CD8+ T Cell-Mediated HLA-A*0201-Restricted Cytotoxicity to Transaldolase Peptide 168 −176 in Patients with Multiple Sclerosis
This information is current as Brian Niland, Katalin Banki, William E. Biddison and Andras of October 7, 2021. Perl J Immunol 2005; 175:8365-8378; ; doi: 10.4049/jimmunol.175.12.8365 http://www.jimmunol.org/content/175/12/8365 Downloaded from
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
CD8؉ T Cell-Mediated HLA-A*0201-Restricted Cytotoxicity to Transaldolase Peptide 168–176 in Patients with Multiple Sclerosis1
Brian Niland,*‡ Katalin Banki,*† William E. Biddison,§ and Andras Perl2*‡
Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168–176 (LLFSFAQAV, TALpep) ex- hibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8؉ T cells specific for TALpep was increased in PBMC of HLA-A2؉ MS patients, as compared with HLA-A2؊ MS patients, HLA-A2؉ other neurological disease patients, and HLA-A2؉ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8؉ T cells, and precursor frequency of TAL-specific IFN-␥-producing T cells was increased in each of seven HLA-A2؉ MS patients tested. Stimulation by ؉ TALpep or rTAL of PBMC from HLA-A2 MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lym- Downloaded from phoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8؉ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR V repertoire analysis revealed use of the TCR V14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR V14 on the cell surface. Moreover, Ab to TCR V14 abrogated cytotoxicity by
HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in http://www.jimmunol.org/ patients with MS. The Journal of Immunology, 2005, 175: 8365–8378.
ultiple sclerosis (MS)3 is considered to be an autoim- relapsing EAE have shown that different encephalitogenic mole- mune disease leading to progressive loss of oligoden- cules or epitopes within them are selected that are compatible with drocytes and demyelination in the CNS. In the acute the heterogeneity of the immune response in MS, suggesting that M ϩ ϩ stage of disease, lesions contain macrophages, CD4 and CD8 T disease initiation and relapse episodes are induced by different Ags cells, and Ig deposits, suggesting that the demyelination process is (13, 14). Although cell-mediated mechanisms may have a primary mediated by the immune system (1–4). The inflammatory picture role in EAE, augmentation of humoral immunity within the CNS by guest on October 7, 2021 of early lesions, which is followed by a progressive gliosis, also is a well-recognized feature of MS (15). A breakdown of the suggested that the pathological process may be initiated by infec- blood-brain barrier would allow Abs to enter the CNS and cause tious agents and then self-perpetuated by a cross-reactive autoim- demyelination by complement activation. In fact, complement may mune process (5–10). Although the Ag(s) driving this self-destruc- be directly involved in death of oligodendrocytes (16). Most efforts tive process in MS has not been identified (11), the importance of have been focused on myelin basic protein (MBP) and proteolipid myelin-derived Ags was demonstrated by their abilities to elicit an protein that make up as much as 30 and 50% of CNS myelin, MS-like demyelinating disease, experimental allergic encephalo- respectively (17, 18). T cell responses to MBP and proteolipid myelitis (EAE), in various animal models (12). protein, or another oligodendrocyte-specific protein myelin oligo- The major difficulties in applying the EAE model to MS stem dendrocyte protein (MOG), did not differ considerably between from a lack of identification of relevant autoantigen(s). Studies on MS patients and control donors (11). Although oral vaccination with a predefined inducing Ag may successfully prevent and treat disease in animal models (19), a similar approach with MBP in 30 *Department of Medicine, †Department of Pathology, and ‡Department of Microbi- ology and Immunology, State University of New York, College of Medicine, Syra- patients with MS led to no significant clinical improvement (20). cuse, NY 13210; and §Neuroimmunology Branch, National Institute of Neurological Molecular mimicry, i.e., cross-reactivity between self Ags and Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 viral proteins, has been implicated in the initiation of autoimmu- Received for publication August 5, 2005. Accepted for publication September nity and MS. Based on homology to retroviral sequences, a novel 26, 2005. autoantigen, partially encoded by a retrotransposon and selectively The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance expressed in oligodendrocytes at high levels (21), was identified as with 18 U.S.C. Section 1734 solely to indicate this fact. human transaldolase (TAL) (22). TAL is a key enzyme of the 1 This work was supported in part by Grants RO1 DK 49221 from the National pentose phosphate pathway (PPP). Although glucose is largely me- Institutes of Health and RG 2466 from the National Multiple Sclerosis Society. tabolized through the glycolytic pathway and the tricarboxylic acid 2 Address correspondence and reprint requests to Dr. Andras Perl, Department of cycle, the significance of PPP in the brain has long been estab- Medicine, State University of New York, 750 East Adams Street, Syracuse, NY 13210. E-mail address: [email protected] lished. During brain development, PPP provides NADPH for the biosynthesis of lipids (23). The latter is particularly important at 3 Abbreviations used in this paper: MS, multiple sclerosis; AEC, 3-amino-9-ethyl-   carbazole; 2m, 2-microglobulin; EAE, experimental allergic encephalomyelitis; the period of active myelination (24, 25). The overall activity of 125  125  FSC, forward scatter; HC, healthy control; I- 2m, I-labeled 2m; MBP, myelin PPP in the brain declines 5-fold from birth to maturity (26). Al- basic protein; MOG, myelin oligodendrocyte protein; MS, multiple sclerosis; OND, other neurological disease; PPP, pentose phosphate pathway; SSC, side scatter; TAL, though under normal conditions only as little as 1% of the glucose transaldolase; TALpep, TAL peptide 168–176; TCL, T cell line. enters the PPP (27), at times of rapid myelination up to 60% of the
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 8366 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS glucose is metabolized via the PPP (28). Involvement of PPP in TAL- or TALpep-pulsed APC derived from autologous PBMC or myelination provided a physiological explanation for the high EBV-transformed B cells expressed TCR V14 on their cell sur- level of TAL in oligodendrocytes (21–29). PPP also plays an es- face. Pretreatment of TCL lines with TCR V14 mAb abrogated sential role in neutralization of oxygen radicals, and elevated levels their cytotoxicity against HLA-A2-transfected Hmy A2.1 or of TAL expression increase susceptibility to apoptosis signals MO3.13 cells, suggesting that TAL-specific CTL may serve as a (30–32). Effector phase of the demyelination process in MS is therapeutic target to prevent oligodendrocyte destruction in pa- thought to be mediated by reactive oxygen intermediates. Intrale- tients with MS. sional CTLs produce TNF-, which, in turn, induces apoptosis, an oxidative stress-mediated programmed cell death, of oligodendro- Materials and Methods cytes (33). The Fas receptor/ligand system has also been impli- Patient and control cells cated in death of oligodendrocytes (34, 35). Thus, oligodendro- Fourteen patients with MS, eight patients with OND, and eight HC donors cyte-specific expression of TAL is possibly linked to production of were investigated (Table I). Patients with MS were diagnosed according to large amounts of lipids, as a major component of myelin and vul- the criteria of Poser et al. (42). PBMC were isolated from heparinized nerability of the vast network of myelin sheaths to oxygen radicals. venous blood on Ficoll-Hypaque gradient and resuspended in RPMI 1640 Immunohistochemical studies of postmortem brain sections re- medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 mg/ml amphoter- vealed decreased staining by MBP- and TAL-specific Abs in MS icin B, and cultured in a humidified atmosphere with 5% CO2 at 37°C. Cell plaques, indicating a concurrent loss of these Ags from sites of culture products were obtained from Mediatech. PBMC were HLA class I Ag demyelination (29). Patients with MS have TAL Abs in their blood typed by the National Institutes of Health microcytotoxicity (43). Viability and and cerebrospinal fluid (21–29). TAL autoantibodies recognize im- functional capacity of all PBMC samples have been monitored by trypan blue
