Regulatory T cells (Tregs) are a subpopulation of T cells with immuno-suppressive properties. Tregs play a key role in immune response regulation and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Tregs and identified a signature composed of five microRNAs (-21, -31, -125a, -181c and -374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3’ untranslated region (3’ UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had opposite effects on FOXP3 expression. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3’ UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Recent reports have shown that histone deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Treg CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on the binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Treg signature. Valproate treatment induced binding of Ets-1 and Ets-2 transcription factors to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs of Treg signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miR expression profile is not due to an increased expression of FOXP3 but directly results from histone deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in the expression level of miR-21 and miR-31. These data, by allowing a better understanding of the molecular phenomena underlying the number and function of Tregs, could open the door to novel therapeutic approaches in cancer immunotherapy and treatment of autoimmune disorders.

Les cancers épithéliaux de l’ovaire sont la première cause de décès par cancer gynécologique. Dans ce travail, nous nous sommes intéressés aux cancers ovariens séreux de haut grade (HGSOC) et de stade avancé, pour lesquels les options thérapeutiques demeurent limitées. Plusieurs laboratoires, dont le nôtre, ont identifié des groupes moléculaires distincts au sein de ces HGSOC sur la base de données transcriptomiques.Dans toutes ces études, un sous-groupe moléculaire, nommé « fibrose » ou« mésenchymateux », est systématiquement observé et invariablement associé à un pronostic sombre pour les patientes. La signature transcriptomique qui identifie ce groupe de patientes inclue de nombreux gènes impliqués dans le remodelage de la matrice extracellulaire et la composition stromale, suggérant un rôle possible du stroma dans l’agressivité de ce sous-groupe moléculaire particulier d’HGSOC.Par une étude combinant de façon originale l’analyse concomitante de 6 marqueurs de fibroblastes associés au cancer (CAF), nous mettons en évidence pour la première fois l’existence de 4 sous-populations de CAF. De façon intéressante, l’une de ces 4 populations(dite CAF-S1) s’accumule significativement dans le sous-type moléculaire« fibrose/mésenchymateux » des HGSOC. Or, cette population particulière de CAF-S1 présente une activité immunosuppressive, en favorisant non seulement la survie ou l’attraction des lymphocytes T régulateurs, mais également en stimulant leur activation par l’expression du facteur de transcription FOXP3 (Forkhead Box P3). Enfin, nous identifions l’isoforme β de CXCL12 (chimiokine à motif C-X-C ligand 12) comme un acteur majeur de l’identité et de la fonctionnalité de ces CAF-S1 immunosuppresseurs. En effet, CXCL12βs’accumule spécifiquement dans les CAF-S1 et joue un rôle clé dans la fonction immunosuppressive de ces CAF au sein du stroma des HGSOC mésenchymateux.Nos résultats mettent ainsi en évidence un mécanisme expliquant, au moins en partie, le pronostic sombre des patientes atteintes d’HGSOC mésenchymateux. La caractérisation approfondie du stroma de ces tumeurs agressives permet d’envisager de nouvelles stratégies thérapeutiques ciblant à la fois les CAF et les cellules immunitaires dans le but d’améliorer la survie des patientes mésenchymateuses. / Epithelial ovarian cancers are the first cause of death from gynecologic cancer. We focusedour work on high-grade serous ovarian cancer (HGSOC) patients, for who only very fewtherapeutic options exist. In the past, several laboratories, including ours, have identified -based on transcriptomic data- distinct molecular subgroups amongst HGSOC. Interestingly,among these different molecular subgroups, one of them, referred to as “Fibrosis” or“Mesenchymal” is systematically identified and consistently associated with poor patientprognosis in all studies. Transcriptomic signature that defines this specific molecularsubgroup of HGSOC contains mainly genes involved in extracellular matrix remodeling andstromal composition, suggesting a potential role of stroma and Carcinoma-AssociatedFibroblasts (CAF) in this particular “Fibrosis/Mesenchymal” HGSOC subgroup.By combining various technics studying concomitantly 6 different CAF markers, we identifiedfor the first time 4 different subpopulations of CAF in HGSOC. Interestingly, one of thesesub-populations, referred to as CAF-S1, significantly accumulates in the“Mesenchymal/Fibrosis” subgroup of HGSOC. Importantly, we demonstrated that the CAFS1cellular subpopulation exhibits immunosuppressive activities. Indeed, CAF-S1 fibroblastsnot only by attract regulatory T lymphocytes but also promote their survival and activation(assessed by expression of FOXP3). Finally, we uncovered the specific role of the CXCL12βisoform as an important player of CAF-S1 identity and immunosuppressive functions inmesenchymal HGSOC.All together, these results identify a stromal heterogeneity in HGSOC, which has beenbroadly underestimated until now. Moreover, our work demonstrates the accumulation of aCAF subpopulation with immunosuppressive functions in HGSOC mesenchymal patients thatcould account, at least in part, for their poor survival rate. Deep characterization of thestroma may enable us to define new therapeutic options combining CAF-targeting therapiesand immunotherapies, in order to improve survival of HGSOC mesenchymal patients.

