La tolérance des lymphocytes TCD8+ est essentielle pour empêcher l'apparition d'auto-immunité mais représente un obstacle pour le développement de réponses cytotoxiques contre les tumeurs. La lymphodéplétion est utilisée comme adjuvant pour l'immunothérapie par transfert adoptif de cellules T cytotoxiques car elle améliore leur efficacité en favorisant la rupture de la tolérance périphérique. En condition lymphopénique aiguë, les lymphocytes T naïfs prolifèrent en absence apparente de stimulation antigénique et vont acquérir un phénotype et une fonctionnalité semblable aux cellules mémoires (LIP pour Lymphopenia Induced Proliferation). Les cellules mémoires ayant un seuil d'activation inférieur cellules naïves, il a été proposé que la différentiation des cellules TCD8+ potentiellement autoréactives en cellules de type mémoire dans des conditions lymphopéniques pouvait conduire à la rupture de la tolérance. Pendant ma thèse, j'ai étudié si la LIP est nécessaire pour la rupture de la tolérance croisée des cellules TCD8+ chez des souris transgéniques irradiées exprimant un antigène modèle dans les cellules β du pancréas. De manière surprenante, nous avons constaté que le blocage de la LIP ne permet pas d'inhiber l'apparition d'auto-réactivité : les lymphocytes TCD8+ qui ne transitent pas par un stade de différenciation de type mémoire parviennent à se différencier en cellules effectrices suite à la présentation croisée de l'antigène et à migrer vers le pancréas. Néanmoins, la LIP est requise pour induire de l'auto-réactivité lorsque la fréquence de lymphocytes T CD8+ est faible ; non pas à cause du déséquilibre de la population de cellules T régulatrices, mais dû à une nette augmentation du nombre de cellules TCD8+ autoréactives. Ainsi, bien que la LIP améliore les réponses auto- réactives des cellules TCD8+, la différentiation en cellules de type mémoire n'est pas indispensable pour la rupture de la tolérance croisée en condition lymphopénique induite par irradiation. / The immune system has evolved multiple mechanisms of peripheral tolerance to control CD8+ T cell responses and to prevent autoimmunity. However, they also represent a barrier for the development of cytotoxic responses against tumors. Lymphodepleting protocols are currently used as adjuvants for adoptive cytotoxic T cell immunotherapy because they enhance their potency. These protocols are thought to promote the breakdown of peripheral CD8+ T cell tolerance. Under acute lymphopenic conditions, naive T cells proliferate, in the apparent absence of antigenic stimulation at least in part due to a greater availability of the cytokine IL-7. Proliferating CD8+ T cells acquire a phenotype and functionality that is similar to memory cells and are termed memory-like cells. Since memory cells have a lower activation threshold than naïve cells, it has been proposed that differentiation of potentially autoreactive CD8+ T cells into memory-like cells under lymphopenic conditions could drive the breakdown of peripheral tolerance. Here we studied whether lymphopenia induced proliferation and differentiation are required for the breakdown of CD8+ T cell cross-tolerance in irradiated transgenic mice expressing a model antigen in the beta cells of the pancreas. Surprisingly, we found that blocking lymphopenia-induced proliferation and differentiation into memory-like cells did not prevent self-reactivity. CD8+ T cells that did not differentiate into memory-like cells still became effectors upon antigen cross-presentation and migrated to the site of antigen expression. Nonetheless, LIP did enhance CD8+ T cell mediated self-reactivity at low T cell frequencies. This effect could not be explained by a Treg imbalance but by a net increase in autoreactive CD8+ T cell numbers. Thus, although LIP enhances CD8+ T cell anti-self responses, differentiation into memory-like cells is not essential for the breakdown of cross-tolerance under the lymphopenic conditions provided by irradiation.

