La diversité génétique du virus de l'immunodéficience humaine, le VIH-1 responsable de la pandémie du SIDA, représente un challenge dans le développement d'un vaccin qui doit conférer une protection contre différentes formes du virus pour être efficace. L'identification de populations de lymphocytes T CD8+ (CTL) capables de reconnaître des variants peptidiques d'un épitope est donc une étape importante. Dans le modèle singes rhésus, j'ai montré en utilisant des tétramères spécifiques de 9 variants peptidiques d'un épitope qu'une même population de CTL générés par la vaccination, peut reconnaître l'épitope relatif à l'immunogène et un certain nombre de ses variants provenant de diverses formes du VIH-1. Ces études ont également permis de caractériser les populations de CTL spécifiques de chaque variant de cet épitope en analysant l'expression des différents gènes codant pour la chaîne variable β du TCR (Vβ répertoire) et par un large séquençage des régions complémentaires déterminantes 3 (CDR3) du TCRβ. Ces travaux ont montré qu'une vaccination utilisant la séquence du clade C de l'enveloppe du VIH-1 conduit à des réponses divergentes chez 2 singes rhésus Mamu-A*01+. De plus, ces résultats ont mis en évidence que l'usage de certainβes nVe permet pas de déterminer le potentiel cross-réactif des CTL. Par ailleurs, une immunisation utilisant des séquences de l'enveloppe du clade B du VIH-1 peut générer des CTL capables de reconnaître un large nombre de variants de l'épitope testé. L'analyse de 8112 séquences CDR3 du TCRβ a permis de les caractériser. Cependant, les tests fonctionnels ont démontré que bon nombre de ces variants peptidiques stimulent une production suboptimale de cytokines par les CTL générés après vaccination. Ces résultats démontrent que la reconnaissance de variant peptidiques d'un épitope est nécessaire mais pas suffisante pour protéger contre différentes formes du VIH-1 exprimant ces séquences. L'identification de variants peptidiques capables d'induire une réponse fonctionnelle des CTL pourrait contribuer au développement d'un vaccin efficace contre le VIH-1. / The sequence diversity of HIV-1 presents a challenge for the development of an effective vaccine, since such a vaccine must confer protection against diverse forms of the virus. Identifying CD8+ T lymphocytes (CTL) recognizing variants of an epitope sequence is an important step. In the rhesus monkey model, I showed by tetramer binding assays, that the same vaccine elicited CD8+ T lymphocyte populations comparably recognize the epitope relative to the immunogen and a number of variant epitope peptides from diverse forms of HIV-1. During my thesis, I also characterized populations of CD8+ T ymphocytes specific for each variant of the tested epitope by studying their Vβ repertoire and by a large sequencing of the complementarity determining region (CDR3) of the TCRβ. These studies showed that a single clade C env immunization generate CTL with differences in the ability to cross-react to variant epitope in monkeys sharing the same MHC class-Imolecule. Moreover, the data showed that Vβ employed by CTL can not predict the capacity of these CTL to recognize epitopes from diverse HIV-1 isolates. Additionally, I showed that clade B Env immunizations generate populations of CTLrecognizing wild type and a number of variant epitope peptides. However, functional assays demonstrate that many of them stimulate suboptimal cytokine production by the vaccineelicited CTL. These finding demonstrate that the recognition of variant epitope peptides is necessary but not sufficient to protect against different forms of HIV-1. Identifying variant epitopesinducing functional responses of CTL should contribute to the development of an effective vaccine.

