral blood mono- rejection. Our previous studies have shown increased Fas expression by nuclear cells (PBMC) from sheep infected with C. abortus produce more activated decidual CD4þ T cells in molar pregnancy compared with IFN-g at parturition than uninfected controls after in vitro restimulation normal pregnancy and increased FasLþ expression in molar trophoblast. with mitogen. Since C. abortus infects the placenta and also the fetus, we The aim of this study was to investigate whether Fas/FasL-mediated were interested in knowing if the fetal/neonatal immune response could apoptosis of decidual immune cells, particularly T cells, is responsible for contribute to disease pathogenesis. PBMC derived from neonatal lambs maternal immune tolerance in molar pregnancy. Using terminal deox- failed to produce IFN-g, irrespective of the infection status of the ewe. ynucleotidyl transferase (TdT)-mediated nick end-labelling (TUNEL), a Fetal inflammatory cell infiltrates in the placenta contained very few cells significant increase in apoptotic cells was demonstrated in decidua expressing mRNA encoding IFN-g or IL-4, but cells expressing TNF-a associated with molar pregnancy compared with normal early pregnancy were abundant. Phenotypic analysis revealed a predominance of CD14þ (pooled partial and complete hydatidiform moles; P ¼ 0Á0277). Co-label- and MHC class IIþ cells, and relatively few cells of the lymphocytic ling studies with an immunoperoxidase technique showed that the apop- lineage. This did not control C. abortus, as seen by the destruction of the totic cells in molar pregnancy were not activated CD45ROþ immune cells, chorionic epithelium in infected placentas. We hypothesise that inflam- CD3þ T cells, CD56þ endometrial granulated lymphocytes or matory cytokine production contributes to disease pathogenesis of chla- CD14þ CD68þ macrophages. A proportion of the apoptotic cells was mydial abortion. decidualized stromal cells. We conclude that there is no evidence for a

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Reproductive immunology 125 critical role of Fas/FasL-mediated apoptosis of decidual immune cells in cytokine produced by Th2 cells, in the maintenance of normal pregnancy. maternal tolerance in either normal or molar pregnancy. In a longitudinal case-control study, the effect of pregnancy on plasma IL- 10 was investigated. Plasma IL-10 concentration was determined using an ELISA technique in 99 pregnant women sampled at 12, 20 and 35 weeks OP200 gestation, 38 non-pregnant controls sampled in parallel and a subgroup of Plasma levels of interleukin-10 during normal women sampled at 3 days postpartum (n, pregnant 21, non-pregnant 21). pregnancy IL-10 was significantly higher in pregnant women than in non-pregnant controls at 12, 20 and 35 weeks gestation (P < 0Á05, P < 0Á01 and V. A . Holmes, J. M. W. Wallace, W. S. Gilmore, P. McFauly & P < 0Á0001, respectively), and in mothers postpartum when compared H. D. Alexandery to non-pregnant controls (P < 0Á01). There was no effect of gestational NorthernIrelandCentreforDietandHealth(NICHE),Universityof Ulster, time on IL-10. These data suggest that elevated IL-10 is a physiological ColeraineBT521SA,UK, yDepartmentsofObstetricsandGynaecologyand consequence of normal pregnancy, and further advocate a role for IL-10 in Haemoatology, Belfast City Hospital, Belfast BT9 7AB, UK successful pregnancy. We propose that this elevation in IL-10 during Pregnancy is proposed to be a Th2 phenomenon. There is increased pregnancy may benefit the mother, influencing other pathways in vivo, recognition of a role for interleukin-10 (IL-10), an immunomodulatory such as the tissue factor coagulation pathway.

