Expanding the cytokine receptor alphabet reprograms T cells into diverse cell states: RNAseq of orthogonal chimera transduced T cells

Cytokines signal through specific cell surface receptor dimeric pairings that have been selected over the course of evolution to activate canonical JAK/STAT signaling pathways and gene expression programs. However, the potential combinatorial diversity of JAK/STAT cytokine receptor pairings is much greater than what is utilized in nature, raising questions about the untapped biology of alternative 'non-natural' pairings. Here we exploited common γ chain (γc) receptor as a shared signaling hub on T cells and enforced the expression of both natural and non-natural heterodimeric JAK/STAT receptor pairings using an orthogonal cytokine receptor platform, followed by a comparative analysis of the resulting T cell phenotypes in vivo. We tested receptors from γc cytokines as well as interferon, IL-10, and other homodimeric receptor families that do not normally pair with γc or are not naturally expressed on T cells. These synthetic receptors induced unique gene expression programs, and led to distinct T cell fates in tumors, including naturally occurring states (type 2 cytotoxic T (Tc2) cell and type 2 helper T (Th2) cell differentiation driven by orthogonal IL-4R) and synthetic states (myeloid-like phagocytic T cells driven by orthogonal GSCFR). T cells armed with orthogonal IL-22R (o22R) and oGCSFR exhibited transcriptional, and chromatin landscapes associated with stemness and resistance to exhaustion, which enhanced anti-tumor properties. Non-native JAK/STAT signals open a path to diversifying T-cell states beyond those induced by the menu of natural cytokines. WT mouse T cells transduced with orthogonal receptors were sorted using an Aria II sorter (BD Biosciences) at the Stanford Shared FACS Facility. Non-ICD o2R-transduced cells were sorted as controls. Following sorting, cells were recovered in MSA-mIL-2 (100 U ml−1) for 24 hours, subjected to IL-2 starvation for another 24 hours, and then stimulated with 5 μM MSA-oIL2 or 10 nM recombinant cytokines (IL-2, IL-4, IL-7, IL-21, IL-10, IFN-α, Peprotech) for 6 hours. Total RNA was extracted using the Quick-RNA 96 Kit (Zymo Research). mRNA was isolated using poly-T magnetic beads, fragmented, and converted into cDNA using random hexamer primers. After second-strand synthesis, libraries were prepared through end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Libraries were pooled and sequenced on the NovaSeq X Plus Series (PE150).