共表达抑制性受体shRNA的CAR-T细胞对重激活的HIV-1潜伏感染储存库清除作用的研究

Our previous studies have shown that CAR-T cells guided by the single-chain Fv of a broadly neutralizing antibody specifically eradicate the HIV-1 infected cells. However, the efficiency of CAR-T cell is compromised after excessive expansion. Our group identified several checkpiont receptors, such as PD-1, Tim-3 and Lag-3, are significantly up-regulated after long-term expansion through analyzing the transcriptional changes. Therefore, we co-expressed CAR-T moiety with shRNA targeted these inhibitory molecules to augment the potency of CAR-T cells. We demonstrated that the modified CAR-T cells maintained memory-like phenotype and robust proliferation during long-term culture, and induced specific cytolysis of cells expressing HIV-1 Env proteins. In this project, we will utilize in-vitro HIV-1 latency model and humanized mice to examine if the shRNA-CAR-T-expressing cells increase the self-renewal capability, prevent exhausion, and persistently suppress HIV-1 rebound. Furthermore, we will verify whether shRNA-CAR-T cells exert more potency to induce the cytolysis of latency reversing agent (LRA)-reactivated HIV-1-infected CD4+ T lymphocytes isolated from infected individuals receiving suppressive cART. Based on these works, we will optimize a CAR-T cell-based immunotherapy strategy for therapeutic application in efforts to reach a functional HIV cure.

在前期研究中，我们研发了以HIV-1广谱中和抗体为胞外结构的VC-CAR-T细胞可以特异性杀伤HIV-1感染细胞。进一步研究发现，经过长期扩增的CAR-T细胞，杀伤活性明显减弱。通过表达谱分析发现PD-1、Tim-3、Lag-3等抑制性受体表达明显上升。因此本项目拟在VC-CAR的模块上共表达几种shRNA特异性敲除以上受体，从而增强CAR-T细胞的功能。进一步实验显示共表达shRNA的VC-CAR-T细胞可以长期维持记忆性表型，并且更有效地杀伤靶细胞。本项目拟采用分子病毒学技术、免疫学技术和人源化小鼠等方法，旨在证明共表达shPD-1/Tim-3/Lag-3可以使CAR-T细胞自我更新增强、功能耗竭减缓、体内半衰期延长，长期且持续地发挥抗病毒作用，从而在HIV-1感染患者样本中更有效地清除重激活的病毒储存库，并优化出更有效的CAR-T疗法，为功能性治愈HIV-1提供更好的候选策略。

我们发现共表达shPD-1/Tim-3/Lag-3（shPTL）-CAR-T细胞的记忆性表型增加，并且.抑制细胞耗竭的表观遗传学印记；从而有效提高对靶细胞的特异性杀伤和功能性细胞因.子的分泌。我们通过CAR-T细胞进行ATAC-seq和转录组芯片检测，发现shPTL-CAR-T细胞.上CD56基因的转录活跃程度及表达显著升高，伴随抗肿瘤免疫效应分子及抗凋亡分子的.表达增加。实验证明，传统CAR-T细胞过表达CD56后体外杀伤靶细胞和肿瘤浸润能力显著.增强；而在shPTL -CAR-T细胞上敲低CD56后则其特异性杀伤功能明显降低。在此基础上.，我们进一步证明了CD56的同质性相互作用是介导特异性CAR-T功能增强的关键因素。此.外，我们通过高通量药物筛选发现洛伐他汀可以有效拮抗HIV-1病毒蛋白Nef，恢复感染.细胞膜表面的MHC-I分子的表达，从而有效促进自体杀伤细胞对病毒储藏库的清除作用。.以上研究，可以进一步增加了T细胞的特异性杀伤活性，为最大程度上清除HIV-1病毒储.存库提供了理论基础和候选策略。