Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Simultaneous CD8+ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine.

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design.

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.

Characterization of tumor antigen specific CD8+ T cells and semi-invariant Vα24/Vβ11 NKT cells in tumor invaded liver

Summary: Detailed knowledge on tumor antigen expression and specific immune cells is required for a rational design of immunotherapy for patients with tumor invaded liver. In this study, we confirmed that Cancer/Testis (CT) tumor-associated antigens are frequently expressed in hepatocellular carcinoma (HCC) and searched for the presence of CD8+ T cells specific for these antigens. In 2/10 HLA-A2+ patients with HCC, we found that MAGE-A10 and/or SSX-2 specific CD8+ T cells naturally responded to the disease, since they were enriched in tumor lesions but not in non-tumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, suggesting that these CTL were selected in vivo for high avidity antigen recognition, providing the rational for specific immunotherapy of HCC, based on immunization with CT antigens such as MAGE-Al 0 and SSX-2. Type 1 NKT cells express an invariant TCR α chain (Vα24.1α18, paired with Vβ11 in human) and share a specific reactivity to αGalactosylceramide (αGC) presented by CD1d. These cells can display paradoxical immuno-regulatory properties including strong anti-tumor effects upon αGC administration in murine models. To understand why NKT cells were not sufficiently protective against tumor development in patients with tumor invaded liver, we characterized the diversity of Vα24/Vβ11 NKT cells in healthy donors (HD) and cancer patients: NKT cells from HD and patients were generally diverse in terms of TCR β chain (Vβ11) variability and NKT cells from HD showed a variable recognition of αGC loaded CD 1 d multimers. Vα24/ Vβ11 NKT cells can be divided in 3 populations, the CD4, DN (CD4-/CD8-) and CD8 NKT cell subsets that show distinct ability of cytokine production. In addition, our functional analysis revealed that DN and CD8 subsets displayed a higher cytolytic potential and a weaker IFNγ release than the CD4 NKT cell subset. NKT cell subsets were variably represented in the blood of HD and cancer patients. However, HD with high NKT cell frequencies displayed an enrichment of the DN and CD8 subsets, and few of them were suggestive of an oligoclonal expansion in vivo. Comparable NKT cell frequencies were found between blood, non-tumoral liver and tumor of patients. In contrast, we identified a gradual enrichment of CD4 NKT cells from blood to the liver and to the tumor, together with a decrease of DN and CD8 NKT cell subsets. Most patient derived NKT cells were unresponsive upon αGalactosylceramide stimulation ex vivo; NKT cells from few patients displayed a weak responsiveness with different cytokine polarization. The NKT cell repertoire was thus different in tumor tissue, suggesting that CD4 NKT cells infiltrating tumors may be detrimental for protection against tumors and instead may favour the tumor growth/recurrence as recently reported in mice. Résumé en français scientifique : Afin de développer le traitement des patients porteurs d'une tumeur dans le foie par immunothérapie, de nouvelles connaissances sont requises concernant l'expression d'antigènes par les tumeurs et les cellules immunitaires spécifiques de ces antigènes. Nous avons vérifié que des antigènes associés aux tumeurs, tels que les antigènes « Cancer-Testis » (CT), sont fréquemment exprimés par le carcinome hepatocéllulaire (CHC). La recherche de lymphocytes T CD8+ spécifiques (CTL) de ces antigènes a révélé que des CTL spécifiques de MAGE-A10 et/ou SSX-2 ont répondu naturellement à la tumeur chez 2/10 patients étudiés. Ces cellules étaient présentes dans les lésions tumorales mais pas dans le foie adjacent. De plus, ces CTL ont démontré une activité cytolytique forte et spécifique contre les cellules tumorales in vitro, ce qui suggère que ces CTL ont été sélectionnés pour une haute avidité de reconnaissance de l'antigène in vivo. Ces données fournissent une base pour l'immunothérapie spécifique du CHC, en proposant de cibler les antigènes CT tels que MAGE-A10 ou SSX-2. Les cellules NKT de type 1 ont une chaîne α de TCR qui est invariante (chez l'homme, Vα24Jα18, apparié avec Vβ11) et reconnaissent spécifiquement l'αGalactosylceramide (αGC) présenté par CD1d. Ces cellules ont des propriétés immuno¬régulatrices qui peuvent être parfois contradictoires et leur activation par l'αGC induit une forte protection anti-tumorale chez la souris: Afin de comprendre pourquoi ces cellules ne sont pas assez protectrices contre le développement des tumeurs dans le foie chez l'homme, nous avons