Orientadores: Ronei Luciano Mamoni, Maria Heloisa Souza Lima Blotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T02:22:29Z (GMT). No. of bitstreams: 1 Ferreira_MariaCarolina_M.pdf: 3235698 bytes, checksum: 858844d8c2fb54db25e9acd5fcdbb327 (MD5) Previous issue date: 2009 / Resumo: Pacientes com paracoccidioidomicose (PCM) apresentam supressão da resposta imunológica celular, caracterizada por testes de hipersensibilidade do tipo tardio negativos a antígenos do P. brasiliensis, alta taxa de apoptose de linfócitos e alta expressão de CTLA-4 (CD152) e alta produção de citocinas supressoras (IL-10 e TGF-b). Esses dados indicam a possível participação de células T regulatórias (CD4+CD25+ - Tregs) na PCM. O objetivo desse trabalho foi verificar se, e por qual mecanismo as células T regulatórias estão envolvidas nessa imunossupressão. Para isso, analisamos o número, fenótipo, assim como a atividade funcional das células regulatórias do sangue periférico de pacientes com PCM antes e após o tratamento antifúngico efetivo. O número e o fenótipo das células T regulatórias foram avaliados por citometria de fluxo, e os resultados demonstraram que pacientes com PCM ativa (DA) apresentam um maior número dessas células que pacientes tratados (DT) ou controles normais (C). Além disso, observamos uma maior expressão de CD95L, CTLA-4, LAP-1 e GITR em células regulatórias do grupo DA em relação aos grupos DT e C. A expressão do RNAm (quantificado por PCR em tempo real) para FoxP3, IL-10 e TGF-ß em CMSP ex vivo também foram maiores no grupo DA que nos controles e pacientes do grupo DT. Com o intuito de comparar a atividade funcional das células T regulatórias, analisamos o efeito da cocultura com células T regulatórias na resposta proliferativa de CMSP estimuladas com ConA. As células regulatórias do grupo DA exibiram uma atividade supressora maior que as provenientes do grupo DT ou C, de uma maneira dose dependente. Para verificar se o mecanismo por meio do qual as células regulatórias exercem sua atividade é dependente de contato, utilizamos um sistema "transwell". Os resultados demonstraram que a atividade supressora das células T regulatórias do grupo controle é dependente de contato, mas que essa atividade para as células provenientes dos pacientes do grupo DA é apenas parcialmente revertida com esse sistema. A adição de citocinas supressoras recombinantes (IL-10 e TGF-ß), assim como de anticorpos neutralizantes para essas citocinas nas coculturas, mostrou que a produção de citocinas regulatórias pode ser outro mecanismo utilizado pelas células regulatórias do grupo DA. Em conclusão, o número aumentado de células T regulatórias no sangue periférico de pacientes com DA, expressando altos níveis de moléculas associadas à atividade regulatória, ao lado da alta expressão do RNAm para FoxpP3, IL-10 e TGF-b, sugere uma participação dessa população de linfócitos na imunossupressão observada em pacientes com PCM ativa. Além disso, nossos resultados indicam que as células T regulatórias dependem principalmente do contato intercelular para exercer seus efeitos supressores, e que a produção de citocinas supressoras também está envolvida nesse processo. / Abstract: Patients with paracoccidioidomycosis (PCM) present suppression of cellular immune response characterized by negative DTH to P. brasiliensis antigens, elevated apoptosis of lymphocytes, high expression of CTLA-4, and production of IL-10 and TGF-ß. Together these data point towards the involvement of regulatory T cells (CD4+CD25+ - Tregs) in PCM. The aims of this study were to verify whether and how Tregs are involved in this immunosuppression, through the analysis of the number and the phenotype, as well as, the functional activity of Tregs cells from peripheral blood of patients before and after antifungal treatment. The number and phenotype of Tregs cells were evaluated by flow cytometry, and the results showed that PCM patients with active disease (AD) present higher number of Treg cells than patients after treatment (TD) or healthy controls (C). Furthermore, we observed higher expression of CD95L, CTLA-4, LAP-1 and GITR on Tregs from AD group, than in cells from TD and C groups. mRNA expression (quantified by qRT-PCR) for FoxP3, IL-10 and TGF-ß in ex vivo PBMC, were also higher in AD group than in controls and in TD group. In order to compare the functional activity of Tregs, we analyzed the effect of Treg cells on the proliferative response of PBMC stimulated with ConA. Tregs