The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

No description available.

The Fas pathway is critical for the maintenance of normal T cell homeostasis. Humans and mice with defects in this pathway exhibit the accumulation of large numbers of peripheral lymphocytes and lupus-like autoimmunity. A major feature of these organisms is the accumulation of non-NK TCRαβ+CD4-CD8- “double negative” (DN) T cells. While regulatory T cells (Tregs) with the DN phenotype have been extensively characterized in Fas-sufficient mice and humans, limited data exist on the role of DN T cells as Tregs in Fas-deficient animals. In fact, most of the literature suggests that the DN T cells accumulating in Fas-deficiency states are pathogenic, contributing to secondary lymph node enlargement and autoimmune disease. In this body of work, data are presented that illustrate that Fas-deficient lymphoproliferative (LPR) DN T cells can act as Tregs in an interferon γ (IFNγ)- and Fas ligand (FasL)-dependent fashion toward Fas-sufficient T cells. LPR DN T cells needed to be able to secrete and respond to IFNγ in order to upregulate surface FasL, in order to ameliorate GVHD mediated by CD4+ T cells in vivo and to suppress the proliferation of and kill activated CD4+ T cells in vitro. FcRγ, a key molecule involved in innate immune responses, can substitute for CD3ζ in the T cell receptor (TCR) of mouse and human T cells in certain circumstances; in doing so, it is essential for the regulatory function of TCR transgenic DN Tregs. FcRγ-deficient LPR mice were found to have exacerbated T cell accumulation and early mortality. We show that while FcRγ expression was required for LPR DN T cells to regulate CD4+ and CD8+ T cells responding to alloantigens in vitro and in vivo, it does not control autologous lymphoproliferation in LPR mice by supporting the function of a regulatory cell, nor does it affect the rate of proliferation of LPR T cells in vivo. Instead, FcRγ-expressing LPR CD4+, CD8+ and DN T cells were found to be undergoing apoptosis at a high rate in vivo, and in contrast to their FcRγ-deficient counterparts, FcRγ+ LPR DN T cells were capable of undergoing TCR restimulation-induced cell death (RICD). The data presented in this thesis therefore show that LPR DN T cells can exhibit IFNγ-, FasL- and FcRγ-dependent regulatory function, and also illustrate a previously unknown function for FcRγ in controlling the expansion of Fas-deficient T cells. The implications of these data for autoimmune lymphoproliferative syndromes, and normal T cell homeostasis, are discussed.

[Truncated abstract] In the last ten years regulator T (Tr) cells have re-emerged as an integral part of the immune system. Research in this field has rapidly demonstrated the role of these cells in the maintenance of immune homeostasis and their involvement in disease. Tr cells are generated in the thymus as a normal part of the developing immune system. Furthermore, antigen-specific Tr cells are induced in the periphery by a mechanism which is yet to be completely elucidated, but is likely to involve dendritic cells. Tr cells play an important role in autoimmune disease, transplantation tolerance, cancer. Most recently Tr cell involvement has been demonstrated in a growing number of infectious diseases. Tr cell induction was reported in Friend Virus infection at the commencement of this study, and subsequent to publication of our findings have also been identified in FIV and HIV. Murine AIDS (MAIDS) is a fatal chronic retroviral infection induced in susceptible strains of mice by infection with BM5d, a replication defective virus, in a viral mixture which is designated LP-BM5. The manipulation of Tr cells detailed in this thesis and the related publication represent the first reported therapy utilising targeted removal of Tr cells. Chapter 1 summarises the literature relevant to this study up to November 2004. Chapter 2 details the materials and methodologies used in this work. Chapter 3 investigates whether Tr cells are involved in the development of murine AIDS, particularly in the early stages of infection. The data presented in this chapter provides evidence of a population of CD4+ Tr cells which express CD25 on their cell surface and secrete TGF-β, some IL-10 and low levels of IL-4 are induced following infection with LP-BM5. These cells were found to arise by day 12 post infection (pi) by flow cytometry and immunosuppressive cytokine expression was found to peak at day 16 pi indicating a role in the early stages of disease progression. Chapter 4 investigates the effect of therapeutically targeting these induced Tr cells using the antimitotic agent Vinblastine during their induction period. The efficacy of treatment was found to be time dependent and was shown to abrogate disease progression maximally when given at day 14 pi. Treatment with anti-CD4 monoclonal antibody was also found to be efficacious at day 14 pi and confirmed the identity of the Tr cells as being CD4+ T cells. Adoptive transfer studies demonstrated that the return of these cells to a successfully treated host results in renewed MAIDS progression, confirming their role in disease progression

[Truncated abstract] Previous studies in the field of tumour immunology had identified regulatory T (Treg) cells as important suppressors of the anti-tumour immune response as the presence of Treg cells in the peripheral blood of cancer patients was correlated with worse disease outcomes. Other studies had identified Treg cells to be active at sites of immune regulation such as the gut of colitis patients. It was therefore hypothesised that Treg cells would be present and active within tumours to suppress the cellular antitumour immune response. ... This treatment targeting multiple pathways of Treg cell mediated immuno-suppression and resulted in tumour regression in 50% of treated animals. Finally, the anti-tumour immune response is complex and a potentially synergistic multi-modality treatment designed to inactivate intra-tumoural Treg cells but to also induce apoptosis in tumour cells themselves was investigated. Alpha-tocopheryl succinate (α-TOS), an analogue of vitamin E, had previously been shown to induce apoptosis in human MM xenografts implanted into immuno-deficient (nude) mice. Administration of α-TOS was therefore examined as a potentially synergistic treatment to be coupled with Treg cell inactivation. At the previously published doses used to treat immuno-deficient mice, α-TOS was found to be toxic to the immuno-competent mice used in this study. A marked depleting effect on total T cells was seen in the treated animals. The results of this thesis demonstrated the high potential for an adjunct immunotherapy of MM. They did however also highlight the importance of future studies into anticancer therapies to be conducted using clinically relevant tumour models and clinically relevant treatment regimes. The need to consider synergistic multi-modal therapies was also emphasised.