Ͼ 3 Downloaded from munoblotted and three-dimensional epitopes and inhibit enzymatic exclusion ( 95%) and PHA-induced proliferative responses by [ H]TdR incorporation, respectively (29). activity of TAL (36). By constrast, TAL Abs were absent in nor- mal individuals and patients with other autoimmune and neuro- Ags and Abs logical diseases (21) and, under identical conditions, no MBP Abs Full-length rTAL-H protein (clone 1425) was expressed as a fusion protein were found in serum and cerebrospinal fluid of MS patients (29). with GST by pGEX-2T plasmid vector (44), affinity purified through bind- The fact that TAL Abs were absent in controls including patients ing of GST to glutathione-coated agarose beads, cleaved from GST by 1 with other neurological disease (OND) or systemic autoimmune National Institutes of Health unit of thrombin (Sigma-Aldrich), and sepa- http://www.jimmunol.org/ rated from the agarose bead-bound GST by centrifugation (21). Control diseases such as SLE and Sjo¨gren syndrome indicated that the GST Ag was prepared by elution from glutathione-coated agarose beads in autoimmune process targeting TAL may be specific for MS. In 10 mM reduced glutathione and 50 mM Tris-HCl (pH 8.0). The purified addition, TAL elicit proliferation, aggregate formation, and skew- full-length rTAL was quantified by the Bradford assay, analyzed by SDS- ing of the TCR V repertoire of peripheral blood T lymphocytes PAGE and Western blot, and tested for TAL enzyme activity, as earlier Ͼ from patients with MS with respect to MBP as control Ag. The described (22). Synthetic peptides were produced and purified to 99% homogeneity by Genemed (Genemed Synthesis). BSA, Con A, and PHA results suggested that TAL may be a more significant target than were obtained from Sigma-Aldrich. Highly specific polyclonal rabbit Abs MBP of myelin-reactive T cells and of humoral autoreactivity in 169 and 170 directed to the 139-aa-long N-terminal segment of TAL were patients with MS (29). developed earlier (22). by guest on October 7, 2021 Most reports attribute oligodendrocyte cell death, at least in part, Prediction and identification of HLA-A2-binding TAL peptide to the cytotoxic effect of CD8ϩ T cells (37). Adoptively transferred epitopes MBP-specific (38) or MOG-specific CD8ϩ T cells induce severe CNS demyelination in animal models (39). In MS brain lesions, The human TAL amino acid sequence was analyzed for the presence of infiltrating CD8ϩ CTL were reported to outnumber CD4ϩ T cells nonamer peptides with HLA class I-binding motifs using a computer-based algorithm that predicts the stability of HLA-Ag/peptide complexes by 10-fold (2). Also, actively demyelinating lesions of MS brains are ϩ quantitating positive and negative effects on binding of each amino acid enriched for clonally expanded CD8 T cells as compared with within a nonamer peptide (National Institutes of Health BioInformatics & CD4ϩ T cells (40, 41). These myelin-reactive CD8ϩ T cells may Molecular Analysis Section website server: ͗http://bimas.dcrt.nih.gov/mol- ϩ ͘ play a role in recruitment and retention of myelin-reactive CD4 bio/hla_bind/ ). Binding of peptides to HLA-A2 was measured by incor- 125  125  poration of I-labeled 2-microglobulin ( I- 2m) into HLA-A2 H T cells by secreting proinflammatory soluble mediators to promote  chain/ 2m/peptide heterotrimeric complexes and analyzed by gel filtration. and mediate inflammatory response in MS brain. In the present  Stability of HLA-A2 H chain/ 2m/peptide complexes was determined by 125  study, we show that among 14 TAL peptides tested, TAL peptide the rate of dissociation (t1/2 in minutes) of I- 2m at 37°C, as earlier Ͼ 168–176 (TALpep) has high binding affinity for HLA-A2. Full- described (45). TAL peptides with measured t1/2 of 100 min were se- length human rTAL- or TALpep-stimulated CTL from HLA-A2ϩ lected to stimulate CTL. MS patients kill TALpep-pulsed HLA-A2-transfected (Hmy Establishment of TAL-specific TCL A2.1), but not HLA-A3-transfected control target cells. By con- ϩ Ϫ PBMC were incubated in petri dishes precoated with autologous serum for trast, HLA-A2 healthy or OND controls and HLA-A2 control 1 h at 37°C to remove monocytes (46). Nonadherent cells were enriched for donors do not show increased killing of peptide-pulsed Hmy A2.1 CD8ϩ T cells by selective depletion of CD4ϩ cells using mAb-coated targets. HLA-A2-transfected, but not control MO3.13 oligoden- Dynabeads M-450 CD4, according to the manufacturer’s instructions (cat- alogue 111.08; Dynal Biotech). Usually, a CD4 T cell depletion of Ͼ99% droglial cells, expressing high levels of endogenous TAL, were 7 also killed by CD8ϩ CTL of MS patients without peptide pulsing was achieved with Dynabeads M-450 CD4. The resultant effector cells (10 cells per donor) were stimulated with 5 g/ml TAL or TALpep. On day 8, the targets, thus indicating that endogenously processed TAL was and subsequently at regular 7- to 10-day intervals, TCL were restimulated recognized by HLA-A2-restricted CTL. As detected by PE-conju- with autologous APC derived from EBV-transformed B cells or PBMC. gated HLA-A*0201/TALpep tetramer staining, the frequencies of APC were generated by incubation with 5 g/ml TAL or TALpep for 1 h, TALpep-specific CD8ϩ T cells were increased in PBL of HLA- irradiated, and added to effector cells at a 1:1 ratio. In between restimula- tion with APC, 10 U/ml human rIL-2 was also added to the effector cells. A2ϩ MS patients when compared with HLA-A2ϩ OND and healthy controls (HC), or HLA-A2Ϫ MS patients. In response to Generation of B cell lines by EBV infection stimulation by rTAL or TALpep, precursor frequency of IFN-␥- ϩ EBV was harvested from the supernatant of B95-8 cells (47), filtered producing T cells was increased in HLA-A2 MS patients. T cell through a 0.45-m mesh, and stored at Ϫ80°C. A total of 107 PBMC was ϩ lines (TCL) of HLA-A2 MS patients raised by stimulation with infected by EBV by incubation with 3 ml of B95-8 supernatant plus 2 ml The Journal of Immunology 8367
Table I. Clinical data, HLA-A2 status, and cumulative results of MS patients and control donorsa
Donor HLA-A2 Age/Sex Dx Duration (years) Dx/Type Hmy MO3.13 Tetramer ELISPOT
MS1 ϩ 62/F 24 PP ϩϩ ϩ ϩ MS2 ϩ 46/F 14 R/R ϩϩ ϩ ϩ MS3 ϩ 49/M 19 R/R ϩϩ ϩ ϩ MS4 ϩ 49/F 25 R/R ϩϩ ϩ ϩ MS5 ϩ 73/M 32 S ND ϩϩ ϩ MS6 ϩ 61/F 15 SP ND ND ϩϩ MS7 ϩ 50/F 26 SP ND ND ϩϩ MS8 Ϫ 53/F 6 PP ND Ϫ ND ND MS9 Ϫ 46/F 22 SP ND ϪϪ Ϫ MS10 Ϫ 55/F 6 R/R ND ϪϪ Ϫ MS11 Ϫ 44/F 5 R/R ND ϪϪ Ϫ MS12 Ϫ 48/F 4 R/R ND ND ϪϪ MS13 Ϫ 63/F 9 SP ND ND ϪϪ MS14 Ϫ 41/F 7 R/R ND ϪϪ Ϫ OND1 ϩ 83/M 12 PD ND ϪϪ Ϫ OND2 ϩ 77/M 4 Stroke ND ND ϪϪ OND3 ϩ 37/F 24 Epilepsy ND ϪϪ Ϫ OND4 ϩ 59/F 42 Epilepsy ND Ϫ ND Ϫ OND5 Ϫ 82/M 8 PD ND ND ϪϪ Downloaded from OND6 Ϫ 64/M Ͻ1 Stroke ND ϪϪ Ϫ OND7 Ϫ 60/F Ͻ1 Paraplegia ND ND ND Ϫ OND8 Ϫ 67/M 2 ALS ND ϪϪ Ϫ HC1 ϩ 29/M N/A N/A Ϫ ND ND ND HC2 ϩ 58/F N/A N/A ϪϪ ND ND HC3 ϩ 33/F N/A N/A ϪϪ Ϫ Ϫ HC4 ϩ 28/M N/A N/A ϪϪ Ϫ Ϫ
HC5 Ϫ 34/F N/A N/A Ϫ ND ND ND http://www.jimmunol.org/ HC6 Ϫ 32/M N/A N/A Ϫ ND ϪϪ
a Dx, Diagnosis; M, male; F, female; R/R, relapsing/remitting; PP, primary progressive; SP, secondary progressive; S, stable; OND, other neurological disease; PD, Parkinson’s disease; ALS, amyotrophic lateral sclerosis; HC, healthy control; Hmy, HLA-A2-restricted TALpep-specific cytotoxicity to Hmy cells; MO3.13, HLA-A2-restricted TALpep-specific cytotoxicity to MO3.13 cells; Tetramer, frequency of HLA-A*0201/TALpep tetramer-positive cells in PBMC stimulated with TALpep for 7 days; ELISPOT, frequency of IFN-␥-producing cells in PBMC stimulated with TALpep for 7 days; ϩ, values above mean ϩ 3 SD of all healthy and OND controls; Ϫ, values below mean ϩ 3 SD of all healthy and OND controls; N/A, not applicable.