The maturation of cytotoxic T lymphocyte (CTL) effectors from CTL precursors (CTLp) requires specific signals mediated through cellular interactions and soluble factors. The most studied factor is T cell growth factor (TCGF) which is also termed interleukin-2 (IL-2). This lymphokine is produced by T helper cells (TH) and induces activated CTLp to proliferate and differentiate. However, in the absence of mitogen or antigen stimulation, IL-2 alone cannot induce CTL (except in the case of very high cell density). A factor is described that is found in the supernatant of 4-β-phorbol-12-myristate-13-acetate (PMA)-induced EL4 cells that can polyclonally activate CTL in the presence of IL-2. This factor elutes at 27 kilodaltons (KDa) on a G-100 column, and its target cell includes T cells of the Thyl⁺ Lyt2⁺ L3T4⁻ phenotype. The factor increases the frequency of IL-2 receptor expressing cells within a population, thereby increasing the response to IL-2. It is suggested that this factor acts through an alternative pathway of CTL activation which is independent of specific stimulation by antigen. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

When injected with live P815 tumour cells, DBA/2 mice developed cytotoxic cells reactive to the tumour. In addition, T helper cells from tumour-bearing mice enhanced the iri vitro generation of cytotoxic cells from normal DBA/2 spleen cells. The helper cells had the following properties (1) expression of the Thy-1.2 antigen; (2) resistance to y-radiation; (3) specific enhancement of the cytotoxic response to P815; (4) detectability in P815-bearing mice at the peak of cytotoxic cell activity; (5) activity in the early phase of cytotoxic cell activation. In parallel to the development of helper cell activity, suppressor cells were generated which suppressed the cytotoxic response to P815. These suppressor cells were removed by pre-treating mice with low doses of cyclophosphamide. High doses of cyclophosphamide reduced the cytotoxic response to both P815 and C57B1/6 alloantigens. Cyclophosphamide treatment reduced the frequency of cytotoxic precursor cells directed against P815, and an antigen-reactive helper cell involved in interleukin 2 production. Both interleukin 2 and thymocytes from P815-primed mice, restored the cytotoxic response against P815, to normal levels. Twenty six percent of animals primed with tumour cells cleared a challenge dose of P815 faster than unprimed control mice. Of these, 88% survived longer than the control animals. Eighteen percent of the recipients of cells from tumour-primed mice, cleared a challenge dose of P815 faster than mice injected with normal cells. Of these 53% survived significantly longer than control groups given either normal cells or no cells at all. Cells from mice primed to PPD showed significantly enhanced proliferative responses to soluble and P815-bound PPD, when compared with unprimed animals. However, cells from only a few PPD-primed mice showed enhanced cytotoxicity against P815 tumour cells, and PPD-primed cells either did not alter, or suppressed, the cytotoxic response of normal DBA/2 spleen cells, when stimulated with PPD-coated P815. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

O objetivo do estudo foi investigar o construto da tarefa de múltiplas acelerações a partir de parâmetros biomecânicos de saltos verticais. 19 jogadoras da seleção brasileira adulta de basquetebol feminino realizaram o teste de múltiplas acelerações (teste T), salto vertical com contramovimento (SV) e salto vertical com contramovimento precedido de corrida (SVcorrida). Os parâmetros biomecânicos do SV foram, tempo de fase concêntrica (Tcon), pico de força de propulsão (PFP), tempo decorrido entre início da fase concêntrica e o pico de força de propulsão (TPFP), taxa de desenvolvimento de força (TDF) e impulso (I); para SVcorrida, velocidade média de aproximação (Velmédia), tempo total do movimento (Ttotal), pico de força passiva (PFPa), tempo decorrido entre início da fase de apoio e pico de força passiva (TPFPa), pico de força de propulsão (PFP), tempo decorrido entre início da fase de apoio e pico de força de propulsão (TPFPp), Load Rate (LR) e taxa de desenvolvimento de força (TDF). Para análise dos dados, inicialmente, recorreu-se a análise de componentes principais, revelando que 61,3% da variância total do SV foi explicada pelo componente predominantemente temporal (Tcon, TPFP e PFP) e 20,7% pelo componente predominantemente cinético (I e TDF); para SVcorrida, 58,7% da variância total foi explicada pelo componente predominantemente temporal (Ttotal, TPFPa, TPFPp, LR e TDF), 15,4% pelo componente predominantemente cinético (PFPa e PFP) e 12,8% pelo componente velocidade (Velmédia). Adicionalmente, foram reveladas correlações significantes de 0,55 (Tcon), -0,54 (Velmédia) e -0,49 (PFP) com o teste T, evidenciando um construto determinado pela capacidade de se gerar elevada força na fase concêntrica, reduzir o tempo de fase concêntrica e se aproximar do ponto de mudança de direção com elevada velocidade. / The main aim of the study was to investigate the multiple-acceleration task construct from vertical jumps biomechanics parameters. Nineteen female basketball players from Brazilian national team performed the multiple-acceleration test (T test), countermovement jump (VJ) and countermovement jump preceded by run (VJr). The VJ biomechanics parameters observed were: concentric phase duration (CPD), propulsion peak force (PPF), time to reach propulsion peak force (TPPF), rate of force development (RFD) and impulse (I); for VJr the parameters were mean velocity (MV), movement total duration (MTD), passive peak force (PaPF), time to reach passive peak force (TPaPF), propulsion peak force (PPF), time to reach propulsion peak force (TPpPF), load rate (LR) and rate of force development (RFD). Data were first analyzed by principal component analysis. The results showed that 61.3% of VJ variance could be explained by time factor (CPD, TPPF and PPF), and 20.7% by kinetic factor (I and RFD). To VJr, 58.7% of total variance could be explained by time factor (MTD, TPaPF, TPpPF, LR, RFD) and 15.4% by kinetic factor (PPF and PaPF) and 12.8% by velocity factor (MV). Furthermore, significant correlations were found between T test with CPD, MV and PPF (0.55, -0.54 and -0.49, respectively), revealing a T test construct related to the ability of generating high level of force in concentric phase, diminishing time of concentric phase, and to reach the change direction point with greatest possible speed.