[Truncated abstract] Failure to fully reconstitute the immune system is a common clinical problem in HIV patients who were severely immunodeficient before responding to combination antiretroviral therapy (CART). This can manifest as a deficiency in the number or function of CD4+ T-cells and occurs most often in patients who had a nadir CD4+ T-cell count below 100/μl when CART was commenced. Observational studies of large cohorts of HIV patients, such as the D:A:D study, have demonstrated that patients with low CD4+ T-cell counts have increased rates of death compared with patients who have normal CD4+ T-cell counts. Furthermore, individual case studies suggest that impaired recovery of pathogen-specific immune responses during CART is associated with opportunistic infections or disease progression. This thesis addresses possible causes of deficiencies in CD4+ T-cell number or function in HIV patients who were very immunodeficient prior to treatment and are responding (virologically) to CART. Firstly, the role of the thymus in producing naive CD4+ T-cells and the effects of persistent immune activation on the recovery of CD4+ T-cell numbers were assessed in patients with either low or high CD4+ T-cell counts after long-term CART. ... Proportions of antigen presenting cell (APC) subpopulations were examined in HIV patients with low or high CMV-specific CD4+ T-cell responses after long-term CART. HIV patients had significantly lower proportions of plasmacytoid dendritic cells (pDC) than HIV-negative controls. Furthermore, the proportions of pDC were positively correlated with CMV-specific CD4+ T-cell responses in HIV patients. Proportions of myeloid dendritic cells (mDC) were significantly higher in HIV patients than controls, and were also increased in patients with low CMV-specific CD4+ T-cell responses. Proportions of M-DC8+ dendritic cells or CD14+ monocytes did not differ between patients and controls, nor were they associated with CMV-specific CD4+ T-cell responses. Quantitation of cytokine (interferon-α, tumour necrosis factor-α, interleukin (IL) -12, IL-23, IL-15, IL-18 and IL-10) mRNA in unstimulated, purified populations of the APC described above revealed few significant differences between patients with low or high CD4+ T-cell IFN-γ responses to CMV. The only notable difference was the slight elevation of IL-15 mRNA levels in patients compared to controls. Since patients in the high responder group had the highest levels of IL-15 mRNA, this association may reflect the anti-apoptotic properties of IL-15. These studies provide valuable insights into the causes of persistent CD4+ T-cell deficiency and dysfunction in HIV patients on CART and may lead to better monitoring and treatments.