of fresh complete RPMI 1640 medium at 37°C overnight. Next day, 4 ml MS patients, and HLA-A2ϩ MS patients as well as TCL from MS patients
of fresh complete RPMI 1640 medium and cyclosporin A (Sigma-Aldrich) were stained with PE-conjugated tetramers for 30 min at 4°C, washed three by guest on October 7, 2021 was added to a final concentration of 0.5 g/ml and cultures were left times, and resuspended in PBS before analysis on a BD Biosciences LSRII undisturbed for 10–14 days. flow cytometer equipped with 20 mW argon (emission at 488 nm) and 16 mW helium-neon lasers (emission at 634 nm) (BD Biosciences). Dead cells T cell-mediated cytotoxicity assays and debris were excluded from the analysis by electronic gating of forward (FSC) and side scatter (SSC) measurements. Detection of staining with the To assess cytotoxic potential and the MHC class I restriction of the TAL tetramer (FL-2 fluorescence) was matched with emission spectra of FITC- peptide-reactive TCLs, HLA-A2-transfected (Hmy A2.1) and HLA-A3- conjugated CD8 (FL-1) and PE-Cy5-conjugated CD4 Abs (FL-3). Expres- transfected (Hmy A3.1) Hmy lymphoblastoma and HLA-A2-transfected sion of TCR V14 protein on the surface of TAL-specific TCL was as- (MO3.13/A2) and control MO3.13 oligodendroglial cells were used (48). sessed by staining with anti-human TCR V14 mAb (mAb, clone g/ml TALpep for2hat37°C. Then, 106 Target cells were pulsed with 5 CAS1.1.3; Serotec) and isotype control mouse IgG1 mAb ME.05 (Serotec target cells were labeled with 200 Ci of Na 51CrO for 1 h, washed three 2 4 clone ME.05), followed by detection with FITC-conjugated goat times, and seeded at a concentration of 5 ϫ 103 /well U-bottom 96-well anti-mouse IgG. plates. Depending on availability, effectors were added at 2.5:1, 5:1, 10:1, 25:1, and 50:1 E:T ratios. TALpep was added at 10 g/ml during the cytotoxicity assay. Control cultures included peptide-pulsed and unpulsed IFN-␥ ELISPOT assay target cells with peptide or medium alone. Maximal release was measured in the presence of 0.1 M HCl. After incubation for4hat37°C, the super- Millipore MultiScreen IP 0.45- m Immobilon-P hydrophobic polyvinyli- natants were harvested and counted in a gamma counter. Percentage of dene difluoride membrane ELISPOT plates (catalogue MAIPN4510) were ϫ Ϫ coated with 15 g/ml anti-human IFN-␥ capture mAb (Mabtech) overnight cytotoxicity was calculated: 100 ((test cpm spontaneous cpm)/(max- imal cpm Ϫ spontaneous cpm)). at 4°C, washed six times with 200 l/well sterile PBS, and blocked with 100 l/well 10% FBS in RPMI 1640 for4hat37°C. After washing once Cytotoxic activity against adherent MO3.13 oligodendroglial cells (49) 5 was measured by detachment of killed cells from the monolayer. A total of with 10% FBS in RPMI 1640, 10 cells in 100 l of RPMI 1640 with 10% FBS were incubated in each well in triplicates for 20 h at 37°C. Subse- 2500 MO3.13 cells/well of flat-bottom 96-well plates was prelabeled with 3 quently, the ELISPOT plates were washed six times with 200 lofPBS [ H]TdR (ICN Biomedicals) with or without 10 g/ml TALpep and al- ␥ lowed to form nonconfluent monolayer for 24 h. Targets were washed three per well. Next, 1 g/ml biotinylated anti-IFN- detection mAb was added in 100 l of PBS with 0.5% FBS for 20 h at 4°C. On day 4, plates were times, and effectors were added for 24 h. Subsequently, plates were washed six times to remove effectors and killed target cells, and cytotoxicity was washed six times with 200 l of PBS per well, and HRP-conjugated 3 streptavidin was added in 100 l of PBS with 0.5% FBS for1hatroom determined based on [ H]TdR content of remaining viable cells, as earlier described (46). temperature. Plates were washed six times with 200 l of PBS per well and developed with 100 l of 3-amino-9-ethyl-carbazole (AEC) substrate. Flow cytometry AEC substrate included 20 mg of AEC dissolved in 2.5 ml of dimethyl- formamide and supplemented with 47.5 ml of acetate buffer (4.6 ml of 0.1
PE-conjugated HLA-A*0201/LLFSFAQAV tetramer and control HIV N acetic acid, 11.0 ml of 0.1 M sodium acetate, 46.9 ml of dH2O, and 25 peptide tetramers, HLA-A2/HIV pol 464 (HLA-A*0201/ILKEPVHGV), l of 30% H2O2) and was passed through a 0.4- m filter to remove pre- and HLA-A2/HIV gag 77 tetramers (HLA-A*0201/SLYNTVATL) were cipitates. After 4-min incubation with substrate, ELISPOT plates were synthesized at the National Institutes of Health Tetramer Core Facility at washed six times with 200 lofH2O per well. Spots were counted at Emory University. Freshly isolated PBMC from HLA-A2ϩ control donors, 40-fold magnification using a dissection microscope. 8368 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS
Analysis of the TCR V repertoire by RT-PCR proteins, like highly soluble TAL, constitute a major source of peptides presented to CD8ϩ T cells by HLA class I molecules (51). Total cellular RNA was isolated from cultured cells (Qiagen). A total of 3 ϩ g of RNA was reverse transcribed into cDNA with oligo(dT) primer by To investigate the possible role of CD8 T cell responses to TAL 200 U of Superscript reverse transcriptase, according to the manufacturer’s in the pathogenesis of MS, we used a computer-based algorithm protocol (Invitrogen Life Technologies). For analysis of TCR V gene us- that predicts the stability of HLA-Ag/peptide complexes by quan-  age, cDNA aliquots were subjected to PCR using 1 of 24 V sense strand- titating positive and negative effects on binding of each amino acid specific oligonucleotides in combination with an antisense strand-specific C primer (C-REVF, 5-CGGGCTGCTCCTTGAGGGGCTGCG-3) (29). within a nonamer peptide (45). Incorporation of a typically 8- to As an internal control, each PCR included C␣-specific sense (C␣-FWJ, 9-aa-long peptide is required for the stabilization of class I H chain 5-CCCTGACCCTGCCGTGTACCAGCT-3) and C␣ antisense primers and  m complexes and their transport to the cell surface (51). ␣ 2 (C -REVE, 5-GTTGCTCCAGGCCGCGGCACTGTT-3) (29). Amplifica- Binding affinity to MHC class I molecules reliably predicts the tions were conducted in 30 cycles at a denaturing temperature of 94°C (1 min), annealing at 60°C (1 min), and extension at 72°C (2 min). Amplified capacity of a peptide epitope to elicit a CTL response (48–52). products were analyzed by electrophoresis in 1.5% agarose gel. Automated Therefore, the TAL amino acid sequence was analyzed for the densitometry was used to quantify the relative levels of DNA band inten- presence of nonamer peptides with HLA class I-binding motifs sity using a Kodak Image Station 440CF with Kodak 1D Image Analysis (45). Ten