Gene expression profiles of CD4-derived (CAR4) and CD8-derived (CAR8) chimeric antigen receptor T cells after stimulation through the CAR, TCR or both receptors

Chimeric antigen receptor (CAR)-expressing T-cells induce durable remissions in patients with relapsed/refractory B-cell malignancies. CARs are artificial constructs introduced into mature T-cells conferring a second, non-MHC restricted specificity in addition to the endogenous T-cell receptor (TCR). The impact of TCR activation on CAR T-cell efficacy in vivo has important implications for clinical optimization of CAR T-cell therapy, but cannot be systematically evaluated in xenograft models. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T-cell therapy for pre-B cell ALL, we demonstrate loss of CD8 CAR T-cell mediated clearance of leukemia associated with T-cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T-cells demonstrate equivalent cytotoxicity, as compared to CD8 CAR T-cells, and in contrast, retain in vivo efficacy in the presence of TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CAR8 upon dual receptor stimulation compared to CAR4, and indicate inherent differences in T-cell pathways. Chimeric antigen receptor (CAR) T cells express two activating receptors, the CAR and the endogenous T cell receptor (TCR). CAR T cells can be derived from either CD8 or CD4 T cells to generate CAR8 and CAR4 cells, respectively. In vivo, CAR8 and CAR4 cells respond differently when simultaneously stimulated through the CAR and TCR. Murine CAR8 and CAR4 cells were generated with a CAR that recognizes murine CD19 and TCRs that recognize HY-minor histocompatibility antigens. CAR8 and CAR4 T cells were co-incubated for 10 hours with B cells that were either possitive or negative for the expression of CD19 and/or HY-antigen. Through this CAR8 and CAR4 T cells were stimulated through the CAR, the TCR, CAR+TCR (simultaneiously) or neither receptor (CD19 negative, HY-negative B cells). Gene expression profiles of CD4-derived (CAR4) and CD8-derived (CAR8) chimeric antigen receptor T cells after stimulation through the CAR, TCR or both receptors

嵌合抗原受体（Chimeric antigen receptor, CAR）修饰的T细胞可使复发/难治性B细胞恶性肿瘤患者获得持久缓解。CAR是导入成熟T细胞内的人工构建体，可赋予其除内源性T细胞受体（T-cell receptor, TCR）之外的第二种非MHC限制性特异性。TCR激活对CAR-T细胞体内疗效的影响，对于CAR-T细胞疗法的临床优化具有重要意义，但目前无法在异种移植模型中开展系统性评估。
采用针对前B细胞急性淋巴细胞白血病（pre-B cell acute lymphoblastic leukemia, pre-B cell ALL）的免疫健全、同基因小鼠CD19靶向CAR-T细胞疗法模型，本研究证实：当存在TCR抗原时，CD8+ CAR-T细胞介导的白血病清除作用会因T细胞耗竭与凋亡而丧失。CD4+ CAR-T细胞的细胞毒性与CD8+ CAR-T细胞相当，与之相反，其在TCR刺激存在的情况下仍可保留体内疗效。基因表达谱分析证实，相较于CD4来源的CAR-T细胞（CAR4），CD8来源的CAR-T细胞（CAR8）在双受体刺激下的耗竭与凋亡程度更高，同时提示T细胞信号通路存在固有差异。
嵌合抗原受体（CAR）T细胞表达两种激活受体：CAR与内源性T细胞受体（TCR）。CAR-T细胞可分别源自CD8+或CD4+ T细胞，从而生成CAR8与CAR4细胞。在体内，当同时通过CAR与TCR受到刺激时，CAR8与CAR4细胞的应答模式存在差异。
本研究构建了识别小鼠CD19的CAR，以及识别HY次要组织相容性抗原的TCR，以此制备小鼠CAR8与CAR4细胞。将CAR8与CAR4 T细胞与CD19和/或HY抗原表达阳性或阴性的B细胞共孵育10小时，通过该方式可使CAR-T细胞分别通过CAR、TCR、CAR+TCR（同时刺激）或不通过任何受体（CD19阴性、HY阴性B细胞）受到刺激。
本研究对经CAR、TCR或双受体刺激后的CD4来源（CAR4）与CD8来源（CAR8）嵌合抗原受体T细胞开展了基因表达谱分析。