Plusieurs populations de cellules immunitaires possèdent une activité cytotoxique permettant l'élimination de cellules altérées. Cette fonction cellulaire est ainsi déterminante dans le contrôle des infections, des processus tumoraux, ou encore des maladies inflammatoires chroniques. Mon projet de thèse se concentre sur des aspects fondamentaux de l'activité lytique de deux populations de lymphocytes T cytotoxiques : les lymphocytes T CD8+ et les lymphocytes T CD4+ régulateurs de type 1. Pour cela, l'exploration des mécanismes de cette activité a été conduite au travers de deux modèles, pathologique et thérapeutique, à différentes échelles biologiques : au niveau de la population ou de la cellule individuelle, mais aussi différentes échelles d'organisations moléculaires : cellulaire et nanoscopique. Nous avons pu démontrer que l'activité de lyse de lymphocytes T CD8+ cytotoxiques face à un excès de cellules cibles est efficace sur des temps prolongés, reposant sur une capacité individuelle fortement hétérogène à effectuer une lyse multiple. L'importance de cette activité de lyse soutenue a été renforcée par l'identification d'un défaut lytique particulièrement prononcé sur le long- terme chez des lymphocytes T CD8+ issus de patients atteints du syndrome de Wiskott-Aldrich. Ce défaut est lié à une activation réduite de l'intégrine LFA-1 et un délai dans la délivrance du coup létal. De plus, la protéine WASP permet de restreindre LFA-1 de haute-affinité en nanoclusters denses ainsi que de permettre l'organisation en un ring de LFA-1 et la localisation des granules lytiques à l'intérieur de celui-ci. Par ailleurs, les lymphocytes T CD4+ régulateurs de type 1 développés dans le cadre d'une thérapie cellulaire (Ovasave(r)) démontrent une capacité de lyse envers les cellules myéloïdes, en complément d'une activité immunosuppressive sur les lymphocytes T conventionnels. Cette activité est mise en place sur du long-terme, jusqu'à atteindre une efficacité optimale, lié à un délai dans la délivrance du coup létal. De manière surprenante, malgré une spécificité pour l'ovalbumine, l'activité cytotoxique semble être indépendante de l'activation du TCR. En outre, la lyse est granzyme-dépendante mais perforine-indépendante. Ainsi ces lymphocytes T thérapeutiques manifestent une activité cytotoxique alternative. Pour conclure, mon projet de thèse a permis de caractériser une activité de lyse soutenue basée sur une capacité individuelle hétérogène. Cette habilité à soutenir une lyse sur du long-terme implique une stabilité de la synapse, où WASP joue notamment un rôle clé pour l'activation et l'organisation de LFA-1. Les lymphocytes T régulateurs thérapeutiques démontrent aussi une activité de lyse soutenue, cependant les acteurs moléculaires sont non conventionnels. De manière générale, une activité de lyse soutenue permettrait de calibrer une réponse cytotoxique prolongée en rapport à la taille de la population cible, ainsi que le partage avec d'autres fonctions cellulaires comme la sécrétion de cytokines. / During different pathological conditions such as infections, tumoral processes or chronic inflammation diseases, altered cells are eliminated through a cytotoxic activity mediated by several immune cell populations. This cellular function is therefore crucial for carrying out the action of the immune system. My thesis project focuses on fundamental aspects of the lytic activity of two cytotoxic lymphocyte populations: CD8+ T cells and type-1 CD4+ regulatory T cells. To explore the mechanisms of this activity, this study has been driven on two cases, pathological and therapeutic models, at the population and single-cell levels and also at the cellular and nanoscopic scales of the molecular organisation. We have been able to demonstrate that the CD8+ T cell lysis activity against an excess of target cells is effective over prolonged periods, relying on a highly heterogeneous individual capacity to perform multiple lysis. The importance of this sustained cytotoxic activity was reinforced by the identification of a lytic defect, particularly pronounced on a long time period, of CD8+ T cells from Wiskott-Aldrich syndrome patients. This defect is related to a reduced activation of the LFA-1 integrin and delay in the lethal hit delivery. In addition, the WASP protein allows to restrict high affinity LFA-1 to dense nanoclusters as well as the assembly of LFA- 1 ring and the localization of the lytic granules inside this ring. Moreover, type-1 CD4+ regulatory T cells from a cellular therapy (Ovasave(r)) demonstrated a cytotoxic activity toward myeloid cells, additionally to an immunosuppressive activity on conventional T cells. This activity is implemented over long time periods, until reaching optimal efficiency, and is related to a delay in the lethal hit delivery. Surprisingly, despite a specificity for ovalbumin, the cytotoxic activity measured in absence of the antigen suggests a TCR independence. In addition, lysis is not mediated by perforin but is exclusively granzyme-dependent. Thus, these therapeutic T cells exhibit an alternative cytotoxic activity. To conclude, my thesis project permits to characterize a sustained lysis activity relying on a heterogeneous individual capacity. This ability to sustain a lytic activity involves stability of the synapse, where WASP plays a key role towards the activation and organization of LFA-1. The therapeutic regulatory T lymphocytes also demonstrated a sustained cytotoxic activity, however the molecular actors are unconventional. On the whole, sustained lytic activity would be key to the calibration of cytotoxic responses in relation to the size of the target population, as well as sharing with other cellular functions such as cytokine secretion.

