Science in China Series C: Life Sciences © 2007

Science in China Press Springer-Verlag

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2 WENG XiuFang, LIANG ZhiHui, LU XiaoLing, ZHONG MaoHua, LU ShengJun, ZHANG CaiE, DENG Jing, WU XiongWen† & GONG FeiLi Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy. alloreactive T cells, peptide/MHC complex, peptide-specific, long-term mixed lymphocytes culture (LTMLC)

Alloreactive T cells recognize tissue antigens expressed by allogeneic cells and mount vigorous responses, as graft rejection in vivo and mixed lymphocyte reaction in vitro. The direct T-cell allorecognition is the principle player in the initial vigorous immune response to allogeneic cells that causes acute rejection[1,2]. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. In www.scichina.com

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contrast to nominal antigen-specific T cells that recognize antigenic peptides in association with self-MHC molecules, alloreactive T cells recognize non-self MHC Received October 25, 2006; accepted November 27, 2006 doi: 10.1007/s11427-007-0036-y † Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant Nos. 30271201 and 30490241), and the “973” Project of the Ministry of Science and Technology of China (Grant No. 2001CB510008)

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molecules and are present at high precursor frequency[3,4]. The molecular basis for exceptionally high frequency and diversity of T cells responding to allogeneic antigens remains elusive. A number of reports have introduced the alloreactive T cells that can recognize reconstituted class I molecules lacking bound peptide to — explain the strong alloreaction[5 7]. Peptide-independent alloreactive T cells have been described to recognize polymorphic residues located on the surface of allogeneic MHC molecule and are indifferent to bound peptide[7]. A second model, in which alloreactive T cells recognize epitopes that are dependent on both the MHC molecule and specifically bound peptides, also has re— ceived experimental support[8 10]. The nominal antigen-specific T cell, which recognizes the antigenic peptide presented by self-MHC, has been clearly demonstrated to recognize specific peptide-MHC combinations. However, whether peptides contribute to specific epitopes or simply stabilize a particular conformation of MHC molecules for direct recognition by alloreactive T cells remains controversial. Matzinger and Bevan [11] showed foresight by suggesting that the strength of the alloresponse might result from recognition of a diverse range of components associated with allogeneic MHC molecules, and now these components are known to be bound peptides derived from proteins synthesized within the cell. However, the large number of different peptides recognized and low frequency of T cells specific to each epitope hamper the search for alloreactive T cells. Our study was based on the concept that among the alloreactive T cells a significant proportion should be specific to one particular peptide presented by allogeneic MHC molecule. Two HLA-A2 restricted peptides, including a viral and a self-protein origin peptides, were loaded onto HLA-A2 expressing, TAP-deficient T2 cell line respectively, making the T2 cell present a high copy number of a single pMHC. The peptide-pulsed T2 stimulated PBLs samples of HLA-A2 defined individuals in a long-term mixed lymphocyte culture (LTMLC) to induce antigen-specific CTLs. The aim of this study is to determine whether the allgeneic CTLs induced by a single pMHC are the pMHC specific, in the same fashion as nominal antigen-specific T cells are. Since the T2 cell expresses HLA-A2 molecule only, for HLA-A2 positive (HLA-A2+ve) PBLs, a viral peptide pulsed T2 cell presents nominal viral antigen; for HLA-A2 negative (HLA-A2-ve) PBLs, the pMHC 204

formed on the surface of T2 cell is alloantigen. The specificity of the induced CTLs was tested by their specific cytotoxicity and binding ability to specific pMHC-tetramer. Our results indicate that self-peptide binding to HLA-A2 can induce the allogeneic T cell response in the LTMLC, and the resulting alloreactive CTLs recognize peptide-MHC combination in the same way as nominal antigen-specific T cells do.