peptides with high predicted HLA-A2-binding stabili- software (Eastman Kodak). ties, i.e., t1/2 of greater than 100 min at 37°C, were identified in Western blot analysis human TAL (Table II). Binding of TAL peptides to HLA-A2 was 125  A total of 500 ng of rTAL-H protein in 10 l per well was separated by measured by incorporation of I- 2m into HLA-A2 H chain/  SDS-PAGE and electroblotted to nitrocellulose (50). Nitrocellulose blots 2m/peptide heterotrimeric complexes and analyzed by gel filtra- were incubated in 100 mM Tris (pH 7.5), 0.9% NaCl, 0.1% Tween 20, and  tion. Stability of HLA-A2 H chain/ 2m/peptide complexes was Downloaded from 5% skim milk with TAL Ab 169 (22). For detection of rabbit Abs, after determined by the rate of dissociation (t in min) of 125I- mat washing, blots were incubated with HRP-conjugated goat anti-rabbit IgG 1/2 2 (Boehringer Mannheim). In between the incubations, the strips were vig- 37°C, as earlier described (45). Among 14 peptides with predicted orously washed in 0.1% Tween 20, 100 mM Tris (pH 7.5), and 0.9% NaCl. HLA-A2-binding stabilities of greater than 100 min (t1/2) at 37°C, The blots were developed with a substrate comprised of 1 mg/ml 4-chlo- TALpep has the highest binding affinity for HLA-A2. TAL pep- ronaphthol and 0.003% hydrogen peroxide. Ͼ tides with measured t1/2 of 100 min (TALpep and TAL peptide 307–317) were tested for recognition by CTL in HLA-A2ϩ donors TAL enzyme activity http://www.jimmunol.org/ (Table II). TAL enzyme activity was measured by the transfer of the dihydroxyac- To test for the existence of HLA class I-restricted TAL-specific etone three-carbon unit from the donor D-fructose-6-phosphate, to the ac- D CTL activity, PBMC of MS and control donors were prestimulated ceptor -erythrose-4-phosphate, as earlier described (22). Enzyme activity ϩ of 50 ng of rTAL was assayed in the presence of 3.2 mM D-fructose with human rTAL for 1 wk. CTL from HLA-A2 MS patients 6-phosphate, 0.2 mM D-erythrose-4-phosphate, 0.1 mM NADH, 10 gof MS1, MS2, MS3, and MS4 showed specific killing of HLA-A2- ␣ -glycerophosphate dehydrogenase/triosephosphate isomerase at a 1:6 ra- transfected (HMY A2.1), but not HLA-A3-transfected control tar- tio in 1 ml of PBS (pH 7.4) at room temperature by continuous absorbance reading at 340 nm for 20 min. get cells (HMY A3.1) pulsed with TALpep (Fig. 1A). Pretreament of effectors with rTAL was more efficient than synthetic peptides
Statistics in inducing CTL activity against TALpep-pulsed targets (data not by guest on October 7, 2021 Results were analyzed by Student’s t test or Mann-Whitney rank sum test shown). Cytotoxic activity escalated with higher E:T ratios. By ϩ for nonparametric data. Correlation was measured using Pearson’s corre- contrast, effector cells of three HLA-A2 HC (HC1, HC2, HC3) lation coefficient. Changes were considered significant at p Ͻ 0.05. and three HLA-A2Ϫ control donors (HC5; Fig. 1A; HC6, data not shown) did not elicit killing of peptide-pulsed targets. Pulsing of Results targets with HLA-A2-binding TAL peptide 307–317 with an order Detection of HLA-A2-restricted TALpep-specific CTLs in of magnitude lower binding affinity (Table II) did not elicit killing. patients with MS MO3.13 oligodendroglial cells express high levels of endoge- Demyelination in patients with MS is thought to be mediated by a nous TAL activity, ϳ0.2 U/mg protein, as compared with Ϫ0.015 selective destruction of oligodendrocytes (11). CD8ϩ CTL out- U/mg protein in PBMC (22). HLA-A2-transfected, but not control number CD4ϩ T cells near MS lesions up to 10-fold (2). Cytosolic MO3.13 cells were efficiently killed by CD8 T cells prestimulated
Table II. Identification and ranking of potential HLA-A2-binding TAL peptidesa
Position HLA-A2 Binding Sequence Predicted t1/2 (min) Measured t1/2 (min) 168–176 LLFSFAQAV 1750.432 1690* 263–273 LLQDNAKLVPV 734.891 ND 167–176 TLLFSFAQAV 512.603 10 263–271 LLQDNAKLV 510.114 30 325–333 MLTERMFNA 459.665 ND 307–316 KLSDGIRKFA 311.612 ND 297–306 WLHNEDQMAV 265.533 40 307–317 KLSDGIRKFAA 265.293 127* 86–95 KLFVLFGAEI 264.797 30 324–333 RMLTERMFNA 139.573 ND 89–99 VLFGAEILKKI 98.347 ND 252–260 FLTISPKLL 97.486 ND 86–96 KLFVLFGAEIL 68.833 ND 110–119 RLSFDKDAMV 57.537 ND ء a Binding stability was based on calculated half-life (predicted t1/2, in min at 37°C) of peptide-HLA-A2 complexes as earlier described (45). , Values Ͼ100. The Journal of Immunology 8369 Downloaded from http://www.jimmunol.org/
FIGURE 1. A, TAL-specific CTL killing of HLA-A2ϩ Hmy A2.1 target cells pulsed with TALpep (f) with respect to control HLA-A3ϩ Hmy A3.1 target cells pulsed with TALpep (Ⅺ). PBMC (5 ϫ 105/ml) from HLA-A2ϩ MS patients (MS/A2ϩ) and HLA-A2ϩ and HLA-A2Ϫ HC donors were cultured with or without 5 g/ml rTAL. On day 3, 10 U/ml IL-2 was added. On day 7, cells were washed and tested for TAL-specific CTL activity against by guest on October 7, 2021 51 51 Na CrO4-labeled Hmy A2.1 and Hmy A3.1 cells at 5:1, 25:1, or 50:1 E:T ratios. Control cultures included Na CrO4-labeled unpulsed target cells. Values represent percent specific lysis of peptide-pulsed targets over nonpulsed targets. Data show mean Ϯ SD of three parallel experiments. Killing of peptide- pulsed HLA-A2ϩ targets (Hmy A2.1), in comparison with control targets (Hmy A3.1), was significantly increased by CTL of HLA-A2ϩ MS patients at p Ͻ 0.0001). HLA-A2ϩ or HLA-A2Ϫ control donors showed no significant CTL activity against ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) each E:T ratio tested TALpep-pulsed target cells. B, Testing of CTL activity by HLA-A2ϩ MS patients (MS/A2ϩ), HLA-A2Ϫ MS patients (MS/A2Ϫ), and HLA-A2ϩ and HLA-A2Ϫ control donors against MO3.13 oligodendroglioma target cells. As effector cells, PBMC were prestimulated with 5 g/ml TAL for 7 days, washed, and tested for HLA-A2-restricted TALpep-specific CTL activity at 5:1 E:T ratio against [3H]TdR-prelabeled MO3.13 cells. During the prelabeling period, HLA-A2ϩ MO3.13/A2 and control MO3.13 target cells were pulsed with 10 g/ml TALpep. Percentage of cytotoxicity was measured by the detachment of target cells from the monolayer in a 24-h assay. Data represent mean Ϯ SD of three experiments. CTL killing of peptide-pulsed HLA-A2ϩ targets in comparison with unpulsed HLA-A2ϩ targets and unpulsed HLA-A2ϩ targets in comparison with unpulsed HLA-A2Ϫ targets was significantly p Ͻ 0.0001). HLA-A2Ϫ MS control and HLA-A2ϩ HC donors showed no ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) increased in each HLA-A2ϩ MS patient significant CTL activity against TALpep-pulsed or unpulsed target cells.
