Natural and non-natural cytokine receptors generate diverse cell states to enhance T cell therapy

Cytokines augment T cell function, but the nature of the optimal signals are not well defined. We deployed orthogonal cytokine-receptors to diversify the JAK/STAT signaling profiles elicited by orthogonal IL-2. We designed chimeric receptors consisting of the orthogonal IL-2 receptor extracellular domain (ECD) fused to intracellular domains (ICDs) from receptors for other common gamma chain cytokines as well as ICDs from interferon receptor, IL-10 receptor and homodimeric receptor families not expressed on T cells. Such non-natural pairings with the common gamma chain expand the JAK/STAT signaling space in T cells. Natural and non-natural orthogonal chimeric receptors led to distinct T cell fates in tumors, including naturally occurring states (Tc2 differentiation driven by orthogonal chimeric IL4R) and synthetic states (myeloid-like T cells driven by orthogonal chimeric granulocyte colony stimulating factor receptor). The orthogonal chimeric IL22R (o22R) potently activated STAT-1, -3, -4, and -5, imparting T cells with stem-like and effector properties. The pronounced anti-tumor functionality mediated by o22R and oGCSFR was linked to remodeling of the exhaustion-associated epigenome. Our study broadens the cytokine receptor repertoire, providing novel solutions to overcome barriers for using engineered T cells to treat cancer. NSG mice were inoculated s.c. with A375 cells (1 × 106) and received i.v. adoptive cell transfer of 3 × 106 NY-ESO-1 TCR-T cells (WT NY-ESO-1 TCR-T cells, or ho22R, or hoGCSFR expressing NY-ESO-1 TCR-T cells) on day 10 post-tumor inoculation followed by i.p. administration of MSA-hoIL-2 (2.5 × 104 unit  × 3 doses, 2.5 × 104 unit  × 3 doses) or PBS control every other day until day 20. On day 21, mice were sacrificed, tumors were minced and dissociated using a human tumor dissociation kit (Miltenyi Biotec) and a gentleMACS Octo Dissociator (Miltenyi Biotec). Tumor-infiltrating lymphocytes (TILs) were first enriched by density gradient centrifugation against Percoll (GE healthcare), and then stained with surface markers and DAPI (Sigma-Aldrich). CD3+ EGFR+ or CD3+ EGFR+YFP+ T cells were sorted using an Aria II sorter (BD Biosciences) at the Stanford Share FACS Facility for ATAC sequencing.