Background Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that play an important role in the peripheral tolerance mechanisms of the immune system. Their suppressive function on autoreactive T cells can prevent autoimmunity. In type 1 diabetes (T1D), Treg have been inconsistently reported to be impaired in their capability to suppress autoreactive T cells (Tan, et al., 2014; Zhang, et al., 2012). Treg can be thymus derived (tTreg) or generated from naïve CD4+ CD25- T cells in the periphery (pTreg), which exhibit similar suppressive qualities as tTreg. They have also been reported to be actively induced (iTreg) under tolerogenic conditions (Kleijwegt, et al., 2010; Yuan and Malek, 2012). Although several Treg subpopulations have been described, the archetypical Treg express the major markers CD4, CD25 and FOXP3, while CD127 is heavily downregulated. However, activated conventional T cells (Tconv) show a similar phenotype, at least transiently (Miyara, et al., 2009). Since Treg and Tconv have opposing functions and therapeutic indications, it is important to obtain markers that confidently identify bona fide Treg. Scientific aim The aim of my thesis is to define the heterogeneity of human T cells with a specific emphasis to identify bona fide Treg. I examined heterogeneity of this population in healthy controls and T1D patients, as my model disease, and examined how T cells that are exposed to antigen can be defined as Treg or Tconv. Material and Methods For marker phenotyping I used samples from new onset T1D patients (age 7-11 years), autoantibody positive (Aab+) patients and age-matched healthy controls, which were tested by flow cytometry with an array of Treg-associated markers. Separately, freshly isolated CD4+CD25+CD127lo Treg and CD+CD25- Tconv were used for transcriptomic analysis, which was done by RNAseq on isolated whole RNA. For functional analysis of antigen specific gene expression patterns I developed a multi-dye proliferation assay. Treg (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+/lo) were sorted from isolated peripheral blood mononuclear cells (PBMC). I recombined the sorted and proliferation dye stained subsets with CD4- cells to simulate whole PBMC assays and stimulated them with tetanus-, influenza- or auto-antigens (GAD65, proinsulin). Cells were incubated for 5 days and responding proliferating cells as well as non-responding cells were single cell sorted and analyzed by multiplex qPCR. In investigating therapeutic approaches to expand or generate Treg, I examined in vitro approaches for de novo induction of Tregs with tolerogenic dendritic cells (tDCs). The tDCs were differentiated from monocytes either in the presence of 1α,25-OH(2)Vitamin D3 and/or Dexamethasone and matured with lipopolysaccharide. In a multistep assay, naïve T cells were incubated with DCs for two rounds and functional suppression assays were performed. The resultant T cells were analyzed at the DNA, protein, and functional level. Results Substantial phenotypic heterogeneity of peripheral blood CD4+ T cells was observed and documented for three major populations: resting Tconv (CD25-CD127+/lo), activated Tconv (CD25+CD127+) and Treg (CD25+CD127lo) in healthy controls. Despite this, I observed no differences between the Treg subpopulations from new onset T1D patients, Aab+ patients and healthy controls. In addition, there were no differences in the Treg transcriptome of T1D patients and healthy controls by RNAseq. I was, however, able to identify a small set of differentially expressed genes was discovered in Tconv suggesting a role of neutrophils in the onset of T1D. Heterogeneity of antigen-responsive Tconv and Treg was identified by gene expression profiling. I was able to define Treg specific as well as activation specific profiles, and found different expression profiles if T cells are foreign antigen or autoantigen activated and if the responding cells are Treg or Tconv. Genes that define the specific profiles include FOXP3, CD127, several cytokines, transcription factors and activation markers. The manipulation of naïve CD4+CD25- T cells by tDCs led to an unstable CD25+CD127loFOXP3+ phenotype of the generated cells. However, none of the subsequently performed functional assays could confirm that the resultant cells were iTreg or exhausted activated Tconv. In particular, methylation status of the Treg-specific demethylated region (TSDR) was inconsistent with stable Treg, suggesting that so-called tolerogenic protocols may not lead to a long-lived Treg phenotype. Conclusion CD4+CD25+ T cells are heterogeneous. I defined marker combinations that will help distinguish Treg from ex vivo and in vitro activated Tconv cells. With these tools, I was able to show that healthy controls and patients with type 1 diabetes cannot be distinguished by Treg phenotype. Comprehensive single cell analysis of antigen activated T cells provided the most promising avenue for identifying antigen-specific Treg and opens new possibilities to analyze immune therapeutic approaches, particularly when Treg expansion is the therapeutic objective. The findings will be used for monitoring children participating in antigen-based prevention studies in children at risk for T1D. / Hintergrund Regulatorische T Zellen (Treg) sind eine Subpopulation der CD4+ T Zellen, welche eine wichtige Rolle in den peripheren Toleranzmechanismen des Immunsystems spielen. Ihre suppressive Funktion auf autoreaktive T Zellen kann Autoimmunität verhindern. Verschiedene Studien berichteten