with TAL protein or TALpep (Fig. 1B). CTL activity by HLA- from MS3 only exhibited 5.8 Ϯ 1.0% cytotoxicity to HmyA2.1 A2ϩ (MS1, MS2, MS3, MS4, and MS5) and HLA-A2Ϫ MS pa- cells (Fig. 1A). HLA-A2-restricted TAL-specific CTL activity was tients (MS8 and MS9), and HLA-A2ϩ control donor (HC3) against enhanced to 23.6 Ϯ 3.7% when using Dynabead M-450 affinity- MO3.13 oligodendroglioma target cells is shown in Fig. 1B.Inthe purified CD8ϩ T cells of MS3, as effectors (data not shown). Over- absence of pulsing the target cells with peptides, killing by CTL of all, MO3.13 oligodendroglioma cells were more susceptible than MO3.13/A2 with respect to control MO3.1 target cells was signif- Hmy lymphoblastoma cells for detection of TAL-specific HLA- icantly increased in all HLA-A2ϩ MS patients ( p Ͻ 0.0001). Kill- A2-restricted cytotoxicity in each patient ( p Ͻ 0.0001). HLA-A2Ϫ ing by TAL or peptide-stimulated CTL of HLA-A2-transfected, MS patients and HLA-A2ϩ control donors showed no significant but not control MO3.13 cells occurred without pulsing the targets CTL activity against TALpep-pulsed or unpulsed target cells. The with peptides. This indicated that endogenously processed TAL results revealed the existence of TALpep-specific HLA-A2-re- was recognized by HLA-A2-restricted CTL. Pulsing of the targets stricted CTL in patients with MS. Ͻ with TALpep further enhanced killing of MO3.13/A2 cells ( p ϩ 0.02; Fig. 1B). Pretreatment of control MO3.13 target cells with Increased precursor frequency of TALpep-specific CD8 T cells TALpep did not elicit killing by HLA-A2ϩ MS CTL. Although in peripheral blood of MS patients PBL of MS patients MS1 and MS2 exhibited considerable cyto- Multimeric peptide-MHC complexes bind more than one TCR and toxic activities, 24.8 Ϯ 1.8% and 19.2 Ϯ 2.0%, respectively, PBL thus have a relatively slow dissociation rate and allow staining and 8370 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS detection of epitope-specific T cells. Staining with PE-conjugated Generation of HLA-A2-restricted TALpep-specific CD8ϩ TCLs HLA-A*0201/LLFSFAQAV tetramers was measured by FL-2 flu- from MS patients orescence. Frequency of HLA-A2/TALpep/tetramer-binding cells ϩ ϩ Ϫ Freshly isolated PBMC from HLA-A2 MS patients MS2 and was determined in seven HLA-A2 MS patients, six HLA-A2 ϩ ϩ Ϫ MS7 were enriched for CD8 T lymphocytes by depletion of MS patients, three HLA-A2 OND controls, three HLA-A2 ϩ ϩ Ϫ CD4 T cells with Ab-coated magnetic beads. TAL-specific OND controls, four HLA-A2 HC, and three HLA-A2 HC. As CD8ϩ TCL were established by stimulation at regular 7- to 10-day shown in Fig. 2, increased numbers of cells staining with HLA- ϩ intervals with rTAL- or TALpep-pulsed and irradiated APC de- A*0201/LLFSFAQAV tetramer were detected in HLA-A2 MS rived from fresh autologous PBMC or EBV-transformed B cells. patients following rTAL stimulation (0.98 Ϯ 0.07%; p Ͻ 0.01). Functional capacity of TALpep tetramer-staining TCL was as- Higher frequency of HLA-A*0201/LLFSFAQAV tetramer pos- sessed by production of IFN-␥ using the ELISPOT assay. Each of itivity was noted in TALpep-stimulated cells (1.4 Ϯ 0.1%; p Ͻ the TCL tested was maintained in vitro for Ͼ1 year by repetitive 0.0001). In contrast, no significant increase in HLA-A*0201/ stimulation with TAL- or TALpep-pulsed autologous APC derived TALpep/tetramer-PE staining was found in HLA-A2ϩ OND from PBMC (PBMC/APC) or EBV-transformed B cells (EBV/ (0.34 Ϯ 0.11%) and HC (0.36 Ϯ 0.21%). HLA-A2/HIV pol 464 APC). As shown in Fig. 4, A and B, highest frequency of IFN-␥- (HLA-A*0201/ILKEPVHGV) and HLA-A2/HIV gag 77 tet- producing cells was obtained when TCL were exposed to the same ramers (HLA-A*0201/SLYNTVATL) did not show increased Ag used for repetitive stimulation, i.e., TAL-prestimulated cells staining of TAL-stimulated PBMC from HLA-A2ϩ MS patients produced more IFN-␥ in the presence of TAL, while TALpep- (Fig. 2). prestimulated cells produced more IFN-␥ in the presence of TAL-
pep. MS2 TCL stimulated with TALpep PBMC/APC had higher Downloaded from ␥ Increased prevalence of TAL-reactive IFN-␥-producing T cells frequencies of IFN- -producing cells than MS2 TCL stimulated ϭ in MS patients with TAL PBMC/APC ( p 0.022; Fig. 4B). Along the same line, TALpep was more effective than TAL in generating IFN-␥-pro- ␥ CTL produce IFN- that, in turn, enhances HLA class I expression ducing T cells when using EBV APC in both patients MS2 ( p ϭ on human oligodendrocytes (53). Therefore, the production of 0.029) and MS7 ( p ϭ 0.003; Fig. 4B). IFN-␥ by TAL-reactive T cells of MS patients and controls was
Ͼ http://www.jimmunol.org/ ϩ While maintaining these cell lines for 3 years, frequency of assessed. PBMC from seven HLA-A2 MS patients, six HLA- HLA-A2-restricted TALpep-specific T cells was repeatedly A2Ϫ MS patients, three HLA-A2ϩ OND controls, three HLA-A2Ϫ ϩ Ϫ assessed by flow cytometry via staining with PE-conjugated OND controls, two HLA-A2 HC, and one HLA-A2 HC were HLA-A*0201/LLFSFAQAV tetramer (FL-2 channel), anti-FITC- prestimulated for 1 wk with rTAL or TALpep, or PHA and the conjugated CD8 (FL-1 channel), and PE-Cy5-conjugated CD4 frequency of IFN-␥-producing T cells were determined by the 5 mAb (FL-3 channel). As shown in Fig. 4C, TAL PBMC/APC- and ELISPOT assay seeding 10 cells/well (Fig. 3A). There was a TALpep PBMC/APC-stimulated TCL from patient MS2 were ␥ ϩ substantial increase of IFN- spots in all HLA-A2 MS patients double positive for HLA-A*0201/TALpep tetramer and CD8 at after both TAL (40.4 Ϯ 2.7, p ϭ 2.3 ϫ 10Ϫ8) and TALpep Ϫ6 rates of 97.7 and 98.6%, respectively (Fig. 4C). The same HLA-
Ϯ ϭ ϫ by guest on October 7, 2021 stimulation (48.5 6.2, p 2.4 10 ) as compared with A*0201/TALpep tetramer-positive populations were CD4 nega- HLA-A2Ϫ MS patients or HLA-A2ϩ OND controls (Fig. 3B). Ϫ tive. In contrast, HLA-A*0201/TALpep tetramer PE and CD8 In HLA-A2 MS patients, stimulation by TAL resulted in en- FITC double-positive T cells comprised only 40.5 and 60.6% of hanced spot production (7.0 Ϯ 0.8) in comparison with stimu- TAL EBV/APC- and TALpep EBV/APC-stimulated TCL from lation by the TALpep (0.58 Ϯ 0.28; p ϭ 0.0001; Fig. 3B). This patient MS2. Such TCL series were generated from patient MS2 indicated that TAL contains non-HLA-A2-restricted epitopes on two occasions 1 year apart and gave similar results. The outside residues 168–176. nontetramer-staining population in MS2 TCL stimulated with TALpep EBV/APC was shown to be all (99.1%) CD8ϩ/CD4Ϫ. The HLA-A*0201/TALpep tetramer-negative population com- prised two distinct populations, 40.1% CD4Ϫ/CD8ϩ and 14.5% CD4ϩ/CD8Ϫ (Fig. 4C). IFN-␥ production was tested on TCL cultured for 7 wk in cul- ture (Fig. 4, A and B), while HLA-A*0201/TALpep tetramer stain- ing was performed