The flexibility of the TCR allows recognition of a large set of naturally occurring epitope variants by HIV-specific cytotoxic T lymphocytes

Florence Buseyne and Yves Rivière Laboratoire d'Immunopathologie Virale, URA CNRS 1930, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

Correspondence to: F. Buseyne


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pathogens attempt to evade immune recognition by expressing mutated antigens. The present study shows that two mechanisms happen in vivo during the course of HIV infection to limit the escape of antigenic variants from cytotoxic T lymphocyte (CTL) recognition: recognition of several epitope variants by the same TCR and generation of several CTL populations specific for a single epitope but recognizing different variant sequences. We have studied two CTL populations directed towards the HIV-p24gag amino acids 176–184 QASQEVKNW epitope, presented by HLA-B5301. Both CTL populations were derived from a long-term asymptomatic HIV-infected child and they express different TCR. Each of the two CTL recognizes five of the 10 naturally occurring variants. These variants are distinct for both CTL and thus a total of eight variants are recognized. Thus, polyclonality of CTL specific for the same epitope but differing in variant sequences recognized may improve the control of variant viruses' replication in vivo. In addition to cross-recognition of several variant epitopes, promiscuous recognition of exogenous peptides complexed to allogeneic HLA-B molecules occurs, showing that the TCR can tolerate amino acid changes on both the peptide and the MHC molecule. This flexibility of the TCR is probably of great importance for control of viruses with high genetic variability, such as HIV.

Keywords: cytotoxic T lymphocyte, HIV, HLA molecules, human


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In many viral infections, the immune system contains or eradicates the infectious agent. Numerous studies have shown that this is achieved at least in part by cytotoxic T lymphocytes (CTL). High frequencies of CTL have been detected in HIV-infected subjects and there is accumulating evidence for a beneficial role of HIV-specific CTL in natural infection (reviewed in 1). Recently, both transfer of virus-specific CTL and depletion of CD8+ T lymphocytes have provided direct demonstration for the antiviral effect of CD8+ T cells in vivo in HIV and SIV infections (24). It is now widely accepted that an effective vaccine against HIV should elicit virus-specific CTL (5).

CD8+ CTL recognize virus-infected cells by an interaction between the TCR and a viral peptide presented by MHC class I molecules on the surface of the infected cells. Definition of a pathogen's sequences recognized by CTL is critical for vaccine development. X-ray analyses of MHC crystals have given important information on the function of MHC molecules as peptide receptors (6). Biochemical isolation and studies of naturally MHC-associated peptides have determined peptide ligand specificity of HLA class I allelic products (7). Several apparently unrelated MHC allelic products are characterized by strikingly similar specificities in terms of the main anchor residues of their peptide ligands. This observation and quantitative binding assays have shown that several HLA alleles can be grouped in a single functional HLA supertype (8). Knowledge of peptide ligand characteristics has improved the efficiency in definition of new CTL epitopes (9). In addition, they explain the promiscuity and degeneracy of CTL epitopes. The promiscuous recognition phenomenon refers to T cells that bear TCR recognizing a given peptide complexed with different MHC molecules (10). The capacity of a peptide to bind multiple MHC molecules and, consequently, to be immunogenic in the context of individuals from different MHC types has been referred to as degeneracy (10).

A high rate of mutations is a characteristic feature of HIV. Epitope variation can result in the lack of antigen recognition or in the induction of an altered response to the pathogen. However, some mutations do not affect recognition by the CTL. In HIV-infected patients, both evidence of viral escape from HIV-specific CTL recognition and absence of such immune evasion have been reported (1114). Two characteristics of the CTL response may limit the impact of amino acid changes on CTL recognition of the antigenic peptide. Although MHC class I-restricted CTL have sufficient specificity to discriminate between antigenic peptides, there is a degree of flexibility in peptide recognition by the TCR (15). This flexibility allows cross-recognition of peptide ligands with amino acid substitutions, as well as promiscuous recognition of the same peptide complexed to different MHC molecules. The clonality of CTL specific to a single viral peptide has been studied in various viral infections by determination of the TCR {alpha} and ß chain usage or sequence. Most CTL responses to a single epitope are polyclonal, whereas some are highly oligoclonal. As TCR specific for the same peptide can contact different residues of the antigenic peptide, they may have different susceptibility to amino acid variations in this peptide (16). The present study shows that the CTL response can prevent some immune escape by the flexibility of the TCR and by the polyclonality of CTL populations.

