γδ T cells are unconventional T cells that that can recognize infected and transformed cells via their γẟ TCR, thus promoting different immune responses. In addition, several studies showed that γδ T cells are important in the protection against different pathogens in early life, such as human cytomegalovirus (CMV). The diversity of the γδ TCR repertoire is mainly generated in the complementarity determining region 3 (CDR3) where V(D)J recombination takes place. One of the main players in the junctional diversity is the terminal-deoxynucleotidyl-transferase (TdT) enzyme responsible for the random template-independent nucleotide addition at the junction of the joining gene segments.In the mouse model it is established that during development, especially before birth, innate γδ T cell subsets are generated in waves and their generation depends on the type of hematopoietic stem and precursor cells (HSPC). These γδ T cells express a semi-invariant γδ TCR and can acquire a functional program already in the thymus. In human, in contrast, the idea of γδ T cells as innate-like lymphocytes is questioned by recent works showing that the γδ TCR repertoire of human pediatric thymuses and of term-delivery cord blood is highly diverse. Here, by analyzing in detail human fetal and post-natal thymi, we observed striking differences between fetal and post-natal γδ thymocytes at the γδ TCR repertoire and functional level. In contrast to post-natal γδ thymocytes, fetal γδ thymocytes were functionally programmed, expressed low levels of TdT and were highly enriched for invariant/public CMV-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). The rearrangements of these invariant sequences were driven by short-homology repeats at the end of the involved gene segments, as it was observed in the mouse. In addition, we investigated the role of HSPC in the generation of this invariant γδ thymocytes by using an in vitro T cell development system and we showed that only fetal HSPC could generate γδ T cells enriched for the same specific features that were found in the ex-vivo fetal γδ thymocytes. Moreover, we showed that the RNA-binding protein Lin28b, highly expressed in fetal γδ T cells, reprogrammed the term delivery HSPC towards the generation of γδ T cells resembling to their fetal counterpart.In conclusion, we show that the human fetal thymus generates, in a HSPC- and Lin28b-dependent manner, innate invariant γδ T cells with programmed effector functions that might provide protection to the fetus during congenital infections, such as against CMV. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

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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.

Dans le monde, 58 millions de personnes sont chroniquement infectées par le virus de l'hépatite C (VHC). Depuis 2011, l'introduction des antiviraux à action directe a permis la guérison des infections chroniques chez la majorité des sujets traités (~95 %). Toutefois, les traitements sont coûteux et ne protègent pas contre les réinfections, d'où la nécessité de développer un vaccin prophylactique pour freiner efficacement l'épidémie du VHC. Environ 30% des primo-infections sont éliminées spontanément, représentant une occasion unique d'étudier les corrélats de l’immunité protectrice nécessaires pour le développement d’un vaccin efficace. Dans cette thèse, nous avons procédé à la définition des corrélats de l'immunité protectrice au cours des infections par le VHC primaires et subséquentes aux niveaux transcriptomique, clonotypique et fonctionnel à partir d’une cohorte d’utilisateurs de drogues par injection. Le premier objectif était de caractériser le répertoire de récepteurs des cellules T CD8 spécifique de l'épitope immunodominant et cross-réactif NS3 1073-1081 (CINGVCWTV) restreint par HLA-A2 au cours d’une primo-infection aiguë progressant vers une résolution spontanée ou une infection chronique. Nous avons identifié un ensemble de treize clonotypes publics, indépendamment de l'issue de l'infection. Plusieurs clonotypes publics avaient une longue durée de vie après résolution de l’infection et ont proliféré après réinfection par le VHC. En explorant les bases de données publiques, nous avons identifié plusieurs clonotypes partagés avec d'autres épitopes viraux restreints par HLA-A2, mais ils étaient de faible fréquence et de réactivité croisée limitée, suggérant un rôle limité des lymphocytes T CD8 cross-réactifs au cours de l'infection primaire par le VHC. Le deuxième objectif était de caractériser les signatures transcriptomiques longitudinales des cellules mononucléaires du sang périphérique totaux chez huit