Restriction of Epstein-Barr Virus-Specific Cytotoxic T Cells by HLA-A,-B, and-c Molecules

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1 Restriction of Epstein-Barr Virus-Specific Cytotoxic T Cells by HLA-A,-B, and-c Molecules Benjamin P. Chen, Valery Lam, Eric E. Kraus, Robert DeMars, and Paul M. Sondel ABSTRACT: HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytoiytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells. ABBREVIATIONS CTL cytotoxic T cells HLA EBV Epstein-Barr virus IL-2 EBV-LCL EBV-transformed PBL B-lymphoblastoid cell line(s) human leukocyte antigens interleukin 2 peripheral blood leukocytes I N T R O D U C T I O N Cytotoxic T cells (CTL) recognize processed viral antigens in the context of self class I human leukocyte antigen (HLA) determinants [1-3]. Recent crystallographic studies of the class I HLA molecule A2 have revealed a putative antigen binding groove; the binding site is bordered by amino acid residues and of the ~1 and ~2 helices, respectively, of the molecule [4,5]. Thus, virus-specific, HLA-restricted CTL corecognize processed viral antigens bound in the groove and sites on the o~1 and ~2 helices of the HLA molecules. Since the a helices of the class I HLA molecules are polymorphic, various viral peptides could bind to class I molecules, and CTL specific for different peptide and HLA histotopes could be generated. From the Departments of Human Oncology (B.P.C.; E.E.K.; R.D.; P.M.S.) and Pediatrics (P.M.S.) and the Laboratory of Genetics (V.L.; R.D.; P.M.S.), University of Wisconsin, Madison, Wisconsin. Address reprint requests to Paul M. Sondel, M.D., Ph.D., K4/448 UWCCC, 600 Highland Avenue, University of Wisconsin, Madison, WI Received January 4, 1989: revised May 5, Human Immunology 26, (1989) 137 American Society for Histocompatibility and Immunogenetics, /89/$3.5(

2 138 B.P. Chen et al The observation that CTL specific for autologous Epstein,Barr virus transfor, med-b-lymphoblastoid cell lines (EBV-LCL) can be readily generated in vitro from EBV-seropositive individuals of different HLA phenotypes [6,7] suggests that EBV could induce expression of multiple peptidic antigens, each of which may be presented to CTL by HLA molecules. However, it is unl~ely that all peptide/hla combinations are immunogenic; consequently, a preference for specific HLA alleles as restriction elements may occur among individuals. In this study, we have used a panel of class I HLA-loss mutants, derived from a single B-lymphoblastoid clone, LCL 721, to study whether HLA-A and -B molecules can serve equally as restriction determinants for EBV-specific autologous CTL Furthermore, we investigated the possibility that HLA-C molecules car, also bind EBV-associated peptide and stimulate CTL. Since the HLA-loss mutants were derived from one EBV-transformed B-cell clone, they should be identical with regard to major histocompatibility complex (MHC)-encoded molecules except for the HLA molecule(s) lost. Thus CTL populations could be assigned a particular HLA restriction determinant based on the pattern of cytotoxicity against the panel of HLA-loss variants. We found with this type of analysis that EBV-specific CTL generated from one donor could be restricted by either HLA-A, -B, or -C. Furthermore, precursor frequency analysis showed that HLA-B5-specific CTL predominated over those specific for HLA-A2 and -C. MATERIALS A N D METHODS Leukocytes. Peripheral blood leukocytes (PBL) were isolated from heparinized blood from individual AB, the donor of EBV-LCL 721, by Ficoll-Hypaque gradient centrifugation. The PBL were cryopreserved by controlled rate freezing in medium containing 10% dimethylsulfoxide and 10% human serum, and stored at -135 C until further use in the cellular assay. EBV-LCL variants, The primary EBV-LCL line 721 was generated by transformation of B lymphocytes from donor AB with Epstein-Barr virus containing supernatant [8]. The HLA-loss variants of 721 were immunosetected with complement and antisera or monoclonal antibodies following gamma-ray irradiation (300 cgy). Two haplotype-loss variants were studied:.45 possesses only the maternal haplotype [8], while variant.127 bears only the paternal haplotype [9,10]. The class I-loss variant.53 lacks serologically detectable HLA-B5 molecules [11],. 