from AD group exhibited stronger regulatory activity than cells from TD and C groups, in a dose dependent manner. To verify the possible mechanism through which Tregs suppress the cell proliferation we utilized a transwell system, which showed that the contact is mandatory for regulatory activity of Treg cells from C group, but had only a partial involvement in cells from AD patients. The addition of IL-10 and TGF-ß and anti cytokines in the co-cultures showed that the production of immunoregulatory cytokines may be other mechanism used by Tregs. In conclusion, the increased number of Treg cells in peripheral blood of patients with active disease, expressing high levels of regulatory markers and suppressive activity, besides the high expression of Foxp3, IL-10 and TGF-ß mRNAs suggest the potential participation of this population in the immunosuppression observed during the disease. Moreover, our results indicate that Tregs act mainly by contact to exert their inhibitory effects, but the production of immunoregulatory cytokines are also involved. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

The involvement of regulatory T cells (Tregs) in immune homeostasis is now recognized as one of the fundamental mechanisms of immune tolerance. While several different types of Tregs cooperate to establish and maintain immune homeostasis, much current research is focused on defining the characteristics of the CD4⁺CD25⁺ Treg subset, as these cells can mediate dominant, long-lasting and transferable tolerance in many experimental models. The aim of this research was to characterize the biological role of a protein known as forkhead box P3 (FOXP3) that was initially identified as an essential transcription factor for the development of mouse CD4⁺CD25⁺ Tregs, in human CD4⁺ T cells. Following confirmation that, like mouse Tregs, human Tregs also expressed high levels of FOXP3, several approaches were used to investigate the role of this protein in human CD4⁺ T cells. 1) Characterization of endogenous FOXP3 expression in CD4⁺ T cell subsets revealed that this protein is not a Treg-specific marker as was previously thought. Instead, low-level and transient expression was found to be typical of highly activated non-regulatory effector T cells. 2) To generate large numbers of Tregs suitable for cellular therapy, the capacity of ectopic FOXP3 expression to drive Treg generation in vitro was explored. It was found that high and constitutive expression mediated by a lentiviral vector, but not fluctuating expression driven by a retroviral vector, was sufficient to generate suppressive cells. Over-expression strategies were also used to characterize a novel splice isoform unique to human cells, FOXP3Δ2 (FOXP3b). 3) To further probe the requirements of FOXP3 to induce suppressor function, a system for conditionally-active FOXP3 ectopic expression was developed. These studies established that FOXP3 acts a quantitative regulator rather than a “master switch” for Tregs, and that there is a temporal component to its capacity to direct Treg phenotype and function. In summary, this research has significantly expanded the understanding of the biological function of FOXP3 in human CD4⁺ T cells. Based on the potential of these cells to be manipulated for therapy, this work contributes to the field of immunology on both academic and clinical research fronts. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Immunomodulation de l'arthrite expérimentale par les cellules dendritiques tolérogènes. Les cellules dendritiques (DCs) sont des cellules présentatrices d'antigènes jouant un rôle clé dans l'initiation et la modulation des réponses immunitaires. En effet, en parallèle de leur capacité à initier une réponse immunitaire adaptative, les DC sont également impliquées dans les mécanismes de tolérance périphérique. Elles sont utilisées depuis 10 ans maintenant en clinique dans des stratégies thérapeutiques anti-tumorale et leurs propriétés tolérogènes ouvrent aujourd'hui leur champ d'applications à des pathologies autoimmunes, l'asthme et la transplantation afin de restaurer une homéostasie de la réponse immune. Les objectifs de ma thèse ont consisté à :- renforcer le potentiel tolérogène des DCs par manipulation in vitro- tester la capacité de DCs tolérogènes à induire une protection de l'arthrite expérimentale- identifier les mécanismes cellulaires et moléculaires impliqués dans la tolérance induite par les DCs. Mon travail de thèse a permis de montrer