Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

Functional avidity: a measure to predict the efficacy of effector T cells?

The functional avidity is determined by exposing T-cell populations in vitro to different amounts of cognate antigen. T-cells with high functional avidity respond to low antigen doses. This in vitro measure is thought to correlate well with the in vivo effector capacity of T-cells. We here present the multifaceted factors determining and influencing the functional avidity of T-cells. We outline how changes in the functional avidity can occur over the course of an infection. This process, known as avidity maturation, can occur despite the fact that T-cells express a fixed TCR. Furthermore, examples are provided illustrating the importance of generating T-cell populations that exhibit a high functional avidity when responding to an infection or tumors. Furthermore, we discuss whether criteria based on which we evaluate an effective T-cell response to acute infections can also be applied to chronic infections such as HIV. Finally, we also focus on observations that high-avidity T-cells show higher signs of exhaustion and facilitate the emergence of virus escape variants. The review summarizes our current understanding of how this may occur as well as how T-cells of different functional avidity contribute to antiviral and anti-tumor immunity. Enhancing our knowledge in this field is relevant for tumor immunotherapy and vaccines design.

Rapid Perturbation in Viremia Levels Drives Increases in Functional Avidity of HIV-specific CD8 T Cells.

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.

Dissecting TCR-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor antigen peptide variants: crucial role of interaction kinetics on functional outcomes.

Soluble peptide/MHC-class-I (pMHC) multimers have recently emerged as unique reagents for the study of specific interactions between the pMHC complex and the TCR. Here, we assessed the relative binding efficiency of a panel of multimers incorporating single-alanine-substituted variants of the tumor-antigen-derived peptide MAGE-A10(254-262) to specific CTL clones displaying different functional avidity. For each individual clone, the efficiency of binding of multimers incorporating MAGE-A10 peptide variants was, in most cases, in good although not linear correlation with the avidity of recognition of the corresponding variant. In addition, we observed two types of discrepancies between efficiency of recognition and multimer binding. First, for some peptide variants, efficient multimer binding was detected in the absence of measurable effector functions. Some of these peptide variants displayed antagonist activity. Second, when comparing different clones we found clear discrepancies between the dose of peptide required to obtain half-maximal lysis in CTL assays and the binding efficiency of the corresponding multimers. These discrepancies, however, were resolved when the differential stability of the TCR/pMHC complexes was determined. For individual clones, decreased recognition correlated with increased TCR/pMHC off-rate. TCR/pMHC complexes formed by antagonist ligands displayed off-rates faster than those of TCR/pMHC complexes formed with weak agonists. In addition, when comparing different clones, the efficiency of multimer staining correlated better with relative multimer off-rates than with half-maximal lysis values. Altogether, the data presented here reconcile and extend our previous results on the impact of the kinetics of interaction of TCR with pMHC complexes on multimer binding and underline the crucial role of TCR/pMHC off-rates for the functional outcome of such interactions.

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.

In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration.

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated "memory" cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes

The efficient in vitro expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) for use in adoptive immunotherapy represents an important clinical goal. Furthermore, the avidity of expanded CTL populations often correlates closely with clinical outcome. In our study, high-avidity CTL lines could be expanded ex vivo from an antigen-primed animal using low peptide concentration, and intermediate peptide concentrations favored the generation of lower avidity CTL. Further increases in peptide concentration during culture inhibited the expansion of all peptide-specific CD8(+) cells. In contrast, a single amino acid variant peptide efficiently generated functional CTL populations at high or low peptide concentration, which responded to wild-type epitope with the lowest average avidity seen in this study. We propose that for some peptides, the efficient generation of low-avidity CTL responses will be favored by stimulation with altered peptide rather than high concentrations of wild-type epitope. In addition, some variant peptides designed to have improved binding to major histocompatibility complex class I may reduce rather than enhance the functional avidity for the wild-type peptide of ex vivo-expanded CTL. These observations are relevant to in vitro expansion of CTL for immunotherapy and strategies to elicit regulatory or therapeutic immunity to neo-self-antigen when central tolerance has eliminated high-avidity, cognate T cells.

