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Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play an essential role in the immunosuppressive networks that contribute to tumor immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-regulation between MDSC and Treg remain incompletely defined. The current work evaluates the influence of MDSC, expanded in two mouse cancer models, on immunosuppressive Treg. We demonstrate that tumor-induced MDSC endowed with the potential of suppressing conventional T lymphocytes surprisingly impair TGF-β1-mediated generation of induced Treg (iTreg) from naïve CD4⁺ T lymphocytes. Suppression of iTreg generation by MDSC occurs early in the differentiation process, and is cell contact dependent. This inhibition of FoxP3-expressing T lymphocyte differentiation by MDSC does not depend on arginase 1, cystine/cysteine depletion, iNOS/NO, or PD-1/PD-L1 signaling. These findings therefore indicate that MDSC from tumor-bearing hosts have the heretofore unreported ability to restrict some immunosuppressive Treg subpopulations.

TGFβ is an immunoregulatory cytokine that has a pivotal function in maintenance of immune tolerance via the control of lymphocyte proliferation, differentiation and survival. Defects in TGFβ1 expression or in its signaling in T cells correlate with the onset of several autoimmune diseases. However, the early effects of this cytokine on the innate immune system, particularly the dendritic cells (DCs) which play an equally important role in development of immune tolerance, are not well documented in vivo. In the current study, we developed conditional knockout mice with targeted deletion of Tgfbr2 specifically in dendritic cells. DC-Tgfbr2 KO mice developed spontaneous multi-organ autoimmune inflammation with T and B cell activation. Phenotypic analysis of dendritic cells revealed no significant differences in the expression of MHCII and co-stimulatory molecules between control and DC-Tgfbr2 KO mice. However, we found that DCs from DC-Tgfbr2 KO mice were more pro-inflammatory, which exacerbated the severity of disease in a T cell transfer model of colitis. Furthermore, increased IFNγ expression by Tgfbr2-deficient DCs inhibited antigen-specific regulatory T cells (Tregs) differentiation by DCs in the presence of TGFβ. Since DCs play an important role in Treg homeostasis in vivo, we also examined the phenotype of Tregs and observed a significant increase in the frequency and numbers of Foxp3⁺ T cells in both the spleen and MLNs of DC- Tgfbr2 KO mice. Further analysis of these Tregs revealed attenuated expression of Foxp3 and an expansion in the numbers of CD4⁺CD25⁻Foxp3⁺T cells suggesting that the Tregs from KO mice may not be fully immunosuppressive. Adoptive transfer of in vitro differentiated iTregs into 2-3 week old DC-Tgfbr2 KO mice partially rescued the autoimmune phenotype by reducing the frequency of activated T cells and severity of colitis but did not prevent inflammation in other organs. The phenotype of this novel mouse model clearly indicates the importance of TGFβ signaling in DCs in the maintenance of immune homeostasis and prevention of autoimmunity and provides an opportunity to study the pathogenesis of complex disorders such as autoimmune gastritis, pancreatitis, hepatitis and inflammatory bowel diseases.

Dentre as infecções bacterianas, a tuberculose é responsável pelo maior número de casos no mundo. É uma doença freqüentemente fatal quando associada com cepas resistentes e extremamente resistentes às drogas, ao abandono de tratamento e imunossupressão. Dependendo da natureza e da magnitude da resposta do hospedeiro, uma inflamação excessiva, comumente não protetora nos indivíduos susceptíveis, acompanha a progressão da infecção. Nesse sentido, é de grande interesse identificar mecanismos que não somente caracterizem a evolução da infecção como também aqueles que participam no controle do dano tecidual. Neste estudo, nós usamos linhagens de animais com susceptibilidade distinta à infecção por M. tuberculosis com o objetivo de avaliar se a freqüência e a atividade das células T reguladoras são influenciadas por características genéticas do hospedeiro e se essas diferenças poderiam representar um fator de susceptibilidade durante a tuberculose experimental. Nossos resultados mostram que tanto a freqüência como a atividade supressora das células T reguladoras de animais BALB/c estava aumentada, inibindo a produção de IFN-g e IL-2 por células efetoras CD4+CD25-. Essa atividade supressora não parece ser dependente de IL-10 ou TGF-b. Do contrário, a freqüência e a capacidade supressoras das células T reguladoras de animais C57BL/6 diminuíram com a infecção e, como conseqüência, foi verificada uma resposta proliferativa mais intensa, maior produção de IFN-g e IL-17, e uma eficiente restrição no crescimento bacteriano em relação aos animais BALB/c. As células T reguladoras de animais C57BL/6 regularam positivamente a