Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

L’expression d’un transgène grâce au vecteur AAV offre une perspective thérapeutique très prometteuse dans le traitement de maladies monogéniques. Malheureusement, il apparait souvent que des réponses immunes contre le vecteur et le transgène conduisent à un rejet des cellules transduites ainsi qu’à la mise en place d’une mémoire immunitaire spécifique empêchant un nouveau traitement ultérieur. Avec la perspective d’éviter tout rejet immun des cellules transduites, j’ai développé deux approches distinctes. D’une part, nous avons développé un système dans lequel l’expression du transgène est déstabilisée dans les cellules présentatrices de l’antigène grâce à l’ajout de cibles du miRNA 142.3p qui est spécifiquement exprimé dans le système hématopoïétique. Nous avons ainsi montré que la réponse immunitaire contre le transgène était favorisée par la transduction des cellules présentatrices de l’antigène par le vecteur, conduisant à la présentation directe du produit du transgène. En comparant l’initiation des réponses immunes contre plusieurs transgènes modèles, nous avons montré que la réponse immune dirigée contre le transgène pouvait être contrôlée mais que celle-ci dépendait étroitement de l’immunogénicité intrinsèque du transgène en question, c'est-à-dire de la présence d’épitopes reconnus par des lymphocytes T CD4 auxiliaires ainsi que par les lymphocytes B. Une autre approche a concerné l’utilisation de lymphocytes T régulateurs exprimant le facteur de transcription Foxp3 (Treg) et plus particulièrement l’étude de leur mode d’induction in vivo. La présence de Treg conférant une tolérance immunitaire spécifique du transgène a été décrite dans diverses situations et les Treg induits à partir de CD4+ matures (pTreg/iTreg) semblent avoir un potentiel thérapeutique important. Cependant, la population précise de lymphocytes CD4+ à même d’être convertie en Treg n’avait pas été identifiée auparavant. Au cours de mes travaux, analysant la capacité de conversion de cellules naïves, mémoires ou de récents émigrants thymiques, j’ai mis en évidence que le potentiel de conversion des lymphocytes CD4 naïfs issus de souris âgées était diminué et que ceci était dû à une caractéristique intrinsèque des lymphocytes T CD4+ provenant de telles souris. Enfin, nous avons montré que cette faible capacité de conversion des lymphocytes CD4 naïfs en Treg était associée à un rejet de greffe accru dans un modèle de transplantation de peau, montrant que la sénescence peut impacter négativement des protocoles d’induction de tolérance faisant appel à l’induction de Treg en périphérie. / Transgene expression through AAVvectors offers a very promising therapeutic perspective in the treatment of monogenic disorders. Unfortunately, it often appears that the immune responses against the transgene and the vector lead to the rejection of transduced cells and to an establishment of a specific immune memory preventing further processing. With a view to avoid immune rejection of transduced cells, I developed two distinct approaches.First, we have developed a system where the transgene expression is destabilized in the antigen presenting cells by addition of the target miRNA 142.3p which is specifically expressed in the hematopoietic system. We have shown that the immune response against the transgene was favored by transduction of antigen presenting cells with the vector, leading to the direct presentation of the transgene product. Comparing the initiation of immune responses against more transgenes models, we showed that the immune response against the transgene could be controlled but it depended greatly on the intrinsic immunogenicity of the transgène: the presence of epitopes recognized by T helper cells and CD4 by B lymphocytes. Another approach has involved the use of regulatory T cells expressing the transcription factor Foxp3 ( Treg ) and more specifically the study of their mode of induction in vivo. The presence of Treg conferring transgene -specific immune tolerance has been described in various situations and induced Treg from CD4 + mature ( pTreg / iTreg ) appear to have a significant therapeutic potential . However, the precise population of CD4 + lymphocytes capable of being converted into Treg was not identified previously. During my work, analyzing the conversion capacity naive, memory cells or thymic recent emigrants, I highlighted that the potential of conversion of naive CD4 lymphocytes from old mice was decreased and this was due to an intrinsic defect of CD4 + T cells from such mice. Finally, we showed that low conversion ability of CD4 naive Treg was associated with an increased graft rejection in a model of skin transplantation, showing that senescence may negatively impact protocols of tolerance induction using induction of Treg in the periphery.