As células T regulatórias (Tregs) são essenciais para a manutenção da tolerância periférica, prevenção de doenças autoimunes e limitantes nas doenças inflamatórias crônicas. Além disso, essas células exercem um papel fundamental no controle da rejeição de transplantes. Diferentes protocolos mostraram que é possível obter Tregs a partir de células T naive CD4+ in vitro. Para tal, é consenso que o TGF-? e a interleucina-2 (IL-2) são capazes de direcionar as células T naive CD4+ a se tornarem regulatórias após um estímulo antigênico (anti-CD3/CD28). Nosso grupo recentemente notou que, durante a imunomodulação de linfócitos T pelas células estromais mesenquimais (CTMs), estas eram capazes de produzir adenosina que, por sua vez, participa do processo de imunorregulação. Outros trabalhos indicam que as CTMs suprimem a proliferação dos linfócitos T pela geração de Tregs e que as CTMs induzem a geração de Tregs através da regulação negativa da via TCR e da via AKTmTOR. Evidências apontam que a adenosina pode atuar regulando negativamente a via mTOR. Portanto, acredita-se que a adenosina possa participar do processo de geração de Tregs através da modulação da via mTOR. Além disso, estudos recentes indicam que a ativação de receptores de adenosina, mais especificamente A2a, com agentes agonistas, leva ao aumento da produção de células Tregs, enquanto que a utilização de agentes antagonistas destes receptores leva à diminuição da diferenciação de Tregs. Porém, estes estudos mostram a geração de Tregs a partir de células T naive de camundongos. Visto a grande importância das Tregs no contexto imunológico, a produção eficiente de Tregs in vitro tem importância fundamental para o desenvolvimento de novos protocolos terapêuticos para o tratamento de doenças autoimunes e no combate à rejeição de transplantes. Assim, o objetivo central deste trabalho foi avaliar a participação de agonistas e antagonistas de receptores de adenosina na indução de células T regulatórias geradas in vitro (iTreg) pela ativação de células T CD4+ naive isoladas de sangue de cordão umbilical (SCU) humano. Para isso, células mononucleares foram isoladas de bolsas de SCU e as células T naive foram isoladas imunomagnéticamente. Essas células foram ativadas com beads ligadas a anticorpos anti-CD2/CD3/CD28 e cultivadas por cinco dias na presença de IL-2 e diferentes concentrações de drogas agonistas e antagonistas de receptores de adenosina. Em seguida, foram avaliados os principais marcadores de células T regulatorias por meio de citometria de fluxo e o meio de cultura foi coletado ao final da geração para quantificação de citocinas. Além disso, o RNA total foi extraído de todas as condições de cultivo para a análise da expressão de genes envolvidos na geração e desenvolvimento das Tregs, por PCR quantitativo. O potencial de supressão de células T efetoras também foi avaliado. / Regulatory T cells (Tregs) are essential for the maintenance of peripheral tolerance, prevention of autoimmune and limiting diseases in chronic inflammatory diseases. In addition, these cells play a key role in the control of transplant rejection. Different protocols have shown that it is possible to obtain Tregs from naive CD4+ T cells in vitro. To this end, there is consensus that TGF-? and interleukin-2 (IL-2) are capable of directing the naive CD4 + T cells to become regulatory following an antigenic stimulus (anti-CD3/CD28).. Our group recently noted that during the immunomodulation of T lymphocytes by mesenchymal stromal cells (MSCs), they were able to produce adenosine which in turn participates in the immunoregulation process. Other studies indicate that MSCs suppress the proliferation of T lymphocytes by generation of Tregs and that MSCs induce generation of Tregs by downregulation of the TCR pathway and the AKT-mTOR pathway. Evidence indicates that adenosine may act by downregulating the mTOR pathway. Therefore, it is believed that adenosine may participate in the generation of Tregs by modulating the mTOR pathway. In addition, recent studies indicate that activation of adenosine receptors, more specifically A2a, with agonist agents, leads to increased production of Treg cells, whereas the use of antagonistic agents of these receptors