Helminth parasites excel at subverting the host’s immune regulatory pathways resulting in immunosuppressed hosts harbouring chronic infections. This immune suppression forms a major barrier to the acquisition of protective Th2 immunity, both in regard to natural infections and potential vaccinations. At the same time, immune downregulation plays a beneficial role in protecting the host from pathology during chronic infection, and epidemiological links between helminth infections and the amelioration of allergy and autoimmunity diseases indicate that helminth-induced immune suppression can be therapeutically applied to the treatment of these conditions. Foxp3+ regulatory T cells (Treg) play central downregulatory roles in controlling reactivity to self-antigens and preventing autoimmune diseases, as well as in limiting inflammatory responses during infection. Helminths induce dominant Foxp3+ Treg responses that play key roles in inhibiting protective immunity and alleviating immunopathology, and that can protect against allergic inflammation. Thus, Foxp3+ Tregs are a fundamental mechanism of immune regulation during helminth infections, and an understanding of the mechanisms governing the induction of Foxp3+ Treg responses is of principal importance for the design of both prophylactic helminth treatments and therapies for allergies and autoimmunity. However, the nature of the T cell co-stimulatory signals driving Treg generation during helminth infection is largely unclear. Recent evidence suggests that the inducible costimulator (ICOS) contributes to Treg control of autoimmune inflammation. Further, ICOS expression is upregulated by Foxp3+ Treg during infection with the filarial nematode Litomosoides sigmodontis suggesting ICOS is important for Treg during helminth infection. Therefore, we investigated the role of ICOS in helminth-induced Treg responses. Similar to L. sigmodontis infection, Foxp3+ Treg increased ICOS expression in response to infection with the intestinal nematode Heligmosomoides polygyrus and with the blood trematode Schistosoma mansoni. Functionally, ICOS was required for the optimal expansion of lymphoid Treg numbers during early stage H. polygyrus infection and following the onset of the acute egg phase of S. mansoni infection suggesting common pathways for Treg induction by diverse helminth species. Whilst helminth induced proliferation and activation of Foxp3+ Treg was ICOS independent, ICOS was essential for Treg survival in settings of homeostasis and helminth infection. In contrast to the lymph node, Treg responses in the intestinal lamina propria (LP) of ICOS-/- mice were increased due to expanded natural Treg. Following H. polygyrus infection Foxp3+ Helios- CD4+ T cells preferentially expanded in wild-type (WT) mice but not in ICOS deficient mice suggesting ICOS is required for the expansion of adaptive Treg at the site of intestinal nematode infection. Functionally, ICOS supports Treg, but not effector T cells (Teff), H. polygyrus induced IL- 10 production suggesting ICOS differentially regulates Treg and Teff. At the H. polygyrus infection site, ICOS acted to downregulate CD4+ T cell Th2 cytokine production. Conversely, in the reactive lymph node ICOS signalling promoted Th2 immune responses, possibly by maintaining the pool of IL-4 secreting type 2 follicular helper T cells. Thus, ICOS has different effects on Th2 immunity depending on tissue location. Because Th2 immunity governs expulsion of H. polygyrus parasites, the differences in Th2 responses between lymph node and infection site could explain why ICOS deficiency did not impact worm burden. Protective immunity to long-lived helminth infection can be quenched in the initial days of infection by the action of Treg. Whether Treg expand and suppress protective immunity during S. mansoni larvae lung transit has not been investigated. We found that in contrast to H. polygyrus and L. sigmodontis infection, early S. mansoni infection did not induce a Treg response suggesting other mechanisms are employed for immune subversion. During the acute egg-phase of S. mansoni infection, Foxp3+ Treg protect the host from damaging egg-induced hepatic immunopathology. Despite reduced Foxp3+ Treg responses, ICOS deficiency did not impact egg-induced immunopathology. Thus, ICOS co-stimulation contributes to early expansion and the continued maintenance of Treg during helminth infection, both in the local lymph node and at the infection site. ICOS is required for Treg function during helminth infection by promoting IL-10 production, whilst its contribution to Th2 effector immunity is tissue specific. In addition, ICOS is dispensable for protective immunity and pathology during helminth infection. As ICOS controls both positive and negative immune responses and can have opposing roles depending on tissue location, an understanding of the consequences of these contradictory effects will be important when considering targeting ICOS therapeutically.