We have characterized two different CTL populations from the same long-term asymptomatic HIV-infected patient, that recognized an HLA-B5301-restricted epitope from HIVp24gag protein, amino acids 176–184 QASQEVKNW. These two CTL represent unique material as they were generated in the same genetic background, during the course of a natural human infection, and they expressed distinct TCR that recognized the same epitope. Our results show the ability of the TCR to recognize several epitope variants as well as peptides complexed to allogeneic molecules. In addition, the specificities of two CTL populations for variant peptides are clearly distinct. Thus both the flexibility of each TCR and the presence of several CTL populations specific for a single epitope are advantageous for the infected host as they broaden the number of mutant epitopes recognized.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient
Patient EM47 has been followed at Hôpital Necker (Paris, France). The legal guardians gave inform consent before entering the study. This patient was born from an HIV-infected mother. He carried a clade B HIV subtype (17). At time of isolation of CTL 141, he was 10.6 years old, had 582 CD4+/mm3, 1635 CD8+/mm3 and his plasma viral load was <104 HIV copies/ml. He was in CDC stage A and untreated. At the time of isolation of CTL 977, he was 13.6 years old, had 340 CD4+/mm3, 1054 CD8+/mm3 and his plasma viral load was 4x103 HIV RNA copies/ml. He was in CDC stage A, and had received AZT and didanosine for 18 months. At 16 years of age, he was still asymptomatic, had no severe immunosuppression (CDC stage A2) and his viral load was <104 HIV RNA copies/ml.

Viruses and synthetic peptides
Peptides corresponding to the naturally occurring variants of the QASQEVKNW epitope (18) were synthesized by Neosystem (Strasbourg, France) and obtained through Agence Nationale de la Recherche sur le SIDA (Table 1Go). The recombinant vaccinia viruses (rVV) were provided by Transgène (Strasbourg, France) and were described in a previous study (17). VV-GAG-B encodes p55gag from HIVLai (vvTG1144) and VV-GAG-A encodes p55gag from a clade A isolate HIV92UG037 (vvTG6026).


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Table 1. Sequence of the variants of CTL epitope p24 amino acids 176–184 used in this study
 
Cell lines and media
Epstein–Barr virus-transformed B lymphoblastoid cell lines (EBV-B) derived from peripheral blood mononuclear cells (PBMC) by standard techniques were maintained in RPMI 1640 (Whittaker, Gagny, France) supplemented with 2 mM L-glutamine (Gibco/BRL, Cergy-Pontoise, France) and 10% FCS (Dutscher, Brumath, France) (RPMIc). WIE cell line was a kind gift from Dr D. Schendel (Munchen, Germany). C1R cells were grown in RPMIc supplemented with 150 µg/ml Hygromycin B (Coger, Paris, France). C1R cells transfected with HLA-B5301 were a kind gift from Dr Masafumi Takiguchi (University of Kumamoto, Japan). CD8+ T cells were cultured in RPMI 1640 supplemented with 5% human AB serum (ETS, Les Ulis, France), 2 mM L-glutamine, non-essential amino acids (Gibco/BRL), 1 mM sodium pyruvate (Gibco/BRL) and 100 IU/ml rIL-2 (Chiron, Suresnes, France; a generous gift from Dr Lebour). Genotyping of EBV-B lines was performed by Dr F. Gotch (Chelsea and Westminster Hospital, London, UK) using amplification refractory mutation system PCR.

Isolation of HIV-1-specific CTL clones
CTL clones were obtained by seeding PBMC in limiting dilution and peptide-specific stimulations. Isolation of CTL clone 141 was previously described (19). Isolation of CTL 977 was performed 3 years later from the same patient using the same technique (19). They were maintained by stimulations every 2–3 weeks. Autologous EBV-B cell lines were incubated overnight with index peptide at 100 ng/ml, then washed and irradiated (100 Gy). The stimulator to effector ratio was 1:1 and allogeneic irradiated feeder cells were added at a concentration of 106 cells/ml. A panel of TCR Vß-specific antibodies previously used to stain the Vß13+ CTL 141 (19) did not react with CTL 977, providing direct evidence that both TCR were distinct.

