Dissertations / Theses on the topic 'Type 1 regulatory T (Tr1) cells'

La sclerosi multipla (SM) è una malattia degenerativa autoimmune del Sistema Nervoso Centrale (SNC), in cui cellule T CD4 autoreattive attaccano la mielina dei neuroni causando danni al SNC. La SM è anche associata all’infezione del virus Epstein-Barr (EBV), ma il ruolo dei virus nella progressione della SM è dibattuto. Le risposte di cellule T autoreattive e anti-virali sono controllate da popolazioni diverse di cellule T regolatorie, quali le Treg FOXP3+ e le cellule T regolatorie di tipo 1 (Tr1). Entrambe le polazioni sono coinvolte nella SM, ma il ruolo delle cellule Tr1 nella SM in vivo è poco chiaro. L'eomesodermina (Eomes), un fattore di trascrizione che definisce il lineage delle cellule Tr1 e controlla l'espressione del granzima (Gzm)K, ci permette la loro identificazione ex vivo. La soppressione delle risposte immunitarie è mediata da queste cellule dopo attivazione antigenica tantochè la loro specificità antigenica ne è un fattore chiave. Terapie cellulari con cellule regolatorie sono attualmente già in uso clinico per altre malattie immunomediate ma per la SM è ancora necessario determinare quale subset cellulare sia in grado di sopprimere la risposta immunitaria. Lo scopo di questa tesi è capire il ruolo delle cellule Tr1 nella SM, in particolare, analizzare le loro capacità migratorie verso SNC e la loro specificità per antigeni autologhi o virali, al fine di identificare una popolazione adatta per una eventuale terapia cellulare. Pertanto, in questo progetto ho monitorato una coorte di pazienti MS recidivanti-remittenti trattati o meno con Natalizumab ‒ anticorpo che blocca l’ingresso dei linfoci T nel SNC ‒ mediante tecniche di citofluorimetrica multidimensionale. Ho mostrato che nel liquor di pazienti MS attivi le cellule Tr1 GzmK+ Eomes+ sono fortemente e selettivamente arricchite suggerendo un loro ruolo nelle ricadute. Inoltre, le Tr1 risultano ridotte nel sangue dei pazienti MS e con un’alta proliferanzione in vivo, suggerendo che le Tr1 siano reclutate e attivate nel SNC di questi pazienti. Coerentemente, nei pazienti SM trattati con Natalizumab, le Tr1 hanno frequenze e tassi di proliferazione normali. Al contrario, i pazienti SM hanno un numero maggiore di Treg che però sono meno proliferanti in vivo, mentre le CTL restano inalterate. Nei pazienti MS le cellule Tr1 rispondono fortemente e selettivamente all'antigene EBNA-1 associato alla latenza di EBV mentre la risposta è debole negli individui sani. Non si rileva invece alcuna risposta agli antigeni della mielina o del John Cunningham Virus. Inoltre i pazienti trattati con Natalizumab hanno livelli significativamente più elevati di Tr1 specifiche per EBV/EBNA1, suggerendo che le Tr1 EBV-specifiche siano reclutate e/o generate da precursori nel SNC. Le Tr1 mostrano maggiori proprietà antinfiammatorie nei pazienti SM secernendo livelli più alti di IL-10 in risposta a stimolazione policlonale. Infine abbiamo rilevato che nel liquor le Tr1 producono notevoli quantità di IL-10, come avviene anche nel sangue periferico di pazienti SM in risposta a EBV/ EBNA1 . Nel complesso, i risultati supportano l'ipotesi di una risposta immunitaria anti-EBV alterata nel SNC dei pazienti SM, e suggeriscono un doppio ruolo per le cellule Tr1 Eomes: esse possono avere un ruolo benefico nelle ricadute in quanto sono presenti nel SNC e producono la citochina anti-infiammatoria IL-10 ma allo stesso tempo, la specificità per EBV nella fase latente potrebbe essere alla base di una risposta inefficiente al virus e quindi della progressione della MS. Ulteriori indagini sulle cellule Tr1 sono necessarie per comprendere le loro capacità soppressive, i geni coinvolti e il loro ruolo nella SM progressiva.
Multiple sclerosis (MS) is a degenerative autoimmune disease of the Central Nervous System (CNS), where autoreactive CD4+T-cells are believed to attack the myelin sheath of neurons causing CNS damage. MS is also associated with viral infections, in particular with Epstein–Barr Virus (EBV), but the role of viruses in MS progression is debated. Auto-reactive and overshooting anti-viral T-cell responses are controlled by regulatory T-cell subsets, namely FOXP3+Treg and IL-10-producing type 1 regulatory cells (Tr1) cells. Both subsets were proposed to be involved in MS, but the role of Tr1 cells in vivo in MS remains unclear. Eomesodermin (Eomes), a putative lineage-defining transcription factor of Tr1 cells that controls directly the expression of Granzyme (Gzm)K, allows their analysis ex vivo. Notably, in order to suppress immune responses efficiently, regulatory T-cells have to be activated by antigens, and their antigen specificity is a key feature. Cell-therapy with regulatory T-cells was established in other immune-mediated diseases, but the subset that efficiently suppresses pathogenic T-cells in MS needs first to be identified. The aim of this thesis is to understand the role of Tr1-cells in MS, in particular, to analyze their CNS-homing capacities and their specificity for self- or viral-antigens, in order to identify subsets that are suited for MS cell-therapy. Therefore, in this project I monitored a cohort of relapsing-remitting MS patient that were either untreated or treated with Natalizumab ‒ the anti-α4 integrin antibody that block the CNS-homing of lymphocytes ‒ by multidimensional cytometric analysis. I found that GzmK+Tr1 cells ‒ and not FOXP3+Treg or GzmB+CTL (cytotoxic lymphocytes) ‒ are strongly and selectively enriched in the cerebrospinal fluid (CSF) of active MS patients, suggesting a role in relapses. Moreover, Tr1 cells were reduced in the blood of MS patients and were highly proliferating in vivo, suggesting that Tr1 cells are recruited and activated in the CNS of MS patients. Consistently, Natalizumab-treated MS patients showed normal Tr1 frequencies and proliferation rates. Conversely, MS patients had strikingly higher frequencies of Tregs and a reduced in vivo turnover, while CTL were unaltered. To assess ex vivo the antigen specificity, a new assay was successfully established. Tr1 and their putative precursors cells responded strongly and selectively to the EBV latency-associated antigen EBNA1 in MS patients, and not with lytic ones, but responded only weakly in healthy individuals. They also failed to respond to myelin antigens or to the John Cunningham Virus. Interestingly, Natalizumab-treated patients had significantly higher levels of EBV-specific Tr1 cells, suggesting that these cells are recruited to and/or generated from precursors in the CNS. Tr1 cells have enhanced anti-inflammatory properties in MS patients, secreting higher levels of IL-10 in response to polyclonal stimulation. Moreover, we have preliminary evidences that Tr1 cells produce also considerable amounts of IL-10 in the CSF and even in response to EBV/EBNA1 in the blood of MS patients. Overall, our results are consistent with the notion that there is a dysregulated immune response against EBV in the CNS of MS patients, and suggest a dual role for Eomes+Tr1 cells regulating EBV-specific and not myelin-reactive T-cells. A key finding for this project is that Tr1 cells may have a beneficial role in relapses since they are present in the CNS and produce the anti-inflammatory cytokine IL-10. But at the same time, the specificity for EBV in the latent phase could be at the basis of the inefficient response to the virus and therefore of MS progression. In the future a better understanding of Tr1 cell role in MS could lead to novel therapeutic approaches, although further investigations on Tr1 cells are needed to understand their suppressive abilities, the genes involved and their role in progressive MS.