<p>Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) V_H genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated V_H genes display superior survival compared to unmutated cases. Biased V_H gene usage has also been reported in CLL which may reflect antigen selection.</p><p>We performed V_H gene analysis in 265 CLL cases and confirmed the prognostic impact of the V_H mutation status. Preferential V_H gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular V_H gene, V_H3-21, displayed poor outcome despite that two-thirds showed mutated V_H genes. Many of the V_H3-21 cases expressed λ light chains, rearranged a V_λ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the V_H3-21 subset comprises an additional CLL entity.</p><p>To further explore the B-cell receptors in CLL, we analyzed the V_H gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted V_L gene usage as in the V_H3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL.</p><p>Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. <2%, 2-5% and >5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. </p><p>A 1513A/C polymorphism in the P2X₇ receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated V_H genes.</p><p> In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.</p>

Recent studies have shown that the somatic mutation status of the immunoglobulin (Ig) VH genes can divide chronic lymphocytic leukemia (CLL) into two prognostic subsets, since cases with mutated VH genes display superior survival compared to unmutated cases. Biased VH gene usage has also been reported in CLL which may reflect antigen selection. We performed VH gene analysis in 265 CLL cases and confirmed the prognostic impact of the VH mutation status. Preferential VH gene usage was also demonstrated in both the mutated and unmutated subset. Interestingly, CLL cases rearranging one particular VH gene, VH3-21, displayed poor outcome despite that two-thirds showed mutated VH genes. Many of the VH3-21 cases expressed λ light chains, rearranged a Vλ2-14 gene, and had homologous complementarity determining region 3s (CDR3s), implying recognition of a common antigen epitope. We believe that the VH3-21 subset comprises an additional CLL entity. To further explore the B-cell receptors in CLL, we analyzed the VH gene rearrangements and, specifically, the heavy chain CDR3 sequences in 346 CLL cases. We identified six new subgroups with similar HCDR3 features and restricted VL gene usage as in the VH3-21-using group. Our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of CLL. Different cutoffs have been suggested to distinguish mutated CLL in addition to the 2% cutoff. Using three levels of somatic mutations, i.e. &lt;2%, 2-5% and &gt;5%, we divided 323 CLLs into subsets with divergent survival. This division revealed a low-mutated subgroup (2-5%) with inferior outcome that would have been masked using the traditional 2% cutoff. A 1513A/C polymorphism in the P2X7 receptor gene was reported to be more frequent in CLL, but no difference in genotype frequencies was revealed in our 170 CLL cases and 200 controls. However, CLL cases with the 1513AC genotype showed superior survival than 1513AA cases and this was in particular confined to CLL with mutated VH genes. In summary, we could define new prognostic subgroups in CLL using Ig gene rearrangement analysis. This also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of CLL.

La diversité génétique du virus de l'immunodéficience humaine, le VIH-1 responsable de la pandémie du SIDA, représente un challenge dans le développement d'un vaccin qui doit conférer une protection contre différentes formes du virus pour être efficace. L'identification de populations de lymphocytes T CD8+ (CTL) capables de reconnaître des variants peptidiques d'un épitope est donc une étape importante. Dans le modèle singes rhésus, j'ai montré en utilisant des tétramères spécifiques de 9 variants peptidiques d'un épitope qu'une même population de CTL générés par la vaccination, peut reconnaître l'épitope relatif à l'immunogène et un certain nombre de ses variants provenant de diverses formes du VIH-1. Ces études ont également permis de caractériser les populations de CTL spécifiques de chaque variant de cet épitope en analysant l'expression des différents gènes codant pour la chaîne variable β du TCR (Vβ répertoire) et par un large séquençage des régions complémentaires déterminantes 3 (CDR3) du TCRβ. Ces travaux ont montré qu'une vaccination utilisant la séquence du clade C de l'enveloppe du VIH-1 conduit à des réponses divergentes chez 2 singes rhésus Mamu-A*01+. De plus, ces résultats ont mis en évidence que l'usage de certainβes nVe permet pas de déterminer le potentiel cross-réactif des CTL. Par ailleurs, une immunisation utilisant des séquences de l'enveloppe du clade B du VIH-1 peut générer des CTL capables de reconnaître un large nombre de variants de l'épitope testé. L'analyse de 8112 séquences CDR3 du TCRβ a permis de les caractériser. Cependant, les tests fonctionnels ont démontré que bon nombre de ces variants peptidiques stimulent une production suboptimale de cytokines par les CTL générés après vaccination. Ces résultats démontrent que la reconnaissance de variant peptidiques d'un épitope est nécessaire mais pas suffisante pour protéger contre différentes formes du VIH-1 exprimant ces séquences. L'identification de variants peptidiques capables d'induire une réponse fonctionnelle des CTL pourrait contribuer au développement d'un vaccin efficace contre le VIH-1.