Rheumatology

OP201 inflammatory myopathies. A transgenic murine model with increased Elevated serum nitric oxide associated with expression of MHC class I on muscles developed myositis. We hypothe- co-expression of PKC-eta (g) and iNOS in peripheral size that this phenomenon may be a critical event leading to the migration blood monocytes (PBM) from inflammatory arthritic of inflammatory cells and subsequent muscle damage. (IA) patients Methods Immunohistochemistry was performed on muscle biopsy sec- tions from six children with JDM. We analysed the expression of MHC T. Pham, P. Rahman,y Y. Tobin,y M. Khraishi,y S. Hamilton,y class I and infiltrating lymphocytes looking for surface receptors that can C. Alderdicey & V. Richardson bind to MHC. Normal muscle sections from an adult and a child were used Faculty of Medicine, Memorial University, St. John’s NF A1B 3V6, as control. We compared the findings with polymyositis and dermato- yRheumatology Research, St. Clare’s Mercy Hospital, St. John’sNF myositis in adults. The panel of antibodies used included CD3, 4, 8, 16, A1C 5B8, Canada 19, 56, dystrophin, b-2 microglobulin and MHC classes I and II. The lack of key signalling molecules in human monocytes (hMo) may be Anti-dystrophin was used to distinguish atrophic muscle cells from NK the reason why they can not express iNOS and produce NO invitro in cells. response to LPS as do murine monocytes. Our earlier study of PKC Results (1) MHC class I is overexpressed on all muscle sections from expression showed that PKC-Z was absent in hMo but abundant in murine JDM subjects, even in very early cases where inflammatory infiltrate is not monocytes, and that PKC-Z transfected hMo expressed iNOS and pro- apparent. (2) The majority of infiltrating cells in JDM are CD16þ CD68þ duced NO in response to LPS. In this study, we have tested this hypothesis macrophages, whereas those in perivascular regions are T cells. further to establish if PKC-Z and iNOS expression were associated in vivo. Inflammatory arthritic patients with active disease had higher serum NO (206 61 mM, n ¼ 26) than healthy (132 25 mM, n ¼ 6) or osteoarthritic OP203 (OA) (115 30 mM, n ¼ 13) subjects. PBM from 8 out of 10 IA patients Anti-BiP antibodies in the serum of patients with co-expressed iNOS and PKC-Z mRNA but those from OA and healthy autoimmune disease groups did neither. Interestingly, clinically quiet IA patients receiving M. D. Bodman-Smith, V. M. Corrigall, C. Chan & infliximab had similar serum NO (256 41 mM, n ¼ 8) to clinically active G. S. Panayi IA patients on NSAIDS (258 44 mM, n ¼ 5) but higher than OA patients Department of Rheumatology, 5th floor, Thomas Guy House, GKT School suggesting that infliximab does not suppress NO, iNOS nor PKC-Z of Medicine, Guy’s Hospital, London SE1 9RT, UK expression. We propose that PKC-Z may be important in the development of IA-induced iNOS positive phenotype in hMo. We have implicated the human chaperone protein BiP in the pathogenesis of rheumatoid arthritis (RA). Increased immunoglobulin binding to BiP on Western blot analysis was seen. We now describe an ELISA developed OP202 to detect serum antibody reactivity to BiP. Specificity of the assay has been Is MHC class I overexpressed in early juvenile shown by free ligand competition and extensive correlation with other dermatomyositis (JDM)? immunological parameters. We confirm the increased binding of immu- noglobulin to BiP in the sera of a cohort of patients with RA when C. Li, H. Varsani, P. Woo, B. Gao, J. Hortony & L. R. Wedderburn compared to controls. Furthermore, these data show that antibody will Rheumatology Unit CW6, Institute of Child Health, 30 Guilford Street, bind to a non-glycosylated form of BiP since the protein is produced in an London WC1N 1EH, yInstitute of Neurology, Queen Square, London E. coli expression system. We also describe an increase in the binding seen WC1N 3BG, UK in patients with primary Sjo¨gren’s syndrome (SS) when compared to SOX Introduction Overexpressionofmajorhistocompatibilitycomplex(MHC) (a non-autoimmune control population). These data support a role for BiP class I on the surface of myocytes is a well-recognized phenomenon in in RA and a possible link with the pathogenesis of SS.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 126 Rheumatology

OP204 inflammatory process. We obtained samples of peripheral blood and Natural killer cells and joint inflammation synovial fluid from 21 patients with inflammatory arthritis and analysed the frequency and phenotype of the peripheral and synovial NK cells. We N. Dalbeth & M. F. C. Callan found the CD56bright subset of NK cells to be greatly expanded within Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 inflamed joints. Further experiments suggest that this subset of cells is 9DS, UK preferentially recruited from the periphery but may then be activated Dysregulation of cytokine networks plays an important role in maintain- locally. The synovial CD56bright NK cells are very sensitive to combina- ing inflammation in arthritis. The secretion of TNF-a, by cells of the tions of the monokines IL-12, IL-15 and IL-18 and respond by secreting monocyte/macrophage lineage, is pivotal in this process. The mechanisms IFN-g and TNF-a, cytokines that can, in turn, activate macrophages. The involved in activation of the monocyte/macrophage population are less functional properties of these NK cells therefore render them good well understood. The purpose of this study was to ask whether natural candidates for a role in amplifying the production of pro-inflammatory killer (NK) cells are present within inflamed joints and whether they could cytokines in arthritis. interact with monocyte/macrophages and play a role in amplifying the

Transplantation

OP205 followed by replacement with stem cells from an HLA-matched sibling CD40 costimulates human memory T cells and favours donor. Up to 80% of patients develop acute graft versus host disease IL-10 secretion (aGvHD) with considerable morbidity and mortality following transplan- tation. Symptoms are varied and can be difficult to confirm. A serological N. Rogers, I. Jackson, N. Camara, G. Lombardi & marker to predict aGvHD would be more beneficial. PBMC and serum R. Lechler samples were collected pretransplant and at regular intervals following Department of Immunology. Imperial College, London W12 0NN, transplantation. CXCL10 (IP-10) and IFN-g levels were measured in UK serum by cytokine ELISA. Both cytokines were significantly elevated Whilst the most well-defined pathway of costimulation is via the B7 during episodes of aGvHD post-transplant in comparison to patients who family of molecules, a large body of data clearly demonstrates a role for did not suffer GvHD. These levels declined following resolution of the CD40/CD154 pathway. We have studied the role of CD40 as an GvHD. The expression of the CXCL10 receptor, CXCR3, on PBMC is independent costimulatory molecule using HLA-DR1 transfectants co- being assessed on PBMC collected at the same time points post-transplant. expressing either CD80, CD86 or CD40. Functional assays using allo- Our results suggest that IFN-g and CXCL10 may serve as important geneic CD4 cells as responders demonstrate that CD40 is capable of clinical markers for the pathogenesis of aGVHD. costimulating proliferation and cytokine release in the absence of B7. Costimulation by CD40 resulted in significantly enhanced levels of IL-10 compared to B7-mediated costimulation. Maximum responsiveness to OP207 CD40 is detected in the memory subset of CD4 cells. On day 3, the Effects of cyclosporin A in gingival epithelial dominant costimulation is provided by CD40, whilst by day 5, this is cells overshadowed by costimulation provided by CD80 and CD86. Costimu- J. Birraux, M. Thomason, J. A. Kirby & J. J. Taylor lation by CD40 is enough to expand an alloreactive T-cell line. After Departments of Oral Biology, Restorative Dentistry, Surgery, Faculty þ 3 days, CD4 cells cocultured with CD40-expressing transfectants of Medicine, University of Newcastle upon Tyne, Newcastle upon Tyne þ demonstrate up-regulated expression of CD40L, not detected on CD4 NE2 4BW, UK T cells cocultured with transfectants expressing CD80 or CD86 and differing cell surface receptor phenotypes. Thus, CD40 is capable of The immunosuppressive cyclosporin A (CsA) prevents acute graft rejec- functioning independently as a CS molecule; this raises questions con- tion in organ transplant patients but has many side-effects, including cerning the consequences of antigen presentation by tissue parenchymal induction of overgrowth in the gingival tissues. We investigated the effects cells. of CsA on the proliferation and death of oral epithelial cells, and hypothesized that CsA could render cells more resistant to stress. Growth assays demonstrated an antiproliferative effect of CsA at immunosup- OP206 pressive concentrations above 1 mg/ml, in HOK-16B cells and in primary Role of CXCL10 and IFN-c in graft versus host gingival epithelial cells obtained from surgical specimens. This effect was disease reversible at low doses of CsA but not at 10 mg/ml, where only 15–30% of the cells survived after 3-day treatment and 3-day recovery. Also, pre- K. P. Piper, S. Hamilton, A. Lovibond, D. Dunnion, J. Ainsworth, treatment with CsA did not have a significant effect on TNFa-induced S. Donkersley, J. Arrazzi, P. Mahendra, C. Craddock & apoptosis, as measured by caspase-3 activity levels. These results suggest P. A. H. Moss a dose-dependent inhibiting effect of CsA on epithelial cell proliferation. Cancer Research UK, Institute of Cancer Studies, University of However, there was no evidence that CsA can affect TNFa-induced Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK apoptosis. Research is now focused on CsA effect on NF-akBeta binding Stem cell transplantation is widely used for the treatment of haematolo- activity. This might provide an insight on the molecular effects of this drug gical malignancies and involves ablation of patient’s immune system in epithelial cells.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 TSEs 127