Submitted by Heitor Rapela Medeiros (heitor.rapela@ufpe.br) on 2015-03-06T11:29:09Z No. of bitstreams: 2 tese Yone-final-20-12-12.pdf: 714270 bytes, checksum: 2a36b003dfa84355204e28d809e01cff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-06T11:29:09Z (GMT). No. of bitstreams: 2 tese Yone-final-20-12-12.pdf: 714270 bytes, checksum: 2a36b003dfa84355204e28d809e01cff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / A tuberculose pulmonar é um problema de saúde pública mundial e apresenta alta incidência no Brasil. Atualmente, o Brasil ocupa o 19º lugar no ranking dos 22 países que concentram 80% dos casos em todo o mundo. A resposta imune humana contra a tuberculose é especialmente mediada pelos linfócitos T CD4+. Entretanto, muitos estudos ainda são necessários para o entendimento exato do papel de cada citocina no mecanismo de cura da doença. A fim de caracterizar a produção das citocinas TNF-a, IFN-g, IL-10, o óxido nítrico (NO) e a expressão do marcador Foxp3, células mononucleares do sangue periférico (PBMC) de contatos intradomiciliares de indivíduo com tuberculose pulmonar com (CTb) e sem (STb) história prévia de TB foram estimuladas in vitro com antígeno de Mycobacterium tuberculosis (AgTb) e com o mitógeno Concanavalina A por 24 e 48 horas. A dosagem das citocinas foi feita por ELISA de captura e de NO pela quantificação do nitrito (NO2 -). PCR em Tempo Real foi a técnica utilizada para detectar o mRNA para Foxp3. Em 24 horas de cultivo celular com antígeno total de M. tuberculosis (CTb = 10.158,38 ± 7.438,38; STb = 15.083,10 ± 9.292,66), observou-se uma produção significativa de TNF-a (0,05) no grupo STb. Os grupos analisados não apresentaram diferenças na expressão do mRNA do marcador Foxp3. Foi verificado aumento (p= 0,04) entre IL-10 e Foxp3 e correlação negativa (p = 0,009) entre NO (aumento) e Foxp3 (diminuição) após 48h de estímulo com AgTb. Observou-se também no grupo STb em 24 horas de culturas estimuladas com AgTb um resultado semelhante ao grupo CTb (p = 0,03). Portanto, os resultados obtidos sugerem que produção de IL-10, IFN-g e de NO indicam que os indivíduos CTb e STb produziram uma resposta celular específica; porém, sem valores que possam indicar uma maior ou menor susceptibilidade à doença, e que TNF-a, possivelmente funciona como um elemento fundamental para a manutenção do estado de saúde dos comunicantes. A expressão do marcador Foxp3 não está associada diretamente a uma maior predisposição nos indivíduos com história de tuberculose pulmonar.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-04-08T15:09:37Z No. of bitstreams: 2 tese Yone-final-20-12-12.pdf: 714270 bytes, checksum: 2a36b003dfa84355204e28d809e01cff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-08T15:09:37Z (GMT). No. of bitstreams: 2 tese Yone-final-20-12-12.pdf: 714270 bytes, checksum: 2a36b003dfa84355204e28d809e01cff (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / A tuberculose pulmonar é um problema de saúde pública mundial e apresenta alta incidência no Brasil. Atualmente, o Brasil ocupa o 19º lugar no ranking dos 22 países que concentram 80% dos casos em todo o mundo. A resposta imune humana contra a tuberculose é especialmente mediada pelos linfócitos T CD4+. Entretanto, muitos estudos ainda são necessários para o entendimento exato do papel de cada citocina no mecanismo de cura da doença. A fim de caracterizar a produção das citocinas TNF-a, IFN-g, IL-10, o óxido nítrico (NO) e a expressão do marcador Foxp3, células mononucleares do sangue periférico (PBMC) de contatos intradomiciliares de indivíduo com tuberculose pulmonar com (CTb) e sem (STb) história prévia de TB foram estimuladas in vitro com antígeno de Mycobacterium tuberculosis (AgTb) e com o mitógeno Concanavalina A por 24 e 48 horas. A dosagem das citocinas foi feita por ELISA de captura e de NO pela quantificação do nitrito (NO2 -). PCR em Tempo Real foi a técnica utilizada para detectar o mRNA para Foxp3. Em 24 horas de cultivo celular com antígeno total de M. tuberculosis (CTb = 10.158,38 ± 7.438,38; STb = 15.083,10 ± 9.292,66), observou-se uma produção significativa de TNF-a (0,05) no grupo STb. Os grupos analisados não apresentaram diferenças na expressão do mRNA do marcador Foxp3. Foi verificado aumento (p= 0,04) entre IL-10 e Foxp3 e correlação negativa (p = 0,009) entre NO (aumento) e Foxp3 (diminuição) após 48h de estímulo com AgTb. Observou-se também no grupo STb em 24 horas de culturas estimuladas com AgTb um resultado semelhante ao grupo CTb (p = 0,03). Portanto, os resultados obtidos sugerem que produção de IL-10, IFN-g e de NO indicam que os indivíduos CTb e STb produziram uma resposta celular específica; porém, sem valores que possam indicar uma maior ou menor susceptibilidade à doença, e que TNF-a, possivelmente funciona como um elemento fundamental para a manutenção do estado de saúde dos comunicantes. A expressão do marcador Foxp3 não está associada diretamente a uma maior predisposição nos indivíduos com história de tuberculose pulmonar.