leads to a decrease in Treg differentiation.. However, these studies show the generation of Tregs from naive T cells of mice. In view of the great importance of Tregs in the immunological context, the efficient production of Tregs in vitro is of fundamental importance for the development of new therapeutic protocols for the treatment of autoimmune diseases and in the fight against transplant rejection. Thus, the central objective of this study was to evaluate the participation of adenosine receptor agonists and antagonists in induction of regulatory T cells generated in vitro (iTreg) by the activation of naive CD4+ T cells isolated from human umbilical cord blood (SCU). For this, mononuclear cells were isolated from SCU and naive T cells were immunomagnetic isolated. These cells were activated with beads bound to anti-CD2/CD3/CD28 antibodies and cultured for five days in the presence of IL-2 and different concentrations of agonist drugs and antagonists of adenosine receptors. Next, the major regulatory T-cell markers were assessed by flow cytometry and the culture medium was collected at the end of the generation for quantification of cytokines. In addition, total RNA was extracted from all culture conditions for the analysis of the expression of genes involved in the generation and development of Tregs by quantitative PCR. The potential for suppression of effector T cells was also evaluated.

Les cellules lymphoïdes innées de groupe 3 (ILC3) contribuent activement à l’homéostasie intestinale par leur production d’Interleukin-22 (IL-22). Ces ILC3 regroupent 2 sous-populations majeures, les LTi (« Lymphoid Tissue inducer »), caractérisées par l’expression du récepteur au chimiokine CCR6, et les ILC3 exprimant le facteur de transcription (FT) T-bet, qui comprennent une population positive pour le marqueur de surface NKp46, récepteur originalement utilisé pour identifier les ILC de groupe 1 (ILC1). Les ILC1 jouent un rôle prépondérant dans la réponse aux pathogènes intracellulaires et anti-tumorale. Jusqu’à présent, trois populations majeures composent les ILC1 : les lymphocytes cytotoxiques Natural Killer (NK ou ILC1b), qui dépendent largement du FT Eomes et expriment l’intégrine CD49b ; les ILC1 hépatiques et intestinaux, qui dépendent du FT T-bet et expriment CD49a (ILC1a) ; et une population CD49a+ et DX5+ indépendante du FT Nfil3 localisée dans les glandes salivaires ou l’utérus (ILC1ab). Mes travaux visent à comprendre la biologie des ILC exprimant NKp46, ainsi que les facteurs impliqués dans leur développement, leur maturation et leur fonction. La majeure partie de ma thèse se concentre sur les NKp46+ ILC3. Premièrement, nous démontrons un rôle majeur pour le récepteur aux chimiokine CXCR6 dans la localisation des NKp46+ ILC3 dans les villi de la lamina propria intestinale (Satoh-Takayama et al. 2014). Deuxièmement, j’ai mis en évidence que NKp46+ ILC3 pouvait perdre l’expression de NKp46 (Verrier et al. 2016). Déclenchée par le TGFβ, cette perte d’expression est associée à une plus forte capacité à produire de l’IL-22, mais aussi à l’acquisition de marqueurs identifiant les LTi (CCR6, MHC-II), démontrant ainsi la plasticité des NKp46+ ILC3. Enfin, en collaboration avec le groupe de Rachel Golub, nous avons confirmé le rôle présumé de la molécule Notch dans cette plasticité (Chea et al. 2016). Dans ce manuscrit, je discuterai du développement et de l’hétérogénéité des ILC3, ILC1a, ILC1b et ILC1ab. L’ensemble de mes résultats soutient une vision dynamique de la biologie des ILC reflétant l’adaptation de ces cellules effectrices face à leur environnement. En caractérisant les différents acteurs impliqués dans ce processus dynamique, mes travaux pourront servir au développement de thérapies visant à contrôler l’équilibre entre ces différentes populations dans divers pathologies comme le cancer, les infections virales, ou encore les maladies intestinales / Group 3 Innate Lymphoid cells (ILC3) actively maintain mucosal homeostasis through the production of Interleukin-22 (IL-22). ILC3 encompass 2 major populations, LTi (« Lymphoid Tissue inducer »), characterized by the expression of the chemokine receptor CCR6, and ILC3 that express the transcription factor T-bet, which include a