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,à S. J. Davis,à W. O. C. M. Cooksony & M. F. Moffatty University of Essex, Department of Biological Sciences, Wivenhoe Park, ÃNuffield 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 Æ 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,à A. Todd,à 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 ÃPublic 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,à A. Draper,à M. Jones,à J. Harris,à K. Welsh,y A. N. Taylorà & onmental factors, including respiratory infection, may influence the P. Cullinanà immune response and subsequent development of disease. In this study, ÃDepartment 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.