widersprüchlich, dass Treg in Typ 1 Diabetes (T1D) in ihrer Fähigkeit beeinträchtigt sind autoreaktive T Zellen zu supprimieren (Tan et al., 2014; Zhang et al., 2012). Treg können im Thymus differenzieren (tTreg) oder aus peripheren naïven CD4+CD25- T Zellen generiert werden (pTreg), welche ähnliche suppressive Eigenschaften wie tTreg besitzen. Es wurde außerdem berichtet, dass Treg aktiv unter tolerisierenden Konditionen induziert werden können (iTreg) (Kleijwegt et al., 2010; Yuan and Malek, 2012). Obwohl verschiedene Treg Subpopulationen beschrieben wurden, exprimieren die archetypischen humanen Treg die Hauptmarker CD4, CD25 und FOXP3 exprimieren, während CD127 herunterreguliert ist. Jedoch zeigen auch aktivierte konventionelle T Zellen (Tconv) diesen Phänotyp (Miyara et al., 2009). Da Treg und Tconv gegensätzliche Funktionen und therapeutische Indikationen aufweisen, ist es wichtig Marker zu erhalten, die sicher bona fide Treg identifizieren. Fragestellung Das Ziel meiner Arbeit ist es, die Heterogenität von humanen T Zellen zu definieren mit einen spezifischen Fokus bona fide Treg zu identifizieren. Dafür untersuchte ich die Heterogenität dieser Zellpopulation in gesunden Individuen und T1D Patienten, als Krankheitsmodell, und wie T Zellen als Treg oder Tconv definiert werden können wenn sie einem Antigen ausgesetzt sind. Material und Methoden Für das Phänotypisieren habe ich Proben von Patienten mit beginnendem T1D (Alter 7-11 Jahre), Autoantikörper positiven Patienten (Aab+) und gesunden Individuen mittels Durchflusszytometrie auf eine Reihe von Treg-assoziierten Markern getestet. Des Weiteren wurden frisch isolierte CD4+CD25+CD127lo Treg und CD+CD25- Tconv für die Transkriptomanalyse (RNAseq) genutzt, welche mit der Gesamt-RNA durchgeführt wurden. Für die funktionelle Analyse von Antigen-spezifischen Genexpressionsmustern habe ich ein Multifarbenproliferationstest entwickelt. Treg (CD4+CD25+CD127lo) und Tconv (CD4+CD25-CD127+/lo) wurden aus isolierten mononukleären Zellen des peripheren Blutes (PBMC) sortiert. Ich habe die sortierten und gefärbten Zellen mit CD4- Zellen zusammengefügt, um einen Gesamt-PBMC-Test zu simulieren und habe die Zellen mit Tetanus-, Influenza- oder Auto-antigen (GAD65, Proinsulin) stimuliert. Die Zellen wurden für 5 Tage inkubiert und die Antigen-reagierenden und -proliferierenden Zellen sowie die nicht-reagierenden Zellen Einzelzell sortiert und mittels Multiplex qPCR analysiert. Um therapeutische Ansätze zum Expandieren oder Generieren von Treg zu untersuchen, habe ich in vitro Ansätze für die de novo Induktion von Treg durch die Nutzung von tolerisierenden dendritischen Zellen (tDCs) untersucht. Die tDCs wurden von Monozyten in Anwesenheit von 1α,25-OH(2)Vitamin D3 und/oder Dexamethason differenziert und mit Lipoploysaccharid maturiert. Naïve T Zellen wurden in einem Mehrschrittverfahren mit DCs inkubiert. Die resultierenden T Zellen wurden auf DNA, Protein und funktioneller Ebene analysiert. Ergebnisse Substantielle phänotypische Heterogenität von peripheren Blut CD4+ T Zellen wurde in drei Hauptpopulationen in gesunden Individuen beobachtet und dokumentiert: ruhende Tconv (CD25-CD127+/lo), aktivierte Tconv (CD25+CD127+) und Treg (CD25+CD127lo). Weiterführend ergab der phänotypische Vergleich von Patienten mit beginnender T1D, Aab+ Patienten und gesunden Individuen keine Unterschiede in den Treg Subpopulationen. Außerdem zeigten sich keine Unterschiede in den durch RNAseq gemessenen Treg Transkriptomen von T1D Patienten und gesunden Individuen. Jedoch wurde ein kleine Gruppe von differentiell exprimierten Genen in Tconv entdeckt, welche eine mögliche Rolle von Neutrophilen in T1D andeuten. Heterogenität von Antigen-spezifischen Tconv und Treg Antworten wurde durch Genexpressionsanalysen identifiziert. Ich konnte Treg- sowie Aktivierungs-spezifische Muster definieren und verschiedene Expressionsprofile finden, wenn T Zellen durch Fremd- oder Autoantigen aktiviert wurden und ob sie die reagierenden Zellen Treg oder Tconv sind. Folgende Gene waren hauptsächlich in die Profilbildung involviert: FOXP3, CD127, mehrere Zytokine, Transkriptionsfaktoren und Aktivierungsmarker. Die Manipulation von naïven CD4+CD25- T Zellen durch tDCs führte zu einem instabilen CD25+CD127loFOXP3+ Phänotyp der generierten Zellen. Jedoch konnte keiner der weiterführenden funktionellen Analysen unterscheiden, ob die resultierenden Zellen iTreg oder aktivierte erschöpfte T Zellen waren. Insbesondere war der Methylierungsstatus der Treg-spezifisch demethylierten Region (TSDR) nicht konsistent mit einen stabilen Treg Phänotyp, was darauf hinweist, dass sogenannte tolerisiernde Protokolle nicht zu einem langlebigen Treg Phänotyp führen. Schlussfolgerungen CD4+CD25+ T Zellen sind heterogen. Ich habe Markerkombinationen definiert die helfen werden Treg von ex vivo und in vitro aktivierten Tconv Zellen zu unterscheiden. Mit diesen Mitteln war ich in der Lage zu zeigen, dass gesunde Individuen und Patienten mit Typ 1 Diabetes nicht anhand ihres Treg Phänotyps unterschieden werden können. Umfassende Einzelzell-Analysen von Antigen aktivierten T Zellen lieferten den vielversprechendsten Ansatz für die Identifizierung von Antigen-spezifischen Treg und eröffnen neue Möglichkeiten um immuntherapeutische Ansätze zu analysieren, insbesondere wenn Treg Expansion das therapeutische Ziel ist. Diese Erkenntnisse werden zukünftig für das Monitoring von Kindern, mit einem hohen T1D Risiko, genutzt die an Antigen-basierten Präventionsstudien teilnehmen.