O diabetes mellitus tipo 1 (DM-1) é uma doença autoimune órgão-específica caracterizada pela destruição seletiva das células pancreáticas produtoras de insulina. A imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (TACTH) constitui uma alternativa terapêutica recente e promissora para o DM-1 recém-diagnosticado, impedindo a progressão da destruição das células pancreáticas produtoras de insulina e induzindo independência insulínica por um período prolongado na maioria dos pacientes. O princípio dessa terapia baseia-se na eliminação das células autorreativas pela imunossupressão intensa e na reconstituição de um sistema imunológico novo e tolerante após o transplante. Com o objetivo de avaliar a função do timo e sua contribuição na geração do repertório de células T nos pacientes com DM-1 após o TACTH, nesse trabalho foram avaliados os níveis de T cell receptor excision circles (TRECs) em células T do sangue periférico e a diversidade do repertório de células T dos pacientes com DM-1 (n=23) antes e em diversos períodos após o transplante. A quantificação absoluta dos níveis de TRECs (número de moléculas de TRECs/100g de DNA) foi realizada pela técnica de PCR em tempo real e a avaliação do repertório de células T foi realizada pela técnica de TCRBV CDR3 Spectratyping. Dentre os vinte e três pacientes, vinte alcançaram a independência insulínica por períodos variáveis de tempo e três não responderam ao tratamento. Não foi observada a restrição do repertório de células T nos pacientes com DM-1 no período pré-transplante, ou seja, quando recém-diagnosticados. Foram identificadas cinco famílias V (7, 18, 19, 20 e 22) em expansão clonal nos pacientes com DM-1. As famílias V 7, 18, 19, 20 apresentaram-se em expansão clonal antes do transplante e se mantiveram com frequência elevada após o transplante, enquanto a família V 22 apresentou aumento da frequência somente nos períodos mais tardios após o transplante. Nos primeiros meses após o transplante, houve redução do número de moléculas de TRECs e restrição do repertório de células T. Contudo, um ano após o transplante, o número de moléculas de TRECs atingiram valores normais e o repertório de células T apresentou-se com ampla diversidade. Nossos resultados mostraram que o TACTH foi capaz de induzir mudanças na composição do repertório de células T dos pacientes com DM-1 após a terapia de IAD/TACTH, evidenciadas por alterações qualitativas e quantitativas dos picos de CDR3 do TCR, sugerindo a reconstituição de um repertório de células T diverso até dois anos pós-transplante. Embora tenha ocorrido reativação da função tímica após o transplante, evidenciada pelo aumento dos níveis de TRECs de um ano e meio a cinco anos pós-transplante, a diversidade do repertório das células T diminuiu a partir de dois anos e meio pós-transplante, sugerindo uma reconstituição tímica de novo de células T naive que expressam preferencialmente algumas cadeias V. Estas evidências imunológicas poderiam explicar a melhora clínica (independência insulínica) temporária observada na maioria dos pacientes após a terapia de IAD/TACTH. / Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterized by insulin-producing pancreatic cell destruction. High-dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) is a recent and promising therapeutic approach for treatment of T1D, preventing the progress of destruction of pancreatic cells and inducing insulin independence for a prolonged period in most patients. The rationale of the AHSCT is based on the elimination of autoreactive cells by the intense immunosuppression and on the reconstitution of a new and tolerant immune system after transplantation. Aiming at assessing the thymic role in the production of new T cell repertoire in T1D patients after AHSCT, in this study was evaluated the levels of T cell receptor excision circles (TRECs) in T cells of peripheral blood as well as the clonality and diversity of T cell repertoire in T1D patients (n=23) before and several periods after transplantation. The absolute quantification of TRECs levels (number of molecules of TRECs/100ng of DNA) was performed by real-time PCR and the analysis of T cell repertoire was performed by TCRBV CDR3 Spectratyping. Among the twenty-three patients, twenty achieved insulin independence for variable periods and three did not respond to the treatment. The T cell repertoire in T1D patients was not restricted in pre-transplantation, i.e., when newly diagnosed. It was identified five V families (7, 18, 19, 20 e 22) in the clonal expansion in T1D patients. The V families 7, 18, 19, 20 were in clonal expansion before transplantation and maintained with high frequency after transplantation, whereas the V 22 family increased its frequency only in the later periods after transplantation. It was observed that the numbers of molecules of TRECs decreased and the T cell repertoire was restricted in the early months after transplantation. However, the levels of TRECs were normalized and the T cell repertoire showed diversity one year after transplantation. Our results indicate that AHSCT was able to induce changes in the composition of the T cell repertoire of patients after AHSCT, evidenced by qualitative and quantitative changes in the composition of T-cell receptor -chain CDR3 peaks, suggesting the reconstitution of diverse T cell repertoire up to two years after transplantation. Although there was reactivation of thymic function after transplantation, as evidenced by increased levels of TRECs from one and a half year to five years after transplantation, the diversity of the T cells repertoire decreased from two and a half years after transplantation, suggesting a reconstruction of new naive T cells that preferentially express some V chains. These immunological evidences could explain the temporary clinical improvement (insulin independence) observed in most patients after IAD / AHSCT therapy.