In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 employ diverse mechanisms to suppress innate and adaptive immunity, which is critical for maintaining immune homeostasis and self-tolerance. Here, we propose Swap-70 as a novel member of the Foxp3-dependent canonical Treg cell signature. We show that Swap-70-/- mice have increased numbers of Foxp3+ Treg cells with an effector/memory-like phenotype that exhibit impaired suppressor function in vitro, but maintain overall immune homeostasis in vivo. Upon formation of an immunological synapse with antigen presenting cells in vitro, cytosolic SWAP-70 protein is selectively recruited to the interface in Treg cells. In this context, Swap-70-/- Treg cells fail to downregulate CD80/CD86 on osteoclast precursor cells by trans-endocytosis and to efficiently suppress osteoclastogenesis and osteoclast function. These data provide first evidence for a crucial role of SWAP-70 in Treg cell biology and further highlight the important non-immune function of Foxp3+ Treg cells in bone homeostasis mediated through direct SWAP-70-dependent mechanisms.

Resting conventional T cells (Tconv) can be distinguished from T regulatory cells (Treg) by the canonical markers FOXP3, CD25 and CD127. However, the expression of these proteins alters after T-cell activation leading to overlap between Tconv and Treg. The objective of this study was to distinguish resting and antigen-responsive T effector (Tconv) and Treg using single-cell technologies. CD4+ Treg and Tconv cells were stimulated with antigen and responsive and non-responsive populations processed for targeted and non-targeted single-cell RNAseq. Machine learning was used to generate a limited set of genes that could distinguish responding and non-responding Treg and Tconv cells and which was used for single-cell multiplex qPCR and to design a flow cytometry panel. Targeted scRNAseq clearly distinguished the four-cell populations. A minimal set of 27 genes was identified by machine learning algorithms to provide discrimination of the four populations at >95% accuracy. In all, 15 of the genes were validated to be differentially expressed by single-cell multiplex qPCR. Discrimination of responding Treg from responding Tconv could be achieved by a flow cytometry strategy that included staining for CD25, CD127, FOXP3, IKZF2, ITGA4, and the novel marker TRIM which was strongly expressed in Tconv and weakly expressed in both responding and non-responding Treg. A minimal set of genes was identified that discriminates responding and non-responding CD4+ Treg and Tconv cells and, which have identified TRIM as a marker to distinguish Treg by flow cytometry.

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.

Thesis advisor: Thomas Chiles / Thesis advisor: James Kim / The purpose of this thesis is to describe some of the research I conducted in Dr. Markmann’s laboratory at Massachusetts General Hospital in the field of transplantation immunology. The first portion provides background information on the immune system and its different components, eventually providing an in-depth look at regulatory T cells, and their role in transplant immunology. The second portion of the thesis is dedicated to my experiments. This part presents the materials and methods used, the previous findings that led to my experiments, the data analysis and results, and a conclusive discussion. The research I did that is included in this thesis pertains to regulatory T cells. It is believed that a potential pathway to prolonging allograft survival is to drive antigen-specific CD4+Foxp3- T cells to convert to CD4+Foxp3+ T cells. I studied the effects of retinoic acid (RA) and anti-CD45RB treatment on Foxp3 expression in CD4+ T cells. These studies were done using different lines of transgenic mice that provided models of antigen-specificity. My in vitro data demonstrated that RA is capable of boosting TGF-β-mediated Foxp3 upregulation in a synergistic manner and that anti-CD45RB is also able to increase Foxp3 expression. In vivo RA experiments were inconclusive, and due to some misfortune and time constraints in vivo anti-CD45RB experiments were not conducted. Research funded by the following sources: 2R56AI048820, 5R01AI057851, and 5K01DK079207. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

