A continuous landscape of signaling encodes a corresponding landscape of CAR T cell phenotype

Abstract 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 proteins 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. 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. Competing Interest Statement CLM has equity in Link Cell Therapies, is on the board of directors for Link Cell Therapies, consults with Link Cell Therapies, Ensoma, Grace Science, Nektar, RedTree Venture Capital, and Immatics. CML has research funding from Tune therapeutics and multiple patents for CAR T cells and other immunotherapies. KGD is an inventor on a pending patent application for technology described in this work. Footnotes A label in Figure 2F has been revised.