produção de TGF-b. No entanto, não regularam negativamente a produção de IFN-g. Além disso, a transferência adotiva de células T reguladoras de animais BALB/c tornou esta linhagem mais susceptível enquanto a transferência de células T reguladoras de animais C57BL/6 afetou discretamente o número de UFC nessa linhagem. Reforçando nossos dados, recentemente, foi descrito por Torcia e colaboradores (2008) que a deficiência funcional de células T reguladoras pode explicar a resistência distinta à malária em diferentes grupos étnicos da África. Nesse contexto, a ativação de células T reguladoras pode representar um importante aspecto a ser ressaltado no desenvolvimento de medidas profiláticas e de terapias imunológicas para doenças infecciosas em indivíduos susceptíveis. / Tuberculosis is responsible for the highest number of cases of bacterial infection in the world, which are frequently fatal, especially when associated with multiply drug-resistant and extremely resistant strains, treatment abandonment and immune suppression. Depending on the nature and magnitude of the host response, an excessive inflammation follows the progression of infection. In this way, it is of great interest to identify mechanisms that not only determine differences in infection outcome, but also those that are most influential in controlling host tissue damage. In this study, we used mouse strains with distinct levels of susceptibility to M. tuberculosis infection to test the hypothesis that the frequency and activity of Treg cells are influenced by genetic background and these differences might represent a susceptibility factor for tuberculosis. Our results show that either the frequency or the suppressor activity of BALB/c Treg cells was enhanced, inhibiting proliferation, IFN-g and IL-2 production by CD4+CD25- responsive cells. This suppressor activity seems not be dependent on IL-10 or TGF-b. In contrast, the frequency and suppressor capacity of C57BL/6 Treg cells decreased and, as a consequence, a more intense proliferative response, higher production of IFN-g and IL-17, and a more efficient restriction of bacterial growth compared to BALB/c mice were observed. C57BL/6 regulatory T cells up regulated TGF-b secretion. However, they did not down regulated IFN-g secretion. In addition, adoptive transfer of BALB/c Treg cells made this strain more susceptible to infection while the transfer of C57BL/6 Treg cells affected only discretely the CFU numbers in this strain. To reinforce our data, recently, it was reported by Torcia and coworkers that a functional deficiency of regulatory T cells may explain distinct resistance to malaria in different ethnic groups of Africa. In this context, activation of Treg cells may become an important issue to be addressed in the development of vaccines and immune modulators for the prophylaxis and therapy of infectious diseases in susceptible subjects.

Notre équipe a démontré précédemment que les cellules dendritiques (DC) traitées par l’Acide Mycophénolique (DC-MPA) induisaient des lymphocytes T (LT) CD4+ régulateurs (iTreg). Dans ce travail, nous avons démontré que les iTreg supprimaient l’allocytotoxicité des LT CD8+ indépendants des LT CD4+. D’autre part, nous avons montré que des LT préactivés en conditions inflammatoires induisaient via CTLA-4 la synthèse d’IL-12 par les DC. Le blocage de CD28 dans les mêmes conditions inhibait la production d’IL-10 et d’Interferon-g mais n’affectait pas celle d’IL-12. Enfin, des LT préactivés par des DC allogéniques différenciées en présence d’IL-10 (DC-10) étaient hyporéactifs, mais de façon surprenante, étaient dénués d’activité régulatrice. Ce travail a montré pour la première fois chez l’homme la capacité des Treg induits à réguler l’allocytotoxicité des LT CD8+. Il suggère aussi que CTLA-4 exprimée par des LT préactivés orientait la réponse immunitaire vers Th1, via un ligand sur la DC, non commun avec CD28. / This work has studied several aspects of the regulation of the immune response in human. Works in our laboratory have previously demonstrated that Mycophenolic Acid-Treated Dendritic Cells (MPA-DC) induced CD4+ regulatory T cells (iTreg). In this work and for the first time in human, we demonstrated that allospecific iTreg suppressed helpless CD8+ T-Cells allocytotoxicity. On the other hand, we have shown that preactivated T-Cells in inflammatory conditions induce IL-12 synthesis by DC through CTLA-4 engagement. Blockade of CD28 in this interaction inhibited the production of IL-10 and Interferon-g without affecting that of IL-12. Finally, allogeneic DC differentiated in the presence of IL-10 (DC-10) induced T cell hyporesponsiveness without any regulatory activity. This work has highlighted the ability of allospecific iTreg to regulate CD8+ T-Cells allocytotoxicity and suggested that the engagement of CTLA-4 expressed by preactivated T cells polarized the immune response towards Th1, via a ligand on DC, different from that of CD28.

