C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

CD28 is a co-stimulatory 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 further understand the role of this kinase, we engineered a novel CAR incorporating an ITK-binding motif (PYRP), which enhances ITK recruitment, increases Y218 phosphorylation, and boosts IL-2 secretion, and improves antitumor efficacy <i>in vivo</i>. 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.