at a later stage, after 58 wk in culture (Fig. 4C), indicating that repetitive stimulation with TAL led to a predomi- nant recognition of TALpep. TCL from HLA-A2ϩ MS patient MS7 were only stimulated with EBV-transformed B cell APC. TAL EBV/APC- and TALpep EBV/APC-stimulated MS7 TCL contained 32.4 and 43.5% TAL- pep tetramer and CD8 double-positive T cells, respectively (Fig. 4C). Similar to the findings in patient MS2, the nontetramer-stain- ing population in MS7 TALpep EBV/APC TCL was 99.4% FIGURE 2. Flow cytometry of cell surface staining with PE-conjugated CD8ϩ/CD4Ϫ, while the MS7 TAL EBV/APC TCL contained HLA-A*0201/TALpep (LLFSFAQAV, TALpep) and PE-conjugated 23.8% tetramer-negative CD4ϩ/CD8Ϫ cells (Fig. 4C). These find- HLA-A*0201/HIV pol 464 (ILKEPVHGV, HIV pol) tetramers. PBMC ϩ Ϫ ings indicated that stimulation with TAL-pulsed EBV/APC ex- from seven HLA-A2 MS patients, six HLA-A2 MS patients, three ϩ ϩ ϩ Ϫ panded tetramer-negative CD8 and CD4 T cells in both HLA- HLA-A2 OND controls, three HLA-A2 OND controls, and two HLA- ϩ A2ϩ HC were prestimulated for 1 wk with either rTAL or TALpep (LL- A2 MS patients. None of the TAL- or TALpep-stimulated TCL FSFAQAV) and stained with the tetramers for 30 min at 4°C. Data show of patients MS2 and MS7 showed significant staining with HLA- mean Ϯ SD of the percentage of tetramer-positive cells for each donor A2/HIV pol 464 and HLA-A2/HIV gag 77 control tetramers .(p Ͻ 0.001. (Ͻ0.5%; data not shown ,ءء ;p Ͻ 0.01 ,ء .group The Journal of Immunology 8371
FIGURE 3. Assessment of the precursor frequency of TAL-specific IFN-␥-producing cells by ELISPOT as- say. PBMC of seven HLA-A2ϩ MS patients, seven HLA-A2Ϫ MS patients, four HLA-A2ϩ OND controls, four HLA-A2Ϫ OND controls, four HLA-A2ϩ HC, and one HLA-A2Ϫ HC were prestimulated for 1 wk with 5 g/ml TAL or TALpep (LLFSFAQAV, TALpep), or PHA, as positive control. Subsequently, PBMC were in- cubated on anti-human IFN-␥-coated ELISPOT polyvi- nylidene difluoride plates for 20 h at 37°C. A, Repre- sentative ELISPOT plate detecting IFN-producing cells in HLA-A2ϩ MS patients (MS2 and MS3), HLA-A2Ϫ ϩ
MS patients (MS10 and MS11), and HLA-A2 and Downloaded from HLA-A2Ϫ OND and HC. B, Frequency of TAL-specific IFN-␥-producing cells in 2 ϫ 105 PBMC from seven HLA-A2ϩ MS patients, seven HLA-A2Ϫ MS patients, four HLA-A2ϩ OND controls, four HLA-A2Ϫ OND controls, and four HLA-A2ϩ HC following stimulation with TAL, TALpep, and PHA, using three replicate wells for each donor. Spots were counted under a dis- http://www.jimmunol.org/ secting microscope. Data represent mean Ϯ SD of spots .p Ͻ 0.001 ,ءء ;p Ͻ 0.01 ,ء .per patient group by guest on October 7, 2021
TAL- and TALpep-specific TCL express the TCR V14 gene sponding to the CDR3 of TCR V14 (Fig. 5B). Both MS2 TAL To determine clonal distribution of TAL- and TALpep-specific PBMC/APC TCL and MS2 TALpep PBMC/APC TCL had the  TCL, the repertoire of TCR V gene usage was investigated by identical TCR V 14 sequence through all regions, including  RT-PCR using 24 TCR V primer sets (29). Twenty of 24 and 19 CDR3. Although the TCR V 14 sequences of EBV/APC-fed  of 24 TCR V genes were detected in unstimulated PBMC from TCL were not identical, they all shared the J 2.1 motif EQFF  MS patients MS2 and MS7, respectively (Fig. 5A). TAL PBMC/ at aa positions 65–68, which corresponded to a similar J 2.7 APC- and TALpep PBMC/APC-stimulated MS2 TCL, both com- motif EQYF in MS2 TCL stimulated by TAL- or TALpep- prised of HLA-A*0201/TALpep tetramer/CD8 double-positive T pulsed PBMC/APC. cells, expressed the TCR V14 gene exclusively (Fig. 5A). More-  over, all TCL stimulated with TAL- or TALpep-pulsed EBV/APC Expression of TCR V 14 protein on the surface of TAL-specific expressed the TCR V14 gene. Three of four EBV/APC-stimu- TCL lated TCL also contained one additional TCR V RT-PCR prod- Anti-human TCR V14 mAb (Serotec clone CAS1.1.3) was used uct. TCR V19 was also detected in MS7 TCL stimulated with to test for the expression TCR V14 on the surface of TAL-spe- TAL- or TALpep-pulsed EBV/APC, which may represent T cells cific HLA-A2ϩ TCL (Fig. 6A). IgG1 mAb ME.05 (Serotec clone recognizing EBV Ags (54). ME.05) was used as isotype control. A total of 97.5 and 99.4% of The TCR V14 RT-PCR product from each TCL and PBMC TAL PBMC/APC- and the MS2 TALpep PBMC/APC-stimulated of MS2 and MS7 was sequenced. Variations in the translated MS2 TCL expressed the TCR V14 Ag, respectively. A total of amino acid sequences were confined to residues 54–70, corre- 51.9 and 57.3% of TAL EBV/APC- and TALpep EBV/APC-fed
FIGURE 4. A, Assessment of IFN-␥ production by PBMC and TAL- and TALpep-stimulated TCL cultured for 7 wk from HLA-A2ϩ MS patients MS2 and MS7 by ELISPOT assay. TCL were generated by stimulation with APC derived from freshly isolated PBMC or autologous EBV-transformed B cells ,p Ͻ 0.001. C ,ءء ;p Ͻ 0.01 ,ء .pulsed with TAL or TALpep. B, Diagram indicates mean Ϯ SD of IFN-␥-producing spots formed by 2 ϫ 105 cells/well Flow cytometry of PBMC and TCL from HLA-A2ϩ MS patients MS2 and MS7 and HC PBMC cultured for 58 wk. Cells were stained with PE-conjugated HLA-A*0201/TALpep tetramer, FITC-conjugated CD8, and PE-Cy5-conjugated CD4 Abs. Dead cells and debris were excluded from the analysis by electronic gating on FSC and SSC measurements. Percentages of positive-staining cells are shown in each dot plot. The results are representative of two independent experiments. 8372 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021 The Journal of Immunology 8373 Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021 8374 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS
MS2 TCL expressed TCR V14, respectively. MS7 TAL EBV/ outnumber CD4ϩ T cells (2); actively demyelinating lesions in MS APC TCL and MS7 TALpep EBV/APC TCL also exhibited sig- brain are enriched for clonally expanded CD8ϩ T cells, as com- nificant TCR V14ϩ populations, 29.2 and 38.5%, respectively. pared with CD4ϩ T cells (40, 41); and oligodendrocyte cell death Small, but significant TCR V14 staining was seen in PBL of is mostly attributed to the cytotoxic effect of CD8ϩ T cells (37). HLA-A2ϩ MS patient MS2, as compared with HLA-A2ϩ HC Adoptively transferred MBP-specific (38) or MOG-specific CD8ϩ PBL. No positive staining was noted with the ME.05 IgG1 isotype T cells induce severe CNS demyelination in animal models (39). control Ab (Fig. 6A). Double staining of TAL EBV/APC- and Although MBP-specific CD8 T cells were detected in human PBL, TALpep EBV/APC-fed MS2 and MS7 TCL revealed exclusive there was no difference in cytotoxicity of MBP 110–118 peptide- binding of HLA-A*0201/TALpep tetramer to cells expressing stimulated CD8 T cells between healthy donors and MS patients TCR V14 (Fig. 6B). (56, 57). Recently, increased