O diabetes mellitus tipo 1 (DM-1) é uma doença autoimune órgão-específica caracterizada pela destruição seletiva das células pancreáticas produtoras de insulina. A imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (TACTH) constitui uma alternativa terapêutica recente e promissora para o DM-1 recém-diagnosticado, impedindo a progressão da destruição das células pancreáticas produtoras de insulina e induzindo independência insulínica por um período prolongado na maioria dos pacientes. O princípio dessa terapia baseia-se na eliminação das células autorreativas pela imunossupressão intensa e na reconstituição de um sistema imunológico novo e tolerante após o transplante. Com o objetivo de avaliar a função do timo e sua contribuição na geração do repertório de células T nos pacientes com DM-1 após o TACTH, nesse trabalho foram avaliados os níveis de T cell receptor excision circles (TRECs) em células T do sangue periférico e a diversidade do repertório de células T dos pacientes com DM-1 (n=23) antes e em diversos períodos após o transplante. A quantificação absoluta dos níveis de TRECs (número de moléculas de TRECs/100g de DNA) foi realizada pela técnica de PCR em tempo real e a avaliação do repertório de células T foi realizada pela técnica de TCRBV CDR3 Spectratyping. Dentre os vinte e três pacientes, vinte alcançaram a independência insulínica por períodos variáveis de tempo e três não responderam ao tratamento. Não foi observada a restrição do repertório de células T nos pacientes com DM-1 no período pré-transplante, ou seja, quando recém-diagnosticados. Foram identificadas cinco famílias V (7, 18, 19, 20 e 22) em expansão clonal nos pacientes com DM-1. As famílias V 7, 18, 19, 20 apresentaram-se em expansão clonal antes do transplante e se mantiveram com frequência elevada após o transplante, enquanto a família V 22 apresentou aumento da frequência somente nos períodos mais tardios após o transplante. Nos primeiros meses após o transplante, houve redução do número de moléculas de TRECs e restrição do repertório de células T. Contudo, um ano após o transplante, o número de moléculas de TRECs atingiram valores normais e o repertório de células T apresentou-se com ampla diversidade. Nossos resultados mostraram que o TACTH foi capaz de induzir mudanças na composição do repertório de células T dos pacientes com DM-1 após a terapia de IAD/TACTH, evidenciadas por alterações qualitativas e quantitativas dos picos de CDR3 do TCR, sugerindo a reconstituição de um repertório de células T diverso até dois anos pós-transplante. Embora tenha ocorrido reativação da função tímica após o transplante, evidenciada pelo aumento dos níveis de TRECs de um ano e meio a cinco anos pós-transplante, a diversidade do repertório das células T diminuiu a partir de dois anos e meio pós-transplante, sugerindo uma reconstituição tímica de novo de células T naive que expressam preferencialmente algumas cadeias V. Estas evidências imunológicas poderiam explicar a melhora clínica (independência insulínica) temporária observada na maioria dos pacientes após a terapia de IAD/TACTH. / Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterized by insulin-producing pancreatic cell destruction. High-dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) is a recent and promising therapeutic approach for treatment of T1D, preventing the progress of destruction of pancreatic cells and inducing insulin independence for a prolonged period in most patients. The rationale of the AHSCT is based on the elimination of autoreactive cells by the intense immunosuppression and on the reconstitution of a new and tolerant immune system after transplantation. Aiming at assessing the thymic role in the production of new T cell repertoire in T1D patients after AHSCT, in this study was evaluated the levels of T cell receptor excision circles (TRECs) in T cells of peripheral blood as well as the clonality and diversity of T cell repertoire in T1D patients (n=23) before and several periods after transplantation. The absolute quantification of TRECs levels (number of molecules of TRECs/100ng of DNA) was performed by real-time PCR and the analysis of T cell repertoire was performed by TCRBV CDR3 Spectratyping. Among the twenty-three patients, twenty achieved insulin independence for variable periods and three did not respond to the treatment. The T cell repertoire in T1D patients was not restricted in pre-transplantation, i.e., when newly diagnosed. It was identified five V families (7, 18, 19, 20 e 22) in the clonal expansion in T1D patients. The V families 7, 18, 19, 20 were in clonal expansion before transplantation and maintained with high frequency after transplantation, whereas the V 22 family increased its frequency only in the later periods after transplantation. It was observed that the numbers of molecules of TRECs decreased and the T cell repertoire was restricted in the early months after transplantation. However, the levels of TRECs were normalized and the T cell repertoire showed diversity one year after transplantation. Our results indicate that AHSCT was able to induce changes in the composition of the T cell repertoire of patients after AHSCT, evidenced by qualitative and quantitative changes in the composition of T-cell receptor -chain CDR3 peaks, suggesting the reconstitution of diverse T cell repertoire up to two years after transplantation. Although there was reactivation of thymic function after transplantation, as evidenced by increased levels of TRECs from one and a half year to five years after transplantation, the diversity of the T cells repertoire decreased from two and a half years after transplantation, suggesting a reconstruction of new naive T cells that preferentially express some V chains. These immunological evidences could explain the temporary clinical improvement (insulin independence) observed in most patients after IAD / AHSCT therapy.