1 Materials and methods 1.1 Peptides and stimulator cells Peptides corresponding to the HLA-A2 restricted immunogenic epitopes of self-protein origin Tyr369-377 (YMDGTMSQV)[12], EBV origin LMP2A426-434 (CLGGLLTMV)[13] and HIV origin Gag77-85(SLYNTVATL) [14] were synthesized and purified to >90% homogeneity by reverse phase HPLC. The peptides were dissolved in DMSO, and diluted in RPMI1640 medium. The final concentrations of these synthetic peptides were 1 mg/ mL. The T2 cell line[15,16], which is deficient in TAP, was propagated in RPMI1640 medium containing 10%FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified atmosphere, with 5% CO2 at 37℃. As stimulator cells in the LTMLC, T2 cells were pulsed with the synthetic peptide at the final concentration of 40 μg/mL, incubated for 3 h in a CO2 incubator and washed extensively with PBS (pH 7.4) to eliminate unbound peptides. Subsequently the cells were inactive by irradiating (200 Gy) and the peptide binding efficiency was detected. T2 cell pulsed with the peptides of LMP, Tyr or HIV is named T2/LMP, T2/Tyr or T2/HIV, respectively. 1.2 Generation of peptide-specific T cell clones Peripheral blood was obtained from four normal HLA-A2-ve donors and four HLA-A2+ve donors. HLA typing was performed by SSP-PCR[17]. The HLA-A allotypes of the four HLA-A2-ve donors are A11, A69; A24, A3; A23, A33; A10, -. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Hypaque density 1.077 g/mL), washed twice in sterile PBS and suspended in CTL medium consisting of RPMI1640, 25 mmol/L HEPES, 2 mmol/L L-glutamine, and 10% human AB serum; and then placed in sterile tissue culture dishes for separation of adherent from nonadherent cells by culturing over-

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night at 37℃. The nonadherent cells (i.e., PBLs) were plated into 24-well plates at 5×106 /well. The remaining nonadherent cells were irradiated, frozen and then thawed before being used as feeding cells. PBLs (5×106/well) were stimulated with irradiated T2 cells pulsed with peptide. In the first stimulation, co-culture was established at the ratio of 25 PBLs to 1 T2 cell in 24-well plates for 7 days in CTL medium. For the second stimulation, the ratio changed to 10︰1. On day 10, IL-2 was added to the culture medium and the final concentration was maintained at 20 U/mL. On day 14, the third stimulation was carried out at the same ratio as the second. On day 21, the cells were harvested by centrifugation. 1.3 HLA-peptide tetrameric complexes and flow cytometry HLA-peptide tetrameric complexes were produced as previously described[18,19]. In brief, the HLA-A2 heavy chain was modified by deletion of the transmembrane domain and COOH-terminal addition of a sequence containing the BirA enzymatic biotinylation site[20]. The HLA-A2 heavy chain and β2-microglobulin (β2m) were produced using a prokaryotic expression system (pET/ HLA-A*0201, pET/ β2m plasmids), purified and refolded in vitro by limiting dilution with the HLA-A2 restricted peptides (Tyr369-377, LMP2A426-434 and Gag77-85). The refolded complexes were purified and biotinylated by BirA (Avidity, Denver, CO) in the presence of biotin and ATP. The biotinylated product was separated from free biotin by gel filtration. Tetramers were assembled by mixing biotinylated protein complexes with Exavidin-PE (Sigma, E4011) at a molecular ratio of 8︰1. Tyr369-377/HLA-A2 tetramer is named Tyr-eramer, LMP2A426-434 and Gag77-85/HLA-A2 tetramers as LMP-tetramer and HIV-tetramer, respectively. Bulk alloreactive CTLs were incubated on ice with 10 μg/mL tetrameric complexes. After 30 min incubation, cells were washed extensively with PBS containing 1% FCS. FITC-CD8 Ab (BD Biosciences) was added, and the samples were incubated on ice for further 30 min. After extensive washing, samples were fixed with PBS containing 2% formaldehyde. Double-color analysis was performed with tetramer-PE and CD8-FITC Ab, using a FACSCalibur (Becton Dickinson, Heidelberg, Germany).

1.4 Cytotoxicity assay Cytotoxicity of the co-cultural bulk was tested with the nonradioactive enzyme immunoassay (EIA), using CytoTox 96 (Promega, USA). The T2 cells pulsed with different peptides were used as target cells, which were washed and added to round-bottom 96-well plates as the final concentration of 2×104 cells/well. Co-cultural bulk was added at different effector:target (E:T) ratios ranging from 25︰1 to 5︰1. Specific lysis was calculated as (experimental release−spontaneous release)/(maximum release−spontaneous release) ×100%. 1.5 Statistical analysis Statistical analysis among experimental groups was performed by ANOVA, and Turkey’s test was used to compare individual groups. A value of P<0.05 was considered statistically significant.