precursor frequency of MBP 111– 119-reactive CD8 T cells was found in a minority of MS patients  TCR V 14 mAb inhibits killing of oligodendroglioma and (58). In contrast, the present data show that the precursor fre- lymphoblastoma target cells by TAL-specific HLA-A2-restricted quency, cytotoxic activity, and IFN-␥ production of TALpep-spe- CTL cific CD8 T cells are increased in each of seven HLA-A2ϩ MS To assess the involvement of TCR V14 in killing by TAL-spe- patients as compared with seven HLA-A2Ϫ MS patients, four cific CTL, effector cells were preincubated with or without mouse HLA-A2ϩ OND patients, four HLA-A2Ϫ OND patients, and four anti-human TCR V14 mAb or mouse isotype control IgG1 mAb, HLA-A2ϩ and two HLA-A2Ϫ healthy donors. and subsequently added to MO3.13 oligodendroglioma or Hmy Patients with MS exhibit TAL-specific T cell proliferation and lymphoblastoma target cells at 10:1 E:T ratio. The TCR V14 have Abs to TAL in their blood and cerebrospinal fluid, whereas Downloaded from mAb dramatically reduced the cytotoxicity by all TAL- and TAL- TAL Abs are absent in normal individuals and patients with other pep-specific TCL against both types of target cells (Fig. 7). MS2 autoimmune and neurological diseases (21, 29). Meanwhile, under TAL PBMC/APC TCL and MS2 TAL pep PBMC/APC TCL ex- similar conditions, Abs to MBP were not found in serum and ce- hibited the highest cytotoxicity against TALpep-pulsed Hmy/A2 rebrospinal fluid of MS patients (29). These results suggested that targets (91 and 97%, respectively) and TALpep-pulsed TAL may be a more significant target than MBP of myelin-reac- MO3.13/A2 targets (90 and 93%, respectively; Fig. 7). TCR V14 tive T cells and of humoral autoreactivity in MS. Demyelination in mAb reduced cytotoxicity against TALpep-pulsed Hmy/A2 and patients with MS is thought to be mediated by a selective de- http://www.jimmunol.org/ MO3.13/A2 targets by an average of 59 and 72%, respectively struction of oligodendrocytes by cytotoxic effect of CD8ϩ T ( p Ͻ 0.0001). Additionally, TCR V14 mAb inhibited the cyto- cells (2, 37, 41, 59). Cytosolic proteins, like highly soluble toxicity against the nonpeptide-pulsed MO3.13/A2 target cells. A TAL, constitute a major source of peptides presented to CD8ϩ total of 66% killing by MS2 TAL PBMC/APC TCL and 75% T cells by HLA class I molecules (51). Among 14 peptides with killing by MS2 TALpep PBMC/APC TCL of MO3.13/A2 targets predicted HLA-A2-binding stabilities of greater than 100 min  was reduced by 70% after addition of anti-human TCR V 14 mAb (t1/2) at 37°C, TALpep has the highest binding affinity for ( p Ͻ 0.0001). Cytotoxic activities of EBV/APC-fed TCL were HLA-A2 and it is specifically recognized by CD8ϩ CTL in  ϩ similarly reduced by TCR V 14 mAb. IgG1 isotype control mAb HLA-A2 MS patients. by guest on October 7, 2021 did not influence the cytotoxicity of TCL against either types of HLA-A2-transfected, but not control MO3.13 oligodendroglial target cells (Fig. 7). cells, expressing high levels of endogenous TAL with respect to Hmy cells, were efficiently killed by TAL-specific CD8 T cells Discussion without pulsing the target cells with TALpep. This indicated that Previous studies from this laboratory, pursuing the goal to iso- endogenously processed TAL was recognized by HLA-A2-re- late autoantigens containing epitopes cross-reactive with viral stricted CTL. Pretreatment of the targets with TALpep further en- proteins, demonstrated that an autoantigen, partially encoded by hanced killing of MO3.13/A2, but not control MO3.13 cells. TAL- a retrotransposon and selectively expressed in oligodendrocytes specific CTL activity was observed in each of seven HLA-A2ϩ at high levels (21), corresponds to TAL (22), a rate-limiting MS patients, as compared with the mean of three HLA-A2ϩ OND enzyme of the pentose phosphate pathway (PPP). The purified patients, three HLA-A2ϩ healthy donors, or six HLA-A2Ϫ MS full-length rTAL was found to be functional in the TAL enzyme patients. The results clearly showed the presence of TALpep-spe- assay by showing a sp. act. of Ͼ16 U/mg protein (22–36). As cific HLA-A2-restricted CTL in patients with MS. Furthermore, references, normal human lymphocytes contain a TAL activity staining with HLA-A*0201/TALpep (LLFSFAQAV) tetramer re- of 0.015 U/mg protein (22). By contrast, TAL activity in human vealed a higher frequency of TAL-specific CTL in TAL- or TAL- oligodendroglioma cell lines M03.13 (49) and HOG (55) was pep-stimulated PBL of seven HLA-A2ϩ MS patients in com- Ͼ0.2 U/mg protein, indicating that high expression of TAL parison with HLA-A2ϩ OND and HC. The frequency of may be retained in oligodendrocytes undergoing malignant IFN-␥-producing T cells was also increased in all seven HLA-A2ϩ transformation. MS patients following stimulation with TAL or TALpep. In HLA- In this study, we document the existence and increased preva- A2Ϫ MS patients, the frequency of IFN-␥-producing T cells was lence of TAL-specific HLA-A2-restricted CD8ϩ CTL in patients moderately increased following stimulation by TAL (7.0 Ϯ 0.8/ with MS (Table I). In MS brain lesions, infiltrating CD8ϩ CTL 105 cells) in comparison with stimulation by the TALpep (0.58 Ϯ
FIGURE 5. A, RT-PCR analysis of the TCR V gene repertoire in unstimulated PBMC and TAL- or TALpep-stimulated TCL of HLA-A2ϩ MS patients. Lanes contain the RT-PCR products of 24 TCR V sense primers combined with C antisense primer and C␣ product serving as internal control. First lane of each panel contains 123-bp m.w. ladder. Top lanes contain RT-PCR products with TCR V 1–12 sense primers, while bottom lanes of each gel contain RT-PCR products with TCR V 13–24 sense primers. Arrows indicate location of TCR V14. B, Alignment of deduced amino acid sequences of TCR V14 CDR3 domains from TCL of HLA-A2ϩ MS patients stimulated with TAL or TALpep. As controls, sequence of unstimulated PBMC from the HLA-A2ϩ MS patients was included. The aa 1–55 correspond to the canonical V14 sequence. CDR3, D region, J region, and C region sequences and designations are indicated. CDR3 sequences are shown from the conserved cysteine of V14 to the hypervariable N(D)N-J junction. The Journal of Immunology 8375