Fine structural variations of alphabetaTCRs selected by vaccination with natural versus altered self-antigen in melanoma patients.

Immunotherapy of cancer is often performed with altered "analog" peptide Ags optimized for HLA class I binding, resulting in enhanced immunogenicity, but the induced T cell responses require further evaluation. Recently, we demonstrated fine specificity differences and enhanced recognition of naturally presented Ag by T cells after vaccination with natural Melan-A/MART-1 peptide, as compared with analog peptide. In this study, we compared the TCR primary structures of 1489 HLA-A*0201/Melan-A(26-35)-specific CD8 T cells derived from both cohorts of patients. Although a strong preference for TRAV12-2 segment usage was present in nearly all patients, usage of particular TRAJ gene segments and CDR3alpha composition differed slightly after vaccination with natural vs analog peptide. Moreover, TCR beta-chain repertoires were broader after natural than analog peptide vaccination. In all patients, we observed a marked conservation of the CDR3beta amino acid composition with recurrent sequences centered on a glycyl-leucyl/valyl/alanyl-glycyl motif. In contrast to viral-specific TCR repertoires, such "public" motifs were primarily expressed by nondominant T cell clonotypes, which contrasted with "private" CDR3beta signatures frequently found in T cell clonotypes that dominated repertoires of individual patients. Interestingly, no differences in functional avidity were observed between public and private T cell clonotypes. Collectively, our data indicate that T cell repertoires generated against natural or analog Melan-A peptide exhibited slightly distinct but otherwise overlapping and structurally conserved TCR features, suggesting that the differences in binding affinity/avidity of TCRs toward pMHC observed in the two cohorts of patients are caused by subtle structural TCR variations.

Induction of circulating tumor-reactive CD8+ T cells after vaccination of melanoma patients with the gp100 209-2M peptide.

Patients with stage I-III melanoma were vaccinated with the modified HLA-A2-binding gp100(209-2M)-peptide after complete surgical resection of their primary lesion and sentinel node biopsy. Cytoplasmic interferon-gamma production by freshly thawed peripheral blood mononuclear cells (direct ex vivo analysis) or by peripheral blood mononuclear cells subjected to 1 cycle of in vitro sensitization with peptide, interleukin-2, and interleukin-15 was measured following restimulation with the modified and native gp100 peptides, and also A2gp100 melanoma cell lines. Peptide-reactive and tumor-reactive T cells were detected in 79% and 66% of selected patients, respectively. Patients could be classified into 3 groups according to their vaccine-elicited T-cell responses. One group of patients responded only to the modified peptide used for immunization, whereas another group of patients reacted to both the modified and native gp100 peptides, but not to naturally processed gp100 antigen on melanoma cells. In the third group of patients, circulating CD8 T cells recognized A2gp100 melanoma cell lines and also both the modified and native peptides. T cells with a low functional avidity, which were capable of lysing tumor cells only if tumor cells were first pulsed by the exogenous administration of native gp100(209-217) peptide were identified in most patients. These results indicate that vaccination with a modified gp100 peptide induced a heterogeneous group of gp100-specific T cells with a spectrum of functional avidities; however, high avidity, tumor-reactive T cells were detected in the majority of patients.

Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen

Human cancer vaccines are often prepared with altered "analog" or "heteroclitic" antigens that have been optimized for HLA class I binding, resulting in enhanced immunogenicity. Here, we take advantage of CpG oligodeoxynucleotides as powerful vaccine adjuvants and demonstrate the induction of high T cell frequencies in melanoma patients, despite the use of natural (unmodified) tumor antigenic peptide. Compared with vaccination with analog peptide, natural peptide induced T cell frequencies that were approximately twofold lower. However, T cells showed superior tumor reactivity because of (i) increased functional avidity for natural antigen and (ii) enhancement of T cell activation and effector function. Thus, novel vaccine formulations comprising potent immune stimulators may allow to circumvent the need for modified antigens and can induce highly functional T cells with precise antigen specificity

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Tumor-reactive, SSX-2-specific CD8+ T cells are selectively expanded during immune responses to antigen-expressing tumors in melanoma patients.