Les lymphocytes T régulateurs (Tregs) jouent un rôle dans la grossesse précoce, localement dans l'utérus et de façon systémique, dans les organes lymphoïdes secondaires et dans le sang. Dans le tissu utérin, j'ai identifié une nouvelle population de Tregs effecteurs-mémoire résidant dans le tissu (uregTrm). Avant la grossesse, uregTrm ont un profil transcriptionnel et un phénotype unique en réponse au micro-environnement utérin. Au début de la grossesse, les uregTrm prolifèrent et expriment de nouveaux gènes impliqués dans des fonctions trophiques telles que le remodelage de la matrice extracellulaire, l'hypoxie et la vasculogenèse. La fonction trophique d'uregTrm a été comparée aux mécanismes développés par les Tregs dans l’infiltrat tumoral de mélanome. Dans les organes lymphoïdes secondaires et le sang, les Tregs en circulation contribuent à une grossesse réussie en favorisant la tolérance materno-foetale. Nous avons montré que ces Tregs orchestrent la régulation des réponses immunitaires dans l'utérus. En outre, les Tregs agissent en trois étapes. Tout d'abord, uregTrm et les Tregs des ganglions lymphatiques drainants auto-spécifiques contrôlent rapidement une réponse auto-immune qui aurait pu être déclenchée par la libération de débris cellulaires en raison de la prolifération de l'endomètre. Deuxièmement, uregTrm contribuent au besoin de remodelage du tissu utérin pour la placentation. Dans un troisième temps, accompagnant l'augmentation de la masse foetale, les Tregs inductibles aident à contrôler la réponse immunitaire anti-foetale. Ceci met en évidence la spécialisation des Tregs dans les tissus et souligne l’importance des Tregs pendant la grossesse et la pathologie. / Regulatory T cells (Tregs) play a role during early pregnancy locally in the uterus and systemically, in the secondary lymphoid organs and blood. In the uterine tissue, I identified a novel population of tissue-resident effectors-memory regulatory T cells (uregTrm). Before pregnancy, uregTrm have a unique transcriptional profile and phenotype in response to the uterine microenvironment. During early pregnancy, uregTrm expand and express new genes implicated in trophic functions such as extracellular matrix remodeling, hypoxia and vasculogenesis. uregTrm trophic function was compared to the mechanisms developed by Tregs from melanoma tumour infiltrate. In the secondary lymphoid organs and blood, circulating Tregs contribute to successful pregnancy by promoting maternal-fetal tolerance. We showed that these Tregs orchestrate down regulation of immune responses in the early pregnant uterus. Besides, Tregs act in three stages. First, self-specific uregTrm and draining lymph nodes Tregs rapidly contain an autoimmune response that could have been triggered by release of cell debris due to the endometrium tissue high proliferation. Second, uregTrm contribute to the increased need of uterine tissue remodeling for placentation. In a third stage, accompanying the fetal mass increase, inducible Tregs help to control the anti-fetal immune response. This further highlights the specialization of Tregs in tissues and underline the relevance of Tregs during pregnancy and disease.

