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Le récepteur à l’érythropoïétine (EPOR) peut être muté dans les érythrocytoses congénitales tandis que des mutations de MPL, récepteur à la thrombopoiétine, sont observées dans certains néoplasmes myéloprolifératifs (NMP). L’érythrocytose congénitale touche exclusivement les progéniteurs érythroïdes et se traduit par une polyglobulie isolée. Des mutations d’EPOR sont décrites dans environ 12% des cas. La première partie de cette thèse reposait sur l’étude fonctionnelle d’une mutation d’EPOR, jamais décrite, c.1300dupC (p.Gln434Profs*11). Ce mutant est responsable, dans les cellules primaires et les lignées cellulaires, d’une hypersensibilité majeure à l’EPO qui n’est pas due à la perte de sites de régulation négative du signal ou à un défaut d’internalisation du récepteur, contrairement aux données de la littérature, mais à une stabilisation d’EPOR à la membrane cellulaire par probable changement conformationnel. Dans la seconde partie, un variant d’EPOR Pro488Ser a été étudié au sein d’une famille de NMP. Seule une activation spontanée faible de STAT5 a pu être mise en évidence dans les lignées cellulaires. Des modèles murins et/ou d’iPSC seront développés dans l’hypothèse d’une coopération entre EPOR P488S et JAK2V617F. Enfin, dans la dernière partie de ce travail, nous avons réalisé une étude fonctionnelle de 2 mutations rares de MPL, Tyr591Asn et Ser204Pro, identifiées chez 3 patients TE triples négatifs. Seul un gain de fonction faible a été mis en évidence, suggérant que ces mutations doivent être associées à d’autres anomalies génétiques pour entrainer l’apparition d’un phénotype. / EPOR mutations are observed in Primary familial and congenital polycythaemia (PFCP) while MPL mutations are found in myeloproliferative neoplasms (MPN). PFCP is an inherited disorder of erythroid progenitor cells resulting in elevated erythrocyte mass. Several mutations of the erythropoietin receptor (EPOR) gene have been associated with PFCP. They are all leading to a premature STOP codon and the truncation of the cytoplasmic COOH-terminal of EPOR. To examine the role of EPOR mutations in the pathogenesis of PFCP, we studied a new EPOR mutation, c.1300dupC (p.Gln434Profs*11). This mutation induced, in primary cells and cell lines, a major hypersensitivity to EPO. This phenotype was not due to the loss of negative regulation domains or an internalisation default, contrary to the current hypothesis, but rather due to conformational modification inducing the stabilisation of the mutant at the cell membrane.In the second part of this work, an EPOR mutation, Pro488Ser, was studied in a myeloproliferative neoplasms (MPN) family. Only a mild spontaneous STAT5 activation was observed in cell lines. Murine models and/or iPSC will be developed in order to test the hypothesis of a cooperation between EPOR P488S and JAK2 V617F. In the last part of this project, 2 rare MPL mutants, Tyr591Asn and Ser204Pro, identified in 3 triple negative ET patients were functionally studied. A weak gain of function was observed, suggesting that these mutants have to be associated to other genetic abnormalities to develop a phenotype.

Yes / Effective suppression of JAK–STAT signalling by the inducible inhibitor “suppressor of cytokine signalling 3” (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3- interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1. / This work was supported by project grants to T.M.P. from the Chief Scientist Office (ETM/226), British Heart Foundation (PG12/1/ 29276, PG 14/32/30812), and a National Health Service Greater Glasgow and Clyde Research Endowment Fund (2011REFCH08). P.F.P. was supported by the National Institutes of Health grant DK097708. J.J.L.W. was supported by a doctoral training studentship from the Biotechnology and Biological Sciences Research Council Doctoral Training Programme in Biochemistry and Molecular Biology at the University of Glasgow (BB/F016735/1). N.A. was supported by a Saudi Government PhD Scholarship. This work was also supported in part by equipment grants to T.M.P. from Diabetes UK (BDA 11/0004309) and Alzheimer’s Research UK (ARUK-EG2016A-3).