population expressing the surface marker NKp46, a receptor originally used to identify group 1 ILC (ILC1). ILC1 plays a major role in the defense against intracellular pathogens and anti-tumoral responses. Three major ILC1 populations have been identified: the cytotoxic lymphocytes « Natural Killer » (NK or ILC1b), which largely rely for on the transcription factor Eomes their generation and express the integrin CD49b; hepatic and intestinal ILC1 that depends on the T-bet transcription factor and express CD49a (ILC1a); and a population that expresses CD49a and CD49b (ILC1ab) and populates the salivary gland and the uterus, which is independent of the transcription factor Nfil3. My work aimed to understand the biology of NKp46 expressing ILC, as well as factor involved in their development, maturation and function. The major part of my work focuses on NKp46+ ILC3. First, we demonstrate a major role for the chemokine receptor CXCR6 in their localisation in the lamina propria villi (Satoh-Takayama et al. 2014). Second, I showed that NKp46+ ILC3 could lose NKp46 expression (Verrier et al. 2016). Induced by TGFβ, this loss of expression was associated with higher IL-22 production and by the acquisition of markers identifying LTi (CCR6, MHC-II), demonstrating NKp46+ ILC3 plasticity. Finally, in collaboration with Rachel Golub’s group, we confirmed a putative role for Notch-signaling in this plasticity (Chea et al. 2016). In this manuscript, I will discuss the development and the heterogeneity of ILC3, ILC1a, ILC1b and ILC1ab. All the results I generated support a dynamic vision of ILC biology, which reflects how they adapt in response to environmental cues. By characterizing the different actors involved in this dynamic process, my work could be used to design therapies aiming at controlling the equilibrium between these different populations in diverse pathologies such as cancer, viral infection, or intestinal diseases

Les primates non-humains (PNH) sont un important réservoir de pathogènes et notamment de rétrovirus. Plusieurs agents infectieux ont émergé dans la population humaine à partir de ce réservoir animal, comme par exemple le virus de l’immunodéficience humaine ou le virus T lymphotrope humain (HTLV) de type 1 qui se sont répandus mondialement et causent de graves pathologies. L’émergence de rétrovirus chez l’Homme nécessite plusieurs étapes passant par la transmission primaire du virus du PNH à l’Homme, la persistance du virus dans l’organisme,la transmission secondaire inter-humaine et enfin sa diffusion dans la population. Mes deux projets de thèse ont porté sur l’étude de deux rétrovirus qui ont un potentiel d’émergence chez l’Homme, le virus foamy simien (SFV) et le virus T-lymphotrope de type 4, dans des cohortes d’individus à risque vivant au Cameroun et au Gabon. Les SFV sont des rétrovirus ubiquitaires chez de nombreux PNH. Plus d’une centaine de cas d’infection d’humains par des SFV ont été observés à ce jour, l’origine étant un contact(principalement par morsure) avec un PNH. L’infection est chronique et semble asymptomatique.De plus, aucune transmission secondaire n’a été détectée à ce jour. Le laboratoire a pu isoler, chez deux individus camerounais, deux souches de SFV de gorille et a constaté une forte variabilité génétique au niveau du gène d’enveloppe. Nous avons donc étudié la variabilité du gène d’enveloppe de SFV de gorille mais également de chimpanzé infectant une soixantaine de chasseurs camerounais et gabonais et une trentaine de PNH. Nous avons pu mettre en évidence la co-circulation de souches de SFV présentant des variants moléculaires du gène d’enveloppe différents chez les gorilles et les chimpanzés. Ces mêmes souches peuvent être transmises à l’Homme à l’occasion de morsures, les deux variants pouvant être transmis simultanément. Ces variants diffèrent au niveau d’une région de 753 pb située dans la région codant la glycoprotéine de surface, au niveau du domaine de liaison au récepteur. Ils pourraient ainsi avoir des propriétés fonctionnelles différentes, notamment au niveau de l’élicitation de la réponse immunitaire de l’hôte. Ces variants sont potentiellement issus d’événements de recombinaison.HTLV-4 a été détecté chez un unique individu, un