Les cellules T régulatrices Foxp3+ (Treg) représentent une sous-population T CD4 cruciale pour le maintient de l'immuno-tolerance. Mieux comprendre la biologie des Treg, leur hétérogénéité, leur développement, leur mécanisme d’action et les facteurs assurant leur survie en périphérie reste un objectif majeur. L'objectif de ce travail de thèse était de mieux définir les mécanismes impliqués dans le contrôle de l’homéostasie Treg et d’évaluer l’éventuelle contribution des perturbations de l’homéostasie Treg en pathologie humaine.Dans la première partie de ce travail de thèse nous avons essayé de finalement définir dans le modèle murin le rôle joué par l'IL-7 dans le contrôle de l’homéostasie Treg. Nous avons montré que l'expression de CD127, la chaîne alpha du récepteur à l'IL-7, est finement régulée à la surface des Treg et qu'elle dépend de leur activation ainsi que de leur localisation tissulaire. Nous avons démontré que l’expression de CD127 par les Treg activées est fonctionnelle, identifiant ces cellules comme cibles potentiels de l'IL-7. En utilisant des modèles murins présentant une altération de la voie de signalisation IL-7/IL-7R et des modèles de transfert adoptif, nous avons obtenu une démonstration définitive du rôle direct de l'IL-7 dans la régulation du nombre de cellules Treg. Enfin, nous avons démontré que la signalisation IL-7 optimise la capacité de ces cellules de réagir à l'IL-2, une cytokine importante dans la régulation de l’homéostasie Treg. Dans la deuxième partie de ce travail, nous avons étudié l’homéostasie Treg dans le contexte de l'infection par le VIH. Cette étude a bénéficié de l’accès à des patients au cours de la primo infection et des HIV contrôleurs. Nous avons montré que les Treg effecteurs plus que les Treg naïves sont affectés par l'infection par le VIH. De plus, nous avons montré que le nombre des effecteurs Treg corrélant inversement avec les réponses T CD8 spécifiques, offrant une preuve ex vivo de l'implication des Treg dans l'immunité anti-VIH. / Regulatory T cells (Treg) represent a crucial CD4 T cells subset involved in maintenance of immune-tolerance. Since their first description important efforts have been undertaken to better understand their biology, their development and their mechanisms of action. However, little is known about factors controlling Treg peripheral homeostasis. The aim of this thesis work was to better define mechanisms involved in governing Treg homeostasis and to investigate the eventual contribution of perturbation of Treg homeostasis in human disease. In the first part of this thesis work we tried to define in the murine system the role played by IL-7 in governing Treg homeostasis. We showed that Treg surface expression of CD127, the IL-7 receptor alpha chain, is finely regulated as it depends on their activation as well as on their tissue localization. More importantly, we demonstrated that Treg do express functional levels of CD127, identifying these cells as potential target of IL-7. Using both genetically modified murine models of altered IL-7 signaling and adoptive transfer models, we obtained definitive evidence for a direct role of IL-7 in governing Treg cell numbers. Finally, we demonstrated that IL-7 signaling in Treg optimizes their capacity to react to IL-2 an important cytokine regulating Treg homeostasis. In the second part of this work we investigated Treg homeostasis in the context of HIV infection. Employing for the first time in HIV infection a novel consensus Treg identification strategy and applying it to different groups of HIV infected patients, including primary infected patients and HIV controllers, we showed that HIV infection is characterized by an early and long lasting alteration of Treg homeostasis. In particular we demonstrated that effector rather than naive Treg are affected by HIV infection. Moreover, we showed that effector Treg numbers inversely correlated with HIV specific CD8 T cells responses, providing ex vivo evidence of Treg involvement in HIV immunity.

Regulatory T cells (Treg) play a crucial role in controlling immune homeostasis. Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Treg. While several mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain unknown. The Treg pool consists of highly diverse subpopulations, indicating that there is a potential to optimise Treg-targeted therapies if disease-relevant mechanisms can be established. Microarray data from our lab suggests a marked upregulation of the integrin αv as well as the IL-33 receptor ST2 in Treg retrieved from the inflamed central nervous system (CNS) during experimental autoimmune encephalomyelitis compared to peripheral lymphoid organs. These two molecules were further investigated within this PhD project with the aim to understand their role in Treg function during chronic inflammatory disease. αvβ integrins have been reported to be needed for effector T cell migration to inflamed sites through binding of extracellular matrix components and are involved in TGF-β activation by a variety of cell types. Conditional knockout mice lacking the integrin αv specifically in Foxpγ+ Treg were generated to address the role of αv integrins on regulatory T cells in inflammatory disease. αv-/- Treg showed a deficiency in activating latent TGF-β, but were able to suppress responder T cell proliferation in vitro as well as in vivo. αv-/- Treg were also able to migrate to the inflamed CNS during EAE and resolve disease. However, αv-/- Treg were detected at significantly lower numbers and proportions in the inflamed gut during