144 lacks HLA-A2 [9], and. 184 has lost both HLA-A2 and B5 [12]. Variant. 180 expresses reduced amounts of HLA-A, -B, and -C but does not express HLA-DP, -DQ, or -DR [13,14]. Phenotypes of these cell lines are shown in Table I. Establishment of primed cytotoxic T-celllines. Ten million PBL from individual AB, from whom LCL 721 was derived, were primed with irradiated (8000 cgy) variant.45 cells or variant. 180 cells in 10 ml supplemented medium [RPMI supplemented with 10/zg/ml streptomycin, 100 U/ml penicillin, 2mM L-glutamine, and 25 mm HEPES (all from Flow Lab., McLean, VA) and 10% pooled, heat-inactivated, nontransfused, human male serum (Pelfreeze, Brown Deer, WI)]. On day 14, the primed cells were harvested, adjusted to 10 x 106 cells/ml, and restimulated with 1 x 106 irradiated.45 or. 180 cells in the presence of 100 U/ml of interleukin 2 (IL-2) (a gift from the Cetus corporation, Emeryville, CA). Seven days after restimulation, primed cells were harvested and cryopreserved in

3 Restriction Determinants for CTL 139 T A B L E 1 HLA phenotypes of EBV-LCL variants derived from EBV-LCL 721 H L A p h e n o t y p e E B V - L C L variants H a p l o t y p e A B C ~ D R D P D Q R e f M a t e r n a l 2 5 C m M a t e r n a l C m M a t e r n a l Crn M a t e r n a l C m b M a t e r n a l 2 5 C m , P a t e r n a l 1 8 C p , 1 0 Two expressed HLA-C alleles are present in LCL 721, but repeated complement-dependem cytotoxicity tests performed by four typing laboratories with panels of standard typing alloantisera have yielded inconsistent results. Cm denotes the HLA-C allele in the maternm haplotype of LCL 721, and Cp denotes the allele in the paternal haplotype. b The serologically detectable amounts of A2 and B5 of this mutant are greatly reduced due to a mutation affecting the posttranscriptional expression of these molecules. aliquots of 2 x 106 cells/ml at -135 C for later cellular assays. These two bulk cultures were designated as AB.45 and AB. 180, respectively. Isolation of cytotoxic T-cell clones. Cytotoxic T-cell clones were isolated from cultures plated at limiting dilution [15]. Briefly, 14-day primed T cells from bulk culture AB.180 were diluted in supplemented medium containing 50 U/ml of IL-2 so that an average of every three wells (each receiving 0.1 ml) would receive one cell. Irradiated variant LCL cells (8000 cgy) were used as feeder/stimulator cells at cells/ml. T-cell clones were expanded in supplemented medium containing 100 U/ml IL-2 and aliquots were cryopreserved until later use. Cell-mediated lysis (CML). Bulk primed T cells or T-cell clones were assayed for specific killing in a standard 4-hr 5iCr release assay. Effector cells were diluted in supplemented medium to yield the effector cell-to-target cell ratios described in each experiment. Target cells were labeled with 250/zCi of ~Cr (New England Nuclear, Boston, MA) in 0.3 ml supplemented medium for 2 hr. The target cells were washed two times in medium, diluted to 5 x 104 cells/ml, and 100/zl of target cells was added to microwells containing 100/zl of effector cells. The plates were centrifuged at 500 x g for 10 min and incubated in a humidified incubator containing 5% CO2. After 4 hr, supernatants were collected with Skatron harvesting frames (Sterling, VA) and 51Cr release counted in a gamma counter. Percent cytotoxicity is calculated using the formula: experimental release - spontaneous release %cytotoxicity = x 100% maximum release - spontaneous release Spontaneous release and maximum release values were determined by incubating target cells in supplemented medium or 0.1% cetrimide detergent (Sigma Chemical, St. Louis, MO) respectively. Results are means -+ standard deviation of triplicate determinants. Split culture analysis of limiting dilution cultures. Aliquots of the AB.45 CTL line were thawed and cultured in 100 U/ml of IL-2 for 48 hr without feeder cells.