The SSX-2 gene encodes a tumor-specific antigen expressed in neoplasms of various histological types. By analyzing a tumor-infiltrated lymph node of a melanoma patient bearing an SSX-2-expressing tumor, we have recently identified the first SSX-2-derived CD8(+) T-cell epitope, that corresponds to peptide SSX-2(41-49), and is recognized by specific CTL in an HLA-A2 restricted fashion. Here, we have used fluorescent HLA-A2/SSX-2(41-49) peptide multimeric complexes to analyze the response to SSX-2(41-49) in melanoma patients and healthy donors. Multimer(+) CD8(+) T cells were readily detected in the majority of patients bearing SSX-2-expressing tumors and, at lower proportions, in patients with nonexpressing tumors and healthy donors. Importantly, isolated A2/SSX-2(41-49) multimer(+) CD8(+) T cells exhibited a large functional heterogeneity in terms of antigen recognition and tumor reactivity. SSX-2-specific CTLs isolated from tumor-infiltrated lymph node of antigen-expressing patients as well as from the corresponding peripheral blood mononuclear cells exhibited high functional avidity of antigen recognition and efficiently recognized antigen-expressing tumors. In contrast, SSX-2-specific CTLs isolated from patients with undetectable responses in the tumor-infiltrated lymph node, as well as from healthy donors, recognized the antigen with decreased functional avidity and were not tumor reactive. Together, these data indicate that CD8(+) T-cell responses to SSX-2(41-49) frequently occur in SSX-2-expressing melanoma patients and suggest that SSX-2(41-49)-specific CTLs of high avidity and tumor reactivity are selectively expanded during immune responses to SSX-2-expressing tumors in vivo.

