45 ℃热疗通过 HSP70差异表达维持 CAR-T 细胞的免疫表型与杀伤功能

The 45 ℃ hyperthermia maintains the immune phenotype and cytotoxic function of CAR-T cells through differential HSP70 expression

摘要:

目的 探究45 ℃热疗通过热休克蛋白70（HSP70）差异表达调控嵌合抗原受体T（CAR-T）细胞功能及 其对肿瘤细胞选择性效应的相关机制。方法 以 Raji 淋巴瘤细胞系和人源 CAR-T 细胞为体外模型，分别进行 45 ℃热疗15 min处理（实验组）与37 ℃对照（对照组）。采用CCK-8法检测细胞活性，流式细胞术分析CAR-T细胞 亚群（CD4 + /CD8 + 比例及CD3 + 表达）；共聚焦显微成像及LDH释放实验评估CAR-T细胞杀伤活性；蛋白质免疫印 迹检测HSP70表达水平，并通过siRNA敲低与慢病毒过表达实验验证HSP70对CAR-T细胞热耐受性的调控作用。 结果 45 ℃热疗后，CAR-T细胞活性保留率超过95%，CD4 + /CD8 + 比例以及CD3 + 表达与对照组相比差异无统计 学意义（P > 0.05），CAR-T细胞对Raji细胞的靶向识别与杀伤效能未明显下降，热疗后CAR-T细胞中HSP70的相对 表达量为 Raji细胞的 4.3倍（P<0.001）；功能验证表明，敲低 HSP70可显著降低 CAR-T细胞在 45 ℃热疗后的存活 率，而过表达HSP70可增强其热耐受性（P < 0.05）。结论 45 ℃热疗条件下，CAR-T细胞可维持其免疫表型与杀伤 功能，其热耐受性可能与HSP70差异表达相关。

Abstract:

Objective To investigate into the mechanism by which 45 ℃ hyperthermia regulates the function of chimeric antigen receptor T (CAR-T) cells and their selective effects on tumor cells through differential expression of heat shock protein 70 (HSP70). Methods Human CAR-T cells and the Raji lymphoma cell line served as in vitro models. Cells were treated with 45 ℃ hyperthermia for 15 min (experimental group) or maintained at 37 ℃ (control group), respectively. Cell viability was evaluated using the CCK-8 assay; CAR-T cell subsets (CD4 + /CD8 + ratio and CD3 + expression) were analyzed by flow cytometry; the cytotoxicity of CAR-T cells was assessed by confocal microscopy and lactate dehydrogenase (LDH) release assays; HSP70 expression levels were determined by Western blotting, and its function was verified by gene knockdown and overexpression experiments. Results Following exposure to 45 ℃ hyperthermia, the viability of CAR-T cells remained above 95%. No statistically significant differences were observed in the CD4 +/CD8 + ratio or CD3 + expression (P > 0.05). The target recognition and cytotoxic efficacy of CAR T cells against Raji cells were not significantly decreased. The relative expression level of HSP70 in CAR-T cells after hyperthermia was 4.3-fold higher than that in Raji cells (P < 0.001). Functional validation showed that knockdown of HSP70 significantly reduced the survival rate of CAR-T cells following 45 ℃ hyperthermia, whereas HSP70 overexpression enhanced their thermotolerance (P < 0.05). Conclusion Under 45 ℃ hyperthermia conditions, CAR-T cells maintained their immunophenotype and cytotoxic function, and their heat tolerance potentially attributable to differential HSP70 expression.