étudié la diversité des cellules NKT Vα24/Vβ11 d'individus sains (IS) et de patients cancéreux. Les cellules NKT peuvent être sous-divisées en 3 populations : Les CD4, DN (CD4- /CD8-) ou CDS, qui ont la capacité de produire des cytokines différentes. Nos analyses fonctionnelles ont aussi révélé que les sous-populations DN et CD8 ont un potentiel cytolytique plus élevé et une production d'IFNγ plus faible que la sous-population CD4. Ces sous-populations sont représentées de manière variable dans le sang des IS ou des patients. Cependant, les IS avec un taux élevé de cellules NKT ont un enrichissement des sous- populations DN ou CDS, et certains suggèrent qu'il s'agit d'une expansion oligo-clonale in vivo. Les patients avaient des fréquences comparables de cellules NKT entre le sang, le foie et la tumeur. Par contre, la sous-population CD4 était progressivement enrichie du sang vers le foie et la tumeur, tandis que les sous-populations DN ou CD8 était perdues. La plupart des cellules NKT des patients ne réagissaient pas lors de stimulation avec l'αGC ex vivo et les cellules NKT de quelques patients répondaient faiblement et avec des polarisations de cytokines différentes. Ces données suggèrent que les cellules NKT CD4, prédominantes dans les tumeurs, sont inefficaces pour la lutte anti-tumorale et pourraient même favoriser la croissance ou la récurrence tumorale. Donc, une mobilisation spécifique des cellules NKT CD4 négatives par immunothérapie pourrait favoriser l'immunité contre des tumeurs chez l'homme. Résumé en français pour un large public Au sein des globules blancs, les lymphocytes T expriment un récepteur (le TCR), qui est propre à chacun d'entre eux et leur permet d'accrocher de manière très spécifique une molécule appelée antigène. Ce TCR est employé par les lymphocytes pour inspecter les antigènes associés avec des molécules présentatrices à la surface des autres cellules. Les lymphocytes T CD8 reconnaissent un fragment de protéine (ou peptide), qui est présenté par une des molécules du Complexe Majeur d'Histocompatibilité de classe I et tuent la cellule qui présente ce peptide. Ils sont ainsi bien adaptés pour éliminer les cellules qui présentent un peptide issu d'un virus quand la cellule est infectée. D'autres cellules T CD8 reconnaissent des peptides comme les antigènes CT, qui sont produits anormalement par les cellules cancéreuses. Nous avons confirmé que les antigènes CT sont fréquemment exprimés par le cancer du foie. Nous avons également identifié des cellules T CD8 spécifiques d'antigènes CT dans la tumeur, mais pas dans le foie normal de 2 patients sur 10. Cela signifie que ces lymphocytes peuvent être naturellement activés contre la tumeur et sont capables de la trouver. De plus les lymphocytes issus d'un patient ont démontré une forte sensibilité pour reconnaître l'antigène et tuent spécifiquement les cellules tumorales. Les antigènes CT représentent donc des cibles intéressantes qui pourront être intégrés dans des vaccins thérapeutiques du cancer du foie. De cette manière, les cellules T CD8 du patient lui-même pourront être induites à détruire de manière spécifique les cellules cancéreuses. Un nouveau type de lymphocytes T a été récemment découvert: les lymphocytes NKT. Quand ils reconnaissent un glycolipide présenté par la molécule CD1d, ils sont capables, de manière encore incomprise, d'initier, d'augmenter, ou à l'inverse d'inhiber la défense immunitaire. Ces cellules NKT ont démontré qu'elles jouent un rôle important dans la défense contre les tumeurs et particulièrement dans le foie des souris. Nous avons étudié les cellules NKT de patients atteints d'une tumeur dans le foie, afin de comprendre pourquoi elles ne sont pas assez protectrice chez l'homme. Les lymphocytes NKT peuvent être sous-divisés en 3 populations: Les CD4, les DN (CD4-/CD8-) et les CD8. Ces 3 classes de NKT peuvent produire différents signaux chimiques appelés cytokines. Contrairement aux cellules NKT DN ou CDS, seules les cellules NKT CD4 sont capables de produire des cytokines qui sont défavorables pour la défense anti-tumorale. Par ailleurs nous avons trouvé que les cellules NKT CD4 tuent moins bien les cellules cancéreuses que les cellules NKT DN ou CD8. L'analyse des cellules NKT, fraîchement extraites du sang, du foie et de la tumeur de patients a révélé que les cellules NKT CD4 sont progressivement enrichies du sang vers le foie et la tumeur. La large prédominance des NKT CD4 à l'intérieur des tumeurs suggère que, chez l'homme, ces cellules sont inappropriées pour la lutte anti-tumorale. Par ailleurs, la plupart des cellules NKT de patients n'étaient pas capables de produire des cytokines après stimulation avec un antigène. Cela explique également pourquoi ces cellules ne protègent pas contre les tumeurs dans le foie.

