Determining intracellular signaling events that control CAR T cell death in vivo.

项目摘要/摘要 这个F31应用程序的目标是研究促进肿瘤特异性死亡的细胞内信号事件 体内的T细胞。为了实现适当的免疫动态平衡，效应T细胞和调节性T细胞的数量 牢房必须受到严格控制。癌症是调节性T细胞可以抑制效应器T的一种疾病状态 细胞和防止消除肿瘤。克服免疫抑制和耐受性 对于癌症，已经在临床上引入了过继T细胞疗法，包括过继转移CAR(嵌合体 抗原受体)T细胞。CAR T细胞含有具有肿瘤抗原特异性的TCR，它是TCR的信号成分 TCR(CD3 Zeta链)以及一个或多个共刺激结构域(如CD28)。CAR T细胞是有效的 对某些癌症，特别是血液病的治疗非常有效。一 然而，问题是CAR T细胞通常不会在体内持续存在，特别是对于实体瘤来说，而且一直是 被证明经历了激活诱导的细胞死亡(AICD)。目前，对CAR T细胞的机制的了解 死亡和促进CAR T细胞存活的策略是有限的。我们发现了一种新的AICD形式 当效应性T细胞在抗CD3和抗CD28包被板上被刺激时。此过程依赖于 PICA(P53诱导CD28依赖的T细胞凋亡)。我们发现效应器T细胞会死亡 而Foxp3+调节性T细胞通过转化生长因子-β信号途径对PICA产生抗性。我们的数据也 显示被大量抗原表达细胞(&gt；1：20)刺激的效应器CAR T细胞经历细胞 死亡。在这种高剂量抗原刺激下存活下来的CAR T细胞是Foxp3+。因此，我们假设 当高剂量抗原刺激时，CAR T细胞会经历PICA。阐明CAR-T的这种作用机制 细胞死亡可以帮助我们更好地设计出在体内持续存在并更有效地杀伤的CAR T细胞。在调查过程中 在效应器T细胞中，我们发现RAS激活剂RASGRP1蛋白在效应器中高度升高 反复刺激后的T细胞。Treg维持低水平的RASGRP1，这依赖于转化生长因子-β 发信号。我们目前还不知道在高剂量抗原中存活的CAR T细胞的功能和表型 如果RASGRP1在体外和体内促进CAR T细胞死亡。目标1将 确定在体内实体瘤刺激下存活的CAR T细胞是否为Foxp3+和功能抑制细胞， 由于转化生长因子-β信号转导。目标2将确定RASGRP1是否促进CAR T细胞的死亡，以及如果设计一种 显性阴性RASGRP1的CAR在体内可挽救CAR T细胞死亡，促进肿瘤杀伤。总的来说， 我们希望这项研究能够阐明CAR T细胞死亡的机制，从而有助于促进CAR T细胞 实体瘤的存活率。此外，这项研究将测试一种新的汽车结构的使用，我们预测这种结构将 提高CAR T细胞的存活率，并可能为未来临床使用的CAR T细胞的设计提供参考。

PROJECT SUMMARY/ABSTRACT The goal of this F31 application is to investigate intracellular signaling events that promote death of tumor-specific T cells in vivo. To achieve appropriate immune homeostasis, populations of effector T cells and regulatory T cells must be tightly controlled. Cancer is one disease state where regulatory T cells can suppress effector T cells and prevent elimination of tumors. To overcome immune suppression and tolerance that occurs during cancer, adoptive T cell therapies have been introduced clinically, including adoptive transfer of CAR (chimeric antigen receptor) T cells. CAR T cells contain TCRs with tumor antigen specificity, a signaling component of the TCR (CD3 zeta chain) as well as one or more co-stimulatory domains (such as CD28). CAR T cells are efficiently activated and have been very effective for the treatment of some cancers, especially hematologic cancers. One problem, however, is that CAR T cells often do not persist in vivo, especially for solid tumors, and have been shown to undergo activation induced cell death (AICD). Currently, knowledge of the mechanisms of CAR T cell death and strategies to promote CAR T cell survival are limited. We discovered a novel form of AICD that occurs when effector T cells are stimulated on anti-CD3 and anti-CD28 coated plates. This process is dependent on p53 and is termed PICA (p53-induced CD28 dependent T cell apoptosis). We found that that effector T cells die during PICA, but that Foxp3+ regulatory T cells are resistant to PICA through TGF-β signaling. Our data also show that effector CAR T cells stimulated with large numbers of antigen-expressing cells (>1:20) undergo cell death. The CAR T cells that survive this high dose antigen stimulation are Foxp3+. We therefore hypothesize that CAR T cells undergo PICA when stimulated with high dose antigen. Elucidating this mechanism of CAR T cell death can help us better design CAR T cells that persist and kill more efficiently in vivo. While investigating PICA in effector T cells, we discovered that the protein RasGRP1, a Ras activator, is highly elevated in effector T cells after repeated stimulation. Tregs maintain low levels of RasGRP1, and this is dependent on TGF-β signaling. We currently do not know the function and phenotype of CAR T cells that survive high dose antigen stimulation from solid tumors in vivo, and if RasGRP1 promotes CAR T cell death in vitro and in vivo. Aim 1 will determine if CAR T cells that survive stimulation by solid tumors in vivo are Foxp3+ and functionally suppressive, due to TGF-β signaling. Aim 2 will determine if RasGRP1 promotes death in CAR T cells, and if engineering a CAR with dominant negative RasGRP1 can rescue CAR T cell death and promote tumor killing in vivo. Overall, we expect this study to illuminate mechanisms of CAR T cell death, which can aid in promoting CAR T cell survival in solid tumors. Additionally, this study will test the use of a novel CAR construct, which we predict will enhance CAR T cell survival and could inform future design of CAR T cells that are used clinically.