C-terminal CD28 phosphorylation, pY218, modulates IL-2 secretion and therapeutic effect of CAR-T cells.

CD28 is a costimulatory component of several second-generation chimeric antigen receptor (CAR)-T cells, providing signals essential for T cell proliferation, survival, and cytokine secretion. However, the specific contribution of individual CD28 intracellular motifs to CAR-T cell function remains incompletely understood. Here, we identify tyrosine 218 (Y218) in the CD28 cytoplasmic domain as a critical regulatory site and demonstrate that its phosphorylation is essential for optimal CAR-T cell activity. Using a 218F mutant, we show that loss of Y218 phosphorylation leads to impaired IL-2 production and abrogates antitumor efficacy. Transcriptomic profiling of 218F CAR-T cells revealed increased expression of IL-17A, IL-17F, and related cytokines, suggesting a shift toward a pro-inflammatory Th17-like phenotype that may contribute to dysfunction. Mechanistically, we demonstrate that the interleukin-2-inducible T cell kinase, ITK, mediates Y218 phosphorylation. To translate this mechanistic insight into improving CAR design, we engineered a novel CAR incorporating a synthetic ITK-binding motif (PYRP). This novel design enhances ITK recruitment, increases Y218 phosphorylation, boosts IL-2 secretion, and improves antitumor efficacy in vivo. Our findings underscore the functional relevance of Y218 phosphorylation in modulating CAR-T cell fate and reveal a strategy to fine-tune CAR signaling through targeted kinase recruitment to enhance therapeutic efficacy.