Utilistions d'anticorps monoclonaux est une partie prometteuse de la thérapie du cancer. À ce jour, il existe plus de 30 anticorps monoclonaux approuvés pour la thérapie contre le cancer. Plus de 350 anticorps se situent également dans différentes phases du développement clinique. La mésothéline est l'une des cibles les plus prometteuses pour l'immunothérapie. La mésothéline est présente à des niveaux relativement faibles dans les cellules mésothéliales de la plèvre, du péritonéum et du péricarde normaux, mais est fortement exprimée dans un certain nombre de cancers différents, y compris les mésothéliomes, le cancer de l'estomac, les carcinomes à cellules squameuses, le cancer de la prostate, le cancer du pancréas, le cancer du poumon et le cancer de l'ovaire. La mésothéline est une glycoprotéine liée au glycosylphosphatidylinositol (GPI) synthétisée sous la forme d'un précurseur de 69 kDa et transformée de façon protéolytique en une forme sécrétée à 30 kDa (anciennement appelée Facteur de potentialisation des mégacaryocytes (MPF)) et une forme liée à la membrane de 40 kDa. Par ailleurs, il peut être clivé par une protéase et peut produire une forme de mésothéline soluble. Il a été déjà montré que cette forme soluble de mésothéline agit comme un ligand et neutralise les anticorps thérapeutiques ciblant la mésothéline. Par conséquent, les anticorps ne pouvaient pas atteindre les cellules cancéreuses et reste inefficaces. Dans notre travail, nous avons décidé de développer un anticorps discriminant spécifique à la forme associée à la membrane pour surmonter l'antagonisme produit par les formes solubles de mésothéline. Pour ce but, nous avons utilisé une nouvelle méthode d'immunisation de souris, que nous avons d'abord toléré la souris avec une mésothéline soluble et ensuite ré-immunisée avec des cellules exprimant la mésothéline. En utilisant la technologie de phage display, nous avons obtenu près de 150 clones de ciblant mésothéline dans 34 familles de VH-CDR3 parmi lesquelles nous avons identifié seulement 2 familles qui se lient à la mésothéline membranaire avec une affinité élevée et ne reconnaissent aucune autre forme soluble de mésothéline. Ici, nous proposons qu'ils puissent être des bons candidats pour être utilisés pour la thérapie contre le cancer de qui permet de passer à travers la barrière de mésothéline soluble. Pour démontrer leur efficacité pour une utilisation thérapeutique, nous avons construit une CAR avec le sc-Fv d'un anticorps discriminant de la forme membranaire. / Antibody based immune treatment is a promising component of cancer therapy. To date there are more than 30 approved monoclonal antibodies for cancer therapy. More than 350 antibodies are also in different phases of clinical development. Mesothelin is one of the most promising targets for immunotherapy. It is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in a number of different cancers, including mesotheliomas, stomach cancers, squamous cell carcinomas, as well as prostate, pancreatic, lung, and ovarian cancers. Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesized as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form (formerly referred to as Megakaryocyte Potentiating Factor (MPF)) and a 40 kDa membrane-bound form. Besides that it can be cleaved by a protease leading to the production of a soluble, shedded, form of mesothelin. It has already been shown that this soluble form of mesothelin acts as a ligand and neutralizes the mesothelin targeting therapeutic antibodies. Therefore antibodies could not reach cancer cells and remained inefficient. In our work we decided to develop discriminating antibodies specific to a membrane associated form so as to overcome the antagonism produced by soluble forms of mesothelin. To this aim we used a novel method of mouse immunization, in which we first tolerized the mouse with soluble mesothelin before immunization with mesothelin expressing cells. By using phage display technology we obtained nearly 150 mesothelin recognizing clones in 34 VH-CDR3 families, among which we identified only 2 families that bind membrane mesothelin with high affinity and do not recognize any other soluble form of mesothelin. Here we suggest that this Fab can be effective candidates to be used for mesothelin expressing cancer therapy being allowed to pass through the soluble mesothelin barrier. To show their efficacy for therapeutic use we constructed a CAR with the sc-Fv of a membrane-form discriminating antibody