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.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8⁺ T cells.

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

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.

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX? vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Analysis of viral- and tumor antigen-specific CD4 T cell responses in healthy donors and melanoma patients

Résumé Des tentatives pour développer des traitements anti-cancéreux basés sur l'utilisation d'antigènes tumoraux ont commencé il y a plus de 10 ans. Depuis quelques années, un certain intérêt s'est portée sur une sous-population particulière des cellules du système immunitaire, les lymphocytes T CD4. Ces cellules jouent un rôle central dans les réponses immunitaires tant contre les virus que contre les cellules tumorales. Comme d'autres lymphocytes T, ces cellules sont activées de manière spécifique en reconnaissant un morceau d'antigène, appelé peptide. Ces peptides proviennent soit de protéines des cellules de l'hôte, soit des protéines étrangères (virus ou bactéries) soit de cellules transformées (cellules tumorales) et sont présentés aux lymphocytes T par des molécules du soi appelées CMH (complexe majeur d'histocompatibilité). Dans le cas des lymphocytes T CD4, ces molécules sont plus précisément des molécules du CMH de classe II (CMH II). Mis à part l'intérêt porté aux réponses médiées par les lymphocytes T cytotoxiques, un intérêt croissant pour les lymphocytes T CD4 s'est développé à cause de la place centrale qu'occupent ces cellules dans les réponses immunitaires. L'identification d'épitopes présentés par des molécules du CMH de classe II dérivés d'un grand nombre d'antigènes tumoraux, ainsi que le développement de techniques permettant de suivre les réponses immunitaires, offre des opportunités pour étudier de manière quantitative et qualitative les lymphocytes T CD4 spécifiques pour un antigène particulier chez des patients cancéreux. De plus, ces épitopes permettent d'induire des réponses médiées par les lymphocytes T CD4 et CD8 chez ces mêmes patients. Dans ce travail, notre premier but était de valider l'utilisation de multimères formés par des complexes peptide:molécules de CMH de class II (pCMH II) pour quantifier la réponse des cellules T CD4 dirigée contre l'épitope HA307-319 dérivé de la protéine hémaglutinine du virus de la grippe et présenté par HLA-DRB1*0401. En analysant des échantillons provenant de volontaires sains ayant reçus un vaccin contre la grippe, nous avons pu démontrer une expansion et une activation transitoires des lymphocytes T CD4 spécifiques pour le peptide HA307-319 après vaccination. De plus, les multimères pCMH II nous ont permis d'analyser plus en détails hétérogénéité des cellules T CD4 spécifiques pour le peptide HA307-319 présents dans le sang périphérique d'individus sains. Par la suite, notre but a été d'analyser les réponses des lymphocytes T CD4 spécifiques pour l'antigène Melan-A chez des patients atteints de mélanome métastatique. Nous avons tout d'abord démontré la présence de cellules T CD4 spécifiques pour l'épitope Melan-A51-73, présenté par HLA-DRBl*0401, qui avait déjà été préalablement décrit. Ensuite, nous avons décrit et