Cytotoxic assays
CTL assays were conventional 4-h 51Cr-release assays as previously described (17). EBV-B cells were infected with rVV 16 h before the assay, labeled for 1 h with 3.7 MBq of Na251CrO4 (NEZ030; NEN, Le Blanc Mesnil, France), washed 3 times and used as target cells. For peptide assays, target cells were labeled for 1 h with Na251CrO4, washed and synthetic peptides were added just before mixing target cells and effector cells. Target cells (5x103) were incubated in triplicate with effector cells, at various E:T ratios, in round-bottom 96-well plates, in 200 µl RPMIc supplemented with 10 mM HEPES (Gibco/BRL). The plates were incubated at 37°C for 4 h and then centrifuged at 150 g for 5 min. Next, 50 µl of supernatant from each well was mixed with 100 µl of scintillation liquid (81150401; Perkin-Elmer, Evry, France) and 51Cr was measured in a ß-counter (Microbeta 1450; Perkin-Elmer). Maximal release was obtained from targets lysed by 5% Triton X-100 + 1% SDS. Spontaneous release was obtained from targets incubated with medium alone. Assays in which the spontaneous 51Cr release exceeded 35% were discarded. Percent specific lysis was calculated as 100x(experimental release – spontaneous release)/(maximal release – spontaneous release). The CTL responses against the HIV antigens were considered positive if they exceeded the mean of control target lysis by 3 SD and by at least 10%. EC50 (50% effective concentration) is defined as peptide concentration required to obtain 50% of maximal lysis.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Eight out of 10 naturally occurring variant sequences from the p24gag amino acids 176–184 epitope are recognized by CTL 141 and 977
CTL clone 141 was derived from a long-term asymptomatic HIV-infected child EM47 (19). A second CTL population, 977, was derived from the same patient 3 years later. Both CTL recognized the same epitope, but expressed a different TCR Vß fragment as determined by FACS analysis. Ten variant sequences of the epitope have been reported and were tested for recognition by CTL 141 and 977 (Table 1Go) (18). The sequences corresponded to optimal epitope as truncated peptides were not recognized (data not shown). The two specific CTL populations recognized a different set of variant sequences (Fig. 1A and BGo). Peptide titration curves revealed that CTL 141 preferentially recognized the sequence corresponding to the clade B consensus, followed by clade A consensus sequence and minor variants (Fig. 2AGo). The respective EC50 were 0.18, 4.8, >1000, 599 and 56 ng/ml for peptides 25-17B, 25-17A, 25-17D, 25-17M1 and 25-17M3. In contrast, CTL 977 recognized consensus clade A sequence much more efficiently than the consensus clade B sequence (EC50 were 0.18 and 47.5 ng/ml for peptides 25-17A and 25-17B respectively). EC50 were 0.52, 3.39 and 0.61 ng/ml for peptides 25-17C, 25-17F and 25-17M5 (Fig. 2BGo). Our data show that two distinct CTL populations derived from the same patient 3 years apart recognized different variants of the same epitope and their affinity for epitope variants varied over a wide range.



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Fig. 1. Recognition of CTL epitope variants by CTL 141 and 977. Recognition of CTL epitope variants was tested in a 51Cr-release assay (see Methods). Autologous EBV-B cells were mixed with CTL 141 (A) or CTL 977 (B) and variant peptides were added at 1 µg/ml. Percentages of specific lysis at an E:T ratio of 3:1 are presented.

 


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Fig. 2. Peptide titration curves of CTL epitope variants recognized by CTL 141 and 977. Ten-fold dilutions of peptides were tested in a 51Cr-release assay, as described in Fig. 1Go. Percentages of specific lysis at an E:T ratio of 1:1 are presented for CTL 141 (A) and CTL 977 (B).

 
HLA-B5301 molecule presents the p24gag amino acids 176–184 CTL epitope
Partially matched target cells were used in a 51Cr-release assay to determine the HLA restricting molecule. First, C1R cell line (HLA-3501low, C0401) transfected with HLA-B5301 was more efficiently lysed than parental C1R by CTL 141 and 977 (Fig. 3A and BGo). Secondly, both effectors recognized with similar efficiency EBV-B cell line EM59 (B5301+C0401) and EBV-B cell line EM17 (HLA-B5301+HLA-C0401+) (Fig. 3C and DGo). These experiments strongly suggest that HLA-B5301 was the restricting element of CTL 141 and 977.