TSEs

OP208 OP209 PrPc expression on cells of the immune system in The cellular targeting of PrP in the peripheral lymphoid scrapie susceptible and resistant sheep system following infection with a range of TSE strains M. Davies, G. Barnard, L. Hopkins, M. Syy & I. Mcconnell D. Best, K. L. Brown & M. E. Bruce Centre for Veterinary Science University of Cambridge, Cambridge CB3 Institute for Animal Health, Ogston Building, West Mains Road, 0ES, UK, yCase Western Reserve University, Cleveland, OH 44120, USA Edinburgh, EH7 3JF, UK PrPc is expressed on the surface of many immunological cells. The altered Previous studies using the ME7 strain of scrapie have shown that mature prion isoform (PrPsc) accumulates in the lymphoreticular system (LRS) follicular dendritic cells (FDCs) are important for neuroinvasion and early in TSE disease development. We hypothesize that cell interactions replication of infectivity in lymphoid tissues. However, there may be within lymphoid microenvironments may result in quantitative and qua- cellular-targeting differences between TSE strains in the peripheral litative changes in PrPc expression and may be linked to the accumulation lymphoid system, as there are known to be in the CNS. This may have of PrPsc on follicular dendritic cells in the LRS. Using lymphatic implications for the development of therapeutic strategies. We examined cannulation in sheep our studies show that PrPc expression by B cells the cellular targeting of PrPSc for seven TSE strains from early stage depends on the stage of B-cell differentiation and activation. Recirculating infection through to the terminal stage of disease. VM or C57BL mice memory B cells in afferent lymph express high levels of PrPc compared to were challenged intracerebrally with a 1% brain homogenate of the 87V, non-recirculating naive B cells. It is suggested that B-cell activation and 22A, ME7, 79A or 139A scrapie strains and the 301V or 301C BSE differentiation may provide the PrPc substrate necessary for conversion of derived strains. Normal brain injected controls were included for com- PrPc to PrPsc in scrapie. Afferent lymph dendritic cells also express very parison. Mice were serially killed at specific time points following high levels of PrPc. The relationship of surface PrPc on cells of the infection and spleens taken for immunocytochemical and PET blot immune system to soluble PrPc in blood and lymph is also being studied. analysis for the detection of PrPSc. PrPSc was first detected at 3 weeks Currently our studies suggest that there is considerable heterogeneity in post-infection, from the spleen of 79A challenged mice, and was asso- the levels of PrPc expression on cells of the immune system related to their ciated with FDCs. Studies are continuing to determine the timing and stage of differentiation and activation. cellular localization of PrPSc in the other TSE strains used in the study.

