Bryostatin enhances CD20 CAR-T therapy efficacy against B-cell lymphoma by overcoming trogocytosis-mediated antigen loss

Introduction Trogocytosis, 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. Methods CD20 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 (CD20 low , CD20 mid , CD20 hi ), 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. Results Upon 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. Conclusion Bryostatin 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.