Unlike other helper T cells, the co-stimulatory ligands responsible for T regulatory type-1 (Trl) cells differentiation remain undefined. Understanding the molecular interactions driving peripheral Trl differentiation is important because Trls potently regulate immune responses, by IL-10 production. In this study we show that co-stimulation of human naïve CD4+ cells through the CD97-CD55 interaction drives Trl activation, expansion and function. T cell activation and expansion was equipotent with CD55 or CD28 co-stimulation, however CD55 co-stimulation resulted in two IL-10 secreting populations. The majority of the IL-10 was secreted by the minor, Trl population (IL-10high IFN-y- IL-4-, <5% cells) that express Trl markers CD49b, LAG-3 and CD226. This Tr 1 phenotype was not re-stimulated by CD28. But on CD55 re-stimulation, Trls proliferated and maintained their differentiated IL-10 high phenotype. The Trls significantly suppressed effector T cell function in an IL-10 dependent manner. The remaining (>95%) cells adopted a Thl- like IFN-y + phenotype. However, in contrast to CD28 derived This, CD55 derived This demonstrated increased plasticity with the ability to co-express IL-10 when re-stimulated through CD55 or CD28. These data identify CD55 as a novel co-stimulator of human Trls and support a role for alternative co-stimulatory pathways in determining the fate of the growing number of T helper populations. In this study we also show a defect in Trls in the autoimmune disease Multiple Sclerosis (MS). In response to CD55 costimulation, naïve CD4+ cells from a cohort of MS patients did not differentiate into Trl cells as normal, however the CD55 induced Thl response was unaffected. These patients showed persistent lack of an IL-10h1gh Trl population on primary and secondary CD55 costimulation. Also, MS patients mounted stronger IFN-y responses compared to healthy controls. These data demonstrate an altered immune balance in MS and highlight a defect in the Trl response as a contributing factor of this change. Overall, this study demonstrates that CD55 acts as a potent co-stimulator and activator of human naive CD4+ cells resulting in the differentiation of a discrete Trl population that inhibits T cell function in an IL-10 dependent manner and maintains the Trl phenotype upon re-stimulation, in healthy individuals. However, there is a defect in this normal Tr 1 response in the autoimmune condition Multiple Sclerosis.

IL-10-producing CD4+ type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T cell responses mainly via cytokine dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)- dependent mechanism that requires HLA class I recognition, CD54/Lymphocyte Function-associated Antigen (LFA)-1 adhesion, and activation via CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1 cell activation, is necessary for defining Tr1 cell target specificity. We also showed that high frequency of GZB expressing CD4+ T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10- producing CD4+ T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions which lead to selective killing of target cells and possibly bystander suppression. Adaptive type 1 regulatory T (Tr1) cells are suppressor cells characterized by the production of IL-10 in the absence of IL-4 and by the ability to suppress immune responses mainly by the release of IL- 10 and TGF-β. Despite several efforts for the detection of molecular markers of Tr1 cells have been made, so far Tr1 cell identification relies on cytokine production profile. Moreover, to date no master regulator gene for Tr1 cells have been defined. To identify Tr1 cell specific surface biomarkers, master regulator genes, and molecules involved in their suppressive functions, we performed a gene expression profiling to compare the gene expression of ex vivo isolated Tr1 cell clones compare to Th0 cell clones, in resting state and upon TCR activation. Results demonstrated that Tr1 cells signature is of anti-inflammation, anti-proliferation, and immuno-modulation. In addition, we identified surface molecules that could be useful to identify Tr1 cell population. Interestingly, several transcription factors resulted differentially expressed in Tr1 cells compared to Th0 cells, which may represent master regulators of Tr1 cells.

Objectifs : Les lymphocytes T régulateurs de par leur rôle primordial dans l'homéostasie de la réponse immune sont des cellules idéales pour une immunothérapie antigène-spécifique dans les maladies auto-immunes. Les lymphocytes T régulateurs de type 1 ou Tr1 sont caractérisées par une forte sécrétion d'IL-10, cytokine qui joue un rôle déterminant dans leur capacité à supprimer des réponses immunes pathologiques dans différents contextes. L'objectif de ma thèse est d'évaluer le potentiel thérapeutique de cellules Tr1 spécifiques du collagène de type II (col-Treg) dans deux modèles de polyarthrite rhumatoïde (PR) chez la souris. Méthode : Les clones Col-Treg ne possèdent pas de marqueurs membranaires spécifiques mais sont caractérisés par un profil cytokinique particulier (IL10highIL4negIFN-γint) et par leur capacité de suppression in vitro. Tout comme les Tregs naturels, ils expriment une quantité importante de GITR, de CD39 et de Granzyme B. Une simple injection de cellules Col-Treg réduit l'incidence et les symptômes cliniques de l'arthrite à la fois de manière préventive et curative, avec un impact significatif sur les anticorps anti-collagène de type II. En outre, l'injection de Tr1 antigène spécifique in vivo diminue de manière significative la prolifération des cellules T antigène spécifique. Conclusion : Nos résultats démontrent le potentiel thérapeutique des cellules Col-Treg dans deux modèles d'arthrite expérimentale prouvant que les cellules Col-Treg représente une nouvelle approche thérapeutique de choix pour le traitement des patients atteint de polyarthrite et réfractaires aux traitements actuels
Objectives : Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an ideal target for therapies to suppress inflammation in an antigen-specific manner. Type 1 Treg cells (Tr1) are defined by their capacity to produce high levels of IL10, which contributes to their ability to suppress pathological immune responses in several settings. The aim of my PhD was to evaluate the therapeutic potential of collagen type II-specific Tr1 (Col-Treg) cells in two models of rheumatoid arthritis (RA) in mice. Methods : Col-Treg clones were isolated and expanded from Collagen-specific TCR transgenic mice. Their cytokine secretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells was evaluated after adoptive transfer in collagen-antibodies- and collagen-induced arthritis models. The in vivo suppressive mechanism on effector T cell proliferation was also investigated. Results : Col-Treg clones are characterized by a cytokine profile (IL10highIL4negIFN-γint) and mediate contact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 and Granzyme B. Single infusion of Col-Treg cells reduced incidence and clinical symptoms of arthritis both in preventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection of antigen-specific type 1 Treg cells decreases significantly the proliferation of antigen-specific effector T cells in vivo. Conclusion : Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providing evidence that Col-Treg could be an efficient cell-based therapy for RA patients refractory to current treatments

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology." Discipline: Microbiology and Immunology; Department/School: Medicine.