Tumour immunology

OP210 OP211 Isolation of anti-VEGF-receptor-2 blocking antibody Regulatory cell-mediated immunosuppression in using phage display technology Hodgkin lymphoma (HL) T. Lam, Z. Gao, C. Ettelaie, J. Hetherington,y A. Maraveyasy & N. A. Marshall, L. R. Munro, R. N. Barker & M. A. Vickers J. Greenman Department of Medicine and Therapeutics, University of Aberdeen, The University of Hull, Postgraduate School of Medicine, Hull HU6 7RX, Foresterhill, Aberdeen AB24 2ZD, UK yDepartment of Uro-Oncology, Castle Hill Hospital, Hull HU16 5JQ, UK Although most patients with HL are cured with current treatment regi- We developed a simple subtractive panning protocol using commercial Fc mens, the pathogenesis of this disease remains obscure. HL is character- chimera to isolate VEGF-R2 blocking antibodies from a phage display ized by rare malignant Reed Sternberg (RS) cells in a reactive infiltrate of library as part of a novel antiangiogenic treatment strategy. VEGF-R2- lymphocytes (ILs). In 30% of cases, the RS cells contain EBV, which specific single-chain antibodies (scFv) were isolated from the Nissim expresses high levels of LMP1. In normal subjects, LMP1 induces large naive semisynthetic library after six rounds of positive panning against numbers of Tr1 cells. We now report a survey of Th function in 47 PBMC recombinant VEGF-R2/Fc chimera immobilized on immunotubes, and samples and 14 lymph nodes of patients with HL. PBMC responses one round of negative panning against VEGF-R1/Fc chimera suspended in (cytokine secretion and proliferation) to mitogen, recall antigen, primary solution, coupled to magnetic beads. Selection of VEGF-R2-binding antigen and LMP1 peptides were significantly suppressed compared to clones was performed by ELISA with VEGF-R1/Fc chimera and human healthy donors, although dominant response to LMP1 was still the IgG1 kappa (k) as negative control antigens. Seven clones were identified induction of Tr1 cells. In striking contrast to the findings in PBMC and partially characterized by PCR analysis of the CDR3 insert and BstN- and non-malignant ‘reactive’ lymph nodes, ILs were anergic to all 1 digestion. Western blot analysis confirmed that all clones produced antigens tested. Mixing PBMC and ILs showed that the anergic effect soluble scFv. Binding properties towards endothelial cells expressing was dominant over PBMC responses, and was partially inhibited by anti- VEGF-R2 are being determined by FACS and Western blotting of primary IL-10, preventing cell–cell contact and anti-CTLA-4. Flow cytometry human umbilical vein endothelial cells. Endothelial cytotoxicity is being showed ILs contained populations of both CD4þ IL-10þ and CD4þ determined using the blocking antibody in combination with metronomic CD25þ cells. We therefore propose that the dominant T cell function dose of estramustine. This strategy represents a new and promising in HL is suppression, which explains the lack of an effective Tc response chemotherapy approach. against the malignant RS cells.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 128 Tumour immunology

OP212 CD57þ, CD161þ T cells) in marrow of patients in remission, following Hepatocyte growth factor protects c-met expressing B haematopoietic malignancy (HRM), compared to non-malignant marrow cell lines from apoptotic death obtained from patients with breast cancer (BCM). Significantly larger proportions of T cells in HRM expressed CD56 than in BCM (9Á72 versus G. Skibinski 3Á32%of marrowT cells,P < 0Á0001). CD57 expression wasmorecommon Queen’s University, Department of Clinical Biochemistry, Institute of on HRM T-cells (23Á07 versus 8Á27%, P ¼ 0Á03), and they were also more Clinical Science, Grosvenor Road, Belfast BT12 6BJ, UK likely to express activation markers CD69 (19Á07 versus 4Á56%) and HLA- The relative sensitivity of neoplastic cells to DNA damaging agents is a DR(24Á71versus8Á66%,P ¼ 0Á01forboth).SignificantlyfewerTcellswere key factor in cancer therapy. We show that pretreatment of Burkitt V 24TCRþ inHRMthanBCM(0Á35 versus0Á75%,P ¼ 0Á02), despitemore lymphoma cell lines expressing c-met protooncogene with hepatocyte CD3 þ CD161 þ NT cells being Va24þ in HRM (38Á38 versus 14Á73% of growth factor (HGF) protects them from death induced by DNA damaging CD161þ T cells, P ¼ 0Á01). Our results show that the composition of the T agents commonly used in tumour therapy. This protection was observed in cell population in human bone marrow is altered following haematopoietic assays based on morphologic assessment of apoptotic cells and DNA malignancy. Furtherstudymayclarify whether thesedifferences result from fragmentation assays. The protection was time- and dose-dependent, or influence the development of the malignancy. maximal protection requiring preincubation with 100 ng/ml of recombi- nant HGF for 48 hr. Western blotting analysis and flow cytometric studies OP215 revealed that HGF inhibited doxorubicin and etoposide induced decreases Blockade of the a a integrin: effects on cytotoxicity of in the levels of the antiapoptotic proteins Bcl-XL and, to a lesser extent, E 7 bladder cancer cells Bcl-2 without inducing changes in the proapoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the micro- J. Cresswell, M. J. Henry, W. K. Wong & J. A. Kirby environment of neoplastic cells may contribute to the development of a Department of Surgery, The Medical School, University of Newcastle, chemoresistant phenotype. Newcastle upon Tyne NE2 4HH, UK

