Role of NOTCH1 Signaling in Engineering CD4+ T Cells for Cancer Immunotherapy

项目总结 目前利用激动型抗CD3和-CD28设计T细胞用于癌症免疫治疗的方法 用于激活T细胞的单抗，可促进T细胞的增殖，但不能概括其他T细胞。 由抗原提呈细胞传递的决定命运的信号。其中一个这样的信号轴，Noch通路， 控制CD4+T细胞效应器功能的获得，并强烈影响行为。研究凹槽信令 在嵌合抗原受体(CAR)T细胞的生产过程中，我们开发了一种使用抗CD3/CD28的培养系统 单抗包裹小球和平板包裹激动型NOTCH1单抗同时诱导T细胞活化 和凹槽信号。当转移到CD19+Raji淋巴瘤的NSG小鼠体内时，CD19特异性的 NOTCH1刺激的(N1)CD4+CAR T细胞表现出明显的增殖优势，而不是对照组(Ig) 细胞。同时给予IgGCD8+CAR T细胞和N1 CD4+CAR T细胞的荷瘤小鼠表现出 与给予免疫球蛋白CD8+和免疫球蛋白CD4+CAR细胞的小鼠相比，这两个亚群的扩增效果都更好。 从而快速清除肿瘤并保护其免受肿瘤的再次攻击。这些数据表明， NOTCH1激动剂可以代表过继T细胞治疗的显著改进，但其机制 Noch信号是如何改善T细胞抗肿瘤功能的，目前尚不清楚。已知凹槽 诱导芳烃受体(AhR)转录活性。药物对AhR活性的抑制作用 在N1培养过程中，CD4+CAR T细胞减少了N1细胞和表面免疫球蛋白G细胞之间的特征差异 体外再刺激后的表型、细胞因子的产生和增殖。我假设NOTCH1 激动剂组织依赖于AhR的转录改变，从而增加增殖细胞因子 在CD4+CAR T细胞中产生，促进CD4依赖的增殖，从而改善CAR T细胞 功效。为了验证这一假设，我将评估AhR抑制和激活在N1和 体外和体内免疫球蛋白CD4+CAR T细胞，并鉴定N1、Ig G中决定命运的转录事件 用散装和单细胞RNA测序的方法抑制了N1个CD4+CAR T细胞。总而言之，这些 实验将研究Noch和AhR信号在产生优越的CAR T细胞中的作用 并建立NOTCH1激动剂提高CAR T细胞治疗效果的机制， 为将这一充满希望的生物医学进步转化为免疫治疗诊所奠定了基础。

PROJECT SUMMARY Current methods for engineering T cells for cancer immunotherapy use agonistic anti-CD3 and -CD28 monoclonal antibodies (mAb) to activate T cells, which induce their proliferation but do not recapitulate other fate-determining signals delivered by antigen-presenting cells. One such signaling axis, the NOTCH pathway, controls CD4+ T cell effector function acquisition and strongly influences behavior. To study NOTCH signaling during chimeric antigen receptor (CAR) T cell production, we developed a culture system using anti-CD3/CD28 mAb-coated beads and plate-coated agonistic NOTCH1-specific mAb to induce simultaneous T cell activation and NOTCH signaling. When transferred into NSG mice bearing CD19+ Raji lymphoma, CD19-specific NOTCH1-agonized (N1) CD4+ CAR T cells displayed a marked proliferative advantage over control (IgG) cells. Tumor-bearing mice given both IgG CD8+ CAR T cells and N1 CD4+ CAR T cells demonstrated superior expansion of both subsets compared to mice given IgG CD8+ and IgG CD4+ CAR T cells, resulting in rapid tumor clearance and protection from tumor re-challenge. These data demonstrate that NOTCH1 agonism could represent a significant improvement to adoptive T cell therapy, but the mechanisms by which NOTCH signaling improves T cell anti-tumor function are currently not understood. NOTCH is known to induce aryl hydrocarbon receptor (AhR) transcriptional activity. Pharmacologic inhibition of AhR activity during N1 CD4+ CAR T cell culture reduced characteristic differences between N1 and IgG cells in surface phenotype, cytokine production and proliferation upon in vitro restimulation. I hypothesize that NOTCH1 agonism organizes AhR-dependent transcriptomic changes that augment proliferative cytokine production in CD4+ CAR T cells, promoting CD4-dependent proliferation that improves CAR T cell efficacy. To test this hypothesis, I will assess the functional effects of AhR inhibition and activation in N1 and IgG CD4+ CAR T cells in vitro and in vivo, and characterize fate-determining transcriptional events in N1, IgG and AhR-inhibited N1 CD4+ CAR T cells using bulk and single-cell RNA sequencing. Collectively, these experiments will investigate the utility of NOTCH and AhR signaling in the generation of superior CAR T cell products and establish the mechanisms by which NOTCH1 agonism improves CAR T cell therapeutic efficacy, laying the groundwork for translation of this promising biomedical advance into immunotherapy clinics.