Microbiology and Immunology
Ph.D.
Interleukin-27 (p28/EBI3) is an immunomodulatory cytokine expressed by activated antigen presenting cells. Although first discovered to be involved in Th1 cell differentiation, further studies demonstrated the immunosuppressive functions of IL-27 including inhibition of Th2 and Th17 differentiation, development of a tolerogenic phenotype in dendritic cells (DC), and promoting type 1 regulatory T cells (Tr1). The anti-inflammatory effects of IL-27 have been demonstrated in vivo in murine models of parasitic infections and autoimmune diseases. Despite the prevalence of studies detailing the induction of IL-27 expression and the role of IL-27 in Tr1 differentiation, little is known about factors that negatively regulate IL-27 expression and Tr1 differentiation. Prostaglandin E2 (PGE2), a lipid mediator abundant at inflammatory sites, was shown to act as a proinflammatory agent in models of inflammatory/autoimmune diseases primarily by promoting CD4 Th1/Th17 differentiation. Here we describe a novel proinflammatory mechanism for PGE2 through the inhibition of IL-27 production in conventional dendritic cells (cDC) and the inhibition of Tr1 differentiation. PGE2 inhibits IL-27 production in bone marrow-derived DC and macrophages, as well as in splenic cDC, through EP2/EP4 receptors, induction of cAMP, and downregulation of IRF1 expression and binding to the p28 IL-27 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-β, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, PI3K, or MAPKs. We observed similar inhibition of p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. In addition to the inhibition of IL-27 production in APCs, PGE2 also directly affects Tr1 differentiation by reducing IL-27-induced CD4+CD49b+LAG-3+Foxp3- Tr1 cells and IL-10 production. The inhibitory effect is mediated by EP4 and induction of cAMP in differentiating CD4 T cells. IL-27-induced Tr1 differentiation and function depends primarily on the sustained expression of c-Maf in addition to AhR and Blimp-1. PGE2 significantly reduced expression of c-Maf without affecting AhR and only marginally reducing Egr-2/Blimp-1 expression. The effects of PGE2 on Tr1 cells are independent of STAT1/STAT3 signaling and of IL-21 signaling. In addition, the effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. The effects of PGE2 on both IL-27 production and IL-27-induced Tr1 differentiation represent novel proinflammatory mechanisms of PGE2.
Temple University--Theses

Les facteurs de transcription de la superfamille des récepteurs nucléaires jouent de multiples rôles dans le développement et la fonction des lymphocytes T régulateurs (TREG). Les TREG sont des cellules régulatrices/suppressives qui contrôlent les réponses d’autres types cellulaires et l’homéostasie locale des tissus.Comme les TREG sont actives au sein de divers organes, tant à l’homéostasie qu’en conditions inflammatoires,ils doivent répondre à la fois aux contexte local au sein du tissus et à un environnement immunologiquement agressif tout en préservant leurs propriétés tolérogéniques au cours du temps. Ces caractéristiques apparemment antinomiques sont contrôlées par un réseau transcriptionnel complexe au sein duquel le facteur de transcription FOXP3 joue un rôle prédominant. Au cours des dernières années, de nombreuses études se sont intéressées aux TREG présent dans les tissus non lymphoïdes (NLT). Ces populations ont été étudiées aussi bien à l’homéostasie qu’en conditions inflammatoires dans diverses pathologies. Des facteurs de transcriptions spécifiques d’un tissus ou d’une fonction déterminées ont été mis en évidence et leur rôle régulateur dans le développement, l’activation, la migration et l’immunosuppression a été caractérisé. RORa est un récepteur nucléaire qui contrôle le développement cérebellaire et hépatique, le métabolisme systémique, la différenciation des lymphocytes auxiliaires TH17, des cellules lymphoïdes innées (ILC) de type 2 et 3. RORa est fortement exprimé dans les TREG des NLT, y compris dans le tissus adipeux viscéral (VAT), l’intestin et la peau. . . .Ces populations de TREG exprimant RORa ont été associées à diverses pathologies. Cependant seule une étude récente a été consacrée à leur rôle précis. L’implication de RORa dans de nombreuses fonction, sa forte expression au sein des TREG des NLT nous a poussé a étudier le rôle de ces TREG exprimant RORa dans diverses pathologies. Dans ce butit, nous avons généré des souris spécifiquement déficientes pour RORa au sein des TREG (RORaFoxp3/Foxp3 ). Nous avons émis l’hypothèse que RORa contrôle le développement ou la fonction des TREG en conditions homéostatiques et dans des pathologies inflammatoires des NLT. Aussi nous avons caractérisé le phénotype des animaux RORaFoxp3/Foxp3 et en particulier les TREG du VAT à l’homéostasie, où la réponse de type 2 est protectrice et dans un modèle d’obésité (et d’insulino-résistance) induit par l’obésité (DIO) dans laquelle nous avons mis en évidence un rôle protecteur important des TREG exprimant RORa dans ces deux conditions expérimentales. Nous également étudié la contribution de ces cellules dans un modèle d’inflammation allergique (AAI) induite par un acarien (HDM) caractérisé par une forte réponse de type 2 et montré une aggravation de la pathologie. Pour étudier le mécanisme moléculaire de l’action de RORa au sein des TREG, nous avons procédé à une analyse transcriptomique des cellules isolées dans diverses conditions expérimentales in vivo et in vitro et avons étudié le rôle de RORa dans les modifications épigénétiques au sein des TREG en caractérisation l’acétylation des histones dans le génome entier. Cette étude nous a permis de mieux appréhender comment les TREG étaient régulées par un facteur nucléaire à l’homéostasie et en conditions inflammatoires. Les récepteurs nucléaires représentent des cibles thérapeutiques intéressantes compte tenu de leur action pléiotropique et de leurs ligands de petite taille. Compte tenu de l’importance des TREG dans l’homéostasie tissulaire et dans de nombreuses pathologies, cibler de tels facteurs au sein d epopulations cellulaires spécifiques représente une stratégie prometteuse dans le case de RORa et des TREG
Transcription factors of the nuclear receptor superfamily have a vast influence on development and function ofregulatory T cell (TREG) cells. TREG cells are suppressive immune cells of adaptive immune system. Their mainfunctions are control of inflammatory response mounted by other immune cells and maintenance of localtissue homeostasis. As TREG act at various sites of the body and both in homeostatic and inflammatory state,they need to adequately respond to local tissue-specific cues as well as adapt to aggressive immuneenvironments while preserving their long-lasting tolerogenic properties. This is achieved by weaving complextranscriptional networks, converging at transcription factors with various coordination functions, the mainbeing forkhead box P3 (FOXP3). During last few years, many studies focused on TREG cells found innon-lymphoid tissue (NLT). These populations of TREG are examined in the contexts of homeostasis and manyinflammatory diseases, and tissue- or function-specific transcription factor (TF) were assigned to some ofthem as regulators of development, activation, proliferation, stability, migration and suppressive functions.Retinoic acid receptor-related orphan receptor alpha (RORa) is a nuclear receptor, which controls cerebellumdevelopment, liver and whole-body metabolism and differentiation of T-helper (TH)17, type 2 innate lymphoidcells (ILC2) and type 3 innate lymphoid cells (ILC3). RORa is highly expressed in NLT TREG, includingpopulations in visceral adipose tissue (VAT), intestine and skin, and gets more and more mentions in thearticles dedicated to TREG in NLT. These RORa-expressing populations of TREG were all shown to be involvedin various pathologies. However, RORa role in TREG was directly addressed only once in a recent study. It’sactive involvement in various processes, high expression in NLT TREG and lack of knowledge make RORa anattractive target for investigation, to deepen current view of homeostasis control by TREG and thus betterunderstand mechanisms of development of associated diseases. To attain these objectives, a mouse strain withTREG-specific RORa deficiency was generated. Our central hypothesis is that RORa controls development orfunction of TREG cells in homeostasis of NLT and potentially in inflammatory diseases. For studying a role ofRORa in NLT TREG during control of tissue homeostasis, in particular, VAT TREG, we have charachterizedphenotype of untreated RORaFoxp3/Foxp3 mice and challenged mice with a model of diet-induced obesity(DIO). In both cases we have found an important role of TREG-expressed RORa. To further investigate a roleof RORa in TREG during pathologies and it’s contribution to various types of immune response we have testedan involvement of RORa in TREG in the model of allergic pathology, namely house dust mite (HDM)-inducedallergic airway inflammation (AAI) model.To elucidate molecular mechanisms of RORa action in TREG cells, we have performed gene expression profilingof TREG cells from examined tissues and conditions in vivo, as well as in vitro. We also have studied a role ofRORa in epigenetic landscape of TREG cells in vitro by probing histone acetylation marks genome wide. As aresult of this study, we have gained a broader understanding of TREG control by nuclear receptors and TF ingeneral in homeostatic conditions and during inflammation. Nuclear receptors proved to be useful targets fortherapeutic agents thanks to their versatile functions inside the cell and to ligand-dependency. Given thecrucial importance of TREG cells in organismal homeostasis and their involvement in numerous pathologies,targeting particular cues inside these cells may be a powerful tool in new treatment strategies. Results of ourstudy might serve as a basis for development of novel pharmaceutical agents targeting RORa