The aEa7 integrin (CD103) is expressed by intraepithelial lymphocytes of OP213 the bladder epithelium and lamina propria. This integrin binds to the Heat shock protein 27, protein kinase C and Ki67 as epithelial adhesion molecule E-cadherin, which is down-regulated during possible predictors of clinical outcome in prostate bladder cancer progression. We propose that the interaction between cancer CD103 and E-cadherin is important for lymphocyte cytotoxicity of bladder cancer cells and therefore loss of E-cadherin may be a mechanism A. Sharples, S. M. Andrew, E. M. I. Williams,y K. B. O’Hare,z of tumour immune escape. Expression of CD103 was induced on per- I. L. McDowall & C. S. Fostery ipheral blood lymphocytes in a mixed lymphocyte reaction supplemented Department of Biological Sciences, Chester College, Parkgate Road, with TGFa. Approximately 50% of CD3þ cells and CD56þ cells were Chester CH1 4BJ, yLiverpool University, Liverpool L69 4BX, zSchool of induced to express CD103. Antibody blockade with an anti-CD103 Health, University of Teeside, Middlesborough TS1 3BA, UK monoclonal antibody significantly reduced adhesion between lympho- Prostate cancer is the second most common cancer diagnosed amongst cytes and E-cadherin expressing bladder cancer cells (P ¼ 0Á0068). Anti- men in England and Wales. Independent markers predictive of the CD103 blockade also significantly reduced cytotoxicity against the behaviour of tumours are urgently required. Sections of 183 prostatic bladder cancer cells in a redirected 51Cr release assay (P ¼ 0Á0006). adenocarcinomas were assigned a Gleason Grade and immunohistology The CD103-E-cadherin interaction is important for adhesion to and used to assess expression of Hsp27, PKC and Ki67 with the KS-300 image cytotoxicity of bladder cancer cells by CD103þ lymphocytes. This analysis program. Survival was assessed using Kaplan–Meier analysis and population consisted of CD3þ and CD56þ cells, suggesting that loss a Cox regression model. Poor survival was associated with negative or of E-cadherin during tumour progression may impair natural and T cell- high expression of PKC, low expression was associated with longer mediated antitumour cytotoxicity. survival times (P ¼ 0Á05). Expression of Hsp27 displayed similar patterns, but the differences between groups were not statistically significant. OP216 Increased expression of Ki67 was associated with poor survival, e.g. Effect of COX-2 inhibition on immune responses to 5 years survival was 82Á1% for cases with low expression compared to colon carcinoma cells 40Á4% of cases with high expression. All antigens were significant predictors in the Cox model. Assessment of expression of Ki67, PKC A. J. Walmesley, A. J. M. Watson & S. E. Christmasy and Hsp27 has potential for the development of a prognostic index and Department of Medicine, University of Liverpool, Liverpool L69 3BX, could have an important role in tumour management. yDepartment of Immunology, University of Liverpool, Liverpool L69 3GA, UK COX-2 is overexpressed in colorectal tumours, suggesting that it has a role OP214 in colorectal carcinogenesis. Products of COX-2 (the prostaglandins) are Altered NT cell phenotype in human bone marrow also thought to suppress the immune response to tumour antigens. COX-2 following haematopoietic malignancy inhibition was investigated on T-cell proliferation in a mitogenic response and from tumour-bearing mice. Splenocytes were isolated from control J. Dean, L. Golden-Mason, A. D. Hill,y D. McCarthyz & mice and mice that had undergone tumour induction with MC26 cells, C. O’Farrelly regression following HSV-TK suicide gene therapy and no tumour Education & Research Centre, Departments of ySurgery and recurrence. Splenocytes were treated with either Con-A or irradiated zHaematology, St. Vincent’s Hospital, Elm Park, Dublin 4, Ireland MC26 cells, and the COX-2 inhibitor Rofecoxib. Proliferation was NT cells (NK marker-positive T cells) are known to play a critical measured by 3H-thymidine incorporation after 3 or 7 days. Rofecoxib anticancer role in murine bone marrow, where up to 50% of local T cells increased splenocyte proliferation in a mitogenic response by 50% at are NK1Á1þ. Whether their human counterparts perform a similar role is concentrations above 1 mM. Irradiated MC26 cells induced a 60% increase not known. This study aimed to examine NT cell populations (CD56þ, in control splenocyte proliferation, enhanced to 100% by 1 mM Rofecoxib.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Tumour immunology 129

Splenocytes from treated mice showed a 300% increase in proliferation in peptide within Tie-2 that induces PBMC proliferation and a˜IFN secretion response to irradiated MC26 cells, amplified to 450% by 1 mM Rofecoxib. in a range of unimunized individuals. The PBMC proliferative response A specific immune response is generated to tumour cells in mice that have was cloned and generated a CD8þ CD45ROþ CD56þ T cell-clone. Within undergone HSV-TK gene therapy. COX-2 inhibition enhances this effect, the 20-mer peptide, there are potential class I and II epitopes. We propose possibly by inhibition of PGE2 production. that the donors PBMC proliferated to both CD4 and CD8 epitopes and that the CD4 response was fundamental in the generation of the CD8 clone. The expression of both CD45RO and CD56 suggests that it is an effector OP217 CTL clone that coexpresses NK receptors. Recent evidence has suggested CD8þ CD45ROþ CD56þ T cell-clone specific for a that these cells are the most efficient cell killers. We are currently mapping receptor overexpressed on tumour endothelium (Tie-2) the epitope and are assessing the clones ability to kill endothelial cells overexpressing Tie-2. The clone could be stimulated by antigen fed to J. M. Ramage, D. T. Patton, R. S. Moss, I. Spendlove & L. G. Durrant dendritic cells, but only if Tie-2 was presented as an Fc fusion protein and CR UK Academic Unit of Clinical Oncology, Nottingham University, City not by Tie-2 alone. Furthermore, stimulation by Tie-2Fc was blocked by Hospital, Hucknall Road, Nottingham NG5 1PB, UK an antibody to the Fc receptor (CD64). This implies that the receptor was A novel target for a cancer vaccine would be receptors, such as Tie-2, responsible for uptake and allowing class I presentation of the CD8 overexpressed on tumour endothelium. We have identified a 20-mer epitope.