2 Results 2.1 The peptides used in this study can bind to HLA-A2 molecules The human cell line T2 is TAP deficient, and accordingly, expresses a reduced amount of HLA-A2 and no other HLA allele on the cell surface. The TAP-deficiency in the T2 cell line causes most MHC class I (i.e., HLA-A2) molecules to remain empty or to associate with low-affinity peptides[15,16]. In the presence of exogenous presentable peptides, HLA-A2 expression significantly increases as empty or unstably assembled HLA-A2 molecules are stabilized through binding of the peptide. By the external addition of peptides, empty molecules can be stabilized and low-affinity peptides replaced, leading to a high copy number of a single peptide-MHC complex on the T2 cell, which is expected to stimulate the PBLs effectively. As shown in Figure 1(b), the untreated T2 cell expressed a small amount of HLA-A2 molecules. The amount of HLA-A2 was increased significantly after being pulsed with the LMP peptide (Figure 1(c)). The percentage of positive cells was increased from 5.27% to 24.35%. The T2 cells pulsed with Tyr or HIV showed similar results (data not shown). 2.2 The LTMLC system is effective to expand viral antigen-specific CTL in vitro for syngeneic PBLs In order to test if our LTMLC system is effective to expand the antigen-specific CTLs, PBLs from four HLA-

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Figure 1 The amount of HLA-A2 molecules in the T2 cells was increased after being pulsed with the LMP peptide. Cells were stained with the HLA-A2 specific monoclonal antibody BB7.2 and FITC-anti-mouse IgG, and quantified by cytometry. (a) 721.221 cells were negative of HLA class I molecules and used as a negative control; (b) some T2 cells without any treatment expressed a few HLA-A2 molecules, the percentage of positive cells was 5.27%; (c) T2 cells pulsed with peptide (LMP) up-regulated the HLA-A2 expression, the percentage of positive cells increased to 24.35%.

A2+ve donors were stimulated with the viral peptide (LMP2A226-434) or self-peptide (Tyr369-377) pulsed T2 cells according to the protocol described above. After being co-cultured with the LMP-pulsed T2 (T2/LMP), the PBLs of HLA-A2+ve individuals were induced to generate LMP/HLA-A2 complex-specific CTLs (CTLT2/LMP), and the specificity of the bulk CTLs was revealed by both cytotoxicity assay and their ability to bind to the specific tetramer (LMP-tetramer). As shown in Figure 2(a), the CTL-T2/LMP showed an obvious cytotoxicity against the specific target, i.e., the LMPpulsed T2 cell, while Effector/Target ratio is 10︰1, but relatively low killing activity towards the HIV-pulsed T2 (T2/HIV) (26.52%±3.72% vs 7.01%±0.87%, P< 0.001). Apparently, the cytotoxicity of CTL-T2/LMP is peptide-dependent. Meanwhile, the bulk CTL-T2/LMP can be stained more frequently by the LMP-tetramer

compared to that by the HIV-tetramer(0.98%±0.33% vs 0.05%±0.01%, P=0.0014). As shown in Figure 3, the LMP-tetramer stained CD8+ T cells in the bulk CTLT2/LMP from four HLA-A2+ve donors ranges from 0.5% to 2%, while the HIV-tetramer stained CD8+ T cells in the same bulk ranges from 0.04% to 0.07%. A self-peptide of Tyrosinase369-377 (Tyr) was also used in our LTMLC system to induce CTL-T2/Tyr. The bulk CTL-T2/Tyr showed similarly low specific lysis activity against the Tyr-pulsed T2/Tyr to that against T2/HIV (Figure 2(b)). 2.3 Allogeneic CTLs induced in vitro by T2/Tyr exhibit a peptide-dependent cytotoxicity and specific Tyr-tetramer binding ability For the PBLs of HLA-A2-ve individuals, the T2/Tyr presents a high copy number of the Tyr–HLA-A2 com-

Figure 2 Viral peptide is able to elicit specific CTL response while binding to the syngeneic HLA molecule in vitro. Two bulk CTL lines from long-term mixed lymphocyte culture with T2/LMP (a) and T2/Tyr (b) in four HLA-A2+ve individuals were analyzed for their specific cytotoxic lysis. The CTL-T2/LMP and CTL-T2/Tyr against T2/HIV were set for control. An elevated specific lysis was observed for CTL-T2/LMP against T2/LMP in the tested samples of four individuals, compared to the CTL-T2/Tyr against T2/Tyr and T2/HIV control.