chasseur camerounais, aujourd’hui décédé.En 2014, le réservoir simien a été identifié comme étant les gorilles. Nous avons recherché la présence de HTLV-4 chez 300 individus camerounais et gabonais qui avaient été mordus par un PNH. Nous avons identifié deux chasseurs gabonais infectés par HTLV-4, qui avaient été mordus par des gorilles 9 à 15 ans avant d’être prélevés. Nous confirmons donc la présence de HTLV-4 infectant de manière persistante des humains et étendons sa répartition au Gabon. Nous suggérons très fortement une origine zoonotique de ces infections. L’une des souches isolées est divergente par rapport aux souches déjà connues et permet donc de définir le sous-type B deHTLV-4.Ces travaux confortent la notion que les PNH, et notamment les gorilles, sont une source importante d’agents infectieux pour l’Homme. Des études supplémentaires seront nécessaires pour mieux caractériser l’infection chez l’Homme et notamment l’éventuelle pathogénicité et transmissibilité de ces deux virus. / Non-human primates (NHPs) are an important reservoir of pathogens, including retroviruses. Several retroviruses have emerged in human population from NHP reservoir, like the human immunodeficiency virus and the human T-lymphotropic virus type 1 who have spread globally and are the causative agents of serious pathologies. During my PhD, I interested in two retroviruses who have an emerging potential in human population, the simian foamy virus (SFV) and the human T-lymphotropic virus type 4 (HTLV-4), in cohorts of individuals at risk in Central Africa. SFV are retroviruses ubiquituous in NHPs. A hundred of human infections with SFV are known, originating from contacts with NHP. The infection is chronic, asymptomatic although no secondary transmission has been observed yet. We showed that two envelope molecular variants of SFV are co-circulating in gorilla and chimpanzee populations. These strains can be transmitted to humans through bites. The variants differ in the receptor binding domain on the envelope and could have different functional properties. HTLV-4 had been detected in a single individual (a cameroonese hunter) and the simian reservoir idenfied as gorillas. We have detected two gabonese hunters infected with HTLV-4, who had been bitten by gorillas. Then we confirm the presence of HTLV-4 in humans in Central Africa. One of the strains is divergent and defines the prototype of a new subtype of HTLV-4

Le mélanome est un cancer de la peau dont l’incidence est en continuelle augmentation dans le monde entier. Des progrès récents dans la compréhension des mécanismes cellulaires complexes régulant l’immunité du cancer ont conduit à l’élaboration de nouvelles stratégies visant des checkpoints spécifiques de la régulation des réponses immunes. Ipilimumab est un anticorps thérapeutique dirigé contre la molécule CTLA-4. Il a permis, chez les patients atteints de mélanome métastatique, d’augmenter la survie globale et a fait l’objet d’une approbation de la FDA en 2011. Cependant cette thérapie ne semble bénéficier qu’à un nombre restreint de patients et souvent de manière retardée. L’identification de marqueurs immunologiques précoces associés à la réponse clinique et à la survie s’avère donc être nécessaire afin d’apporter des éléments d’orientation. Dans ce contexte, nous avons suivi de manière longitudinale, prospective et retrospective une cohorte de 77 patients atteints de mélanome métastatique. Nous nous sommes intéressés dans un premier temps aux molécules sériques reflétant soit l’importance de l’envahissement tumoral (LDH, S100B et MIA) soit l’implication de mécanismes d’échappement immunitaire (MICA soluble et anticorps anti-MICA). Nous montrons une association entre des faibles concentrations sériques de LDH et S100B, soutenues après première et deuxième dose d’ipilimumab, et la réponse au traitement et la survie. De plus, des niveaux élevés de MICA solubles avant le traitement sont associés à une fréquence plus faible de complications à type d’auto-immunité. Nous avons de plus suivi les populations lymphocytaires avec une attention particulière portée sur les compartiments naïf et mémoires T, les facteurs de transcription impliqués dans la différentiation des lymphocytes T