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 (Tregs), expressing the transcription factor Foxp3, form a key component of peripheral immune tolerance, guarding against auto-aggressive immune responses. Multiple Sclerosis is an inflammatory and demyelinating disease of the central nervous system (CNS) which is largely believed to be mediated by immune components reacting to the self myelin antigens that insulate the nerve fibres. Recent investigations have reported that regulatory T cells are dysfunctional in MS patients; therefore enhancing the regulatory T cell responses in MS is an attractive therapeutic target. Using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) we have attempted to develop disease-relevant Treg-based therapies to prevent disease induction. This required an understanding of the antigenic-reactivity of Tregs during disease. Results described in this thesis show that a proportion of Tregs in the draining lymph nodes and CNS were reactive to the disease initiating antigen(s) and could suppress in vitro responses of naïve T cells bearing transgenic T cell receptors, recognising the same antigen. Adoptive transfer of antigen-reactive Tregs suppressed disease induced with the same antigen, but also reduced disease induced with a distinct myelin antigen. Peptide-based tolerance using a high affinity MHC binding peptide analogue expanded and maintained antigen-reactive T cells which were tolerant to antigenic restimulation, although these cells did not express Foxp3. Peptide-treated mice showed reduced incidence of disease relapses during EAE induced against a distinct myelin antigen. Thus, while EAE and MS will involve a polyclonal effector T cell response to many antigens, therapeutic targeting of Tregs reactive against one CNS component may be sufficient to reduce disease. Endogenous expression of myelin autoantigen did not grossly alter the response of antigenreactive Tregs in the periphery. However, expression of endogenously derived viral superantigen enhanced the proportion of superantigen-reactive Foxp3+ Tregs in the periphery. This observation was extended using exogenous superantigen, suggesting that prolonged exposure to low dose (super)antigen tips the balance of the immune system in favour of regulation. This has implications for the ability to successfully fight infection, as well as for the limitation of autoaggressive responses and may contribute to the understanding of the hygiene hypothesis.

T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.

Toxoplasmosis is an important cause of congenital disease, and it is one of the most common opportunistic infections in patients with acquired immunodeficiency syndrome. The need for a reliable experimental model of this infection is crucial not only for achieving a better understanding of the patho-physiology of the disease, but also for developing better methods for evaluating new therapeutic regimens. The purpose of the present study was to investigate the role of CD4+CD25+ T regulatory lymphocytes in mice infected with Toxoplasma gondii. T regulatory (Treg) cells have been shown to play an important role in our immune system in controlling the activity of other T lymphocytes. These cells are differentiated from other T lymphocyte populations based on the co-expression of CD4 and CD25 and expression of the Foxp3 gene. The results of several recent studies have suggested that certain pathogens may be able to increase their survival in the host by exploiting T reg cell activity. T regulatory cells have been shown to control the persistence of the protozoan parasite, Leishmania major, in mice; however, this population of cells plays only a limited role during murine infection with Trypanosoma cruzi. In the present study we have investigated the role of Treg cells during murine infection with the ME49 strain of T. gondii. In vivo depletion of Treg cells was accomplished by injecting mice with a monoclonal antibody (Mab) isolated from the 7D4 rat hybridoma cell line. This Mab is specific for the interleukin-2 receptor chain (also known as CD25). Female Swiss Webster mice of approximately 6-7 weeks of age were depleted of Treg cells by intraperitoneal injection of 400µg of Mab, mice were injected once 7days prior to infection, and a second time 1 day prior to infection, with 20 tissue cysts of T. gondii. Mouse weight and tissue cyst numbers were monitored to evaluate the impact of Treg depletion on the outcome of infection. Our results suggest that depletion of Treg cells has little measurable impact during the acute stage of infection with the ME49 strain of T. gondii. Further studies will be required to determine what role, if any, these cells play in the chronic stage of murine toxoplasmosis.