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Fig. 3. Recognition of index peptide presented by partially matched cell lines to CTL 141 and 977. Ten-fold dilutions of peptide were tested on various cell lines in a 51Cr-release assay, as described in Fig. 1Go. The index peptides were those recognized with maximal efficiency by the CTL: peptide 25-17B and 25-17A for CTL 141 and 977 respectively. Autologous EM47 EBV-B cells were included in each assay. C1R cells transfected or not with HLA-B5301 were tested for recognition by CTL 141 (A) or CTL 977 (B). Partially matched EBV-B cells expressing or not HLA-B5301 and HLA-C0401 were tested in parallel for recognition by CTL 141 (C) or CTL 977 (D).

 
However, C1R cells and WIE EBV-B cells that expressed HLA-B3501 and C0401, but not HLA-B5301, were lysed by effectors when pulsed with peptides (Fig. 3AGo–D). Recognition of WIE cells, homozygous for HLA-B3501, was clearly reduced for CTL 977, whereas they were well recognized by CTL 141. For CTL 141 EC50 of index peptide 25-17B was only 10 times higher on WIE than on autologous EM47 EBV-B cells. In contrast, when rVV were used to endogenously express the epitope, C1R cells transfected with HLA-B5301+ were efficiently recognized by both CTL but not the parental C1R cells that are HLA-B3501+ (Fig. 4AGo–F). What is more, EM17 cells (HLA-B5301+) but not WIE cells (HLA-B3501+) were able to present the endogenously synthesized epitope to CTL (Fig. 4GGo–L). From these results, we conclude that HLA-B5301 was the restricting molecule presenting p24gag amino acids 176–184 CTL epitope to both CTL, 141 and 977. Presentation of exogenous peptides by WIE or C1R cells may be viewed as promiscuous presentation of the peptides by HLA-B3501. Indeed, HLA-B3501 and B5301 molecules are very similar and differ from each other by only 5 amino acids (2022).



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Fig. 4. Recognition of endogenously expressed epitope presented by a partially matched cell line to CTL 141 and 977. Target cells were infected by control VV or by rVV encoding the p55gag from HIVLai (VV-GAG-B) or HIV62UG037 (VV-GAG-A), including CTL epitope 25-17B and 25-17A respectively. Results obtained with VV-GAG-B are shown for CTL 141 (A–C and G–I) and results obtained with VV-GAG-A are shown for CTL 977 (D–F and J–L). Target cells were autologous EBV-B cell line EM47 (A, D, G and J), C1R cells (B and E), HLA-B5301 transfected C1R cells (C and F), EM17 EBV-B (H and K) and WIE cell line (I and L).

 
Promiscuous presentation of p24gag amino acids 176–184 CTL epitope by HLA-B35 allelic variants
The first observation of promiscuous presentation of the epitope by both HLA-B5301 and B3501 led us to search for other related HLA-B molecules able to present the antigenic peptides to both CTL populations. More than 30 EBV-B cell lines of known HLA genotype were tested for lysis by both 141 and 977 CTL at high peptide concentration (1 µg/ml; Fig. 5Go). For some targets, data were confirmed by testing 10-fold peptide dilutions and presentation of endogenously synthesized epitope (Table 2Go). Lysis of the five HLA-B5301+ cell lines was close to that of autologous cells (Fig. 5Go). Minimal peptide concentration giving significant lysis was 0.1 ng/ml and only the HLA-B5301+ matched cell lines presented endogenous peptide (Table 2Go). These data confirmed previous results and showed that HLA-B5301 was the most efficient HLA-presenting molecule, and the only one able to present endogenously synthesized epitope.



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Fig. 5. Recognition of index peptides presented by several EBV-B cell lines to CTL 141 and 977. Target cells were mixed with effectors and index peptides at a final concentration of 1 µg/ml as described in Fig. 3Go. To normalized results from different experiments, they are expressed as the percentage of recognition compared to recognition of autologous target cells at an E:T ratio of 3:1: [100x(% lysis of cells loaded peptide – % lysis of cells incubated with medium)/(% lysis of EM47 EBV-B cells loaded peptide – % lysis of EM47 EBV-B cells incubated with medium)]. (A) Effectors cells were CTL 141 and target cells were coated with peptide 25-17B. (B) Effectors cells were CTL 977 and target cells were coated with peptide 25-17A.