Paracoccidioides brasiliensis é um fungo dimórfico patogênico agente etiológico da paracoccidioidomicose (PCM), uma micose endêmica no Brasil. A busca por alternativas para reduzir o tempo de tratamento da PCM levou ao desenvolvimento de uma vacina de DNA contendo a sequência do peptídeo P10 de P. brasiliensis. Neste trabalho, avaliamos a geração de células T de memória e células T reguladoras em camundongos imunizados com esta vacina de DNA antes e após o desafio com o fungo, através da análise de seus esplenócitos e linfócitos pulmonares por citometria de fluxo. Os resultados mostram um aumento no percentual de células T reguladoras e de memória no baço e pulmões dos animais imunizados antes e depois de 30, 60 e 120 dias do desafio em comparação com os grupos controle e não imunizado. Outro experimento revelou que o modelo experimental da PCM in vivo é capaz de induzir a expressão de ROR&#947t. Este estudo mostra que nossa vacina de DNA contra a PCM gera células com fenótipo de reguladoras e de memória, caracterizando seu potencial para o tratamento desta micose. / Paracoccidioides brasiliensis is a dimorphic fungal pathogen that is the etiological agent of paracoccidioidomycosis (PCM), a mycosis endemic in Brazil. The search for new alternatives to reduce the duration of PCM treatment led to the development of a DNA vaccine encoding the peptide P10 of P. brasiliensis. Presently, we evaluated the generation of memory and regulatory T cells in mice immunized with this DNA vaccine, before and after the challenge with the fungus by analizing their splenocytes and pulmonary lymphocytes by flow cytometry. The results show an increase in the percentage of regulatory and memory T cells on spleens and lungs of immunized mice before and after 30, 60 and 120 days of challenge compared with the control and untreated groups. Another experiment revealed that the PCM in vivo infection model is capable of inducing ROR&#947t expression. This study demonstrates that our DNA vaccine against PCM generates cells with a regulatory and memory phenotype, which shows its potencial in the treatment of this mycosis.

Globalement, un tiers de la population mondiale est infectée par Mycobacterium tuberculosis, l'agent infectieux de la tuberculose (TB). Fort heureusement, seuls 5 à 10 % des individus infectés développent un jour une TB active. Les individus non malades restent cependant infectés à vie, on parle d'infection latente. Chaque année, 8-10 millions nouveaux cas de tuberculose active sont recensés et M. tuberculosis est responsable de 1,5 à 2 millions de décès. Depuis plus d'une décennie, M. tuberculosis s'est étroitement associé à l'infection par le virus de l'immunodéficience humaine. Cette alliance néfaste représente une importante menace pour les pays en voie de développement, car ces 2 pathogènes déciment les forces vives de ces populations. Il faut malheureusement rajouter à ce triste tableau une fréquence grandissante de souches multi-résistantes, voire extensivement multi-résistantes. Face à ces souches, les avancées thérapeutiques du siècle dernier sont pratiquement réduites à néant. <p>Considérant ces données, il est désormais crucial d'améliorer nos outils de dépistage de l'infection latente, de diagnostic de la maladie active, de prévention (vaccins) et de traitement. Pour atteindre ces objectifs, une des pistes est la caractérisation détaillée des réponses immunitaires. En comparant les réponses immunitaires des sujets infectés de manière latente à celles liées à la maladie active, nous pourrons peut-être comprendre certains mécanismes de protection. L'étude des réponses immunitaires induites par la « Heparin-Binding-Hemagglutinin » (HBHA) s'est faite dans cet objectif. La HBHA est une adhésine exprimée par le complexe M. tuberculosis. Elle est impliquée dans la dissémination extrapulmonaire du bacille et constitue donc un facteur de virulence. Par ailleurs, une vaccination de souris par seulement 3 doses de 5 µg de HBHA suffit à protéger de l'infection avec une efficacité comparable à celle du vaccin BCG. Chez l'homme, les sujets sains mais infectés développent d'importantes sécrétions d'interféron-gamma (IFN-γ) en réponse à cet antigène, alors que la majorité des patients tuberculeux ne le font pas. Cette différence est importante pour comprendre une des raisons d'échappement de M. tuberculosis au contrôle immunitaire. La HBHA est une protéine méthylée et la méthylation s’avère essentielle pour ses propriétés immunoprotectrices. <p>Nos travaux présentés ici se sont axés sur deux éléments de la réponse immunitaire à la HBHA chez l'homme :d'une part, l'exploitation de la réponse périphérique d'IFN-γ à la HBHA comme outil de dépistage de l'infection latente et, d'autre part, l'étude des raisons de la faible sécrétion d'IFN-γ spécifique de la HBHA lors de la maladie active.