4 140 B.P. Chen et ai. Viable cells were harvested and serially diluted into microwells; 24 replicate wells of each cell concentration (i.e., 300, 250, 200, 150, 125, 100, 75, 50, and 25 cells/well) were plated. Each microwell also received irradiated.45 variant cells and 50 U/ml of IL-2. Ten days after the initiation of the cultures, each microwell culture was split into five replicates and each replicate set was assayed for cytotoxicity against separate 51Cr labeled populations of different HLA-loss variant EBV-LCL, i.e.,.45,.53,.144,.184, and.180. For each population of target cells, the mean spontaneous release plus 3 standard deviations of each target cell line was used to separate positive from negative wells. Estimates of the precursor frequencies were determined by minimum chi square method from the Poisson's distribution relationship between the logarithm of the percentage of noncytolytic (negative) cultures and the number of cells initially plated per well. Details of minimum chi square analysis of limiting dilution results and the statistical significance of differences have been described by Taswell [ 16J. Monoclonal antibodies fmoabs). The hybridomas for the monomorphic anti-hla class I MoAb PA2.6 and the anti-hla-dr MoAb L243 were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). Ammonium sulfate-precipitated ascites fluids were extensively dialyzed against phosphate buffered saline (PBS) and diluted for use in the inhibition studies. Phenotyping of T-cell clones. Indirect immunofluorescence was done with anti- CD3, -CD4, and -CD8 MoAbs (Becton Dickinson, Mountain View, CA) and developed with a fluorescein-tabeled anti-mouse immunoglobulin antibody (Coulter, Hialeah, FL). Five thousand cells were analyzed for each marker on an Ortho cytofluorograph system model 50 flow cytometer (Ortho, Raritan, NJ). RESULTS Preference for HLA-B5 by CTL-Specific for EBV-LCL To determine the restriction determinants for CTL specific for autologous LCL 721, the T-cell lines AB.45 and AB.180 were tested against a panel of class 1 HLA-loss target cells derived from LCL 721 described in Table 1. Both cultures killed the maternal haplotype-bearing variant.45, but not the paternal haplotype-bearing mutant. 127 or autotogous PBL target cells (Table 2). These results established that the CTL lines primed against a complete (AB.45) or partial (AB.180) maternal haplotype recognize EBV antigens in the context of HLA determinants of the maternal haplotype;.127 and AB-PBL target cells were lysable, as they were killed by an alloreactive bulk culture PS.ABLcL. Among the five variants expressing the maternal-haplotype determinants, the HLA-B5- bearing variants.45,.144, and.180 were lysed more extensively than the HLA-A2-bearing variant.53. The comparable susceptibility of these HLA-loss variants to lysis by the alloreacnve culture PS.ABLcL shows that the preferential lysis of HLA-B5-expressmg target cells was not due to differential lysability of these LCL target ceils. Split culture analysis of the AB.45 culture plated at limiting dilution documented that the majority of LCL-specific CTL precursors are HLA-B 5-restricted. As shown in Figure 1 and Table 3, the relative frequencies of CTL killing the B5-bearing target cells are 1/25 for.45, 1/39 for 144, and 1/28 for.180, compared with 1/385 for the A2-bearing. 53 targets. These results indicate that the greater destruction of HLA-B5-bearing target cells than of A2-bearing target