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 182-202). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Type 1 diabetes (T1D) is etiologically considered as an autoimmune disease, where insulin-producing β-cells are damaged by autoimmune attacks. Regulatory T (Treg) cells are immune homeostasis cells. In the present thesis I aimed to investigate the role of Treg cells and other immune cells in the early development of T1D. In order to do that, we first determined which immune cells that are altered at an early stage of the T1D development. We found that dendritic cells and plasmacytoid dendritic cells induce the initial immune response. Next, we investigated the role of Treg cells in multiple low dose streptozotocin (MLDSTZ) induced T1D and in NOD mice. We found that the numbers of Treg cells were increased in both MLDSTZ and NOD mice when the MLDSTZ mice were hyperglycemic. However, the increased Treg cells showed a decreased production of anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) and an increased expression of pro-inflammatory cytokines (IFN-γ and IL-17a). These results revealed that Treg cells switch their phenotype under T1D conditions. IL-35 administration effectively prevented the development of, and reversed established MLDSTZ induced T1D. Treg cells from IL-35 treated mice showed an increased expression of the Eos transcription factor, accompanied by an increased expression of IL-35 and a decreased expression of IFN-γ and IL-17a. These data indicate that IL-35 administration counteracted the early development of T1D by maintaining the phenotype of the Treg cells. Furthermore, IL-35 administration reversed established T1D in the NOD mouse model by maintaining the phenotype of Treg cells, seemingly by inducing the expression of Eos. Moreover, the circulating level of IL-35 was significantly lowered in both new onset and long-standing T1D patients compared to healthy controls. In addition, patients with T1D with remaining C-peptide had significantly higher levels of IL-35 than patients lacking C-peptide, suggesting that IL-35 might prevent the loss of β-cell mass. In line with this hypothesis, we found that LADA patients had a higher proportion of IL-35+ tolerogenic antigen presenting cells than T1D patients. Subsequently, we determined the proportions of IL-35+ Treg cells and IL-17a+ Treg cells in T1D patients with diabetic nephropathy (DN), which were age, sex and BMI matched with healthy controls and T1D patients. The proportion of IL-35+ Treg cells was decreased in DN and T1D patients, but IL-17a+ Treg cells were more abundant than in healthy controls. Furthermore, we found that the number of Foxp3+ Treg cells was increased in the kidneys of MLDSTZ mice. However, infiltration of mononuclear cells was seen in kidneys of these mice. In addition, kidney tissues of IL-35 treated MLDSTZ mice did not show any mononuclear cell infiltration. These results demonstrate that IL-35 may be used to prevent mononuclear cell infiltration in kidney diseases. Our findings indicate that the numbers of Foxp3+ Treg cells are increased in T1D, but that these Treg cells fail to counteract the ongoing immune assault in islets and kidneys of hyperglycemic mice. This could be explained by a phenotypic shift of the Treg cells under hyperglycemic conditions. IL-35 administration reversed established T1D in two different animal models of T1D and prevented mononuclear cell infiltration in the kidneys by maintaining the phenotype of Treg cells.

Treg cells counter-balance autoreactive immune cells in healthy individuals. In the absence of Treg cells, multi-organ immune diseases manifest. Thus, Treg cell defects are suspected to contribute to T1D. Currently, it remains unclear whether Treg cell defects are responsible for the manifestation of T1D. Thus, we hypothesized that Treg cell defects results in T1D. We observed that T reg cells are present in normal frequencies in the thymus and peripheral immune system of diabetes-prone mice. Furthermore, thymic and peripheral T reg cells are operative and mediate regulation by suppressing effector T (Teff) cells in the pancreatic lymph nodes and pancreas. Specifically, regulation corresponded with increased frequencies of Treg cells and reduced in diabetogenic T cells in both sites, and controlled insulitis. However, as the immune response against beta cells of the pancreatic islets progresses in prediabetic mice, peripheral Treg cells wane in function and cannot sustain sufficient Teff cell regulation in the pancreatic lymph nodes and pancreas. In summary, this study characterizes areas of T reg cell immunoregulation of T1D; and highlights a Treg cell dysfunction, not a quantitative Treg cell defect, manifesting in the peripheral immune system prior to diabetes onset but following T cell activation, which contributes to the escape of autoreactive T cells.

In this dissertation we investigate the role of avidity in the T cell selection process by examining the impact of signal modulation on T cell and/or NKT cell development. Projects discussed herein (including peptide, anti-CD1d, and hydrocortisone (HC) therapy) examine how changes in avidity can be used to explore potential therapies for Type 1 diabetes mellitus (T1DM). In the case of peptide therapy, we find that fetal thymic organ culture (FTOC), treated with exogenous diabetes related GAD peptides, lose their ability to generate T cell responses to GAD treatment peptides. Also, peptide therapy is shown to inhibit T1DM in vitro (ivT1DM) and in vivo. The abnormally high level of GAD peptides that are presented during peptide therapy treatment are thought to increase avidity between peptide specific T cells and selecting cells during thymic education, leading to increased negative selection of those T cells. In the case of anti-CD1d, FTOC from C57BL/6 (B6) and non-obese diabetic (NOD) mice, when treated with 10 μg/mL of anti-CD1d, show divergent responses to treatment. In response to anti-CD1d, "normal" B6 FTOC shows decreased T cell development and NKT production. Conversely, "poor signaling" NOD mice show no major impact on general T cell development but instead show increases in NKT cell production. Also, treatment with anti-CD1d is shown to inhibit diabetes in our ivT1DM model. These effects are thought to be due to increases in avidity generated through anti-CD1d related increased TCR expression. Changes in avidity caused by anti-CD1d treatment are thought to generate increased negative selection in B6 FTOC, while the same avidity increases are thought to increase positive selection (without increasing negative selection) in "poor signaling" NOD FTOC. In the case of HC treatment, B6 FTOC treated with HC show changes in T cell yield, maturity, and TCR Vβ usage. Research with HC indicates that signal inhibitors have the capacity to change T cell development in a dose and time dependent manner. Based on this work, selection signal inhibitors or enhancers may have the capacity to change T cell development in a fashion that decreases autoimmune T cells and/or enhances regulatory NKT cell development.