l'efficacité de la vaccination de souris arthritiques avec des DCs immatures conservant leurs propriétés tolérogènes in vitro et in vivo, grâce au traitement préalable avec un agent immunosuppresseur, la rapamycine. L'injection répétée de DCs immatures induit la génération de lymphocytes T régulateurs CD4+ CD49b+ sécrétant de l'IL-10 ayant de fortes capacités immunosuppressives. Ce projet a permis de mettre en évidence l'efficacité des DCs dans le traitement d'une pathologie autoimmune déjà établie et l'implication d'une population cellulaire régulatrice originale. / Tolerogenic dendritic cells for immumodulation in experimental arthritis.Dendritic cells (DCs) are the most potent antigen-presenting cells that play critical roles in the initiation and regulation of immune responses. Based on their tolerogenic properties, DCs offer potential as therapeutic tools to ameliorate or prevent graft rejection or graft-versus-host disease, or to treat autoimmune disorders.The objectives of my PhD consisted to:- reinforce the tolerogenic potential of DCs by in vitro handling.- assess the capacity of such tolerogenic DCs to induce a protective response in experimental autoimmune arthritis- identify cellular and molecular mechanisms implied in the tolerogenic DCs-induced protectionOur results suggest that, in contrast with conventional DCs, the rapamycin-conditioned iDCs maintain their tolerogenic potential upon injection in inflammatory settings and are able to dampen an already Th1-primed immune response, conferring a protection from arthritis. The protection of the mice was associated with an expansion of the IL-10-secreting CD49b+ Treg in the spleen and liver of the injected mice and a decrease of the Th1 immune response. These results underscore the therapeutic potential of tolerogenic DCs in an established autoimmune disease as well as the anti-inflammatory potential of the CD49b+ Treg cell population induced following DC vaccination.

La grande originalité de mon projet réside dans la génération de cellules stromales mésenchymateuses (MSCs) à partir de cellules souches pluripotentes induites humaines (iPS). Je rappellerai les propriétés phénotypiques, de multipotence et immunosuppressives des MSCs et m’attarderai sur leurs différents mécanismes immunomodulateurs. Cependant, leur nombre limité et leur isolation difficile limitent leur utilisation thérapeutique nécessitant une autre source de cellules.Mon travail a donc été de générer et de caractériser des MSCs issues d’iPS (huiPS-MSCs). L'avantage des huiPS-MSCs réside dans leur plus grande disponibilité et la possibilité d'en avoir à volonté. Encore faut-il valider l’intérêt thérapeutique potentiel de ces huiPS-MSCs. Premièrement, mes résultats in vitro montrent que les huiPS-MSCs présentent une activité immunosuppressive sur les lymphocytes T (LT) activés conduisant à une induction de LT régulateurs FoxP3+ fonctionnels. Deuxièmement, dans une approche plus axée sur la thérapie, j’ai analysé in vivo l’activité́ immunosuppressive des huiPS-MSCs dans un modèle de réaction xénogénique de greffon contre l'hôte (souris immunodéficientes NSG injectées avec des LT humains). Je montre clairement, après traitement avec les huiPS-MSCs, une réduction de la proportion de LT humains producteurs de cytokines inflammatoires (IFNγ et TNFα) typiques de la pathologie et l’apparition concomitante de LT présentant un phénotype régulateur (production d’IL10 et expression de FoxP3). La fin de mon travail a été de caractériser moléculairement la régulation de l’expression de PD-L1, une molécule immuno-régulatrice puissante, entre les MSCs issues de la moelle osseuse (BM-MSCs) de donneurs sains et nos huiPS- MSCs. Les huiPS-MSCs ont une expression constitutive de PD-L1, qui est absente sur les BM-MSCs. J’ai analysé les microARNs susceptibles de limiter l’expression de PD-L1, j’ai pu en identifier plusieurs. En mesurant leur expression dans les différentes MSCs à notre disposition, je montre que cette expression est inverse par rapport à celle de PD-L1. J’ai ainsi pu démontrer l’activité immunosuppressive de nos huiPS-MSCs in vitro et in vivo avec une perspective d’induction de tolérance immune, et caractériser la régulation de l’expression de PD-L1, molécule immunosuppressive exprimée par les huiPS-MSCs. / The mesenchymal stromal cells (MSCs) present many features that render attractive as therapeutic cells. Their phenotype, multipotency and immunosuppressive properties are well described. Nevertheless, major