5 Restriction Determinants for CTL 141 TABLE 2 Cytotoxicity on HLA-loss variants by T-cell lines % Cytotoxicity of target cells a Effectors b E :T ~ AB-PBL AB.45 AB (3) 18(1) 65(2) 42(3) 17(2) -2(2) 1(1) 15 70(3) 23(2) 68(3) 60(3) 25(2) -2(1) 2(3) 30 73(2) 28(3) 69(2) 72(3) 30(2) -3(1) 3(1) 8 20(1) 7(1) 18(2) 66(4) 7(1) -2(1) 1(2) 15 29(1) 11(1) 30(2) 86(4) 14(2) -3(1) 7(6) 30 37(1) 16(2) 38(2) 81(3) 15(1) 2(1) 6(2) PS.ABt~L 30 50(2) 53(5) 37(4) 40(1) 52(2) 44(1) 46(3) Results presented are mean % cytotoxicity (- standard deviation) of triplicate cultures of each target cell measured in a 4-hr 5tCr release assay. b AB.45 and AB.180 were bulk cultures of PBL from donor AB primed to autologous EBV-LCL mutants.45 and 180. PS.ABLct was an alloreactive culture established by priming PBL from donor PS to EBV-LCL from donor AB, Effector-to-target cell ratio. cells (Table 2) results, at least in part, from a greater number of B5-restricted precursors in the PBL prior to intentional priming in vitro with B5- and A2-bearing autologous EBV-LCL. B u l k C u l t u r e a n d L i m i t i n g D i l u t i o n A n a l y s i s E v i d e n c e S u p p o r t i n g H L A - C R e c o g n i t i o n A p p r o x i m a t e l y 1 in 120 C T L in A B. 4 5, w h i c h was p r i m e d against a c o m p l e t e m a t e r n a l h a p l o t y p e, can lyse the A 2 - n u l l, B5-null, C m +, class I I - b e a r i n g variant 184 ( Figure 1 and T a b l e 3). S o m e o f the killing o f t h e s e. 184 target cells c o u l d be F I G U R E 1 Minimum X e estimates of frequency (f~c) of CTL restricted by HLA-A2, -B5, or -Cm. For each frequency determination, the lower line represents the upper 95r confidence limit of fmo the middle line represents the estimated fm~, and the upper line represents the lower 95% confidence limit of fmc.,} o l o get.~ "6 o s 2 8 ~u o l o oo3 f=1/39 ", ~ oo, 2's s'o ~ s, &, ~, s o 2s ~o ~ s, o o, 2 s, s o sb ~&,~oa&2~o3oo Cells per Culture

6 142 B.P. Chen et al. TABLE 3 Frequency of CTL restricted by HLA-A2, B5, or Cr~ Target cell Frequency a 9 5 % CI (f)b p (f),.45 1/25 1/32-1/ /39 1 / / /28 1 / / /385 / / / /120 1 / / * AB.45 microtiter well cultures were derived from 24 inocula at each of six different limiting dilut/on cell concentrations and were tested for lysis of target cells. The Poisson's distribution relationship between the number of cells initially pl~ted per well and the proportions of cultures that did not significantly lyse a given kind of target cells were used to estimate the frequency of CTL precursors that were specific for that kind of target cell. 6 95% confidence intervals (CI) for the frequency (f~ are shown. The p value corresponds to the X 2 value of frequency (f). mediated by class II-restricted CTL while other CTL may be restricted by HLA-Cm molecules. Priming of T cells to the DP-, DQ-, DR-nonexpressing variant. 180 also generated a maternal haplotype-restricted bulk CTL population that effected low-level, but significant, lysis of variant.184 (Table 2). This is consistent with HLA-C restriction of some CTL (see discussion). Restriction of CTL Clones by HLA-A, -B, or -C The cytotoxic profiles of 1 t representative clones derived from a bulk AB. 180 culture are shown in Table 4; these clones were selected for extensive study because they expanded to give enough cells for multiple testing. Mutant. 180 stimulator cells were used to avoid the generation of class II-restricted T cells. Clones in group 1 (C1G and A4A) lysed all variants tested except the A2-deficient variants. 144,. 