Natural and non-natural cytokine receptors generate diverse cell states to enhance T cell therapy [scRNA-seq]

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. C57BL/6 mice were inoculated s.c. with B16F10 tumor cells (1 × 10^6), sublethally lymphodepleted by irradiation on day 7, and received i.v. adoptive transfer of 3 × 10^6 Pmel T cells (either untransduced or transduced with mo2R, mo4R, mo20R, mo22R, or moGCSFR) on day 8 post tumor followed by i.p. administration of MSA-oIL-2 (2.5 × 10^4 unit per day) or PBS control every other day until day 16. On day 17, mice were sacrificed, tumors were minced and dissociated using a mouse 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 (Cytiva), and then stained with surface markers and DAPI (Thermo Fisher Scientific). Thy1.1+CD8+ or Thy1.1+CD8+ YFP+ T cells were sorted using an Aria II sorter (BD Biosciences) at the Stanford Share FACS Facility for Single cell RNA sequencing.