Signal transduction and gene induction in lymphocytes

摘要 在这项资助的前一个项目期间，我们研究了CD8+T细胞的转录机制 “衰竭”，在持续的抗原刺激条件下观察到的一种低反应状态。 慢性病毒感染。耗尽的T细胞表现为增殖和细胞因子产生减少，并上调表达。 晚期抑制性细胞表面受体包括CTLA4、PD-1、LAG3和TIM3。这些受体的抗体 逆转T细胞耗竭，并对癌症患者实施T细胞免疫检查点 封锁“，这是一种最近在癌症免疫治疗中非常成功的策略。组合 针对抑制性受体的抗体显示出比单独使用单个抗体更有效的效果， 这与疲惫的T细胞通常表达几种抑制性受体的事实相一致。从而理解 导致抑制受体表达和T细胞“衰竭/功能障碍”的分子机制将 补充和加强针对单个抑制性受体组合的治疗效果。 我们之前已经证明，无能和衰竭的转录程序是由 转录因子NFAT，在其伴侣AP-1缺失的情况下起作用(Fos-jun)。在这些研究过程中，我们 开发了一种工程化的NFAT，CA-RIT-NFAT1，它在以下情况下诱导出疲劳的特征 转化为CD8+T细胞。为了了解这种低反应程序的生物学意义，我们使用了 过继转移肿瘤反应性或非反应性TCR-2抗肿瘤反应的小鼠模型 转基因T细胞或带有嵌合抗原受体的T细胞(CAR-T细胞)。使用这些模型，我们发现 Nr4a转录因子等转录因子家族，作为NFAT的“力竭相关”靶标。我们 进一步表明，缺乏所有三个Nr4a家族成员的肿瘤浸润性CD8+T细胞显示了一个基因 与激活的T细胞和排斥肿瘤的表达谱相似，比对照CD8+T细胞更有效。 在这个应用程序中，我们将检验这样一个假设，即小鼠和人类的CD4+和CD8+肿瘤浸润性T细胞(TIL) 至少部分地由NFAT和Nr4a介导的细胞固有转录程序在功能上沉默 转录因子，以及NFAT诱导的其他转录因子。在目标1中，我们将机械地定义 Nr4a转录因子在CD8+T细胞耗竭中的细胞内源性作用我们将询问TIL中Nr4a的删除如何 克服疲惫，增强效应表型，促进肿瘤消退。在目标2中，我们将 检测其他相关转录因子在CD8+T细胞耗竭中的作用，并用 精疲力竭计划展现了这一点。在目标3中，我们将研究NFAT和Nr4a在原始人中的作用 携带耗竭标记的T细胞或CA-RIT-NFAT1转导的T细胞，以及从人肿瘤分离的TIL中。 我们提出的研究将有助于从机制上更广泛地理解转录机制 在肿瘤浸润性免疫细胞中发挥作用，并可能引发癌症患者免疫疗法的改进。

ABSTRACT In the previous project period of this grant, we investigated the transcriptional mechanisms of CD8+ T cell “exhaustion”, a hyporesponsive state observed under conditions of sustained antigen stimulation in cancer and chronic viral infections. Exhausted T cells show decreased proliferation and cytokine production, and upregu- late inhibitory cell surface receptors including CTLA4, PD-1, LAG3 and TIM3. Antibodies to these receptors reverse T cell exhaustion, and their administration to cancer patients forms the basis for “immune checkpoint blockade”, a strategy that has recently been remarkably successful in cancer immunotherapy. Combinations of antibodies to inhibitory receptors show greater efficacy than administration of individual antibodies alone, consistent with the fact that exhausted T cells typically express several inhibitory receptors. Thus understanding the molecular mechanisms that lead to inhibitory receptor expression and T cell “exhaustion/ dysfunction” would complement and enhance the effects of therapies that target combinations of individual inhibitory receptors. We previously showed that the transcriptional programs of anergy and exhaustion are initiated by the transcription factor NFAT, acting in the absence of its partner AP-1 (Fos-Jun). In the course of these studies, we developed an engineered NFAT, CA-RIT-NFAT1, which induces the characteristic features of exhaustion when transduced into CD8+ T cells. To understand the biological implications of this hyporesponsive program, we used mouse models of anti-tumor responses that involving adoptive transfer of tumor-reactive or unreactive TCR- transgenic T cells or T cells bearing chimeric antigen receptors (CAR-T cells). Using these models, we identified Nr4a transcription factors, and other families of transcription factors, as “exhaustion-related” target of NFAT. We further showed that tumor-infiltrating CD8+ T cells lacking all three Nr4a family members displayed a gene expression profile similar to that of activated T cells and rejected tumors more efficiently than control CD8+ T cells. In this application we will test the hypothesis that mouse and human CD4+ and CD8+ tumor-infiltrating T cells (TILs) are functionally silenced by a cell-intrinsic transcriptional program mediated, at least in part, by NFAT and Nr4a transcription factors, and other transcription factors induced by NFAT. In Aim 1, we will define mechanistically the cell-intrinsic roles of Nr4a transcription factors in CD8+ T cell exhaustion. We will ask how Nr4a deletion in TILs overcomes exhaustion, enhances the effector phenotype and promotes tumor regression. In Aim 2, we will examine the roles of other pertinent transcription factors in CD8+ T cell exhaustion, and define the kinetics with which the exhaustion program unfolds. In Aim 3, we will examine the roles of NFAT and Nr4a in primary human T cells bearing exhaustion markers or transduced with CA-RIT-NFAT1, and in TILs isolated from human tumors. Our proposed studies will contribute to a broad mechanistic understanding of the transcriptional mechanisms operating in tumor-infiltrating immune cells, and may spark improved immunotherapies for cancer patients.