restriction for their clinical use is due to the limited in vitro expansion and low quantity of cells that can be collected from adult tissues. The originality of my project consisted in the generation of mesenchymal stromal cells (MSCs) from human induced pluripotent stem cells (iPS). These huiPS-MSCs could fulfill some of the specification required to improve MSCs use in therapeutic approaches: welldefined and unlimited number of cells with reproducible functional characteristics. In a first approach, I characterized the huiPS-MSCs generated in the laboratory. My results highlight the immunosuppressive activity in vitro of the huiPS-MSCs on T-cell stimulation that induces a switch in T-cell cytokine polarization toward the generation of Treg cells. Secondly, in a more therapy-oriented approach, I analyzed in vivo immunosuppressive activity of huiPS-MSCs in a xenogeneic graft versus host model (NSG immunodeficient mice injected with human T lymphocytes). My data showed significantly reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines (IFNγ and TNFα). By contrast, T cells producing IL-10 and FoxP3+ Treg cells, absent in nontreated animals, were detected in huiPS-MSCs treated mice, confirming the in vitro results of a tolerizing process. The end of my work was to characterize the molecular regulation of the expression of PDL1, an immunoregulatory molecule expressed by the MSCs. Comparing bone marrow MSCs (BM-MSCs) from healthy donors and our huiPS-MSCs, I showed that the huiPSMSCs have a constitutive expression of PD-L1, which is absent on BM-MSCs. Analysing microRNAs that could limit the expression of PD-L1, I could identify several microRNAs which expression is inverse to the expression of PD-L1. For the first time, my results highlight the immunosuppressive activity of huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo and characterize the regulation of PD-L1 expression, an immunosuppressive molecule expressed by the MSCs. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Les cellules dendritiques (DC) sont les cellules professionnelles de la capture et de la présentation antigènique et font l'interface entre l'immunité innée et l'immunité adaptative. Suite à la reconnaissance de divers signaux de dangers (PAMP/DAMP), les DC s'activent et dégradent l'antigène afin de le présenter aux LT naïfs dans les organes lymphoïdes secondaires et ainsi initient une réponse immunitaire immunogène ou tolérogène. Le contrôle de la réponse immunitaire est notamment possible grâce aux lymphocytes T régulateurs. Il en existe deux types : les nTreg qui se différencient dans le thymus et les pTreg qui se différencient dans les organes lymphoïdes secondaires et/ou dans les tissus. Les DC sont indispensables à leur activation et à leur différenciation respectivement. Les Treg possédent un arsenal variés de mécanismes qui leur conférent leur fonction régulatrice : sécrétion de cytokines immunosuppressives (IL-10, TGF-β et IL-35), activité cytotoxique, déprivation de l'environnement en IL-2….Selon la nature des réponses lymphocytaires T CD4 qu'elles induisent, les DC peuvent être classées fonctionnellement en DC activatrices/immunogènes (DCact) versus DC tolérogènes (DCreg). Les DCact sont définies par leur capacité à induire des lymphocytes T CD4 effecteurs et CD8 cytotoxiques. Les DCreg sont caractérisées par leur capacité à induire des Treg qui vont contrôler la réponse immunitaire. Ces DCreg peuvent être induites par des cytokines (IL-10 et TGF- β produites par exemple par certains Treg), sous l'effet sous l'effet de vitamines anti-oxydantes ou de la vitamine D3, de l'acide rétinoïque, par traitement aux glucocorticoïdes (GC) ou encore sous l'effet de facteurs produits par des pathogènes comme la toxine cholérique. Durant mes travaux de thèse je me suis intéressé aux conséquences physiologiques de l'expression de la protéine Glucocorticoid-Induced Leucine Zipper dans les DC. En effet, les DC humaines et murines surexpriment cette protéine sous l'effet des GC, de l'IL-10, du TGF-β, de la mitomycin C, de la rapamycine, de la vitamine D3 et enfin de l'environnement tumoral. Sur des DC dérivées de monocytes, ils avait été montré que GILZ est nécessaire et suffisant pour induire un phénotype (diminution de l'expression membranaire de CD40, CD80, CD86, CMH-II et augmentation de PD-L1 et ILT-3) et une fonction (sécrétion d'IL-10 et diminution de la production de CCL3, CCL5 et CXCL8) tolérogènes chez ces cellules. De