Abstract
Cytokines are critical for guiding the differentiation of T lymphocytes to perform specialized tasks in the immune response. Developing strategies to manipulate cytokine-signaling pathways holds promise to program T cell differentiation toward the most therapeutically useful direction. Suppressor of cytokine signaling (SOCS) proteins are attractive targets, as they effectively inhibit undesirable cytokine signaling. However, these proteins target multiple signaling pathways, some of which we may need to remain uninhibited. SOCS3 inhibits IL-12 signaling but also inhibits the IL-2-signaling pathway. In this study, we use computational protein design based on SOCS3 and JAK crystal structures to engineer a mutant SOCS3 with altered specificity. We generated a mutant SOCS3 designed to ablate interactions with JAK1 but maintain interactions with JAK2. We show that this mutant does indeed ablate JAK1 inhibition, although, unexpectedly, it still coimmunoprecipitates with JAK1 and does so to a greater extent than with JAK2. When expressed in CD8 T cells, mutant SOCS3 preserved inhibition of JAK2-dependent STAT4 phosphorylation following IL-12 treatment. However, inhibition of STAT phosphorylation was ablated following stimulation with JAK1-dependent cytokines IL-2, IFN-α, and IL-21. Wild-type SOCS3 inhibited CD8 T cell expansion in vivo and induced a memory precursor phenotype. In vivo T cell expansion was restored by expression of the mutant SOCS3, and this also reverted the phenotype toward effector T cell differentiation. These data show that SOCS proteins can be engineered to fine-tune their specificity, and this can exert important changes to T cell biology.