mémoires, les cytokines produites et les récepteurs de chimiokines de lymphocytes T. Nos résultats montrent que le taux de lymphocytes avant l’introduction du traitement est un facteur prédictif de meilleure survie et de réponse positive à la semaine 16, indépendamment du taux de LDH et de la corticothérapie. De plus, un effet global de l'ipilimumab sur l'expansion des cellules T mémoires classiques a été observé, associé à la réponse au traitement. En revanche, les fréquences des cellules souches T mémoires (TSCM) récemment décrites diminuent malgré une augmentation de la prolifération, suggérant un processus de différenciation. L'ipilimumab induit aussi l'expansion de lymphocytes T exprimant CXCR3, CCR4 et CCR6, permettant une migration vers la peau et les tissus inflammés, avec augmentation de leur capacité à produire des cytokines effectrices. Une augmentation précoce des cellules T CD8+ exprimant le facteur de transcription Eomes s’est révélée être associée à un contrôle de la maladie. Enfin, et sur la base des résultats précédents, nous avons étudié la capacité des lymphocytes T mémoires de patients à proliférer après stimulation in vitro. Contrairement aux sujets sains, les patients présentent un défaut dans la capacité de prolifération des TSCM qui pourrait être liée à un défaut dans la régulation d’Eomes et Ki-67. Nous proposons ainsi un modèle permettant de suivre de manière précoce les étapes conduisant à la différentiation des TSCM en sous populations mémoires, associées à une réponse clinique sous ipilimumab, et impliquant le facteur de transcription Eomes. / Melanoma is a skin cancer with incidence increasing at dramatic rates worldwide. Ipilimumab, an anti-CTLA-4 therapy developed in the view of counter-balancing the inhibitory role of CTLA-4 in T lymphocytes, was the first immune checkpoint inhibitor demonstrating to extend overall survival in patients with metastatic melanoma, with FDA approval in 2011. However, biomarkers allowing the identification of the subset of patients that will more likely benefit from this immunotherapy or that may allow a good monitoring of patient clinical management during treatment are lacking. The principal objective of this work was to identify potential and early predictive biomarkers of ipilimumab response and/or survival in a cohort of 77 metastatic melanoma patients. Firstly, serum levels of melanoma markers such as LDH, S100B and soluble MICA (and its counter-part anti-MICA antibody), tumour markers associated with tumour development and/or immune escape, were assessed. A correlation between lower baseline levels of LDH and S100B, sustained after the first and second doses of ipilimumab, and treatment response and survival was observed, suggesting their potential utility in treatment monitoring. In addition, higher baseline levels of soluble MICA were found to be associated with a less frequency of immune-related adverse events, which might provide important information for the management of frequent ipilimumab-related adverse events. Secondly, immune markers with a special focus on transcription factors, cytokine secretion and chemokine receptors of T lymphocytes and memory T subsets were assessed. An association between baseline absolute lymphocyte counts and extended overall survival as well as better treatment response was found. In addition, a global effect of ipilimumab on the expansion of conventional memory T cells was observed, which was associated with treatment response. By contrast, frequencies of the recently described stem-cell memory T cells were shown to decrease despite increased proliferation, suggesting a process of differentiation. Additionally, ipilimumab induced the expansion of CXCR3, CCR4 and CCR6-expressing T lymphocytes and effector cytokines secretion capacity. Early increased levels of Eomes-expressing CD8+ T cells were found to be associated with disease control. Lastly, and based on the previous results, we investigated the ability of patients’ memory T cells to proliferate under in vitro stimulation. We found that, in contrast to healthy subjects, patients possess a defect in the ability of stem-cell memory T cells expansion in vitro, that might be related to a defect in Eomes and Ki-67 regulation.