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Figure 3 The increased frequency of specific LMP-tetramer stained CD8+ T cells in the cultural bulk of HLA-A2+ve PBLs co-cultured with T2/LMP after the LTMLC. PBLs samples of four HLA-A2+ve individuals were co-cultured with the T2/LMP. The culture bulks were stained with the anti-CD8 antibody and LMP-tetramer (upper panel) or HIV-tetramer (middle panel) after the LTMLC, respectively. The positive percentage of the bulk CTL-T2/LMP ranged from 0.5% to 2% using the specific LMP-tetramer. The non-specific staining percentage indicated by the HIV-tetramer staining ranged from 0.04% to 0.07% (lower panel).

plex as an allogeneic epitope. After the LTMLC of T2/Tyr and HLA-A2-ve PBLs, the bluk allo-reactive T cells were tested for their cytotoxic specificity and binding ability to specific Tyr-teramer. Figure 4 shows the cytotoxicity assay for the PBLs samples of 4 HLA-A2-ve individuals, at an E︰T ratio of 10︰1. The CTL-T2/Tyr induced from HLA-A2-ve PBLs gave about 30% specific lysis against T2/Tyr, but that against T2/HIV or K562 was reduced to the significantly low level below 10%. This result indicates that allogeneic CTLs induced in vitro by T2/Tyr exhibit a peptide-dependent cytotoxicity, and strongly suggests that allogeneic CTLs specifically recognize the pMHC complex instead of allogeneic HLA molecule alone, in the same way as the viral peptide/self MHC-specific T cells

(such as HLA-A2+ve CTL-T2/LMP) do. The results of peptide-HLA-A2 tetramer staining also show that the allogeneic CTLs induced in vitro by T2/Tyr bear a peptide-dependent, specific tetramer binding ability (Figure 5). The Tyr-tetramer can stain bulk HLA-A2-ve CTL-T2/Tyr, and the percentage of the tetramer positive cells ranges from 0.5% to 2%. However, an irrelevant tetramer, the HIV-tetramer, binds to the same bulk at relatively low frequency ranging from 0.03% to 0.09%. It indicates that pMHC tetramer is able to discriminate peptide specific alloreactive T cells as it is for nominal Ag-specific T cells. As shown in Figure 6, the frequency of specific tetramer positive cells and specific cytotoxicity of HLA-A2-ve CTL-T2/Tyr are similar to those of the HLA-A2+ve nominal Ag-specific T cell

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Figure 4 Self-peptide is able to elicit the peptide-specific T cell response in vitro when binding to an allogeneic MHC molecule. The PBLs samples from four HLA-A2-ve donors (a), (b), (c) and (d) were co-cultured with T2/Tyr, and the bulk CTL lines (CTL-T2/Tyr) were analyzed for their specific cytotoxicity to the T2/Tyr(▲), the T2/HIV(□) and K562(◇) after the LTMLC. The right panel showed the mean value of specific lysis at an E︰T ratio of 10︰1 for the four samples. Obviously, in the HLA-A2-ve bulk CTL lines (CTL-T2/Tyr), there existed HLA-A2/Try complex-specific CTLs, for the cytotoxicity of the bulk against the T2/Tyr (28.07%±2.58%) was more active than that against the T2/HIV (6.87%±1.01%) and K562 (6.37%±0.85%). This result shows that the allogeneic CTLs killed the targets in a peptide-dependant fashion. The elevated cytotoxicity of the cultural bulk was mediated by specific CTL, since no elevated killing activity could be observed when the K562 was used as target.

induced by T2/LMP (A2+ CTL-T2/LMP). It suggests that self-peptide binding to allogeneic HLA and viral peptide binding to self-HLA could elicit specific T cell in vitro with similar efficiency.