plus, les DC GILZhi sécrètent de l'IL-10 et induisent la différenciation de Treg CTLA-4+IL-10+, spécifiques de l'antigène, dont certains expriment le facteur de transcription FoxP3 et qui inhibent la prolifération de LT CD4 autologues. Mes travaux ont pour finalité d'évaluer l'importance physiologique de l'expression de GILZ par les DC in vivo, et en particulier sa contribution à l'induction et au maintien de la tolérance immunologique à l'homéostasie ou dans un contexte pathologique. Pour atteindre ces objectifs, je disposais de deux modèles de souris complémentaires, créés au laboratoire, les souris CD11c-GILZhi, surexprimant GILZ spécifiquement dans les DC, et les souris CD11c-GILZko, conditionnellement déficientes pour GILZ dans leurs DC. Les travaux que j'ai effectués jusqu'à présent ont permis d'établir que la surexpression de GILZ dans les DC leur confère un phénotype tolérogène in vivo (faible expression des molécules de CMH II et production d'IL-10) et est suffisante pour induire une accumulation de Treg à l'homéostasie et une expansion de nTreg suite à une stimulation antigénique. De plus, L'absence de GILZ dans les DC leur confère une capacité de capture antigènique par macropinocytose accrue. In vivo, cet accroissement de la capture est sélectif et ne touche que les DC CD8α+.Finement régulée par des signaux antigéniques, biologiques ou chimiques, GILZ est un puissant régulateur de la balance immunogénicité/tolérogénicité dans les fonctions de la DC. / Dendritic cells (DC) are professionnal antigen-presenting cells and interface between innate immunity and adaptative immunity. After danger signals recognition, DC activate and process antigen to present to naive T cells in the secondary lymphoid organs (SLO) and thus, initiate the immune response, immunogenic or tolerogenic. Control of immunitary response is possible by regulatory T cells (Treg). There are two types of Treg : nTreg that differenciate in the thymus, and pTre that differenciate in SLO and/or in tissues. DC are essential for their activation or their differenciation respectively. Treg possess a large arsenal of regulatory mechanisms : cytokines secretion (IL-10, TGF-B and IL-35), cytotoxic activity, IL-2 environnement deprivation…According to theT CD4 lymphocytary responses, DC are functionnaly classified in activator/immunogenic DC (DCact) versus tolerogenic DC (DCreg). DCact induce effector CD4 T cells and cytotoxic CD8 T cells. DCreg induce Treg controlling the immune response. DCreg can be induce by cytokines (IL-10 and TGF-B produce by Treg for example), by anti-oxidant vitamin or vitamin D3, by retinoic acid, by glucocorticoid (GC) treatment and by various product of pathogens. During my thesis work, I focused on the physiological consequences of GILZ expression in DC. Human and murin DC overexpress GILZ after GC treatment and after treatment with IL-10, TGF-B, mitomicyn C, rapamycin, vitamin D3 and under the influence of the tumoral microenvironnement. On monocytes derived-DC, it has been demonstrated that GILZ is necessary and sufficient to induce a tolerogen phenotype (decrease of CD40, CD80, CD86, MHC-II expression and increase of PD-L1 and ILT-3 expression) and functionnality (IL-1à secretion and decrease of CCL3, CCL5 and CXCL8 production). So, GILZhi DC induce the differenciation of antigen-specific CTLA-4+IL-10+ Treg whose a part express FoxP3 and inhibit the proliferation of CD4 autologous T cells. My work evaluate the physiological importance of GILZ expression by DC in vivo, and more particularlythe contribution of this expression on the induction and the support of the immune tolerance at homeostasis and in pathologic context. To reach my objectives, I haved two complementary murin models, created in the laboratory : the CD11c-GILZhi mice, overexpress GILZ specifically in the DC, and the CD11c-GILZko mice, deficient for GILZ expression in mice. Studies I have done show that overexpression of GILZ in vivo in DC induce a tolerogenic phenotype in these cells (decrease of MHC-II expression and IL-10 production) and is sufficient to induce accumulation of Treg at homeostasis and expansion of nTreg after antigenic stimulation. Thus, lack of GILZ in DC increase their macropinocytic antigen uptake capacity. In vivo, this increase is selective of CD8a+ DC. Being strongly regulated by antigen, biologic or chemicals signals, these work show GILZ like a powerful regulator of the immunogenecity/tolerogenicity balance in the DC functionnalities.