184, and. 127, indicating HLA-A2 restriction of these clones. Clones in group 2 (B4H, C4C, B3F, and BIC) are HLA-B5 restricted, as they lysed the B5-bearing variant. 144 but not the A2-bearing variant. 53 or the B5-deficient variants. 184 and Included in group 3 are clones that lysed all variants tested except the maternal haplotype-loss mutant As in the case of the.180-primed bulk CTL populations described above, the lysis of both the DP-, DQ-, DR-nonexpressin~g variant.180 and the A2-, B5-, Cm ~, class II ~ mutant. 184 by the maternal haplotype-restricted clones in group 3 suggests that they are HLA-C restricted. Monoclonal antibody inhibition studies further confirmed the class I HLA restriction of the 11 clones presented in groups 1-3. As shown in Table 5, all 11 clones killed variant.45, and this killing was completely or partially inhibited by the anti-class I MoAb PA 2.6 but not by the anti-dr MoAb L243; as a control, an HLA-DR-restricted clone AB.45.BIG derived from bulk culture AB.45 (Table 4) was inhibited by MoAb L243. DISCUSSION We have shown that HLA-C molecules, as well as HLA-A2 and -B5, can function as restriction determinants for EBV-LCL-specific CTL. The assignments of HLA determinant restriction to specific clones of CTL were based on their cytolytic

7 Restriction Determinants for CTL 143 TABLE 4 Distinct patterns of HLA restriction of CTL clones % Cytotoxicity of target cells ~ Phenotypes b Clones' E:T a CD3 CD4 CD8 Group 1 C1G 10 48(2) 12(1) - 3 ( 4 ) 72(5) 1(1) 1(2) A4A 10 24(1) 24(1) 4(1) 59(5) 3(2) 0(2) N D N D N D Group 2 Group 3 B 4 H 10 47(2) 4(1) 31(4) 30(2) 2(2) 0(1) C4C 10 21(2) 0(1) 20(2) 74(3) -4(2) 1(2) B3F 10 46(1) 3(1) 37(2) 55(2) 3(1) 1(1) B1C 20 33(2) - 6 ( 4 ) 51(9) 14(1) - 4(2) 3(2) A I F 10 82(4) 73(4) 27(4) 89(4) 37(4) 1(2) N D N D N D A3C 2 40(2) 27(2) 32(3) 45(2) 23(1) 0(1) N D N D N D A3F 3 36(2) 11(1) 19(3) 23(2) 16(1) 0(2) B3D 5 31(2) 36(4) 33(2) 49(2) 14(2) 2(2) N D N D N D E3C 20 35(1) 57(3) 50(2) 45(4) 15(2) 3(3) N D N D N D AB.45 B1G ~ 20 82(4) 71(5) 89(5) - 3 ( 3 ) 90(5) 4(3) PS.ABccc / 20 49(2) 50(3) 54(4) 63(2) 57(2) 33(1) N D N D N D Mean % cytotoxicity (-+standard deviation) of triplicate cultures measured in a 4-hr 51Cr release assay. b The phenotypes of each clone were determined by indirect immunofluorescence of 5000 cells for each marker. ~ Clones were derived from bulk culture AB a Effector-to-target cell ration. ' An EBV-LCL-specific, class II-restricted clone derived from the AB.45 bulk culture. f Lymphocytes from allogeneic donor PS were primed with ABtcL; this polyclonal culture was tested as alloreactive effector to prove lysability of all target cells tested. TABLE 5 Inhibition of cytotoxicity against HLA-loss variants by anti-hla monoclonal antibodies % Cytotoxicity of variant ~ Clones med PA2.6 b L C1G 70(2) ( 7 ) 60(3) 71(2) -6(5) 91(4) -12(2) 1(1) B3F 57(4) 16(5) 40(4) - 5 ( 3 ) 54(3) 41(2) -8(2) 1(1) B4H 67(3) 19(5) 48(5) - 10(2) 57(4) 86(3) - 10(3) 1(1) C4C 15(3) ( 6 ) 10(3) - 4 ( 3 ) 10(4) 81(3) 2(2) 4(1) B1C 20(3) 2(4) 18(5) 0(1) 30(7) 23(2) 5(2) 4(2) A I F 79(5) 19(4) 60(3) 55(6) 24(5) 88(2) 29(4) - 4 ( 3 ) A3F 16(2) 2(1) 13(2) 10(1) 11(1) 19(1) 10(1) 8(8) B3D 22(2) 3(2) 20(4) 26(4) 20(2) 41(2) 14(2) 3(1) E3C 27(1) 7(1) 20(5) 28(3) 25(2) 20(3) 17(2) - 3 ( 1 ) A3C 26(4) - 6 ( 3 ) 19(3) 22(3) 15(2) 27(1) 15(2) 1(3) AB.45.B1G 40(4) 37(5) 6(3) 30(4) 40(3) 2(2) 43(4) 2(3) % Cytotoxicity measured at an effector-to-target cell ratio of 10 : 1 in a 4-hr 51Cr release assay. Results are mean % cytotoxicity (-+standard deviation) of triplicate cultures. b Monoclonal antibodies used were anti-hla-class I PA2.6 and anti-dr L243. Target cells were preincubated in medium containing diluted MoAb for 30 min before the addition of effector cells. MoAb were present for the duration of the assay.

