Le hématopoïétique protéine adaptateur spécifique SLP-76 est un élément clé des voies de transduction du signal en aval du récepteur des cellules T (TCR). Analyse des lignées de cellules T SLP-76 déficient et SLP-76 souris knock-out montré que SLP-76 est indispensable pour le développement des lymphocytes T et de réactivité des cellules T matures. Les travaux antérieurs de notre laboratoire en utilisant des lignées de cellules T a conduit à l'identification et la caractérisation d'une boucle de rétroaction négative impliquant SLP-76, qui module la signalisation TCR-dépendante et l'activation des cellules T. Cependant, la signification physiologique de ce mécanisme de régulation n'a pas été abordé jusqu'à présent. L'objectif de cette thèse est de répondre à la fonction de ce mécanisme de régulation in vivo par l'analyse de la fonction des cellules T dans une souche de souris généré récemment "knock-in", exprimant un mutant SLP-76-S376A. Basé sur les résultats précédents, était attendu ce mutant de perturber la boucle de rétroaction négative SLP-76-dépendante. Je montre ici que l'expression de SLP-76-S376A ne donne pas lieu à des modifications phénotypiques significatives de thymocytes matures ou des sous-ensembles de cellules T, ce qui suggère que ce mutant ne modifie pas de manière significative le développement des cellules T. En outre, aucune différence n'a été observée dans le taux de prolifération ou la mort cellulaire induite par activation de cellules T mutantes par rapport aux témoins, et l'expression des marqueurs d'activation de surface n'a été que légèrement affectées. Cependant, une activation accrue de plusieurs protéines de signalisation a été observée dans les cellules CD4 + et CD8 + T stimulées in vitro, conformément aux résultats précédents dans les lignées de cellules T humaines. In vitro les cellules T CD4 + activés ont montré une augmentation significative de la sécrétion de l'interleukine-4 et -5, tandis que d'autres cytokines comme l'interleukine-2 et l'interféron-γ est apparu insensible. Ces résultats suggèrent que le réglage de la signalisation TCR-dépendante par la boucle de rétroaction négative SLP-76-dépendante peut favoriser réponses de type Th2, avec des conséquences potentielles dans certaines pathologies telles que l'asthme ou les infections parasitaires. Ainsi, nous avons utilisé un modèle expérimental de l'asthme d'évaluer la réponse des cellules eosinophiles in vivo. En effet, les résultats préliminaires ont montré que l'antigène contesté souris mutantes d'asthme induisant recrutent beaucoup plus d'éosinophiles dans les poumons par rapport aux souris témoins. Pris ensemble, ces données suggèrent que les cellules SLP76-S376A T comprennent modulations dans proximale signal de transduction TCR qui peuvent générer un biais vers la production de cytokines Th2 en réponse à la stimulation du TCR. / The hematopoietic specific adapter protein SLP-76 is a key element of signal transduction pathways downstream of the T cell receptor (TCR). Analysis of SLP-76-deficient T cell lines and SLP-76 knockout mice showed that SLP-76 is indispensable for T cell development and for responsiveness of mature T cells. Previous work of our laboratory using T cell lines led to the identification and characterization of a negative-feedback loop involving SLP-76, which modulates TCR-dependent signaling and T cell activation. However, the physiological significance of this regulatory mechanism was not addressed thus far. The aim of this PhD thesis is to address the function of this regulatory mechanism in vivo by analyzing T cell function in a recently generated “knock-in” mouse strain expressing a SLP-76-S376A mutant. Based on previous results, this mutant was expected to disrupt the SLP-76-dependent negative feedback loop. I show here that expression of SLP-76-S376A does not result in significant phenotypic alterations of thymocytes or mature T cell subsets, suggesting that this mutant does not significantly affect T cell development. Furthermore, no differences were observed in proliferation rates or activation-induced cell death of mutant T cells compared to controls, and the expression of surface activation markers was only slightly affected. However, an increased activation of several signaling proteins was observed in both CD4+ and CD8+ T cells stimulated in vitro, in line with previous results in human T cell lines. In vitro activated CD4+ T cells showed a significant increase in secretion of interleukin-4 and -5, whereas other cytokines like interleukin-2 and interferon- appeared unaffected. These results, suggest that tuning of TCR-dependent signaling by the SLP-76-dependent negative feedback loop may favor Th2-type responses, with potential consequences in specific pathologies such as asthma or parasite infections. Thus, we have employed an experimental model of asthma to evaluate the eosinophil response in vivo. Indeed, preliminary results showed that asthma-inducing antigen challenged mutant mice recruit significantly more eosinophils to the lung compared to control mice. Taken together, these data suggest that SLP76-S376A T cells comprise modulations in proximal TCR signal transduction that may generate a bias towards production of Th2-cytokines in response to TCR stimulation.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第18980号 / 工博第4022号 / 新制||工||1619 / 31931 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 楠見 明弘, 教授 安達 泰治, 准教授 井上 康博 / 学位規則第4条第1項該当

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18980号 / 工博第4022号 / 新制||工||1619(附属図書館) / 31931 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 楠見 明弘, 教授 安達 泰治, 准教授 井上 康博 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<p>Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca²⁺ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. </p><p>A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.</p>

Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.