 

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Table 2. HLA class I genotype, presentation of synthetic peptides and p55gag encoding VV to CTL 141 and 977
 
Next, we observed that several HLA-B35+ target cells presented the peptides with efficiency that varied from 27 to 84% of autologous cell recognition for CTL 141 and 30 to 63% for CTL 977. Lysis by CTL 141 of the HLA-B3501+ WIE cell line was similar to that of the HLA-B3502+ EM105. However, higher lysis of the HLA-B3502+ cell line was observed with CTL 977 (Fig. 5Go and Table 2Go). Lysis of the HLA-B3503+ EM109 cell line by both CTL was very low. As both EM105 and EM109 expressed the HLA-B4901 molecules, differences in peptides presentation were probably due to the HLA-B35 allele. Thus, the various HLA-B35 subtypes have different ability to present the peptides to CTL and the two CTL populations differ in their ability to recognize peptides presented by closely related HLA-B35 allelic variants.

Members of the HLA-B7 supertype differed widely in their ability to present the p24gag amino acids 176–184 CTL epitope
For all target cells tested, lysis by CTL was not due to allogeneic recognition, as cells incubated with peptide-free medium were not lysed (data not shown). Most targets were recognized with low efficiency by both CTL (percent of lysis <40% of lysis of autologous cells and minimal peptide concentration to obtain significant lysis equal or >10 ng/ml, Fig. 5Go and Table 2Go). Several members of the HLA-B7 supertype, that includes HLA-B5301, were poorly recognized, such as HLA-B51, -B52, -B55 and -B7801 (see cell lines EM3, EM71, EM40, EM68, EM82 and EM98 in Fig. 5Go and Table 2Go). Three cell lines had intermediate ability to present the peptide to both CTL: EM20 (HLA-B7, -B50), EM60 (HLA-B0704, -B5601) and EM50 (HLA-B13, -B45). EM107 (HLA-B7, -B15) presented peptide with intermediate efficiency to CTL 141 only. Three of these cell lines expressed HLA-B7 that belongs to the same supertype family as HLA-B35 and -B5301 (23). However, HLA-B7+ cell lines differed in their ability to present the peptide to CTL (compare lysis of EM20, EM26, EM40 and EM60 in Table 2Go and Fig. 5Go). These results may be due to HLA-B7 subtype-specific presentation, similar to that observed with the various HLA-B35 subtypes. Alternatively, they may be due to the second HLA-B allele expressed by the HLA-B7+ cell line. Indeed B5601 carried by cell line EM60 also belongs to the HLA-B7 supertype (24), but the second HLA-B56+ cell line (Los 72) was poorly recognized. In conclusion, HLA molecules that belong to the same supertype as HLA-B53 varied widely in their ability to present the antigenic peptides to CTL 141 and 977.

Most of the target cells expressing HLA-B alleles unrelated to HLA-B53 were lysed with low efficiency. However, EM50 (HLA-B13, -B45) cell line was able to present the peptides with moderate efficiency (Table 2Go). Only one subtype has been described for HLA-B45. As the second HLA-B45+ cell line tested (EM71) poorly presented the peptide, presentation by the EM50 cell line was probably due to HLA-B13. Therefore, HLA molecules unrelated to HLA-B5301 or to the HLA-B7 supertype, such as HLA-B13, could present the peptide to HLA-B5301-restricted CTL. On the contrary, HLA-B57 is able to present the same peptide, QASQEVKNW, to HLA-B57-restricted CTL line (25) but not to the CTL described here. The HLA-B57+ cell line EM3 coated with exogenous peptide or infected with rVV expressing Gag was lysed by autologous HLA-B57-restricted CTL (26), but not by HLA-B5301-restricted CTL (Fig. 5Go and Table 2Go). In this case, promiscuous presentation to HLA-B5301-restricted CTL was not limited by ability of the target cell to process, bind and present the epitope, but by the capacity of the TCR to recognize the peptide bound to HLA-B57.

Definitive conclusions on the ability of the HLA-B molecules to present the peptides will require additional experiments with cell lines transfected with a single, well-defined HLA molecule. However, our experiments show that the pattern of HLA molecules recognized by a given TCR only partly matched the HLA molecules that share similar peptide binding motifs. In addition, the two distinct TCR differed in their pattern of promiscuous recognition of allogeneic HLA-B molecules complexed to the peptide.

