构建共表达GSK-3β shRNA的三代CD19-CAR T细胞以改善弥漫大B细胞淋巴瘤化疗后患者CAR-T细胞功能的研究

二代CD19-CAR T细胞在B细胞非霍奇金淋巴瘤（B-NHL）的治疗效果欠佳，我们发现利用化疗后弥漫大B细胞淋巴瘤（DLBCL）患者制备的CAR-T细胞存在增殖能力不足及耗竭增加。GSK-3β在T细胞分化发育中扮演重要角色，通过在三代CD19-CAR T细胞下调其表达，我们发现能同时调控分化及耗竭，并诱导耗竭关键分子TOX表达。本课题通过制备共表达shGSK-3β的三代CD19-CAR T细胞，以期纠正化疗患者来源的CAR-T功能缺陷：①shGSK-3β通过增加早期分化、降低终末耗竭细胞的比例，提高增殖活性及杀伤，弥补数量不足；②shGSK-3β在诱导TOX的表达机制及其在调整耗竭、增强应答及存活的调控作用；③shGSK-3β通过减少IL-10的分泌，下调DLBCL表面PD-L1的表达，逆转CAR-T细胞功能抑制。上述结果将为我们改善目前患者来源CAR-T细胞功能缺陷提供新的解决方案。

The complete remission rate of second-generation of CD19 chimeric antigen receptor (CAR) T cells in treating B-cell non-Hodgkin's lymphoma (B-NHL) was not as robust as that in acute lymphoblastic leukemia. We found that CAR-T cells generated from patients with diffuse large B-cell lymphoma (DLBCL) after chemotherapy were in dysfunctional state: reduced capacity of proliferation and performing with exhaustion. GSK-3β plays an important role in regulating differentiation. By co-expressing GSK-3β shRNA in third generation of CD19-CAR T cells, the TOX expression was upregulated, meanwhile, the differentiation and exhaustion were also affected. Our project aims to repair the dysfunction of CAR-T cell derived from patients after chemotherapy by co-expressing shGSK-3β with CD19-CAR, hoping which that will exerts its role as followings: ①shGSK-3β in 3rd generation CAR-T cells would increase the proportion of early differentiated cells and reduce the proportion of terminally exhausted T cells, resulting in promoting proliferation and killing capability; ② The mechanism of shGSK-3β in inducing TOX expression and its role in reducing exhaustion, enhancing response and maintaining survival; ③ shGSK-3β in CAR-T cells would reduce IL-10 secretion, which would downregulate the PD-L1 expression on DLBCL, resulting in the reversion of CAR-T cell exhaustion. Our results will provide new solutions in improving function of CAR-T cells derived from patients after chemotherapy.