3 Discussion The notions of alloreaction and self-MHC restriction are tightly entangled. On the one hand, there exist experimental data suggesting that T cells are self-restricted: they recognize pathogens presented by self-MHC molecules, but they ignore them if presented by foreign MHC[21,22]. On the other hand, 1% to 10% of T cells are alloreactive: they respond to foreign MHC molecules, i.e., they are not self-MHC restricted. A number of reports have introduced that alloreactive T cells recognize reconstituted class I molecules lacking bound pep— tide[5 7], and TCRs of alloreactive recognize polymorphic residues located on the surface of allogeneic MHC molecules and are indifferent to bound peptide. In this form of allorecognition, all MHC molecules on an allogeneic cell are antigenic, creating a high antigen density that could account for the vigorous response. However, antigen-processing-deficient cells, whose MHC molecules express a very limited range of peptides, are 208

not recognized by the majority of alloreactive T cells[23,24]. Adding peptides to the culture can restore allorecognition, and many alloreactive T-cell clones exhibit selective peptide recognition, indicating that they — are peptide-specific[25 27]. Only a few of the peptides recognized by alloreactive T cells, however, have been — identified[28 30]. It is likely that a large number of different peptides recognized and low frequency of T cells specific to each epitope hamper the search for alloreactive T cells that recognize a particular peptide. The manipulation of the epitope expressed on the APCs or target cells recognized by T cell was the initial step for our study. The HLA-A2+ve, TAP-deficient cell line T2 loaded with the viral peptide or self-peptide in vitro was applied for this purpose. The low level of peptides in the T2 cell line causes most HLA-A2 molecules to remain empty or to associate with low-affinity peptides. By the external addition of peptides, empty molecules can be stabilized and low-affinity peptides replaced, leading to a high copy number of a single peptide-MHC complex on the surface of the T2[15,16]. As shown in Figure 1, the amount of HLA-A2 is increased significantly after pulsed with the peptide. Since the T2 cell expresses HLA-A2 only and no other HLA alleles,

WENG XiuFang et al. Sci China Ser C-Life Sci | April 2007 | vol. 50 | no. 2 | 203-211

Figure 5 The increased frequency of specific Tyr-tetramer stained CD8+ T cells in the cultural bulk of HLA-A2-ve PBLs co-cultured with T2/Tyr after the LTMLC. The PBLs samples of four HLA-A2-ve donors (A, B, C and D) were co-cultured with T2/Tyr, and the cultural bulks were stained with the Tyr-tetramer or the HIV-tetramer after the LTMLC, respectively, and CD8 antibody. The specific Tyr-tetramer stained CD8+T cells appeared more frequently in the HLA-A2-ve bulks compared to that stained with an irrelevant HIV-tetramer. The frequency of the Tyr-tetramer positive cells of the HLA-A2-ve bulks ranged from 0.5% to 2%, while those of the HIV-tetramer stained cells in the same bulk ranged from 0.03% to 0.09%.

Figure 6 The efficiency to elicit specific T-cell responses in vitro by the self-peptide associated with allogeneic HLA is similar to that by the viral peptide with syngeneic HLA. The efficiency to elicit specific T-cell response in vitro was assessed by the frequency of specific tetramer binding cells in the cultural bulk and the cytotoxicity of the bulk against specific target. The frequency of the Tyr-tetramer staining cells in the HLA-A2-ve bulk co-cultured with the T2/Tyr showed a similar range to that of the LMP-tetramer staining cells in HLA-A2+ve bulk co-cultured with the T2/LMP (0.88% VS 0.98%, P=0.66). Both of the two CTL lines showed similarly significant specific cytotoxicity against the corresponding targets (28.07% vs 26.52%, P=0.3676). WENG XiuFang et al. Sci China Ser C-Life Sci | April 2007 | vol. 50 | no. 2 | 203-211