Vγ9Vδ2 T cells are a subset of human T lymphocytes activated by phosphoantigens in a T cell receptor-dependent manner to fight microbial invaders or kill transformed cells. Phosphoantigens are low molecular weight nonpeptidic pyrophosphate containing metabolites produced both endogenously (upregulated in transformed cells) and by microbes. Vγ9Vδ2 T cells are the first T cells generated in the foetus and have programmed functions before encountering the post-partum environment.In this PhD thesis, the aim was to assess the origin of Vγ9Vδ2 T cells in early versus adult life and to evaluate their T cell receptor repertoire and effector potential in the neonatal and infant period. First, human Vγ9Vδ2 T cells were characterised coming from foetal blood and generated by the foetal thymus and then similarities and differences with adult blood Vγ9Vδ2 T cells were identified. The data showed that there is a post-natal thymic output of Vγ9Vδ2 T cells which are different from their foetal counterparts. This finding could help guide the development of cancer immunotherapy strategies aiming to improve the resistance and tenacity of Vγ9Vδ2 T cells which enter an exhaustion state after long encounter with the antigen.Furthermore, human Vγ9Vδ2 T cells were studied early after birth regarding their T cell receptor repertoire and function. At 10 weeks after birth, Vγ9Vδ2 T cells had expanded, and a big part of the Vγ9Vδ2 T cell repertoire was foetal-derived. Additionally, Vγ9Vδ2 T cells had undergone significant functional polarisation toward potent killer effector cells. The expansion and shift in effector functions were not influenced by neonatal BCG vaccination, highlighting the role of environmental exposure upon birth. The data gathered here highlight the unique properties of this innate-like lymphocyte population which can act as a first wave of protection in early life while conventional αβ T cells are not yet optimal. Later in life, another wave of Vγ9Vδ2 T cells arrives from the thymus to expand and populate the adult periphery, providing a possible avenue of new and robust cancer cell killers in the scope of immunotherapy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Epstein-Barr Virus (EBV) is a ubiquitous human virus linked to several diseases, including cancers. CD8 T cells are important for controlling EBV replication. Generation and maintenance of virus-specific CD8 T cells is dependent on specific interaction between MHC-peptide complexes on the infected cell and the CD8 T cell receptor (TCR). Several lines of evidence suggest that the TCR repertoire is an essential component of the CD8 T-cell immune response. The current work focuses on delineating the features of the TCR repertoire that drive the selection of EBV-specific CD8 T cells into the memory phase. We used bulk and single-cell TCRαβ sequencing to analyze the TCR repertoire of human CD8 T cells specific for two immunodominant HLA-A02:01-restricted EBV-derived epitopes: BRLF1109-117 (YVLDHLIVV) and BMLF1280-288 (GLCTLVAML) during the acute and memory phases of primary EBV infection in humans. We showed that persistent EBV-specific clonotypes accounted for only 9% of unique clonotypes but were highly expanded in acute EBV infection and more commonly expressed identifiable features than non-persistent clonotypes. The other 91% of highly diverse unique clonotypes disappeared and were replaced in convalescence by equally diverse “de-novo” clonotypes. We provide evidence suggesting that recognition of BRLF1109-117may be driven by the TCRα. We identified a highly dominant and degenerate BRLF1109-117-specific TCRα sequence, AV8.1-CAVKDTDKLIF-AJ34, that was shared by all donors studied and identified conserved residues within this sequence that were important for antigen recognition. These findings are relevant to current efforts to develop or optimize the efficacy of T cell based therapies or vaccines.