0.28/105 cells; p ϭ 0.0001), suggesting that TAL contains non- HLA-A2-restricted epitopes outside residues 168–176. Repet- itive stimulation of MS2 TCL with TAL or TALpep PBMC- derived APC elicited a single clonal population of TAL-specific CTL double positive for HLA-A*0201/TALpep tetramer PE and CD8 FITC. TAL-specific TCL exhibited markedly en- hanced cytotoxic activity (Fig. 6) and robust IFN-␥ production (Fig. 4). IFN-␥ stimulates HLA class I expression on human oligodendrocytes (53); thus, increased IFN-␥ production by TAL-specific T cells is likely to increase susceptibility of oli- godendrocytes to killing by CTL. Stimulation of CD8 effector cells with TAL elicited a clonal population of HLA-A*0201/TALpep tetramer-positive cells. All TAL and TALpep PBMC/APC-stimulated CTL lines used a unique TCR V14 gene, thus suggesting that TALpep appears to be the dominant HLA-A2-restricted T cell epitope in human TAL. Along this line, all TAL or TALpep EBV/APC-stimulated TCL from patients MS2 and MS7 contained a dominant HLA-A*0201/ TALpep tetramer/CD8ϩ population that expressed TCR V14. In accordance with the presence of HLA-A*0201/TALpep tetramer- Downloaded from negative cells, EBV/APC-fed TCL were oligoclonal, using TCR V9, V7, V4, and V19. Of note, all of these V were also expressed in CD8ϩ TCL obtained from EBV-seropositive HLA- A2ϩ healthy donors stimulated with irradiated autologous EBV- transformed B cells (54). Thus, HLA-A*0201/TALpep tetramerϪ/ CD8ϩ cells in EBV/APC-fed TCL may respond to EBV Ags. TCL http://www.jimmunol.org/ fed by EBV/APC pulsed with TAL, but not TALpep, also com- prised CD4ϩ T cells, 14.5% in MS2 and 23.8% in MS7, respec- tively, suggesting that TAL also harbored HLA class II-restricted epitopes. The TCR V14 RT-PCR products of each TCL as well as PBMC of M2 and M7 were sequenced. Variations in the translated amino acid sequences were confined to residues 54– 70, harboring the CDR3 region of TCR V14 (Fig. 5B). Both by guest on October 7, 2021 MS2 TAL PBMC/APC TCL and MS2 TALpep PBMC/APC TCL had the identical TCR V14 sequence through all regions, including CDR3. Although the TCR V14 sequences of EBV/ APC-fed TCL were not identical, they all shared the EQFF motif at aa positions 65–68. The EQFF motif was preceded by neutral and polar amino acids GTSGY and LSGGY in MS2 TAL EBV/APC TCL and MS2 TALpep EBV/APC TCL, re- spectively. By contrast, the EQFF motif was preceded by highly charged RPRE and GRRD residues in MS7 TAL EBV/APC TCL and MS7 TALpep EBV/APC TCL, respectively. The con- served EQFF motif corresponded to the functionally similar EQYF motif in MS2 TCL stimulated by TAL- or TALpep- pulsed PBMC/APC. The EQFF and EQYF motifs are derived from the J2.1 and J2.7 regions, respectively. The findings indicate that the EQF/YF motif may contibute to recognition of TALpep by TCR V14. Use of the TCR V14 gene by TAL-specific TCL was con- firmed by demonstrating expression of TCR V14 Ag on the sur- face of TAL-specific TCL. Flow cytometry analysis revealed
ing cells is shown above each histogram. The results are representative of FIGURE 6. A, Detection of TCR V14 protein on the surface of TAL- two independent experiments. B, Flow cytometry of TAL EBV/APC- and ϩ specific TCL by flow cytometry. PBMC of HLA-A2 HC (HC4) and MS TALpep EBV/APC-fed TCL from HLA-A2ϩ MS patients MS2 and MS7 ϩ patient (MS2) and TCL of HLA-A2 MS patients MS2 and MS7 were stained by anti-human TCR V14 mAb and PE-conjugated HLA-A2/TAL- stained with anti-TCR V14 mAb (CAS1.1.3) or IgG1 isotype control pep/tetramer. Staining with anti-TCR V14 mAb was detected with FITC- mAb (ME.05), followed by staining with FITC-conjugated secondary goat conjugated secondary goat anti-mouse IgG. TCR V14 expression was anti-mouse IgG. Dead cells and debris were excluded from the analysis by assessed by FL-1 fluorescence (x-axis), and HLA-A2/TALpep/tetramer ϩ electronic gating of FSC and SSC measurements. TCR V14 cell pop- binding was monitored by FL-2 fluorescence (y-axis). Percentages of pos- ulations (filled histograms) are overlayed on histogram of cells stained with itive-staining cells are shown in each dot plot. The results are representa- ϩ isotype control IgG1 (open histograms). Percentage of TCR V14 -stain- tive of two independent experiments. 8376 TAL-SPECIFIC CD8ϩ T CELL CYTOTOXICITY IN MS Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021
FIGURE 7. Inhibition by TCR V14 mAb of TAL-specific HLA-A2-restricted CTL killing of MO3.13 (left column) and Hmy target cells (right column). TAL-specific TCL were preincubated with mouse anti-human TCR V14 mAb or mouse isotype control IgG1 mAb and added to targets at 10:1 .p Ͻ 0.0001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .E:T ratio. Percentage of cytotoxicity values represents mean Ϯ SD of six replicate experiments The Journal of Immunology 8377 exclusive binding of HLA-A*0201/TALpep tetramer to cells ex- 22. Banki, K., D. Halladay, and A. Perl. 1994. Cloning and expression of the human pressing TCR V14 in all TAL-specific TCL, suggesting that TCR gene for transaldolase: a novel highly repetitive element constitutes an integral part of the coding sequence. J. Biol. Chem. 269: 2847–2851. V14 may be uniquely responsible for recognition of TAL in 23. Mayes, P. A. 1993. The pentose phosphate pathway & other pathways of hexose HLA-A2ϩ MS patients. Pretreatment of CTL lines with TCR metabolism. In Harper’s Biochemistry. R. K. Murray, D. K. Granner, V14 mAb profoundly inhibited cytotoxicity against HLA-A2- P. A. Mayes, and V. W. Rodwell, eds. Appleton & Lange, Norwalk, pp. 201–211. 24. Jacobson, S. 1963. Sequence of myelination in the brain of the albino rat. transfected Hmy A2.1 or MO3.13 target cells, establishing the J. Comp. Neurol. 121: 5–29. functional relevance of the TCR V14/TALpep/HLA-A2 trimo- 25. McDougal, D. B., Jr., D. W. Schulz, J. V. Passonneau, J. R. Clark, M. A. Reynolds, and O. H. Lowry. 1961. Quantitative studies of white matter. I. lecular interaction. Therefore, selective elimination of TAL-spe- Enzymes involved in glucose 6-phosphate metabolism. J. Gen. Physiol. 44: cific CTL may prevent oligodendrocyte destruction and serve as a 487–498. novel target for therapeutic intervention in patients with MS. 26. Baquer, N. Z., J. S. Hothersall, P. McLean, and A. L. Greenbaum. 1977. Aspects of carbohydrate metabolism in developing brain. Dev. Med. Child Neurol. 19: 81–104. 27. Balazs, R. 1970. Carbohydrate metabolism. In Handbook of Neurochemistry. Acknowledgments A. Lajtha, ed. Plenum Press, New York, pp. 1–36. We thank Drs. Neil Cashman (Montreal Neurological Institute) and 28. Hotta, S. S. 1962. Glucose metabolism in brain tissue: the hexose-monophosphate Glyn Dawson (University of Chicago) for providing human oligodendro- shunt and its role in glutathione reduction. J. Neurochem. 9: 43–51. glioma cell lines M03.13 (49) and HOG (55), respectively, and 29. Colombo, E., K. Banki, A. H. Tatum, J. Daucher, P. Ferrante, R. S. Murray, P. E. Phillips, and A. Perl. 1997. Comparative analysis of antibody and cell- Paul Phillips for continued encouragement and support. mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis. J. Clin. Invest. 99: 1238–1250. 30. Banki, K., E. Hutter, E. Colombo, N. J. Gonchoroff, and A. Perl. 1996. 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