NOD.B6 Idd3 congenic mice, whose Idd3 locus originates from the autoimmune resistant C57BL/6 mouse, demonstrate a drastic reduction in diabetes onset. In this study we demonstrate that NOD.B6 Idd3 mice show halted disease progression and also show reduced levels of TH1 cells in their peripheral immune system. In vitro , CD4+ CD25- effector T (TEFF ) cells from congenic mice display increased proliferation and IL-2 production compared to wild type NOD mice. However, unfractionated NOD.B6 Idd3 CD4+ T cells maintain a higher TREG:T EFF ratio and proliferate less than their NOD counterparts, suggesting increased immunoregulation. In vivo, the Idd3 locus does not appear to reduce the number of islet-reactive cells or increase the systemic frequency of TREG cells in the immune system of pre-diabetic mice. Instead, reduced infiltration of TEFF cells in the pancreas of NOD.B6 Idd3 mice suggests increased suppression of autoimmune cells at the site of inflammation in vivo. Consistent with this hypothesis, tolerance mechanisms in the NOD.B6 Idd3 mouse are much better at suppressing the proliferation of islet-reactive T EFF cells in the pLN. Furthermore, using a novel strain of BDC.Idd3 mice, which contain islet-reactive TCR transgenic CD4+ T cells and which are homozygous for the B6-derived Idd3 allele, we demonstrate that TEFF cells containing the protective Idd3 allele do not possess reduced diabetogenic potential in vivo. Using an adoptive transfer model for type-1 diabetes, we demonstrate that CD4+CD25+ TREG containing the protective Idd3 allele are intrinsically better suppressors than TREG cells of NOD genotype. Collectively, our data show that the protective Idd3 allele favours immunoregulation in the pancreatic lesion by enhancing TREG cell function. Although the precise mechanism of this protection remains unknown, these findings may ultimately help develop therapeutic strategies to reduce or prevent the autoimmune destruction of pancreatic beta islet cells in type-1 diabetes patients.

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

Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing β cells by autoreactive lymphocytes. CD4+FOXP3+ T regulatory cells (Tregs) are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to T1D. Tregs require the cytokine interleukin-2 (IL-2) for maintenance of their suppressive function, and polymorphic variants in IL-2/IL-2R pathway genes are associated with T1D. Tregs can display plasticity by converting into Th17 cells, and intermediate FOXP3+IL-17+ cells have been identified. We hypothesized that pancreatic β cell destruction in T1D is driven by conversion of autoreactive Treg cells into a Th17 phenotype due to defective Treg IL-2 signaling in T1D subjects, who have polymorphic variants in the IL2RA gene. We assessed by flow cytometry the proportion of Treg and Th17 subsets in peripheral blood mononuclear cells from T1D subjects. The subjects were genotyped to determine whether they had the T1D-associated IL2RArs3118470 CC risk haplotype. Samples from T1D subjects were also obtained before the onset of disease. We found that Tregs are potentially transitioning towards a Th17 phenotype in recent-onset T1D subjects as they have an elevated proportion of FOXP3+IL-17+ cells and Th17 cells in their peripheral blood. We went on to show that T1D subjects with the T1D-associated IL2RArs3118470 CC risk haplotype have Treg cells with IL-2 signaling deficits and an increase in the proportion of IL-17+FOXP3+ cells in their peripheral blood at diagnosis. We did not find changes in the overall proportions of Tregs and Th17 cells in T1D subjects sampled before the onset of diagnosis. However, we observed a subset of CD39-expressing Treg cells were reduced in proportion before disease onset and could act as a biomarker of T1D. In conclusion, we show that defective IL-17-secreting Tregs are involved with T1D pathogenesis in a genetically identifiable subset of subjects, and provide a rationale for the treatment of T1D with therapeutics that target the IL-17 pathway.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Type I diabetes (T1D) is an autoimmune disease in which the insulin producing β cells of the pancreas are selectively targeted for destruction by autoimmune effector T cells. The aberrant effects of these effector T cells may in part be due to a dysfunction in the regulatory T cell (TREG) compartment and currently there is great interest in developing methods to strengthen the immunoregulation of patients with T1D. A potential way to boost immunoregulation in these patients would be the use of adoptive TREG therapy whereby expanded polyclonal TREG are transferred into patients. Although this treatment in mouse models of disease has shown promise it has been found that antigen specific TREG cells are much more efficacious of preventing disease, and can even reverse disease. The translation of these murine experiments into the human setting is however complex, since the generation of large numbers of antigen specific TREG from human patients is currently a major hurdle. One way to remove this barrier is to utilise lentiviral gene transfer technology, which can allow for the transfer of antigen specific T cell receptor (TCR) genes into a desired cell population. Specifically for T1D, it is hypothesised that TREG engineered to express a MHC Class I restricted (MHCI) TCR, although unconventional, would selectively function at the site of inflammation i.e. within the islets. This project, therefore, aims to generate MHCI islet antigen specific TREG with the hypothesis that these would confer islet antigen specific suppression. To test this hypothesis we engineered human CD4+ TREG to express two MHCI islet antigen specific TCRs whilst using a third high affinity pathogenic MHCI TCR as a control. As others have shown, we demonstrate that the control TCR was effectively able to re-direct the antigen specificity of TREG cells through signalling and function. However, we discovered that transfer of autoimmune MHCI TCRs were unable to yield the same results as the control TCR due in part to their natural low affinity for antigen. To circumvent this, we engineered CD4+ TREG to express an MHCI autoimmune TCR along with the CD8αβ co-receptor or a CD8αβ high affinity variant. Using this system, human TREG could be successfully re-directed towards an islet specific peptide and exhibit antigen specific suppression. Thus, this study is the first of its kind to use an autoimmune disease relevant, MHCI TCR to successfully re-direct the Ag specificity of TREG cells.