Virology

OP218 Respiratory virus induced mucin synthesis and secretion C. A. Hewson, M. R. Edbrookey & S. L. Johnston Department Respiratory Medicine, NHLI at St Mary’s, Imperial College, London W2 1PG, UK, yCellular Genomics, GlaxoSmithKline R&D, Stevenage SG1 2NY, UK Asthma and COPD are economically important diseases and acute exacerbations involving rhinoviruses (RV) are common. Mucus hyp- ersecretion is associated with these exacerbations yet, to date, no studies have investigated the link between RV infection and mucus hyper- secretion. The human respiratory epithelial cell-line NCI-H292 was infected with RV-16 to examine its role in stimulation of the three main respiratory mucins, MUC2, MUC5AC and MUC5B. TaqMan1 PCRs, a lectin assay for total mucin and specific ELISAs were used to identify gene expression and protein release. Total mucin release was increased at OP220 24 hr post-infection (42% increase, P < 0Á01) as were both MUC2 and Infection of human cardiac cells by Coxsackie B MUC5AC gene expression at 8 hr post-infection, peaking between 24 viruses and 48 hr (4Á8-fold (P < 0Á01) and 7Á9-fold (P < 0Á05) at peak, respec- G. Orthopoulos, K. Triantafilou & M. Triantafilou tively). MUC5AC protein release was increased in a dose-responsive Insitute of Biomedical and Biomolecular Sciences, School of Biological manner (2Á3-fold P < 0Á01), however, MUC2 protein was undetectable. Sciences, University of Portsmouth, King Henry Building, King Henry I MUC5B was not consistently increased in the studies. This study is the Street, Portsmouth PO1 2DY, UK first to demonstrate a direct response in mucin production due to viral infection. Continuing work will identify the specific cell signalling Viral myocarditis is an inflammatory disease of the heart that is induced involved and if there are conserved mechanisms between different virus primarily by enteroviruses. Among the enteroviruses most commonly types. associated with this disease are the Coxsackie B viruses (CBVs), and in particularly CBV3 and CBV5. Even though, decay accelerating factor (DAF or CD55) and recently Coxsackie–Adenovirus receptor protein (CAR) have been identified as receptor molecules utilized by Coxasckie OP219 B3 virus, the exact receptor molecules that CBV3 and CBV5 utilize in Abstract withdrawn order to infect cardiac cells are not yet known. In this study we have attempted to elucidate the cell surface receptors that lead to the infection and damage of cardiac cells by CBV3 and CBV5 by using human cardiac cells. Our results have shown that these viruses can cause cardiac cell destruction by cytolytic infection of the cardiac cells. Flow cytometric quantification of the viruses binding has been shown to be significant and can not be inhibited by the usage of anti-CD55 and anti-CAR antibodies. The same antibodies could not inhibit CBV5 internalization, thus leading us to believe that Coxsackie B viruses could be utilizing a different set of receptors in order to infect cardiac muscle.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 130 Virology

OP221 enhance in vivo stability. In vitro, DAF-fc blocked CVB infection rever- Soluble receptors abrogate CVB3-induced myocarditis sibly, while CAR-fc CVB neutralization was irreversible. However, the in mice efficacy of each varied widely between strains of CVB3. The ability of i.v. administered DAF-Fc and CAR-fc to inhibit myocarditis in mice follow- B. Spiller, B. Yanagaway & B. M. Mcmanusy ing intraperitoneal infection with a myopathic strain of CVB3 was tested UWCM Medical Biochem, Third Flr. Tenovus, Cardiff CF14 4XX, UK, at 3 days prior to infection, during infection, or 3 days following infection yUBC, Department of Pathology, Vancouver V6E 4J2, Canada and compared to virus alone and sham infected animals. Pathological Coxsackie virus B3 (CVB3) infection is a major cause of myocarditis. The assessment was carried out with H&E sections and virus replication tested Coxsackie–Adenovirus receptor (CAR) has been identified as the primary by in situ hybridization for both positive and negative strand viral RNA. receptor and human decay-accelerating factor (DAF) has been identified Both soluble receptors abrogated the myocarditis, but only if animals were as an accessory receptor for CVB serotypes 1, 3, and 5. We constructed pre- or co-treated. Only CAR-fc was found to additionally abrogate human DAF-fc and CAR-fc fusion proteins to aid in purification and to accompanying virus-induced pancreatitis.