8 144 B.P. Chen et al. profiles against a panel of autologous HLA-loss target cells. These HLA-loss mutants were all derived from a cloned EBV-LCL line 721 and differ from each other in the presence or absence of selected HLA determinants. Therefore, the simultaneous loss of an HLA determinant from a variant and the loss of its susceptibility to killing by a CTL clone allow the assignment of the lost restriction determinant to the CTL clone tested. By using this panel of variant EBV-LCL in a similar analysis, we have previously identified HLA class I-restricted proliferat~ ing T cells in AB. 180 cultures [ 15]. In the present study, we have used a similar method to analyze the specificity of CTL reactive with autologous EBV-LCL. Cytotoxic T-cell clones obtained after priming with LCL deficient in the paternal HLA haptotype (.45 and. 180) did not kill target cells expressing only HLA molecules of the paternal haplotype. Lysis by these clones of LCL variants bearing antigens of the maternal HLA haplotype were inhibited by an anti-hla class I antibody. Killing of the panel of LCL variants established that some of these clones are A2-restricted and some are B5-restricted. Our argument for HLA-C restriction of clones from group 3 (Table 4) is based on two observations: first, EBV-specific CTL clones lyse mutant.184, which was derived from the HLA-A2,-B5,-Cm-expressing mutant.45 by successive deletions of the HLA- A2 and -B5 genes; second, lysis by these clones was blocked by an anti-hla class I, but not by an anti-dr monoclonal antibody. Among the known HLA class l genes, i.e., A, B, C, and E [17], only the A, B, and C gene products are known to be expressed on the cell surface [18]; upon the deletion of A and B genes, only the C determinants remain. Therefore, CTL that can kill A-null, B-null target cells and are blocked by anti-hla class I monoclonal antibody should be C-determinant-restricted. Recent results obtained with AB.180 bulk cultures showed that the killing of 51Cr labeled.180 target cells was inhibited by unlabeled. 180 and. 184 cells but not by the more recently described variant.221 derived from variant.184 [18], which expresses HLA class II, but is deficient in HLA-A, -B, and -C expression; this further confirms the presence of HLA-C-restricted clones in the AB.180 culture (data not shown). These studies have documented a predominance of donor AB-derived autologous EBV-specific CTL that recognize HLA-B5 over autologous CTL that recognize either HLA-A2 or -Cm. Several explanations of this predominance are possible. First, HLA-A2 and -Cm antigens may not bind immunogenic EBV-induced peptides as well as HLA-B5 and consequently may not be as effective in stimulating EBV-LCL-specific CTL. Second, the generation of CTL is also assisted by the interaction of CD8 molecules with class I molecules [19]. If CD8 molecules have stronger interactions with some class I molecules than others, the observed pattern of frequencies for HLA-A2, -B5, and -Cm-restricted CTL could potentially be accounted for. In this regard, in a binding assay designed to study the direct binding of CD8 and HLA molecules [20,21], it was found that HLA-Aw68 molecules, in contrast to 17 other HLA-A and -B molecules, do not bind CD8 molecules [21]. Furthermore, in a family study, Gaston et al. [6] have consistently shown that HLA-Aw68 is a