a curative T cell transfer model of colitis; this led to a quantitative impairment in the ability of αv-/- Treg to cure colitis when compared to wild-type (WT) Treg. Whether this is a deficit in migration, survival, proliferation, or Foxp3 stability, remains to be investigated. IL-33 acts as an alarmin and is best studied as a cytokine released upon tissue damage that induces a potent type 2 immune response by acting on a multitude of immune cells. Expression of the IL-33 receptor ST2 on Treg has recently been associated with positive metabolic parameters in visceral adipose tissue, protection from gut inflammation, and tissue-restorative function in other inflamed tissues such as injured muscle or lung. This project showed that in steady state, ST2+ Treg expressed high levels of several markers which have been associated with potent regulatory function. When stimulated in vitro, ST2+ Treg showed a better survival and expansion rate compared to their ST2- counterparts, even more so in the presence of IL-33. T-bet deficiency in Treg resulted in an increased ST2+ Treg pool, and T-bet-associated cytokine IFN-γ was found to antagonise IL-33-induced expansion of the ST2+ Treg pool in a T-bet-independent manner. When ST2+ and ST2- Treg were tested for their respective suppressive capacity in vivo, ST2+ Treg were able to suppress responder T cell expansion despite being found only at low numbers in secondary lymphoid organs compared to ST2- Treg. However, in a curative model of T cell transfer colitis, ST2+ Treg were less capable of controlling the ongoing immune response than ST2- Treg. A possible explanation for the superiority of ST2- Treg in this setting can be found in the fact that injected ST2- Treg acquired a distribution of ST2 expression reminiscent of WT Treg over the course of disease. On the other hand, an increased starting pool of ST2+ Treg as occurs in T-bet-/- Treg significantly enhanced the capacity of Treg to control colitis compared to WT Treg. In conclusion, both ST2- and ST2+ Treg are likely to have a distinct, non-redundant role in suppressing T cell activation in secondary lymphoid organs and controlling ongoing inflammation in peripheral tissue, respectively.

In multiple sclerosis (MS), pathogenic T effector cells (Teff) are believed to orchestrate immune-mediated destruction of the central nervous system (CNS) myelin sheath. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, CNS infiltration by regulatory T cells (Treg), producing the anti-inflammatory cytokine IL-10, promotes the resolution of disease. Currently, little is understood about how Treg function within the inflamed CNS and on which cells they exert their suppressive function. There is a debate as to whether Treg in MS patients are capable of infiltrating the CNS and if they do, it is unclear whether they are functional. Understanding Treg function in EAE and MS could open up new possibilities for treatment, as Treg could be modulated for immunosuppressive therapy. A key step in the development of EAE (and presumably MS) is the ability of Teff cells to cross the blood brain barrier (BBB) and enter the CNS parenchyma. The hypothesis of this work was that Treg facilitate resolution of the inflamed CNS by preventing entry of the pathogenic T cells into the CNS parenchyma, thus preventing further damage. As such, it is important to understand with which immune cells and CNS resident cells Treg communicate to achieve resolution of disease. The presence of Treg in MS lesions was investigated with double immunohistochemistry (IHC) in frozen post-mortem MS brain tissue. CD4+Foxp3+ Treg were present in a subset of patients and their presence was associated with perivascular retention of CD4+Foxp3- and CD8+Foxp3- T cells. Foxp3+ cells in MS lesions predominantly expressed IL-10, indicating regulatory activity, although low-level production of IL-17, TNF-α, IFN-γ and GM-CSF was observed as well. Generally, analysis of total cytokine expression identified distinct patterns of cytokine production between lesions. Nonetheless, these could not be used to discriminate individual patients. These studies were repeated in C57BL/6 mice in which the Treg population was depleted before onset of EAE to mimic lesions with and without Treg presence, as found in MS patients. An immunofluorescent technique to study up to 5 fluorochromes simultaneously was developed to study antigen presenting cell (APC), Teff and Treg location, spatial relationship and function (as measured by cytokine expression) in the CNS of EAE mice at different stages of disease. Using this technique it was found that CD4+Foxp3- Teff and CD4+Foxp3+ Treg were located within 50-100μm of CD11c+ APC in the CNS of EAE affected mice. CNS Teff and Treg predominantly produced IFN-γ or IL-10, although low levels of IL-17 were detected in Teff and Treg as well. IL-17+ Treg were close to IL-17+ Teff, IFN-γ+ Treg were close to IFN-γ+ Teff, but IL-10+ Treg were not in close proximity to IL-10+ T cells in the CNS during EAE. In conclusion, there is evidence for functional Treg in EAE and MS lesions, supporting the concept of enhancing Treg activity as a clinical intervention. Treg seem to be capable of retaining pathogenic T cells at the blood brain barrier in MS lesions. In addition, studies of cytokine expression in MS lesions indicated that there is no sound basis for patient stratification based on peripheral blood cytokine profile. This thesis advances our understanding of Treg location, function and spatial relationship with other immune cells within the inflamed CNS.