Analysis of antiviral immunity in primary and chronic infections

Several evidences in humans underscored the contribution of CD4 and CD8 T-cell responses in controlling viral and bacterial infections. However, CD4 and CD8 Τ cells have distinct and specific effector functions leading to a hierarchical importance in responding to different types of pathogens. In this context, the present work aimed to investigate distinct CD8 T-cell features potentially influencing T-cell efficacy against viral infection. To achieve this-objective, CD8 Τ cells derived from HIV-infected patients and healthy donors harbouring virus-specific immune responses or immunized with an HTV vaccine candidate were studied. In particular, we performed a comprehensive cross-sectional and longitudinal analysis to characterize the function, the phenotype and the functional avidity of HIV-specific CD8 Τ cells during acute (PHI) and chronic infection and, in particular, we investigated immunological parameters potentially associated with the functional avidity of HIV-specific CD8 Τ cells. In addition, we studied the expression pattern of co-inhibitory molecules and the influence of CD 160 on the functions of CD8 Τ cells in absence of chronic infections. From these analyses we observed that the functional avidity of HIV-specific CD8 T- cell responses was significantly lower in acute than in chronic infection, but was not different between chronic progressive and non-progressive patients. Functional avidity remained low after several years of antiretroviral therapy in PHI patients, but increased in patients experiencing a virus rebound following treatment interruption in association with a massive renewal of the global CD8 complementarity-determining region 3 of the TCR. The functional avidity was also directly associated to T-cell exhaustion. In individuals with no sign of HIV or Hepatitis A, Β or C virus infection, CD8 Τ cells expressed higher levels of co-inhibitory molecules than CD4 Τ cells and this was dependent on the stage of T-cell differentiation and activation. The expression of CD 160 impaired the proliferation capacity and IL-2 production of CD8 Τ cells and was reduced upon CD8 T-cell activation, entitling CD 160 as unique marker of CD8 T-cell exhaustion. The CD 160 blockade restored the proliferation capacity of virus-specific CD8 Τ cells providing a potential new target for immunotherapy. All together, these results expand our knowledge regarding the interplay between the immune system and the viruses. - De nombreuses études chez l'Homme ont mis en évidence la contribution des réponses cellulaires Τ CD4 et CD8 dans le contrôle des infections virales et bactériennes. En particulier, les lymphocytes Τ ont différentes fonctions effectrices spécifiques qui leur confèrent un rôle clé lors d'infections par différents pathogènes. Ce travail vise à étudier différentes caractéristiques des cellules Τ CD8 affectant l'efficacité des réponses cellulaires contre les virus. Pour atteindre cet objectif nous avons étudié les cellules Τ CD8 provenant de patients infectés par le VIH et de donneurs sains avec des réponses immunitaires naturelles ou vaccinales contre des virus. Nous avons effectué plusieurs analyses transversales et longitudinales des fonctions, du phénotype et de l'avidité fonctionnelle des lymphocytes Τ CD8 spécifiques au VIH au cours d'infections aiguës et chroniques; en particulier, nous avons étudié les paramètres immunologiques qui pourraient être associés à l'avidité fonctionnelle. De plus, nous avons investigué le profil d'expression des principales molécules co-inhibitrices et en particulier le rôle du CD 160 dans les fonctions des lymphocytes Τ CD8. Sur la base de ces analyses, nous avons constaté que l'avidité fonctionnelle des cellules Τ CD8 spécifiques au VIH était significativement plus faible lors infections aiguës que lors d'infections chroniques, mais n'était, par contre, pas différente entre les patients avec des infections chroniques progressives et non progressives. L'avidité fonctionnelle reste faible après plusieurs années de traitement antirétroviral, mais augmente chez les patients subissant un rebond viral, et donc exposés à des hautes virémies, suite à l'interruption du traitement. Cette augmentation d'avidité des lymphocytes Τ CD8, liée à un épuisement fonctionnel accru, était quantitativement directement associée à un renouvellement massif du TCR. Indépendamment de l'infection par le VIH, les cellules Τ CD8 expriment des niveaux plus élevés de molécules co-inhibitrices (PD-1, 2B4 et CD 160) par rapport aux cellules Τ CD4 et ceci dépend de leur stade de différenciation et d'activation. En particulier, CD 160 semble être un marqueur clé d'épuisement cellulaire des cellules Τ CD8, et donc une nouvelle cible potentielle pour l'immunothérapie, car a) son expression réduit la capacité proliférative et la production d'IL-2 b) CD 160 diminue suite à 1'activation et c) le blocage de CD 160 redonne la capacité proliférative aux cellules Τ CD8 spécifiques aux virus. - Le système immunitaire est un ensemble de cellules, tissus et organes indispensables pour limiter l'entrée des pathogènes à travers la peau et les muqueuses. Parmi les différentes cellules composant le système immunitaire, les cellules Τ CD4 et CD8 sont fondamentales pour le contrôle des infections virales et bactériennes. Les moyens pour combattre les différents pathogènes peuvent être cependant très variables. Les cellules Τ CD8, qui sont indispensables pour la lutte contre les virus, peuvent avoir différents niveaux de sensibilité; les cellules qui répondent à de faibles quantités d'antigène ont une forte sensibilité. Suite à une première infection virale, les cellules Τ CD8 ont une sensibilité plus faible que lors d'expositions répétées au même virus. En effet, la réexposition au pathogène induit une augmentation de sensibilité, grâce au recrutement et/ou à l'expansion de cellules Τ dotées d'une sensibilité plus élevée. Les cellules Τ CD8 avec une plus haute sensibilité semblent être caractérisées par une perte de fonctionnalité (épuisement fonctionnel associé à une haute expression de molécules dites inhibitrices). En absence d'infection, la fonction des molécules inhibitrices n'est pas encore clairement définie. Les cellules Τ CD8 montrent un niveau d'expression plus élevé de ces molécules par rapport aux cellules Τ CD4. Ceci dépend de l'état des cellules. Parmi ces molécules, le CD160 est associé à l'incapacité des cellules à proliférer et à produire de l'IL-2, une protéine importante pour la prolifération et la survie cellulaire. L'incapacité des cellules exprimant le CD 160 à proliférer en réponse à des virus peut être restaurée par le blocage fonctionnel du récepteur CD 160. Cette étude étoffe notre connaissance du rôle des cellules Τ CD8 ainsi que des conséquences induites par leur épuisement fonctionnel. Ces informations sont fondamentales pour le développement de nouvelles stratégies thérapeutiques et vaccinales.