5 Regulatory T cells (Tregs) are essential for the maintenance of peripheral self-tolerance and prevention of autoimmunity by suppressing the response of self-reactive CD8+ and CD4+ T cells. However, while interactions of Tregs with CD4+ T cells have been extensively studied, their effect on the self-tolerance of CD8+ T cells has not been explored in detail. The main aim of this diploma project was to provide evidence whether and how Tregs prevent autoimmunity induced by CD8+ T cells. We used an experimental mouse model of autoimmune diabetes allowing us to acutely deplete Tregs and titrate the number of self-reactive T cells, self- antigen affinity, and self-antigen doses. We found out that Tregs play an important role in the prevention of CD8+ T-cell mediated autoimmunity. Moreover, we revealed that Tregs suppress both high-affinity T cells that escape negative selection and relatively weakly self-reactive, but numerous, positively selected T cells. Tregs do so by increasing requirement for the number of self-reactive CD8+ T cells required for the autoimmunity induction. Intriguingly, presence of Tregs does not impact threshold for self-antigen. Moreover, for the first time, we showed that Tregs can suppress CD8+ T-cell-mediated autoimmunity in the absence of conventional CD4+ T cells. This means that...

Regulatory T cells (Treg) represent one of the most important mechanisms of immunoregulation. Treg suppress immune reactions and prevent overactivation of the immune system. There is a lot of ways of Treg action described, here we have focused on Treg interference with macrophages. The suppressor capacity of a highly purified Treg population was demonstrated in proliferation assays. The level of suppression of effector T cell proliferation differs depending on the presence of macrophages in the culture. Treg suppression has been significantly higher in the presence of macrophages. These observations led to hypotesis that Treg affect directly macrophages. However, using flow cytometry, reduction of expression of costimulatory molecules on macrophages after culture with Treg was not observed. Macrophages precultured with Treg showed a comparable functionality as macrophages cultured alone. Neither flow cytometry nor live cell imaging revealed any cytotoxic activity of Treg towards macrophages. Despite the presence of macrophages, Treg did not suppress effector cell proliferation in a model, where stronger activation of effector cells was induced. Therefore, a new hypothesis was presented - initially observed higher suppression in the presence of macrophages was probably caused by a qualitatively or...

Type 1 Diabetes (T1D) is an autoimmune disease characterized by islet -specific T cells that infiltrate the pancreas and destroy the β-cells, crippling the necessary supply of insulin to control blood glucose levels. Although the disease can be managed by blood glucose monitoring, insulin injections and strict dietary regimens, there is currently no cure and complications continue to cause significant morbidity and mortality. Immunotherapies designed to inhibit islet-specific T cells are an attractive approach to treat T1D. Antibodies blocking the Interleukin-7/Interleukin-7 Receptor α (IL-7/IL-7Rα) pathway, which is critical for naïve and memory T cell survival, have shown promise in the non-obese diabetic (NOD) mouse model. Systemic administration of anti-IL-7Rα antibodies have prevented and reversed T1D in these models. However, hyperglycemia returned upon cessation of treatment, indicating no durable tolerance was established. Because of these results, I sought to better understand the mechanisms underlying the impact of IL-7Rα blockade on conventional islet-specific T cells and regulatory T cells (Tregs). I hypothesized that IL-7 signaling blockade (1) altered metabolism leading to impaired diabetogenic T cell effector functions and (2) impaired Tregs in peripheral tissues, potentially diminishing the therapeutic activity of anti-IL-7Rα antibodies. The data show that IL-7 signaling blockade impaired mitochondrial respiration independent of calcium signaling and downstream proteins. Impaired respiration shifted T cells to a more inefficient metabolic state. These inefficiencies were associated with diminished pro-inflammatory cytokine production. The cell-intrinsic role of IL-7 signaling in Tregs was assessed by crossing floxed IL-7Rα NOD mice with NOD mice that expressed Cre recombinase exclusively in Foxp3+ Tregs. At the onset of T1D IL-7Rα-deficient Tregs presented in lower frequencies than IL-7Rα-sufficient Tregs in pancreatic lymph nodes, and expressed higher levels of the co-inhibitory receptors PD-1 and TIGIT, indicating a more exhausted phenotype. Importantly, NOD mice with IL-7Rα-deficient Tregs developed T1D earlier. The data presented herein show that IL-7 signaling regulates T cell mitochondrial metabolism, T cell effector function, and identifies a role for IL-7 in the survival and function of Tregs in peripheral tissues during T1D development. This work simultaneously validates the IL-7 pathway as a potential immunotherapeutic target to modulate islet-specific T cells and cautions that unintended effects on protective Tregs must be taken into account for therapeutic strategies.

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