Some epitope variants were presented with greater efficiency by HLA-B35 than by HLA-B5301 to CTL 141
Promiscuous recognition of allogeneic target cells pulsed with index peptide is summarized in Fig. 5Go. At the same time, variant peptides were also tested for recognition on a mismatched target cell line (data not shown). Results were always consistent with those obtained with the index peptide, except in one case: HLA-B3501+ cells presented variants peptides 25-17D, 25-17M1 and 25-17M3 more efficiently than autologous HLA-B5301+ target cells to CTL 141. This phenomenon was confirmed by peptide titration (Fig. 6Go). 25-17D and 25-17M1 peptide concentrations were 1000 times lower on the WIE HLA-B3501+ cell line than on the autologous HLA-B5301+ cell line to reach similar lysis. For peptide 25-17M3, lysis was slightly enhanced on the HLA-B3501+ cell line (Fig. 6Go) and peptide 25-17A was presented with similar efficiency (data not shown). This experiment shows that for one of the two CTL populations, recognition of variant peptides was more efficient on an allogeneic HLA molecule than on the appropriate restricting element, whereas the opposite phenomenon was observed for the index peptide, 25-17B.



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Fig. 6. HLA-B3501 presented epitope variants with greater efficiency than HLA-B5301. Ten-fold dilutions of peptides were tested in a 51Cr-release assay, as described in Fig. 1Go. Percentages of specific lysis by CTL 141 at an E:T ratio of 1:1 are presented for HLA-B5301+ autologous target cells (A) and HLA-B3501+ WIE cell line (B).

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Two CTL populations specific for the same HIV epitope were isolated from a long-term asymptomatic child. They express different TCR and recognize distinct variants of the epitope. These CTL are tolerant to mutations on the antigenic peptide and amino acids variations on the HLA-B-presenting molecule. This study shows that both the flexibility of the TCR and the polyclonality of the CTL response broaden the number of mutant viral sequences recognized by the immune system. Thus both mechanisms may avoid the immune escape of HIV variants in vivo.

We describe here an HLA-B5301-restricted CTL epitope, QASQEVKNW from HIV-p24gag. This epitope does not fit the HLA-B5301 peptide-binding motif. The initial description of the HLA-B3501 and -B5301 peptide binding by pool sequencing of self-peptides eluted from the molecules proposed proline as the only amino acid found at position 2 for both molecules (27). Later, the presence of P, A and V at position 2 was reported for peptides binding to HLA-B3501 (28), and A at position 2 was found for most HLA molecules from the B5 family (29). As HLA-B3501 and -B5301 share the same B pocket, the presence of A instead of P in position 2 in some HLA-B5301-binding peptides is not unexpected. Furthermore, the crystal structure of HLA-B5301 revealed a reduced volume of the B pocket, which may tolerate the presence of the small alanine side chain (22). Thus, the presence of A in position 2 of the epitope p24gag amino acids 176–184 does not fit with the peptide binding motif proposed for HLA-B5301 (27), but is consistent with peptide binding characteristics of the HLA-B7 supertype and with structural characteristics of the HLA-B5301 peptide binding groove.

Some peptides are immunogenic in the context of different MHC molecules. Such degenerate immunogenicity of CTL epitope presented in the context of multiple MHC alleles was described for HLA-A3 and -A11 (10), for distinct HLA-A2 subtypes (30), for HLA-B35 and -B51 (31,32). The p24gag amino acids 176–184 CTL epitope described here was reported to be presented in the context of HLA-B5701 (25). Thus, our study identified another degenerate CTL epitope.

Promiscuous presentation of CTL epitope by several MHC class I molecules to the same CTL clone was previously reported in infectious and cancer diseases. On the contrary, minor differences between two MHC molecules can profoundly affect recognition by the TCR. Several examples of both situations have been described for HIV-specific CTL. Some but not all HLA-B3501 CTL epitopes can be presented by HLA-B5101 (31). CTL from HLA-B5701+ patients recognized epitopes presented by HLA-B5701 and B5801, but not by HLA-B5802 (25,33). In our study, addition of free exogenous peptide induced lysis of almost all cell lines tested, although the level of lysis depended on HLA molecules expressed by the target cell. The specificity of TCR recognition was well illustrated by the differential recognition of three allelic variants of the HLA-B35 molecule. Our experiments allowed the assessment of the biological relevance of the HLA supertypes in the context of presentation of free exogenous peptide to CTL. One main finding of the present study is that promiscuous presentation of the QASQEVKNW epitope did not fully match the HLA-B7 supertype, to which HLA-B5301 belongs. As an example, the EM50 cell line, which expressed HLA-B13 and B45 that are not related to the HLA-B7 supertype, was better recognized than some cell lines expressing HLA molecules related to the HLA-B7 supertype (see Fig. 5Go). Promiscuous recognition of the same epitope in the context of different HLA molecules depends on the ability of the MHC to bind the peptide. The EM3 HLA-B57+ cell line bound, processed and presented the same peptide to HLA-B57-restricted CTL (26). In this case, the low level of promiscuous presentation was not due to the binding capacity of the HLA molecule but rather to the specificity of the TCR. In conclusion, our data showed that the affinity of TCR for the MHC–peptide complex plays a major role in promiscuous recognition of the antigenic peptide, independently of the peptide binding characteristics of the HLA molecule. This study shows that the TCR can tolerate modification of its ligand, either on the peptide or on the HLA molecule. This adaptability may be relevant in vivo to recognize new viral variants.