sujets ayant spontanément résolu deux infections consécutives par le VHC. Nous avons également comparé ces signatures avec un schéma vaccinal composé d'un vecteur à adénovirus de chimpanzé suivi d'un rappel utilisant la vaccine modifiée Ankara, exprimant tout deux les protéines non-structurales du VHC. Nous avons identifié une signature transcriptomique des plasmocytes au cours d'une réinfection aiguë, absente lors de l'infection primaire et après le rappel du vaccin. La résolution spontanée est associée à une expansion rapide des cellules B mémoires spécifiques de la glycoprotéine E2 chez 3 sujets et à une augmentation transitoire des anticorps neutralisants anti- E2 chez 6 sujets. Parallèlement, il y avait une augmentation de l'étendue et de l'ampleur des lymphocytes T spécifiques du VHC chez 7 sujets. En conclusion, nous avons identifié treize clonotypes publics uniques au VHC qui ont proliféré au cours des infections primaire et secondaire. La faible fréquence des clonotypes cross-réactifs suggère qu'ils ne sont pas des déterminants majeurs de l’issue de l’infection. De plus, nous avons observé une augmentation simultanée des réponses des lymphocytes B et T spécifiques du VHC au stade aiguë précoce, suggérant un rôle des deux bras de l’immunité adaptative dans la clairance de la réinfection du VHC. Nos résultats soutiennent l'idée de combiner deux stratégies vaccinales induisant à la fois une immunité à médiation cellulaire et une immunité humorale visant à prévenir les infections chroniques par le VHC. / Worldwide, 58 million individuals are chronically infected with hepatitis C virus (HCV). Since 2011, the introduction of direct acting antivirals enabled the cure of chronic HCV in the majority of treated subjects (~95%). However, direct-acting antivirals treatments are expensive and do not protect against reinfection, urging the need to develop a prophylactic vaccine to efficiently curb the HCV epidemic. Around 30% of acutely infected individuals will spontaneously clear the infection, representing a unique opportunity to study the correlates of immune protection needed to develop a potent vaccine. In this thesis, we proceeded to define the correlates of protective immunity during primary and sub-sequent HCV infections at the transcriptomic, clonotypic and functional levels using longitudinal peripheral blood mononuclear cells samples collected from a cohort of people who inject drugs (PWID). The first aim was to characterize the CD8 T cell receptor repertoire specific to the immunodominant and cross-reactive HLA-A2 restricted NS3 1073-1081 (CINGVCWTV) epitope during acute HCV in PWID progressing to either spontaneous resolution or chronic infection. We identified a set of thirteen public clonotypes in HCV-infected subjects irrespective of infection outcome. Several public clonotypes were long-lived in resolvers and expanded upon reinfection. By mining publicly available data, we identified several TCR clonotypes shared with other HLA-A2 restricted epitopes, but they were of low frequency and limited cross-reactivity, suggesting that they are not major determinants of infectious outcome. The second aim was to characterize longitudinal transcriptomic signatures using total peripheral blood mononuclear cells, as well as T and B cell recall responses in eight subjects who spontaneously resolved two successive episodes of HCV infection. Furthermore, we compared the transcriptomic signatures of primary and secondary resolving HCV infections, with an HCV nonstructural protein vaccine regimen of recombinant chimpanzee adenovirus 3 vector prime followed by modified vaccinia Ankara boost. We identified a plasma cell transcriptomic signature during early acute HCV reinfection that was absent in primary infection and following HCV vaccine boost. Spontaneous resolution of HCV reinfection was associated with rapid expansion of glycoprotein E2-specifc memory B cells in 3 subjects and transient increase in E2-specific neutralizing antibodies in 6 subjects. Concurrently, there was an increase in the breadth and magnitude of HCV-specific T cells in 7 subjects. In conclusion, we identified thirteen new public CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of crossreactive TCRs suggests that they are not major determinants of infectious outcome. Moreover, we observed a concurrent increase of HCV-specific B and T cell responses early during acute HCV reinfection at the transcriptomic and functional levels, suggesting a role for both arms of the adaptive immune response in HCV reinfection clearance. Our results support the combined T and B cell-based vaccine strategy aimed at preventing chronic HCV infections.