La Diabetis tipus 1 (T1D) és una malaltia autoimmunitària crònica caracteritzada per la pèrdua selectiva de les cèl·lules beta productores d'insulina del pàncrees. La seva 1111etiologia no està clara encara, però es creu que té un origen multifactorial que involucra factors genètics, ambientals i estocàstics. Quan es perd la tolerància als autoantígens, es produeix una infiltració dels illots pancreàtics per cèl·lules T CD8+, macròfags, cèl·lules T CD4+ i cèl·lules B, entre altres tipus cel·lulars que orquestren la seva destrucció. A la T1D la pèrdua de tolerància s'ha associat a l'existència de defectes funcionals i baixa freqüència de cèl·lules Treg i cèl·lules iNKT en ratolins NOD. Per tant, aquest estudi analitza la funció reguladora de les cèl·lules iNKT humanes aïllades de controls sans i de pacients amb T1D al debut clínic de la malaltia. Les dades mostren que les cèl·lules iNKT tenien la capacitat per suprimir la proliferació de les cèl·lules T efectores. Curiosament, la supressió era depenent de la secreció de citocina IL-13, fenomen que es va confirmar quan es recuperava la proliferació de les cèl·lules T efectores en bloquejar la IL-13 amb un anticòs monoclonal. En canvi, les cèl·lules iNKT derivades de pacients T1D no presentaven capacitat supressora i aquest defecte funcional es va poder relacionar amb una disminució en la secreció d'IL-13. Atès que les cèl·lules iNKT poden modular la funció d'altres cèl·lules immunes, es va analitzar la seva capacitat per cooperar amb cèl·lules Treg i millorar la seva funció reguladora, prevenint millor la destrucció de les cèl·lules beta. Els resultats van mostrar que les cèl·lules iNKT individus sans tenien un efecte adjuvant en la supressió de cèl·lules T efectores per cèl·lules Treg. L’efecte adjuvant era depenent de la secreció de la citocina IL-13 i independent del contacte cèl·lula-cèl·lula. Per entendre el possible paper d'aquestes cèl·lules reguladores a la T1D, es va analitzar la freqüència, la localització anatòmica i la funcionalitat al pàncrees de pacients amb T1D. La freqüència d’ambdós tipus cel·lulars augmentava al pàncrees al debut de la T1D però gairebé desapareixien al pàncrees dels pacients de llarga evolució. Aquests resultats contrastaven amb els obtinguts a ratolins NOD, on la freqüència d'aquestes dues poblacions seguia augmentant a l'etapa diabètica. La distribució també Les cèl·lules iNKT i Treg presentaven una distribució diferencial dins i al voltant dels illots pancreàtics i al teixit exocrí que canviava amb la progressió de la diabetis. Al debut de la malaltia les cèl·lules Treg eren reclutades als illots pancreàtics, mentre que les cèl·lules iNKT es mantenien preferentment al teixit exocrí. Finalment, les cèl·lules iNKT i Treg aïllades del pàncrees d'un pacient amb T1D eren funcionals, és a dir, que les cèl·lules iNKT eren específiques per al glicosfigolípid αGalCer i les cèl·lules Treg suprimien la proliferació de les cèl·lules T efectores. Per tant, el conjunt de dades mostrava que alteracions en la secreció d'IL-13 per cèl·lules iNKT a l'inici de la malaltia podrien conduir a la progressió de la resposta autoimmunitària a la Diabetis tipus 1. A més, la seva cooperació amb cèl·lules Treg i la presència dels dos tipus cel·lulars a l'òrgan diana de la resposta suggerien que la seva cooperació pot tenir lloc in situ i controlar així la destrucció de les cèl·lules beta pancreàtiques. Per tant, les noves dades sobre la funció reguladora de les cèl·lules iNKT, la identificació de glicoesfingolípids que milloren la secreció d’IL-13 per cèl·lules iNKT i la cooperació de Treg i cèl·lules iNKT, proporcionen un punt de partida per al disseny de noves estratègies terapèutiques que permetin eludir les restriccions de les immunoteràpies amb cèl·lules restringides per MHC.
Type 1 Diabetes is a chronic autoimmune disease characterized by the selective loss of insulin producing pancreatic β cells. Its etiology is unclear yet but is thought to have a multifactorial origin involving genetic, environmental and stochastic factors. When tolerance to autoantigens is lost, there is an infiltration of the pancreatic islets with CD8+ T cells, macrophages, CD4+ T cells and B cells, among other cell types that orchestrate their destruction. In T1D tolerance breakdown has been associated to the existence of functional defects and low frequencies of Foxp3+ Treg and iNKT cells in NOD mice. Therefore this study analyzes the regulatory function of human iNKT cells isolated from PBMCS of healthy controls and T1D patients at disease onset. The data show that iNKT cells have the capacity to suppress the proliferation of T effector cells. Interestingly suppression is dependent on the secretion of the cytokine IL-13, a phenomenon that was confirmed when using a blocking antibody to IL-13 resulted on the recovery of T effector cell proliferation. Conversely, regulation was impaired in iNKT cells derived from T1D patients and this functional defect could be related to a decrease in the secretion of IL-13. Since iNKT cells can modulate the function of other immune cells, we analyzed their capacity to interact with Treg cells and improve their regulatory function to better prevent the destruction of pancreatic β cells. The results showed that healthy individuals’ iNKT cells had an adjuvant effect on the suppression of T effector cells by Treg cells. The contribution of iNKT cells was again dependent on the secretion of the immunoregulatory cytokine IL-13 and independent of cell-cell contacts. To understand the possible role of these regulatory cells in T1D, their frequency, anatomical localization and functionality was studied in the pancreas of patients with T1D. We quantified the number of iNKT and Treg cells at different stages of the disease development both in human and NOD mice samples and we analyzed their distribution in and around the pancreatic islets and the exocrine tissue compartment. iNKT and Treg cells showed a differential distribution at disease onset as Tregs were concentrated inside the pancreatic islets while iNKT cells were mainly located in the exocrine tissue. Further, both populations almost disappeared from the pancreas of long-termT1D patients. These results contrasted with those obtained from NOD mice where the frequency of these two populations continued to rise at the diabetic stage. In addition, iNKT and Treg cells isolated from the pancreas of a T1D patient at disease onset were both functional, that is to say that iNKT cells were specific for the glycosphigolipid αGalCer and the Treg cells suppressed the proliferation of T effector cells. Therefore, the set of data shows that alterations in the secretion of IL-13 by iNKT cells at disease onset could lead to the progression of the autoimmune response in T1D. Their cooperation with Treg cells and the presence of both cell types at the target organ of the autoimmune response suggests that their cooperation can take place in situ controlling the destruction of the pancreatic β cells by autoreactive T cells. Thus, the new data on the regulatory function of iNKT cells, the identification of glycosphingolipid agonists enhancing iNKT cells’ IL-13 secretion and the cooperation of Treg and iNKT cells to regulate autoreactive T cells provide a starting point to the design new therapeutic strategies that bypass the constrains of the MHC-restricted immunotherapies.

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.