Vaccines

OP222 OP224 Efficacy of intranasal boosting in plague immunization Expression of LFA-3 and ICAM-1 in adenovirus-transformed human cells and their S. J. Elvin, J. E. Eyles & E. D. Williamson sensitivity to NK-mediated cytolysis Room A201, Building 07, DSTL Chemical & Biological Sciences, Porton Down, Salisbury SP4 0JQ, UK G. Bottley, G. P. Cooky & G. E. Blair Department of Biochemistry and Molecular Biology, University of Leeds, The recombinant plague subunit vaccine antigens Fraction 1 (F1) and V Leeds LS2 9JT, yMolecular Medicine Unit, St. James’s University were used to immunize BALB/c mice by the i.m. route using the adjuvant Hospital, Leeds LS9 7TF, UK alhydrogel. At various time points over a 12-month period postimmuniza- tion cohorts of mice were boosted by either the i.m. route using rF1 þ rV in We aim to investigate the susceptibility of Adenovirus 12 (Ad12) and alhydrogel or by the i.n. route using rF1 þ rV in PLLA microspheres. Adenovirus 5 (Ad5) transformed human cells to NK cell lysis in vitro Animals were subsequently challenged at different time points post using a human NK cell line, YT. The YT cell line is not inhibited by target boosting with virulent Y. pestis GB by the subcutaneous route. Protection cell expression of MHC class I. This allows the effect of molecules other against challenge conferred by the two boost immunization strategies was than MHC class I to be investigated for their role in determining assessed, to determine if a mucosally administered boost would be as susceptibility to NK cell lysis. We investigated the expression of adhesion efficacious as the regular i.m. boost. molecules on the Ad5 and Ad12 transformed cells using flow cytometry. Despite their susceptibility to NK cytolysis, the Ad12-transformed cells # Crown Copyright 2002 Dstl expressed significantly lower levels of cell surface LFA-3 than either the Ad5 or control transformed cell lines. ICAM-1 could not be detected on OP223 any of the Ad-transformed cells. The results indicate that Ad12 and Ad5 Intradermal and epidermal administration of Y. pestis transformed cells are differentially sensitive to NK cells and that these subunits prime amnestic responses that can protect differences are not due to altered levels of MHC class I. Furthermore, the mice from virulent plague expression of LFA-3 and ICAM-1 on the target cells does not correlate J. E. Eyles, S. J. Elvin, A. Westwood, C. Lebutt, E. D. Williamson, with their sensitivity to NK cells. Current experiments are aimed at S. Somavarapuy & H. O. Aplary identifying the mechanisms that determine the susceptibility of Ad- Dstl, Porton Down, Salisbury, SP4 0JQ, yLondon School of transformed cells to NK cells. Pharmacy, London WC1N 1AX, UK F1 and V were injected intradermally (10 mg each) into female BALB/c OP226 mice. F1 and V (in this case 30 mg each) were also applied to the shaved Enhanced production of antibodies and cytotoxic T skin of mice in solution with 100 mg cholera toxin adjuvant. A third group lymphocytes to a HIV peptide using a novl adjuvant of mice was immunized intranasally with 10 mg F1 and V admixed with 0Á1 mg CT. Mice were boosted by combinations of these routes and serum S. K. Matharu & K. E. Nye antibody responses to F1 and V were evaluated using indirect ELISA. Department of Immunology, Queen Mary, University of London, 38 Little ELISPOT readouts served to confirm the tenet that i.d. injection of F1 Britain, West Smithfield, London EC1A 7BE, UK effects a rapid humoral response. Primary V/F1 serum antibody responses In the search for a more potent and less toxic immunodulator, an aggregate were low and very variable in mice immunized by the transcutaneous structure composed mainly of tomatine (Mr 1034) was composed. Toma- route. However, despite the absence of a notable effector response, it was tine, an alkaloid glycoside, is obtained from the leaves of the wilt-resistant apparent that the transcutaneous route was effective at priming mice for wild tomato species Lycopersicon pimpinellifolium. It has previously been subsequent boosting by other routes. Furthermore, the transcutaneous studied for its ability to augment cytotoxic T lymphocytes and a humoral route could be used to boost mice that had been primed by other routes. immune response using the model antigen, ovalbumin. In this project, we Mice primed transcutaneously and boosted nasally, or primed nasally (or have studied the ability of tomatine to induce a humoral immune response intradermally) and boosted transcutaneously were solidly (10/10) pro- via different routes of immunization as well as a cytotoxic T lymphocyte tected when injected with 4 104 MLDs Y. pestis. immune response. Investigations have been carried out using a synthetic # Crown Copyright 2002 Dstl peptide derived from the CD4 attachment site of gp120, HIV.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 Vaccines 131

OP227 of DC with heat-inactivated (HI) B. mallei induced proliferative responses Recognition of mumps virus by murine CD4þ T cell in in naive T cells. This proliferative response was accompanied by the the absence of humoral responses induction of IL-12 and IFN-g. BALB/c mice were then immunized intradermally with either unstimulated DC, DC stimulated with HI M. Flynn, P. A. Pipkin,y M. A. Afzaly & B. P. Mahon B. mallei or with antigen alone. Mice were bled postimmunization and Mucosal Immunology Laboratory, Institute of Immunology, NUI their IgG isotype profiles were measured. At day 35 postimmunization, Maynooth, Co. Kildare, Ireland, yDivision of Virology, NIBSC, Blanche the mice were challenged via the intraperitoneal route with 107 cfu/mouse Lane, S. Mimms, Potters Bar, Herts, EN6 3QG, UK of B. mallei. Mice were observed daily for signs of illness and 21-days Infection by mumps virus (MuV) results in a serious childhood disease. postchallenge, the spleens of the surviving animals were removed. Pre- Safe, efficacious live attenuated vaccines are available as monovalent, liminary results indicate that mice immunized with HI B. mallei stimu- bivalent or trivalent formulations. Nevertheless, there is a need for lated DC had fewer clinical signs of illness than those animals immunized mucosally delivered subunit vaccines against all MMR components. with unstimulated DC or with antigen alone. Differences in the IgG The MuV hemagglutinin-neuraminidase (HN) protein is a vaccine can- isotypes induced following immunization were also evident. The pre- didate; however, similar molecules can interfere with normal immunity. In dominant IgG3 response induced following immunization with antigen the present study, we characterize the immunogenicity and immunomo- alone was lost in mice immunized with the antigen stimulated DC. Work is dulatory properties of HN. We have cloned and expressed HN, and ongoing to determine the relevance of these observations. examined the immunogenicity of MuV in mice. Initial studies suggested # Crown Copyright 2002 Dstl that MuV was not immunogenic in this model; however, we now demonstrate that MuV and its components exert a subtle influence on the immune systems of non-permissive hosts. A panel of MuV-specificT OP230 cell lines and clones have been established, and used to characterize the Enhanced in vivo immune responses to components of the virus recognized by the cellular immune response. The T cell-dependent antigens via CD40 stimulation phenotype of these clones has also been characterized with regard to T. A. Barr, A. L. McCormick & A. W. Heath surface marker and cytokine expression, and demonstrate that MuV Division of Genomic Medicine, Academic Unit of Infection and Immunity, immunization of mice induces cell mediated, but not humoral immunity. University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK

OP228 There is great potential for novel vaccines based on recombinant proteins Role of CD4þ and CD8þ T cells in protection against and synthetic peptides. Unfortunately, these antigens often lack the biovars of Francisella tularensis immunogenicity of whole, killed pathogens used in traditional vaccines. Thus, there is strong interest in novel immunological adjuvants of low K. F. Griffin, D. G. C. Rees & R. W. Titball reactogenicity, but high potency, to enhance immune responses and realize Dstl Biomedical Sciences, Porton Down, Salisbury SP4 0JQ, UK the potential of these new vaccine strategies. CD40 antibodies have been The Francisella tularensis live vaccine strain (LVS), an attenuated shown to have adjuvant effects when administered at very high doses. palaearctica strain, has been widely administered to humans under These large doses are unpractical and induce a cascade of cytokine release investigational new drug status. LVS can be virulent in mice and is often giving rise to septic shock-like symptoms, as well as splenomegaly and used as a model in studies on immunity to F.tularensis. As the tularensis polyclonal antibody production. Here, we describe a method by which and palaearctica biovars differ in virulence, tularensis being the most CD40 antibody can exhibit potent adjuvant effects at very low doses, virulent, investigating mechanisms of protection using an attenuated greatly enhancing in vivo responses to subunit antigens. The lack of palaearctica model may be inappropriate. We investigated the role of detectable systemic effects indicates that this method may be a powerful CD4þ and CD8þ T cells in protection of LVS-immunized mice against and practical means of enhancing the efficacy of recombinant vaccines. fully virulent HN63 (palaearctica bivar)and Schu4 (tularensis biovar). Following immunization with LVS, mice were treated with either anti- CD4, anti-CD8, anti-CD4þ, anti-CD8 or control antibody. Groups were OP231 then challenged with HN63 or Schu4 and observed for 21 days. Groups Adjuvant function of the peptide binding fragment of challenged with HN63 all survived depletion of either CD4þ or CD8þ T HSP70 cells and 4/5 survived depletion of both T cell populations. In groups z þ þ E. G. McGowan, C. van Dolleweerd, M. Singh, L. A. Bergmeier, challenged with Schu4, 0/5 survived depletion of CD8 T cells or CD4 y þ T. Lehner & C. Kelly CD8 T cells; however, 2/5 survived CD4 depletion. Our results indicate y þ Immunology Unit, Oral Medicine and Pathology, Peter Gorer that although CD8 T cells are not required for survival against infection Department Immunobiology, Guy’s Hospital, London SE1 9RT, UK, with a palaearctica strain, they are required for protecion against the more zLionex Diagnostics, Braunschweig, Germany virulent tularensis strain. Heat shock proteins (Hsp) are highly conserved, acting as chaperones and # Crown Copyright 2002 Dstl adjuvants. We have expressed two recombinant Mycobacterium tubercu- losis Hsp70 proteins in Escherichia coli, the full protein (aa1–610) and the OP229 peptide binding domain, PBD (aa359–610). Using surface plasmon Generation of immune responses to heat inactivated resonance, both Hsp70 proteins were shown to non-covalently associate Burkholderia mallei using bone marrow derived with a peptide derived from the sequence of CCR5. Non-covalently dendritic cells as antigen presenting cells associated Hsp70–peptide complexes were used to immunize C57BL6 mice to stimulate a response to the peptide, examined by ELISA and M. Morton, G. D. Healey & E. D. Williamson splenic T cell proliferative responses. Immunization with the PBD– Dstl, Porton Down, Salisbury SP4 0JQ, UK peptide complex elicited antibodies (Ab) to the peptide which were Dendritic cells (DC) were isolated from mouse bone marrow following skewed towards IgG2a (1 : 64 103) and IgG3 (1 : 32 103), with invitro culture in the presence of GM-CSF and TNF-a. In vitro stimulation IgG1 significantly reduced (1 : 2 103), suggesting Th-1 polarization

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132 132 Vaccines

of the immune response. Ab titres to Hsp70 were reduced and Th-1 and promote a Th1-type immune response. To address this, we have polarization to the peptide was enhanced when the PBD was used as the investigated the effect(s) that coinoculation of viral nucleic acids has on adjuvant compared to immunization with full length Hsp70. Proliferative the immune response to protein antigens in mice. The adjuvant properties responses showed stimulation with the immunizing peptide inducing a of viral RNA (extracted from a murine cell line infected with the stimulation index of 4Á5, compared with 2Á5 when using a control peptide. arenavirus lymphocytic choriomeningitis virus (LCMV) or from LCMV This suggests that the C-terminal fragment of hsp70, acts as an effective virions) and viral DNA (extracted from cells infected with vaccinia virus Th-1 polarizing adjuvant. or from cytoplasmic viral ‘factories’) were compared to those of RNA and DNA derived from uninfected cells. Viral nucleic acids (RNA and DNA) were found to enhance the antibody response to model protein antigens in OP232 mice in particular, promoting the production of antibodies of the IgG2a Do viral nucleic acids have adjuvant properties? and 2b isotypes. In addition, they increased the magnitude of antigen- specific CD8þ T cell responses. Nucleic acids derived from uninfected M. J. Ashton cells had similar but much less marked effects. The mechanism(s) under- The Edward Jenner Institute, Newbury RG20 7NN, UK lying the immunostimulatory effects of viral nucleic acids are currently Protein antigens inoculated alone tend to be poorly immunogenic, being investigated. Preliminary experiments have indicated a role for whereas viral infections tend to elicit a very strong Th1 response. This type 1 interferons, which will be confirmed and extended by future raises the hypothesis that viral components may have adjuvant properties, studies.

# 2002 Blackwell Publishing Ltd. Immunology, 107, Supplement 1, 79–132