The resolution of crystal structures of three different MHC class I-restricted TCR complexed with their respective peptide–MHC ligands has revealed general features of the interaction of the TCR with their ligands (16,34,35). For each of the three TCR–MHC–peptide complexes described, atomic contacts between the peptide and the TCR account for only one-third of the total interactions. In addition, the predominant interactions between the TCR and the MHC molecule are made with conserved residues of the MHC. In our study, recognition of targets by the CTL strictly required the presence of cognate peptide. This observation implies that the peptides were able to bind all MHC molecules studied and that peptide–TCR interactions were critical for recognition of the MHC–peptide complex. The fact that most MHC molecules tested presented the peptide suggests that the conserved MHC residues played a predominant role in the global interactions between the TCR and peptide–MHC complex. Both CTL populations differed in the peptide variants recognized, as well as in the promiscuous recognition of HLA-B mismatched target cells, suggesting that contact residues between the two TCR and the peptide might be distinct, as revealed by crystal structure of two other TCR recognizing the same HTLV peptide complexed with HLA-A2 (16).

In contrast to lysis of peptide-pulsed target cells, no promiscuous presentation of endogenously synthesized epitope complexed to allogeneic HLA-B molecules was observed. This was probably due to the limited amount of epitope presented at the cell surface that may result from competition with other endogenously processed epitopes or from differential intracellular processing. However, another HIV epitope was endogenously processed and presented in association with two HLA molecules (32). In contrast, in HLA-B5301+ cell lines the two endogenously synthesized epitope variants 25-17A and 25-17B were recognized by the CTL. Therefore, TCR promiscuous recognition of variant epitopes will not be limited by antigen processing, unless mutations directly affect binding to the HLA molecule or cleavage by the cellular proteases. It is important to note that some variant peptides are recognized only at very high peptide concentration, such as peptide 25-17D by CTL 141. It is therefore probable that the level of endogenously processed peptide for this variant will be too low to be recognized by this CTL.

Our study showed promiscuous recognition of several naturally occurring sequence variants by two CTL. These two CTL isolated from the same patient displayed only partially overlapping patterns of variant sequence recognition. This observation supports the role of CTL polyclonality as a mechanism to broaden the recognition of various HIV subtypes and this may be particularly relevant for vaccine design aimed at inducing CTL that recognize the various HIV strains present worldwide. They may have been generated at the same time or they may reflect sequential expansion of CTL specific for variants that have emerged over time (36). In addition, our results showed that TCR plasticity played a determinant role in promiscuous presentation of modified peptides or HLA molecules. In conclusion, both the flexibility of the TCR and the polyclonality of the CTL may be advantageous for the host, since they allow recognition of a different set of peptides by the infected host and may thus contribute to the limitation of viral immune escape by mutation of CTL epitopes.


    Acknowledgments
 
We thank Professors S. Blanche and Rouzioux for their support. We are grateful to J.-P. Abastado, H.-G. Rammensee and M. Singh for critical reading of the manuscript. This work has been supported by Institut Pasteur, Agence Nationale de la Recherche sur le SIDA and Y. R. is an Elizabeth Glaser scientist.


    Abbreviations
 
CTL cytotoxic T lymphocyte
PBMC peripheral blood mononuclear cell
rVV recombinant vaccinia virus
VV vaccinia virus

    Notes
 
Transmitting editor: A. Fisher

Received 8 December 2000, accepted 12 April 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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