Abstract A013: Programmable JAK/STAT signaling drives CAR T cells to enhanced functional states

Cytokine signaling is critical to the function of natural immune cells and engineered immune cell therapies such as chimeric antigen receptor (CAR) T cells. It remains unclear how the limited set of signal transducers and activators of transcription (STATs) and other proteinsa activated by these cytokine receptors can encode the observed diversity of immune cell phenotypes. To understand how signaling downstream of cytokines control immune cell phenotype, we sought to map the structure of Janus kinase (JAK)/STAT signaling domains to cell signaling and resulting CAR T cell function. We recombined 14 signaling motifs to construct a library of ∼30,000 constitutively active synthetic cytokine receptors (SCRs) with intracellular domains composed of novel signaling motif combinations that activate different signaling cascades. We experimentally tested ∼450 SCRs which generated a range of CAR T cell memory, cytotoxicity, and proliferation. SCRs with pSTAT1 and 3 signaling generated effector memory CAR T cells, while SCRs with strong pSTAT5 generated effector CAR T cells with potent anti-tumor activity, measured by flow cytometry and phosphoproteomics. Subsequent kinase substrate enrichment analysis (KSEA) identified key differences of kinases associated with cell signaling networks, such as CDKs that are correlated with high proliferative profile of effector CAR T cells. To map the structure-signaling-phenotype landscape we trained models to predict signaling and CAR T cell phenotype that result from varied motif combinations. From neural network predictions we identified features, including strong STAT5 and Shc signaling, that promote unsafe autonomous CAR T cell proliferation. Models also revealed a trade-off between memory and cytotoxicity, with a Pareto front encoded by a continuous change in signaling. These results demonstrate that recombination of a limited set of signaling motifs creates a continuous spectrum of signaling that encodes a corresponding spectrum of cell phenotype. This work synthetically expands the combinatorial space of JAK/STAT signaling and provides a foundation for rational design of CAR T cells with improved cytotoxicity, memory, and safety profiles. Citation Format: Wansang Cho, Jenny Y. Liu, Alex N. Beckett, Erin Craig, Dain R. Brademan, Judith C. Lunger, Peng Xu, Katie Ho, Ethan E. Chen, Antonio Salcido-Alcantar, Lucas E. Sant'Anna, Kamal Obbad, Nakoa Po, Sophia Joy Ong, Elena Sotillo, Robert Tibshirani, Ruth Huttenhain, Crystal L. Mackall, Kyle G. Daniels. Programmable JAK/STAT signaling drives CAR T cells to enhanced functional states [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A013.