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for the PBLs samples of HLA-A2+ve individuals, the external addition of the LMP, the viral peptide, makes the T2 present a viral antigen and induce the nominal Ag-specific T-cell response. For HLA-A2-ve PBLs, the Tyr pulsed T2 cells act as a model of allogeneic antigen and elicit the alloreactive T-cell response, for the Tyr peptide is HLA-A2 restricted, and the Tyr is a constituent component of normal cells. The T2 cell pulsed with an irrelative peptide, HIV origin Gag77-85, which is also HLA-A2 restricted, was used as a control. Since many studies have shown that the HLA-A2 molecules on the T2 cell surface are not entirely empty[16] due to the TAP-independent peptides, the T2 cell without any treatment is not a good control, especially in the alloreaction study. With the manipulated epitope on the T2 cell and the HLA-A2 defined PBLs, a nominal Ag-specific T-cell response and an alloreactive T-cell response in vitro were induced with the LTMLC. The specificity of the resulting CTLs was determined by their cytotoxicity against specific target and their binding ability to specific peptide-HLA tetramer. PBLs samples of four HLA-A2+ve and four HLA-A2-ve individuals were included in this study. For the T-cell responding to peptide binding to self-MHC, the HLA-A2+ve PBLs co-cultured with T2 cells loaded with the viral peptide LMP (T2/LMP) gave rise to the expansion of the nominal LMP-specific CTLs. The latter was demonstrated by an elevated cytotoxicity against the T2 expressing LMP/HLA-A2 complex and an increased frequency of binding to the LMP/HLA-A2 tetramer for the cultural bulk. Meanwhile, the HLA-A2+ve PBLs co-cultured with the self-peptide, the Tyr, pulsed T2, less effective specific CTLs response were observed; for the cultural bulk showed a similar killing activity and tetramer binding ability specific for the Tyr/HLA-A2 complex to those specific for the HIV/HLA-A2 complex, the latter was an irrelevant control set for specificity assay. This would be a reminiscence of the self-tolerance in vivo. For the alloreactive T-cell response, the PBLs of HLA-A2-ve donors were co-cultured with the Tyr, a self-peptide, pulsed T2 cells (T2/Tyr). The expanded alloreactive bulk also showed the Tyr/HLA-A2 complex-specific cytotoxicity and tetramer binding ability, as the bulk killed the Tyr pulsed T2 cells much more effectively than the HIV pulsed T2, and bound to the Tyr-tetramer more frequently than to the HIV-tetramer. The results indicate that a viral peptide binding to a 210

self-MHC, as well as a self-peptide binding to an allo-MHC, is able to elicit a pMHC-specific CTL response in vitro. However, the percentage of specific tetramer positive cells and the strength of cytotoxicity were not parallel to scale among the individuals. Since specific pMHC-tetramer binding ability is not necessary to account for the specific lysis, it is possible there exist pMHC-tetramer positive cells with varying specific cytotoxic activity. It has been reported that in some instances alloreactivity may not depend on the recognition of a precise self-peptide but on a MHC class I epitope influenced by the peptide. Based on the results of our study, we argue that there exists a relatively large proportion of pMHC-specific alloreactive T cells in the co-culture bulk, although the existence of peptide-independent alloreactive T cells cannot be excluded completely in our study. The cytotoxicity assay and tetramer-binding test for the bulk after the LTMLC reveal that alloreactive T cells (HLA-A2-ve CTL-T2/Tyr) are peptide-specific, which means that the alloreactive T cells recognize the pMHC ligand in the same fashion as the nominal Ag-specific T cell lines (HLA-A2+ve CTL-T2/LMP) do. All the results strongly suggest that alloreative T cells recognize epitopes that are dependent on both the MHC molecule and bound peptide. TCRs interact with allogeneic MHC in a manner that is almost indistinguishable from conventional recognition of antigenic peptide presented by self-MHC[31]. Our observations support the hypothesis that the strength of alloresponse might result from recognition of a diverse range of peptides presented by allogeneic MHC molecules, and the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The expansion of pMHC-specific CTL in allogeneic PBLs with the method of the LTMLC is of potential significance for tumor immunotherapy. It has been postulated that peptide-specific, allorestricted — CTLs might be useful for tumor immunotherapy[32 35], since tumor rejection by the immune system is often inefficient because of tolerance toward the tumor tissue in cancer patients[36]. The approach involving in vitro stimulation of T cells restricted toward an MHC molecule that is not present during their negative selection might therefore offer the possibility of isolating CTLs against self or foreign peptides. The adoptive transfer of allorestricted CTLs with known specificity would not

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We thank LIANG Bing, Dr. CHEN XueLing, Dr. LI JiaNan, WU WenHua and CHEN GuoAn for their kind assistances in this work.

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WENG XiuFang et al. Sci China Ser C-Life Sci | April 2007 | vol. 50 | no. 2 | 203-211

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