caractérisé 2 nouveaux peptides issus de Melan-A qui sont présentés aux cellules T CD4 par différentes molécules du CMH de clans II. Des cellules spécifiques pour ces deux épitopes ont été trouvées chez 9/ 16 patients analysés. De plus, des multimères pCMH II chargés avec un des épitopes nous ont permis de détecter ex vivo des lymphocytes T CD4 spécifiques pour Melan-A dans le sang périphérique d'un patient atteint de mélanome. Mis ensemble, tous ces résultats suggèrent une potentielle utilisation des multimères pCMH II pour analyser en détail les lymphocytes T CD4 spécifiques d'antigènes définis. Cependant, le suivi ex vivo de telles cellules ne semble être possible que dans des cas bien particuliers. Néanmoins, les nouveaux épitopes issus de Melan-A et présentés par des molécules du CMH de classe II que nous avons décrits dans cette étude aideront à étudier plus en détails les lymphocytes T CD4 spécifiques pour Melan-A chez des patients atteints de mélanome, un sujet d'étude sur lequel peu de résultats sont à ce jour disponibles. Summary Attempts to develop cancer vaccines based on molecularly defined tumorassociated antigens were initiated more than 10 years ago. Apart from CTLmediated anti-tumor immunity, interests are. now focused on CD4 T cells that are central players of immune responses. The identification of MHC class-II-restricted epitopes from numerous tumor antigens together with the development of monitoring tools offers the opportunity to quantitatively and qualitatively study antigen-specific CD4 T lymphocytes in cancer patients and to induce both CTL and T helper responses in cancer patients. In this work, we first aimed at validating the use of peptide:MHC class II complex (pMHC II) multimers to quantitate the CD4 T cell response against the hemagglutinin-derived epitope HAso~-si9 from influenza virus presented by HLA-DRBl*0401. By analysing samples from healthy volunteers vaccinated with ananti-influenza vaccine, we could demonstrate a transient expansion and activation of HA-specific CD4 T cells after treatment. Moreover, pMHC II multimers helped us to study the heterogeneity of HAspecific CD4 T cells found in peripheral blood of healthy individuals. Then, we aimed to analyse Melan-A-specific CD4 T cell responses in metastatic melanoma patients. We first demonstrated the presence of CD4 T cells specific for the previously described Melan-A51_73 epitope presented by HLA-DRB 1 *0401 in peripheral blood of those patients. Second, we described and characterised 2 new Melan-A-derived peptides that are presented by different MHC II molecules to CD4 T cells. Specific cells for these epitopes were found in 9/ 16 rnelánoma patients analysed. In addition, pMHC II multimers loaded with one of the two epitopes allowed us to detect ex vivo Melan-A-specific CD4 T cells in peripheral blood of a melanoma patient. Together, these results suggest a potential use of pMHC II multimers in analysing in detail antigen-specific CD4 T cells. However, ex vivo monitoring of such cells will be possible only in particular conditions. Nevertheless, the new Melan-A-derived MHC II-restricted epitopes described here will help to study in more detail Melan-A-specific CD4 T cells in melanoma patients, a field where only scarce data are available.