<p> <p>L'évaluation de la sécrétion périphérique d'IFN-γ en réponse à la HBHA a permis de démontrer rétrospectivement que celle-ci permet de détecter plus de 90 % des sujets réagissant positivement à l'injection intradermique de tuberculine. De manière intéressante, l'utilisation d'un test commercial, le QuantiFERON TB Gold IT (QFT-IT) n'a permis de détecter que la moitié des sujets infectés sains. De notre point de vue, le QFT-IT ne peut être recommandé seul pour le dépistage systématique de l'infection latente par M. tuberculosis. De manière parallèle, un test de stimulation basé uniquement sur la sécrétion d’IFN-γ suite à une stimulation à l'ESAT-6, composant du QFT-IT, n'a pas permis d'augmenter la sensibilité, ni d'ajouter une plus-value au test basé sur la HBHA. A l'instar de l'intradermoréaction à la tuberculine, le dépistage de la maladie active reste décevant que ce soit par l'utilisation de la HBHA ou de l'ESAT-6.<p>La TB active est caractérisée par une basse sécrétion périphérique d'IFN-γ en réponse à la stimulation par la HBHA. Cette faible sécrétion est cependant réversible, puisque un traitement efficace permet d'atteindre des taux d'IFN-γ significativement plus élevés. Ceci nous démontre qu'il s'agit d'une suppression associée à la phase active de l'infection. Nous avons d'abord évalué l'importance de la modulation de la sécrétion d'IFN-γ en réponse à la HBHA par 2 cytokines immunomodulatrices, l'interleukine-10 (IL-10) et le Transforming-Growth-Factor-Beta (TGF-ß). De manière intéressante, alors que ces 2 cytokines sont associées à l'infection par M. tuberculosis, la HBHA n'est inductrice ni d'IL-10, ni de TGF-ß. Les lymphocytes T régulateurs (Treg) expriment 2 marqueurs d'intérêt :le CD25, composant du récepteur à l'IL-2, et Foxp3, un gène régulateur majeur des cellules Treg. Ces cellules sont décrites comme suppressives de réponses immunitaires déclenchées par des antigènes du Soi et du non-Soi. Nous avons montré que la proportion de lymphocytes Treg périphériques est augmentée en cas de TB active. Par ailleurs, nous avons également démontré que ces cellules suppriment la sécrétion d'IFN-γ et la prolifération induite par la HBHA après stimulation des cellules mononucléées sanguines périphériques de patients tuberculeux in vitro. Cependant, la réponse anti-HBHA des patients tuberculeux, qui est démasquée par la déplétion des lymphocytes Treg, n'est pas dirigée contre des épitopes protecteurs. En effet, la méthylation n'influence pas leur sécrétion d'IFN-γ. De ce point de vue, les lymphocytes Treg sont impliqués dans la maladie tuberculeuse et influencent négativement les réponses dirigées contre un antigène protecteur. Cependant, il semble que la TB active soit également associée à une ignorance d'épitopes protecteurs.<p>Enfin, nous avons également démontré qu'il était possible d'induire des lymphocytes Treg au départ de cellules sanguines périphériques de sujets infectés sains. En effet, la stimulation in vitro des cellules sanguines périphériques en présence de BCG et de TGF-ß est un moyen rapide pour induire l'apparition de lymphocytes Treg fonctionnels in vitro. Ceci nous interroge quant aux rôles des lymphocytes Treg dans la pathogenèse de la maladie. En effet, un excès de TGF-ß circulant est observé dans certaines conditions cliniques à haut-risque de TB post-primaire. De ce point de vue, les lymphocytes Treg pourraient être des acteurs déterminant dans la perte du contrôle à long terme de l'infection et, par là, pourraient être des cibles thérapeutiques d'intérêts lors de l'infection par M. tuberculosis. /Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). It is estimated approximately one third of the World’s population is infected with M. tuberculosis. Fortunately, only 5 to 10 % of the infected individuals will develop the disease throughout their life. However, the other healthy infected individuals remain infected for life: this is the latent TB infection (LTBI). Every year, 8 to 10 million new cases of TB are recorded globally, and about 2 to 3 million of people die from the disease. During the last several decades the co-infection of M. tuberculosis and the human immunodeficiency virus have worsened the picture. This dreadful association currently affects mostly the poorest people of the World. Unfortunately, bad news never stands alone. We now witness increasing emergence of multi-drug-resistant and even of