Le myélome multiple (MM) est une hémopathie maligne, caractérisée par la prolifération au niveau de la moelle osseuse de plasmocytes tumoraux sécrétant le plus souvent une immunoglobuline monoclonale. Malgré de récents progrès thérapeutiques, cette maladie reste aujourd’hui incurable avec une survie médiane d’environ 6 ans. Cependant, il existe une grande hétérogénéité pronostique parmi les patients qui est associée à différentes anomalies génétiques. Parmi celles-ci, la translocation t(4;14) (p16;q32), retrouvée dans 15% des MM, définit l’une des formes de MM actuellement la plus grave. Malgré le rôle potentiel des deux oncogènes FGFR3 et MMSET qu’elle dérégule, les mécanismes moléculaires permettant d’expliquer la gravité des MM avec t(4;14) ne sont pas clairement élucidés. En mettant en œuvre des approches de biologie moléculaire, de séquençage à haut débit et de tests fonctionnels sur une très large série de MM t(4;14), mes travaux ont cherché à identifier les événements génétiques associés au mauvais pronostic de ce sous-groupe de malades. Les résultats montrent l’implication du point de cassure au sein du gène MMSET dans le pronostic des patients. Ils ont également permis de définir le paysage particulier des mutations génétiques affectant les MM t(4;14) en mettent en évidence les fréquences relativement élevées des altérations dans les gènes ATM/ATR et PRKD2. Enfin, ils ont identifiés PKD2 comme une cible thérapeutique et démontré qu’un inhibiteur des PKDs, kb NB 142-70, bloquait la prolifération des cellules de MM in vitro. L’ensemble de ces résultats jette un éclairage nouveau sur la physiopathologie des MM t(4;14) et ouvre la voie vers de nouvelles approches thérapeutiques. / Multiple myeloma (MM) is a hematological malignancy characterized by the proliferationof plasma cells in the bone marrow usually secreting a monoclonal immunoglobulin. Despite recenttherapeutic advances, the disease remains incurable, with a median survival of approximately 6years. However, there is considerable clinical heterogeneity between patients, with differentoutcome primarily associated with discrete genetic abnormalities. Among these, the t (4;14) (p16;q32), which is found in 15% of MM, defines one of the most serious subgroups of MM. Despite thepotential role of two deregulated oncogenes (FGFR3 and MMSET), the molecular mechanisms thatexplain the severity of MM with t (4;14) have not been clearly elucidated. By implementing theapproaches of molecular biology, high throughput sequencing and functional tests on a wide rangeof MM t (4;14), my project sought to identify genetic events associated with the poor prognosis ofthis subgroup of patients. The results showed that the site of the translocation breakpoints withinthe MMSET gene affect patient outcome. They also profiled the landscape of specific geneticmutations affecting t (4;14) MM, and revealed a relatively high frequency of alterations in the ATM/ ATR genes and PRKD2. Finally, this project has identified PKD2 as a therapeutic target anddemonstrated that an inhibitor of PKDs (kb NB 142-70) blocks the proliferation of MM cells in vitro.All these results throw new light on the pathophysiology of MM t (4;14) and opens the way to newtherapeutic approaches.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Matrix metalloproteinases (MMPs) are known for their role in extracellular matrix remodeling, but their role in regulating intracellular immune cell function is unknown. We reported that MMP inhibition down regulated T cell proliferation in response to alloantigens and autoantigens; but the direct role of MMP involvement in T cell activation has not been reported. Methods: MMP deficient or MMP sufficient wild-type CD4+ or CD8+ T cells from C57BL/6 mice were treated with SB-3CT, a specific inhibitor of MMP2 and MMP9, stimulated with anti-CD3 Ab, alone, or with IL-2 or CD28. Cellular activation and cytokine profiles were examined. A mouse model of antigen specific T cell mediated lung injury was used to examine MMP inhibition in antigen-specific T cell mediated lung injury. Results: SB-3CT (1-25μM) induced dose-dependent reductions in anti-CD3 Ab-induced proliferation (p<0.0001). Compared to wild-type, MMP9-/- CD4+ and CD8+ T cells proliferated 80-85% less (p<0.001) in response to anti-CD3 Ab. Compared to untreated or wild-type cells, anti-CD3 Ab-induced calcium flux was enhanced in SB-3CT-treated or MMP9-/- CD4+ and CD8+ T cells. Cytokine transcripts for IL-2, TNF-α and IFN-γ were reduced in both CD4+ and CD8+ MMP9-/- T cells, as well as in SB3CT treated CD4+ T cells. MMP inhibition dampened antigen-specific T cell mediated lung injury. Conclusions: Although known to be functional extracellularly, the current data suggest that MMPs function inside the cell to regulate intracellular signaling events involved in T cell activation. T cell targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases. - David S. Wilkes, M.D., Chair.