Background. Islet amyloid polypeptide (IAPP, also known as amylin) is a 37-amino acid peptide principally co-secreted with insulin from the beta-cells of the pancreatic islets. Some of the physiological actions of human amylin (hIAPP) include glucose regulation, suppression of appetite and stimulation of renal sodium and water reabsorption. Amylin deficiency and diminished post-prandial amylin response have been reported in advanced stages of type 1 and type 2 diabetes. In autopsy specimens of type 2 diabetes, amyloid is found in 40--90% of cases. During the characterization of islet morphology of aged hIAPP transgenic mice, I observed pathological features suggestive of immune dysregulation. Review of literature also suggested possible immuno-modulating functions of human amylin in in vitro experiments. Since autoimmunity and innate immunity are implicated in aging and diabetes, I explored the immunological role of amylin with particular focus on CD4+CD25+ T regulatory cells and toll-like receptors (TLR) which are known mediators of autoimmunity and innate immunity respectively.
Conclusions. Human amylin may play an important role in modulating immunity mainly through stimulating CD4+CD25+ Treg cells, decreasing PLN and altering expression of TLR-4 and cytokines. If these findings are confirmed in in vivo model, human amylin has the potential to become a novel and promising therapy to prevent and reverse autoimmune disease such as autoimmune type 1 diabetes.
Hypothesis. Human amylin may have immunomodulating effects which may have implications on pathogenesis of autoimmune type 1 diabetes.
Materials and methods. Male hemizygous hIAPP transgenic mice (n=32) and their nontransgenic littermates (n=20) were fed with normal chow and studied longitudinally up to 18 months of age with measurement of plasma insulin, glucose and amylin at regular intervals. Detailed oral glucose tolerance test, intra-peritoneal insulin tolerance test, insulin and amylin protein expression were examined at 3, 7, 12 and 18 months of age. Histological changes of pancreas and spleen including changes in CD4+CD25+ T regulatory cells and cytokines were examined at 12 and 18 months.
Objectives. (1) I systemically characterized the morphological, functional and immune regulatory role of human amylin in aged hIAPP transgenic mice which include metabolic profiles, plasma levels of amylin and insulin as well as morphological changes of pancreatic lymph nodes (PLN). (2) I then examined splenic expression of TLR-4 associated changes in cytokines (TNF-alpha, TGF-beta, and IL-6). (3) I also examined the expression level of receptor activity modifying proteins (RAMPs) in pancreas and spleen. (4) I finished by investigating the role of human amylin on stimulating CD4+CD25+ T regulatory (Treg) cells in hIAPP transgenic mice and peripheral blood monocytes (PBMC) from healthy subjects.
Results. (1) With aging, the hIAPP transgenic mice demonstrated increased plasma amylin, decreased plasma insulin, reduced insulin to amylin ratio and improved insulin sensitivity (p<0.05). (2) The aged hIAPP transgenic mice showed changes in immune function as indicated by: (a) Reduced number and size of PLN (p<0.05). (b) Decreased expression level of TLR-4 in splenocytes (p<0.05). (c) Increased expression of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) protein but decreased level of IL-6 in splenocytes (p<0.05). (3) The changes in the levels of immune cytokines such as IL-1, IL-2, IL-4, IL-10, IL-17, interferon-gamma and GM-CSF were similar between hIAPP transgenic and nontransgenic mice (p>0.05). (4) The levels of RAMP1, RAMP2, and RAMP3 were higher in the spleen of hIAPP transgenic mice than nontransgenic mice (p<0.05). (5) The hIAPP transgenic mice showed higher percentage of CD4+CD25+ Treg cells compared with nontransgenic littermates. Treatment with human amylin, but not rat amylin, increased the percentage of FoxP3+CD4+CD25+ Treg cells in both splenic T lymphocytes of hIAPP transgenic mice and PBMCs of healthy subjects ex vivo (p<0.05).
He, Lan
Adviser: Juliana C.N. Chan.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 176-199).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Autoimmunity develops over an extended period of time as the result of an amalgamation of genetic, environmental, and immunologic events. Though the precise etiological factors leading to most autoimmune disease are awaiting consensus, a common thread of the autoimmune paradigm is the inappropriate activation of tissue-specific immune cells by one or more autoantigen, which begins the destruction of the tissue. To prohibit immunopathology and fine-tune the immune responses in healthy individuals, the stimulatory activities of effector/memory T (Teffs) cells must be counteracted by the suppressive mechanisms of regulatory T cells (Tregs). Thus, the potential to modulate the ratio between Teff and Tregs in autoimmune patients has been widely investigated with high hopes to permanently cure certain autoimmune diseases such as type 1 diabetes militus (T1D). Autoantigen therapies, which attempt to induce Tregs to suppress pathogenic effector cells in an autoantigen-specific manner, have shown efficacy in preventing T1D in mice, but have largely failed in clinical trials. One approach to improve the effectiveness of islet autoantigen vaccinations is to combine them with an additional modulator of the T cell response which favors a regulatory phenotype. In the work presented here, we asked whether the addition of anti-interleukin-7 receptor-alpha (anti-IL-7Rα) monoclonal antibodies (mAbs) to islet autoantigen immunizations would modulate the T cell response and prevent T1D in non-obese diabetic (NOD) mice. It was found that anti-IL-7Rα mAbs reduced the absolute numbers of islet antigen-specific T cells when immunized with islet peptide in conjunction with the commonly used vaccine adjuvant alum. Such treatments were also observed to increase nonspecific IL-2, IFN-𝛾, and IL-10 cytokine production, resulting in no improvement of T1D onset prevention. In another approach, we generated a conjugate vaccine by conjugating islet autoantigens to the immunogenic carrier protein, Keyhole Limpet Hemocyanin (KLH). We found that islet antigen-KLH (Ag-KLH) vaccination resulted in significant expansion of the desirable antigen-specific Tregs. Further, Ag-KLH immunization successfully delayed, and in some cases entirely prevented, T1D onset in NOD mice. Indicating that KLH-conjugated vaccine may represent a promising approach for future autoantigen therapies against autoimmunity. Interestingly, administration of anti-IL-7Rα mAbs did not improve these outcomes. To the contrary, we again observed excessive nonspecific cytokine production induced by IL-7Rα blockade that inhibited the beneficial effects of Ag-KLH vaccination. Taken together, we concluded that the addition of anti-IL-7Rα mAbs did not improve the efficacy of autoantigen vaccinations to prevent T1D. Significant work still remains to better characterize and isolate the beneficial effects of anti-IL-7Rα mAbs to treat autoimmunity.

Type 1 diabetes (T1D) is an autoimmune disease leading to destruction of insulin-secreting pancreatic -cells. Environmental factors e.g. exposures to infections, dietary components play a substantial role in etiopathogenesis of T1D and are responsible for rapid increase of T1D incidence in past decades, preferentially in developed countries. Despite long record of T1D research no causative cure or efficient prevention exists. While gluten displays proinflammatory properties, gluten-free diet (GFD) has been documented by several studies as a strong diabetes- preventive environmental factor in spontaneous animal models of T1D, mostly in NOD mouse. The aim of this thesis is to better characterize effects of GFD on the immune system of NOD mouse. Using flow cytometry, we compared effects of GFD vs standard (STD) Altromin diets on NK cell subsets, Tregs, as well as other regulatory cell subsets and their cytokine profile in prediabetic SPF NOD females that were exposed to the diets since "in utero". A reference diabetes incidence in NOD females in our SPF facility kept on STD and GFD was recorded. Diabetes-preventive capacity of GFD were tested by using the NOD-SCID model of diabetes transfer, in which splenocytes from at-onset NOD females kept on GFD or STD were transferred to NOD-SCID recipients....

Type 1 diabetes (T1D) is an autoimmune disease with multifactorial aetiology that involves an attack of self-reactive cytotoxic CD8 lymphocytes on insulin-producing beta cells in the pancreas. In the T1D pathophysiology, both innate and adaptive immunity mechanisms cooperate in the development of inflammation leading to autoimmune destruction. Autoreactive T lymphocytes are the canonical destructors of the beta cells, and B cells produce autoantibodies; the innate immunity cells are considered the initiators of the pathological autoimmune reaction by promoting T and B cell activation. Here, we provide evidence of both innate and adaptive immunity cell types dysregulation in patients with T1D, and that these changes occur before the onset of the disease. The changes in T regulatory lymphocytes (Tregs) and B cell subpopulations occur already in asymptomatic T1D first-degree relatives. During the first year after the onset of the disease, there is a gradual decrease in the neutrophil numbers in the periphery, which probably infiltrate the pancreas. We have focused more closely on the innate immunity dysregulation and its contribution to T1D pathogenesis. Initially, we describe that neutrophil products called neutrophil extracellular traps (NETs) are able to induce IFNγ-producing T cells through...

Type 1 diabetes (T1D) is an autoimmune disease, whose incidence is rising every year, and its prevention or a cure does not exist. T1D is influenced by multiple genetic factors but environmental factors represent the major contributor to the recent almost epidemic increase of T1D incidence worldwide, primarily in developed countries. Amongst these factors belong for example enteroviral infections, microbiota dysbiosis or gluten-free diet (GFD). GFD has been proven to have a protective effect in NOD mice, which is a spontaneous model of T1D, and a beneficial effect on glycemic control in humans, when administered after T1D onset. This diploma thesis examined changes of regulatory and potentially regulatory T-cells and their cytokines in peripheral blood mononuclear cells (PBMC) of T1D children, who underwent 12-month intervention trial of GFD. Secondly, the thesis assessed if the influence of GFD on immune regulatory functions can be transferred by colonization of germ-free NOD mice with gut microbiota of these children. We have found that intervention with GFD increases percentage of Tr1 cells and IL-10 producing CD4+ T-cells in PBMC of T1D children. Furthermore, the beneficial effect on immune regulation can be at least partially transferred to NOD mice by the colonization with human microbiota...

La contribution des DCs dans l’initiation et la perpétuation des maladies auto-immunes est bien établie. Chez la souris Non Obèse Diabétique (NOD), modèle spontané du diabète de type 1 (DT1), plusieurs travaux ont rapportés des anomalies phénotypiques et fonctionnelles des DCs. Les DCs sont parmi les premières cellules qui infiltrent les ilots pancréatiques, produisent des quantités excessives de cytokines pro-inflammatoires et contribuent à l’activation des lymphocytes T auto-réactifs (Teff). Cette capacité accrue des DCs à activer les Teffs est régulée par plusieurs voies de signalisation intracellulaire. STAT5 est parmi les facteurs de transcription critiques dans la régulation des gènes associés au développement, la maturation et les fonctions des DCs. La prédisposition au DT1 chez la NOD est déterminée par plusieurs régions de susceptibilités au diabète (idd1-20). De façon intéressante, le gène Stat5b est localisé dans la région de susceptibilité idd4 chez la souris NOD suggérant son implication dans le développement du diabète. En effet des études récentes ont identifiés un dysfonctionnement dans la voie de signalisation JAK-STAT5 chez les souris NOD, y compris la présence d’une mutation (L327M) au niveau du domaine de liaison à l’ADN de Stat5b qui altère sa liaison à l’ADN. Par ailleurs, les études réalisées dans notre laboratoire ont montré que le conditionnement des DCs au GM-CSF ou à la TSLP, qui activent la voie de signalisation Jak-Stat5, constitue une voie potentielle d’immunothérapie chez la souris NOD. Ces données suggèrent un rôle central de Stat5b dans la régulation des fonctions tolérogènes des cellules du système immunitaire. Nous avons généré un modèle de souris NOD transgéniques (NOD.CD11cStat5b-CA) exprimant de façon constitutive la forme active de STAT5B de la souris C57BL/6 spécifiquement dans les DCs. Nos résultats ont montré que ces souris transgéniques ont développées une protection totale contre le diabète auto-immun. Cette résistance au diabète à long terme est associe à l’acquisition des fonctions tolérogènes par les Stat5b-CA.DCs qui se manifestent par un phénotype mature tolérogène, marquées par une forte expression de molécules immunorégulatrices (PD-L1 et PD-L2) et une grande production de cytokines anti-inflammatoire (TGF-β) et une baisse significative de la production de cytokines pro-inflammatoires (IL-12p70, TNF-α et d’IL-23). Par ailleurs, nous avons mis en évidence le rôle de STAT5B dans la régulation à la hausse d’IRF4 et l’implication du complexe STAT5B/EZH2 dans le contrôle de la régulation à la baisse d’IRF8. En effet, cette régulation différentielle de l’expression des gènes Irf4 et Irf8 est accompagnée du développement d’une sous population CD11c+ CD11b+ DCs. Nos études ont démontré que le potentiel tolérogène des Stat5b-CA.DCs à rétablir et à maintenir la tolérance périphérique du système immunitaire vis-à-vis des auto-antigènes est associe à leur grande capacité d’induire la conversion et l’expansion des Tregs ainsi que la différentiation de deux populations cellulaires régulatrices Th2 et Tc2. Nous avons aussi démontré in vivo qu’une injection intraveineuse unique de Stat5-CA.DCs (spléniques ou générés de la moelle osseuse) ou de Tregs des souris transgéniques NOD.CD11cStat5b-CA a induit une protection totale contre le diabète chez les souris NOD receveuses. Notre étude apporte donc une évidence claire que la correction du défaut de la voie de signalisation Jak-Stat5b au sein des DC chez la souris NOD induit une protection à long terme contre le diabète. Finalement, cette voie de signalisation peut constituer une cible thérapeutique éventuelle non seulement dans le contexte du diabète de type 1 mais également dans d’autres maladies auto-immunes.
The contribution of DCs in the initiation and progression of autoimmune diseases is well established. Several studies have reported that phenotypic and functional abnormalities of DCs, in Non Obese Diabetic (NOD), contribute to spontaneous type 1 diabetes (T1D) development. DCs are among the first cells that infiltrate the pancreatic islets, produce excessive amounts of pro-inflammatory cytokines, and contribute to the activation of T effector cells (Teff). This increased ability of DCs to activate Teff is regulated by several intracellular signaling pathways. STAT5 is among the critical transcription factors in the regulation of genes associated with the development, maturation and functions of DCs. The predisposition to T1D in NOD is determined by several regions of susceptibility to diabetes (idd1-20). Interestingly, the Stat5b gene is located in the idd4 susceptibility region in NOD mice suggesting its involvement in the development of diabetes. Recent studies have identified a dysfunction in the Jak-Stat5 signaling pathway in NOD mice, including the presence of a mutation (L327M) at the DNA-binding domain of Stat5b which alters its binding to DNA. Furthermore, previous studies from our laboratory have shown that the GM-CSF- or TSLP-conditioned DCs, which activate the Jak-Stat5 signaling pathway, is a potential pathway for immunotherapy in NOD mice. These data suggest a central role for Stat5b in the regulation of tolerogenic functions of the immune cells. Here, we generated a transgenic NOD mouse model (NOD.CD11cStat5b-CA) that constitutively express the active form of STAT5B from the C57BL/6 mouse specifically in DCs. Our results showed that these transgenic mice are completely protected against autoimmune diabetes. This long-term diabetes protection is associated with the acquisition of tolerogenic functions by Stat5b-CA.DCs, that exhibit a mature tolerogenic phenotype, overexpression of immunoregulatory molecules (PD-L1 and PD-L2) and produce anti-inflammatory cytokines (TGF-β) and a significantly decrease their production of pro-inflammatory cytokines (IL-12p70, TNF-α and IL-23). Moreover, we have highlighted the role of STAT5B in the upregulation of IRF4 and also the involvement of the STAT5B/ EZH2 complex in downregulation of IRF8. This differential regulation of the Irf4 and Irf8 genes expression is accompanied by promoting the development of CD11c+CD11b+ DC subset. Furthermore, we demonstrated that the tolerogenic Stat5b-CA.DCs were able to restore and maintain peripheral immune tolerance to autoantigens, which is associated with their high ability to induce conversion and expansion Tregs and to promote Th2 and Tc2 immune deviation. We also demonstrated that a single intravenous injection of Stat5-CA.DCs (splenic or bone marrow derived dendritic cells) or Tregs from transgenic mice NOD.CD11cStat5b-CA halted ongoing diabetes in recipient NOD mice. Thus, our study provides clear evidence that the correction of the Jak-Stat5b signaling pathway defect in DC of NOD mice induces long-term protection against diabetes suggesting that signaling pathway can be a potential therapeutic target not only in the context of type 1 diabetes but also in other autoimmune diseases.