Elevated levels of the cytokine interleukin-18 (IL-18) are found in many chronic inflammatory disorders, including inflammatory bowel disease (IBD). However, the role of IL-18 in mucosal immunity and inflammation is not well understood. At mucosal and environmental interfaces, Th17 cells have been shown to contribute to protection from pathogenic infection. In contrast, regulatory T (Treg) cells maintain intestinal homeostasis by preventing aberrant inflammatory responses to the resident microbiota. We demonstrate that under homeostatic conditions, colonic Th17 cells highly express IL-18 receptor (IL-18R1) and that intestinal epithelial cell production of IL-18 acts directly on CD4⁺ T cells to limit colonic Th17 differentiation. Furthermore, whilst IL-18R1-signalling is dispensable for induction of colitis, we observed a critical role for IL-18R1-signalling in Foxp3⁺ Treg mediated control of colitis. Together, these studies demonstrate that the intestinal epithelium regulates colonic CD4⁺ T cell responses through production of the cytokine IL-18.

Les lymphocytes T régulateurs CD4+Foxp3+ (Treg) sont indispensables au contrôle des cellules T auto-réactives et au maintien de la tolérance immune en périphérie. Ils sont sélectionnés positivement dans le thymus, possèdent un répertoire TCR extrêmement diversifié suggérant une reconnaissance d’antigènes variés, requièrent une signalisation continue via leur TCR pour maintenir leurs fonctions en périphérie, fonctions qu’ils exercent avec une certaine spécificité de tissu. Ils contrôleraient donc spécifiquement les lymphocytes T auto-réactives en reconnaissant comme eux des antigènes du soi. Mon travail de thèse s’est articulé autour de deux axes visant à revisiter ce paradigme de l’autoréactivité des Treg, un point essentiel qui concerne les propriétés de reconnaissance antigénique de ces cellules. Afin de ne pas biaiser nos résultats avec des TCR artificiels, non issus de Treg, nous avons choisi d’étudier les Treg polyclonaux de souris normales. Dans une première série d’expériences, nous avons pu montrer que l’activation de lymphocytes T auto-réactifs anti-mâle dans une souris mâle conduisait à un remodelage du répertoire TCR des Treg, avec un usage préférentiel des Vβ5 et Vβ12. L’absence d’amplification clonale ainsi que des données récentes de la littérature démontrant l’amplification de sous-populations de Treg par des rétrovirus endogènes au cours de réponses immunitaires chroniques, nous ont conduit à conclure que l’activation de lymphocytes T auto-réactifs pourrait réactiver des rétrovirus endogènes et donc l’expression de super-antigènes capables d’amplifier les Treg portant certains Vβ. Dans le second volet de mon travail de thèse, nous avons caractérisé le niveau d’autoréactivité des Treg in vivo. En procédant à une comparaison systématique avec des Treg ayant des niveaux d’allo-réactivité différents et connus, nous avons montré que, de manière inattendue, les Treg polyclonaux possèdent un niveau étonnamment faible d’autoréactivité. Seule une fraction d’entre eux était capable de recevoir un signal TCR et des expériences de quantification ont révélé que cette fraction de cellules auto-réactives ne devait pas excéder 1% du répertoire total des Treg. L’ensemble de nos résultats suggèrent donc que les Treg sont faiblement réactifs en périphérie et/ou sont dirigés contre des antigènes endogènes rarement représentés. / No abstract available

Zöliakie ist durch eine dysregulierte Immunreaktion auf die in Getreiden enthaltene Proteinfraktion Gluten charakterisiert. Die Assoziation der Erkrankung mit Polymorphismen in immunregulatorischen Genen weist auf eine Rolle von regulatorischen T-Zellen im Krankheitsgeschehen hin. Foxp3+ regulatorische T-Zellen haben eine essentielle Bedeutung für die Aufrechterhaltung der intestinalen Immunhomöostase und die Limitierung von Autoimmunität. In der vorliegenden Arbeit wurde eine 2005 bis 2010 diagnostizierte Gruppe von 51 Kindern und Jugendlichen mit Zöliakie untersucht. Diese Gruppe wurde mit 51 geschlechts- und altersadaptierten Kontrollen ohne