extensively-multi-drug-resistant M. tuberculosis strains. Against these strains current therapeutics are virtually useless. <p>The development of new tools for prevention (vaccines), diagnostics and treatment is crucial. In order to fulfill these objectives, detailed studies on the immune responses is one of the main tracks to explore. Indeed, the comparison of immune responses in LTBI subjects with those in TB patients may provide some clues to understand immune mechanisms of protection. Studies of the immune responses that are specific to Heparin-Binding-Hemagglutinin (HBHA) may be one of these clues. HBHA is an adhesin, which is expressed by the micro-organisms of the M. tuberculosis complex. It largely contributes to the extrapulmonary dissemination of the tubercle bacilli. Hence, HBHA may be qualified as an important virulence factor. Furthermore, vaccination of mice with three doses of only 5 µg HBHA each affords the same level of protection as vaccination with BCG. In humans, peripheral blood mononuclear cells (PBMC) from LTBI subjects secrete significant levels of IFN-γ in response to HBHA, whereas PBMC from TB patients do not. This discrepancy may be a cornerstone in the understanding of some of the mechanisms underlying the immune escape mediated by M. tuberculosis. HBHA is a methylated protein, and the methylation is crucial for its immuno-protective properties. <p>This work focused on 2 major issues of the HBHA-specific immune response in humans: the use of the peripheral IFN-γ secretion in response to HBHA as a diagnostic tool for LTBI and the analysis of the underlying mechanisms to the low IFN-γ secretion during active TB.<p> <p>In our study, the measurement of HBHA-specific IFN-γ secretion resulted in the detection of more than 90 % of the tuberculin-skin-test (TST) positive LTBI. Strikingly, the QuantiFERON TB Gold IT (QFT-IT), a commercial test, failed to identify those LTBI subjects in more than 50 % of the cases. Therefore, we cannot recommend the use of QFT-IT alone instead of the TST for the detection of LTBI. Similarly, a test relying on the detection of IFN-γ secretion upon ESAT-6 stimulation, one of the antigens used in the QFT-IT, was not sufficiently sensitive for the LTBI detection, nor did it improve the sensitivity or the specificity of the HBHA-based test. In contrast to the diagnosis of LTBI, the tests based on HBHA- or ESAT-6-induced IFN-γ secretions displayed poor sensitivity for the diagnosis of active TB.<p>During active TB, the HBHA-specific IFN-γ secretion in the periphery is low. However, this weak secretion is reversible upon effective treatment, as the IFN-γ response to HBHA is increased after completion of chemotherapy. This is strongly suggestive of an immune suppression during active disease. Therefore, we have first evaluated the role of two immunomodulatory cytokines, interleukin-10 (IL-10) and Transforming-Growth-Factor-Beta (TGF-ß), in the suppression of the HBHA-specific IFN-γ secretion. We found that neutralization of neither IL-10 nor TGF-ß with specific antibodies induced HBHA-specific IFN-γ secretion by PBMC of TB patients in vitro. In contrast, depletion of regulatory T cells (Treg) that express 2 major markers, CD25, a constituent of the IL-2 receptor, and Foxp3, a master regulatory gene, resulted in increased HBHA-specific IFN-γ secretion by the PBMC of TB patients. These cells are known to be involved in the suppression of immune responses to both Self and non-Self antigens. We further show that the size of the peripheral Treg cell population increases during active disease. In addition to suppressing the HBHA-specific IFN-γ secretion these cells suppress T cell proliferation in response to HBHA in vitro. However, even after depletion of the Treg cells, the uncovered HBHA-specific immune responses are not directed to the methylated epitopes during TB disease. <p>Finally, we show that Treg cells can be induced (or expanded) from the PBMC of LTBI subjects. Stimulation of those PBMC with BCG in the presence of TGF-ß resulted in a quick appearance of functional Treg cells in vitro. This observation strongly suggests a role of Treg cells in the pathogenesis of TB, in particular in the progression of latency to reactivation. Interestingly, excessive concentration of TGF-ß, associated with various clinical conditions, is high risk factor for post-primary TB. Thus, Treg cells may result in the loss of immune control against latent M. tuberculosis infection. Therefore, Treg cells may represent potential therapeutic targets during M. tuberculosis infection. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished