Data Sheet 4_Bryostatin enhances CD20 CAR-T therapy efficacy against B-cell lymphoma by overcoming trogocytosis-mediated antigen loss.pdf

IntroductionTrogocytosis, an active membrane transfer process, impairs the therapeutic efficacy of CAR-T cells by inducing antigen loss from tumor cells. This study investigated whether Bryostatin, a PKC modulator derived from marine organisms, could enhance CD20 CAR-T cell activity by up-regulating the CD20 antigen on tumor cells and promoting T cell activation, differentiation and function.
MethodsCD20 antigen expression and trogocytosis-mediated membrane transfer were assessed by flow cytometry and immunofluorescence following co‑culture of CD20 CAR‑T cells with Raji or BALL‑1 cells. Trogocytosis‑positive (Trog⁺) CAR‑T cell cytotoxicity and fratricide by fresh CAR‑T cells were evaluated by ELISA. Proteomic profiling compared metabolic features of Trog⁺ and Trog⁻ CAR‑T cells. Using flow‑sorted BALL‑1 subsets with differential CD20 expression (CD20low, CD20mid, CD20hi), we examined how antigen density affects CAR‑T persistence and killing. Finally, Bryostatin‑mediated CD20 upregulation and its therapeutic impact on CAR‑T efficacy were tested in vitro and in a murine subcutaneous lymphoma model.
ResultsUpon contact with Raji or BALL-1 cells, CD20 CAR‑T cells underwent trogocytosis, leading to marked loss of tumor‑cell CD20 and impaired cytotoxicity of trogocytosis‑positive (Trog⁺) CAR‑T cells, which also became susceptible to fratricide. CD20 antigen density positively correlated with CAR‑T killing efficacy. Proteomic analysis revealed that Trog⁺ CAR‑T cells exhibited enriched activity in ribosome biogenesis, mRNA surveillance, and RNA catalysis, suggesting elevated protein synthesis alongside exhaustion features. Key MEK/ERK‑related transcription factors (c‑JUN, TCF7) linked to T‑cell activation were downregulated in Trog⁺ cells. In both in vitro and mouse lymphoma models, Bryostatin potentiated CD20 CAR‑T‑mediated tumor suppression. Mechanistically, bryostatin upregulated CD20 expression in tumor cells via the MEK/ERK pathway and enhanced c‑JUN/TCF7 levels in CAR‑T cells, promoting their tumor infiltration.
ConclusionBryostatin enhances CD20 CAR‑T efficacy by counteracting trogocytosis‑driven antigen loss and upregulating CD20 expression, providing a promising strategy to overcome antigen escape in lymphoma therapy.

引言：胞啃作用（Trogocytosis）是一种主动的膜转移过程，可通过诱导肿瘤细胞丢失抗原而削弱嵌合抗原受体T细胞（CAR-T）的治疗效果。本研究探讨了源自海洋生物的蛋白激酶C（PKC）调节剂苔藓抑素（Bryostatin）是否可通过上调肿瘤细胞表面CD20抗原、促进T细胞活化、分化与功能，增强CD20嵌合抗原受体T细胞的活性。
方法：将CD20嵌合抗原受体T细胞与Raji细胞或BALL-1细胞共培养后，采用流式细胞术与免疫荧光法检测CD20抗原表达及胞啃作用介导的膜转移过程；采用酶联免疫吸附试验（ELISA）评估胞啃作用阳性（Trog⁺）CAR-T细胞的细胞毒性以及新鲜CAR-T细胞介导的同胞杀伤效应；通过蛋白质组学分析对比Trog⁺与Trog⁻ CAR-T细胞的代谢特征；利用流式分选获得CD20表达水平各异的BALL-1细胞亚群（CD20low、CD20mid、CD20hi），探究抗原密度对CAR-T细胞存续与杀伤功能的影响；最后，在体外及小鼠皮下淋巴瘤模型中验证苔藓抑素介导的CD20上调作用及其对CAR-T细胞治疗效果的影响。
结果：当CD20嵌合抗原受体T细胞与Raji细胞或BALL-1细胞接触后，会发生胞啃作用，导致肿瘤细胞CD20显著丢失，同时胞啃作用阳性（Trog⁺）CAR-T细胞的细胞毒性受损，且其对同胞杀伤效应更易感。CD20抗原密度与CAR-T细胞杀伤效能呈正相关。蛋白质组学分析显示，Trog⁺ CAR-T细胞在核糖体生物发生、mRNA监视及RNA催化通路中富集，提示其蛋白质合成水平升高且伴随T细胞耗竭特征。与T细胞活化相关的关键丝裂原活化蛋白激酶激酶/细胞外调节蛋白激酶（MEK/ERK）通路转录因子（c-JUN、TCF7）在Trog⁺细胞中表达下调。在体外及小鼠淋巴瘤模型中，苔藓抑素均可增强CD20嵌合抗原受体T细胞介导的肿瘤抑制效应。其作用机制为：苔藓抑素通过MEK/ERK通路上调肿瘤细胞CD20的表达，并升高CAR-T细胞内c-JUN/TCF7的水平，从而促进CAR-T细胞向肿瘤组织浸润。
结论：苔藓抑素可通过抵消胞啃作用介导的抗原丢失并上调CD20表达，增强CD20嵌合抗原受体T细胞的治疗效果，为淋巴瘤治疗中克服抗原逃逸提供了极具潜力的策略。