Single Cell Analysis of T Cell Responses to Antigen

T细胞免疫动态平衡是活化和调节之间的平衡。在资助的最初阶段，我们在单个细胞水平上对T细胞的激活进行了详细的研究。在这次更新中，我们现在将重点放在画面的另一半，T细胞调节。我们的目标是寻求关于CD4+CD25+抑制T细胞中IL-2R信号转导的新的初步数据，以及脂质磷酸酶PTEN在调节这一途径中的潜在作用。 CD4+CD25+抑制性T细胞对免疫系统的动态平衡是必不可少的。值得注意的是，与效应器CD4+T细胞不同，尽管表达IL-2R的全部3条链，但CD4+CD25+T调节细胞不能单独对IL-2刺激作出反应。我们的数据表明，CD4+CD25+T细胞通过以下途径对IL-2产生反应：(1)在不分裂的情况下进行原始细胞转化；(2)通过诱导BclxL蛋白获得对凋亡的抵抗。对CD4+CD25+Tregs中IL-2R信号转导的详细生化研究表明，STAT-5的完整和正常的磷酸化，但PI-3K靶标Akt或p70S6激酶的磷酸化缺失。令人惊讶的是，我们发现PI-3K本身被IL-2激活，这表明在Treg中PI-3K靶标的下游调控。更多的数据表明，Tregs结构性地表达高水平的脂磷酸酶PTEN，这是PI-3K信号转导的调节因素，而Tregs的TCR刺激，已知诱导IL-2反应，完全阻断PTEN的表达。 联合并新启动IL-2介导的PI-3K下游信号转导。这些数据表明，T Regs中的IL-2R信号与正常的T效应细胞明显不同，并强烈暗示PTEN是该过程中不可或缺的一部分。基于这些数据，我们有三个目标： 在目标1中，我们将确定TCR激活后PTEN下调的动力学。 接下来将使用药物抑制剂以及存在TCR信号缺陷的小鼠和细胞系来确定调节这一过程的信号机制。在目标2中，我们将使用T细胞中PTEN部分和完全缺陷以及可诱导缺陷的小鼠，以确定PTEN在调节细胞发育和体外功能中的作用，特别是对IL-2的需求和反应。 最后，在目标3中，我们将使用自身免疫和移植模型来研究PTEN在Treg生物学中的体内作用。

T cell immune homeostasis is a balance between activation and regulation. In the initial period of funding, we performed detailed studies of T cell activation at a single cell level. In this renewal, we now will focus on the "other half' of the picture, T cell regulation. Our aims will pursue new preliminary data on IL-2R signal transduction in CD4+CD25+ suppressor T cells, and the potential role of the lipid phosphatase PTEN in regulating this pathway. CD4+CD25+ suppressor T cells are essential for homeostasis of the immune system. Strikingly, unlike effector CD4+ T cells, CD4+CD25+ T regs fail to proliferate in response to IL-2 stimulation alone, although they express all 3 chains of the IL-2R. Our data show that CD4+CD25+ T regs are responsive to IL-2 by: (1) undergoing blast transformation without cell division; and (2) acquiring resistance to apoptosis with induction of Bcl-xL protein. Detailed biochemical studies of IL-2R signal transduction in CD4+CD25+ T regs demonstrate intact and normal phosphorylation of STAT-5 but absence of phosphorylation of the PI-3K targets Akt or p70S6 kinase. Surprisingly, we find that PI-3K itself is activated by IL-2, suggesting downstream regulation of PI-3K targets in Tregs. Additional data demonstrate that Tregs constitutively express high levels of the lipid phosphatase PTEN, a regulation of PI-3K signal transduction, and that TCR stimulation of Tregs, which is known to induce IL-2 responsiveness, completely blocks PTEN expression in association and newly enables IL-2 mediated PI-3K downstream signaling. These data show that IL-2R signaling in T regs is distinctly different from that of =normal" T effector cells, and strongly implicates PTEN as an integral part of that process. Based on these data we have three aims: In aim #1, we will determine the kinetics of PTEN downregulation following TCR activation. Pharmacologic inhibitors as well as mice and cell lines with defects in TCR signaling will next be used to determine the signaling mechanisms which regulate this process. In aim #2 we will use mice with partial and complete deficiencies of PTEN in T cells, as well as with inducible deficiencies, to determine the role of PTEN in regulatory cell development and in vitro function, in particular on IL-2 requirements and responsiveness. Finally, in aim #3, we will employ an autoimmune and a transplant model to study the in vivo role of PTEN in Treg biology.