nonpreferred restriction determinant. These two studies support the possibility that qualitative differences in the HLA binding of CD8 molecules could bias the CTL repertoire. At present, it is not known if HLA-C can bind CD8 molecules, and the effect of CD8 on HLA-C-restricted CTL remains speculative. Third, the allele-specific preference might not relate to either the quantitative interaction of either CD8 or the EBV determinant with the class I antigen, but rather to the number of T cells with receptors specifically recognizing the EBV

9 Restriction Determinants for CTL 145 antigens associated with the specific HLA allele A variety of germ line and somatic factors may potentially influence the repertoire of T-cell recognition, as measured by the number of cells with receptors for specific antigens [22]. Quantitatively, HLA-C molecules are also present in lower number than -A and -B molecules. The C molecules, therefore, would be less accessible than A and B molecules on cells expressing all three molecules; this could also lower the magnitude of the response by HLA-C-restricted CTL. Our success in detecting HLA-C-restricted CTL could be attributed to the use of a DP,DQ,DR-nonexpressing variant.180, which also has greatly reduced cell surface expression of A2 and B5 molecules [13,14], as stimulator cells and EBV as antigen. With the loss of class II molecules and decreased A2 and B5 on the HLA-deletion mutant 180, HLA-C molecules would be more accessible to CTL. The use of EBV-LCL as stimulator cells could also contribute to the detection of HLA-C-restricted CTL. EBV-LCL of various HLA phenotypes are very immunogenic for autologous PBL [6,7], suggesting that there are multiple T-cell epitopes or one major antigenic peptide that can be presented by many HLA alleles, including HLA-C. At present we cannot distinguish these two possibilities because only one ligand has been identified using a synthetic peptide [23]; a peptide corresponding to residues of the latent infection membrane protein of EBV [23,24] has been shown to be presented by HLA-A1 molecules [23]. Results of Dill et al. [25] demonstrating the restriction of influenza-specific CTL in HLA-Cw3 transgenic mice support our hypothesis that HLA-C-restricted CTL can be generated by making HLA-C more accessible to CTL. In conclusion, the use of HLA-loss mutants has allowed us to demonstrate the immunodominance of HLA-B5 in an autologous CTL anti-ebv-lcl response in donor AB, suggesting quantitative and qualitative differences between HLA molecules in the presentation of EBV-associated immunogens. We also isolated HLA-C-restricted CTL clones The cytotoxic activity of the HLA-C-restricted clones is inhibited by an anti-hla class I monoclonal antibody. The isolation of the HLA-C-restricted CTL clones will allow comparative studies of the interaction of accessory molecules, such as CD8, with CTL restricted by HLA-C and various HLA-A and -B alleles. ACKNOWLEDGMENTS This work was supported by NIH grants AI P30-CA14520 (the Wisconsin Clinical Cancer Center). (R.D.), CA (P.S.), and REFERENCES 1. Townsend ARM, Gotch FM, Davey J: Cytotoxic T cells recognize fragments of the influenza nucleoprotein. Cell 42:457, Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ: The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44:959, Gotch F, McMichael A, Smith G, Moss B: Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes. J Exp Med 165:408, Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506, 1987.

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