Das Ziel dieser Arbeit war, die funktionellen Charkteristika von humanen antigenerfahrenen CD4-T-Zellen in Relation zur Expression von IL-7Ra und CCR7 zu untersuchen. Im Rahmen dessen wurden zwei verschiedene experimentelle Ansätze wurden gewählt: Zur Analyse von Populationen, die während einer Primärantwort auftreten, wurden antigenerfahrene CD4-T-Zellen in vitro mit TSST-beladenen dendritischen Zellen stimuliert. Der zweite Ansatz bestand darin, zirkulierende antigenerfahrene CD4-T-Zellen entsprechend ihrer CCR7- und IL7R-Expression zu isolieren, um die heterogenen zirkulierenden T-Zellen zu untersuchen. Die Experimente zeigen, daß IL7RhiCCR7+ T-Zellen Charakteristika zentraler Gedächtniszellen besitzen. IL7RlowCCR7–identifiziert hingegen Zellen mit Effektor-T-Zellmerkmalen. Dementsprechend wiesen IL7RlowCCR7– T-Zellen ein stark verringertes Zellüberleben auf, waren stark beeinträchtigt in darin in Anwesenheit von homöostatischen Zytokinen zu proliferieren und exprimierten nur wenig IL-2. Im Gegenzug überlebten IL7RhiCCR7+ T-Zellen gut, reagierten auf homöostatische Zytokine, sekretierten IL-2 und expandierten nach antigenspezifischer Stimulation. Interessanterweise erkannten ex vivo isolierte IL7Rlow T-Zellen vornehmlich persistierende Antigene. Dies weist darauf hin, daß diese Zellen chronisch aktiviert sind. In vitro konnte demonstriert werden, daß die funktionelle Ausprägung der den zentralen Gedächtniszellen ähnlichen IL7RhiCCR7+ T-Zellen deutlich von der Stärke der Stimulation während der Generierung der IL7RhiCCR7+ T-Zellen abhängt. Dieser Umstand stützt die Hypothese, daß die Quantität der Signale während der primären Stimulation die Richtung der Zelldifferenzierung bestimmt und ein solcher Mechanismus zur Heterogenität der zentralen Gedächtnis-T-Zellen in vivo beiträgt. Zusammenfassend kann man feststellen, daß, sich die Marker CCR7 und IL7R in Kombination als ein wertvoll zur Identifizierung von CD4-Gedächtnis- und –Effektor-T-Zellen erweisen / The aim of this work was to elucidate the functional characteristics of human antigen-experienced CD4 T cells according to the expression of IL7RAlpha and CCR7. Two different approaches were used: in order to analyze the subsets occurring during a primary response, antigen-experienced CD4 cells were analyzed in vitro after priming with the superantigen TSST. The signal strength of TCR-stimulation during the priming was modulated in order to understand how the levels of stimulation might influence the generation of memory-like T cells. The second approach was to isolate circulating CD4 T cells expressing different combinations of CCR7 and IL7R in order to analyze the heterogeneous pool of antigen-experienced cells found under steady state conditions ex vivo. The results revealed that both in vitro and ex vivo the IL7RhiCCR7+ T cell subset corresponds to cells with TCM characteristics whereas IL7RlowCCR7– identifies cells with effector characteristics. Correspondingly, IL7RlowCCR7–CD4 T cells showed low survival rates, impaired proliferation in the presence of homeostatic cytokines and a low IL-2 production, IL7RhiCCR7+ CD4 T cells survived well, responded to homeostatic cytokines, secreted IL-2 and expanded upon antigenic stimulation. Notably, ex vivo isolated IL7Rlow T cells preferentially recognized under steady state conditions persistent antigens, suggesting that these cells are chronically activated. Finally, it was demonstrated that IL7RhiCCR7+ TCM-like cells generated in vitro acquired different functional properties depending on the strength of stimulation during the priming. This fact supports the concept that the amount of signal received during the priming influences the cell fate decision contributing to the heterogeneity of the TCM pool in vivo. In summary, despite some differences observed between in vitro and ex vivo CD4 T cell subsets, the combination of the markers CCR7 and IL7R is useful to distinguish memory- from effector-like CD4 T cells.