Zöliakie verglichen. Es wurden anamnestische, paraklinische und histologische Daten mit der Verteilung von CD3+Foxp3+ regulatorischen T-Zellen in der Dünndarmschleimhaut untersucht. Patienten mit Zöliakie wiesen eine leichte Anämie, jedoch keine signifikante Wachstums- und Gewichtsentwicklung auf, was die oligosymptomatische Verlaufsform in der Gesamtkohorte unterstreicht. Es konnte gezeigt werden, dass CD3+Foxp3+ regulatorische T-Zellen bei Zöliakie-Patienten vermehrt in der Dünndarmschleimhaut akkumulieren. Weiterhin korreliert die Häufigkeit CD3+Foxp3+ regulatorischer T-Zellen sowohl mit dem Schweregrad der Schleimhaut-schädigung (gemessen an der Marsh-Oberhuber-Klassifikation, dem Zotten-Krypten Verhältnis oder der Zahl der intraepithelialen Lymphozyten) als auch mit den Titern Zöliakie-spezifischer Antikörper. Die Akkumulation CD3+Foxp3+ regulatorischer T Zellen lässt sich partiell als Folge einer Anreicherung von CD4+ T-Zellen auf Kosten CD8+ T-Zellen erklären. Die Daten weisen darauf hin, dass Foxp3+ regulatorische T Zellen sekundär als Folge des gluteninduzierten Entzündungsprozesses in der Schleimhaut akkumulieren, diesen offensichtlich aber nicht effektiv begrenzen. Die mögliche Assoziation der Immundysregulation der Zöliakie mit Foxp3+ regulatorischen T-Zellen ist damit nicht durch eine numerische Reduktion sondern wahrscheinlich durch partielle funktionelle Defekte bedingt.

Indiana University-Purdue University Indianapolis (IUPUI) / Airway inflammation is the most common and important component of respiratory diseases, such as influenza and asthma. Severe influenza A virus infection typically triggers detrimental lung inflammation with massive immune cell infiltration and hyper-production of cytokines and chemokines. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor known for its function in bone formation and spermatogenesis, in regulating antiviral immune responses and immunopathology. Nmp4-deficient mice are protected from influenza induced immunopathology and body weight loss. While having no effects on viral clearance or CD8/CD4 T cell or humoral immune responses, Nmp4 deficiency in either lung structural cells or hematopoietic cells significantly reduces the recruitment of neutrophils and monocytes to the lungs. Furthermore, NMP4 binds to the promoters and/or conserved non-coding sequences of the chemokine genes Ccl2 and Cxcl1 and upregulates their expression in mouse lung epithelial cells and macrophages. These chemokines attract monocytes and neutrophils to the airway, resulting in exaggerated airway inflammation and collateral lung damage. Another transcription factor forkhead box P3 (FOXP3) is critical for the development of regulatory T cells (Tregs) that function to control immune responses. Unlike human FOXP3 gene that encodes two major isoforms, a full length (FOXP3-FL) isoform and a short isoform lacking the exon 2 region (FOXP3-ΔE2), mouse Foxp3 gene only encodes Foxp3-FL isoform. We generate Foxp3-ΔE2 mice to study its function and find that Tregs expressing the Foxp3-ΔE2 isoform have intrinsic defects, thus allowing intensified adaptive immune responses without changes in innate immunity against influenza infection. In a model of chronic asthma, mice expressing only the Foxp3-ΔE2 isoform have significantly increased allergic airway inflammation and elevated production of allergen-specific IgE compared with mice expression the Foxp3-FL isoform. Mechanistically, Tregs expressing the Foxp3-ΔE2 isoform are less stable and prone to trans-differentiation into effector Th9-like cells, which are closely associated with the pathogenesis of asthma. These data suggest that the two Foxp3 isoforms have different functions in regulating airway immune responses. Overall, we have defined the important roles of both transcription factors NMP4 and FOXP3 in regulating airway inflammation. / 2022-08-17

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19626号 / 医博第4133号 / 新制||医||1016(附属図書館) / 32662 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 清水 章, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM