Transcriptional profiling of IL6R-deficient CD8 T cells isolated from subcutaneous MC38 tumors treated with anti-PD-L1 therapy

Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. In multiple syngeneic mouse tumor models, we found that blockade of IL-6 signaling (using an IL6R-blocking antibody) synergized with anti-PD-L1 therapy to drive potent anti-tumor CD8 T cell responses and tumor rejection. To better characterize the cell-intrinsic effects of IL-6 signaling in tumor-reactive CD8+ T cells during anti-PD-L1 therapy, we generated mice with genetic IL6R deficiency restricted to CD8 T cells by crossing IL6R.loxp and E8i.CD8.Cre mice (CD8?IL6R mice). Compared to WT littermate controls, we found that CD8?IL6R mice had stronger respones to anti-PD-L1 therapy in terms of improved CD8 T cell function (e.g. increased production of IFN? and TNF, measured by flow cytometry) and enhanced tumor control, suggesting that direct IL-6 signaling in CD8 T cells is sufficient to impair anti-tumor immunity. In this study we aimed to characterize the phenotype of IL6R-deficient CD8 T cells in more detail via whole-transcriptome profiling. CD8?IL6R and WT littermates were implanted with MC38 tumors in the right flank; when tumors reached ~150mm3 in volume, animals were randomized to isotype control or anti-PD-L1 treatment. CD8 T cells were FACS-purified from tumor tissue 7 days later and profiled by bulk RNAseq. Compared to cells from WT mice, CD8 T cells from CD8?IL6R mice showed increased expression of interferon-driven gene signatures, increased expression of cell cycle genes, and increased expression of genes critical for oxidative phosphorylation. In contrast, WT cells had higher expression of genes associated with naive and memory precursor cells. Thus, IL-6 signaling in tumor-reactive CD8 T cells limits their capacity to differentiate into potent anti-tumor effectors. Overall design: Total CD8 T cells from digested MC38 tumor tissue were sorted by FACS from mice with the following genotypes: IL6R.wt/wt_E8i.CD8.Cre+ (WT) and IL6R.loxp/loxp_E8i.CD8.Cre+ (IL6R.ko). Tumors were harvested 1 week after start of treatment with isotype control or anti-PD-L1 (clone 6E11) antibodies (10 mg/kg), dosed on day 0 and day 4. N=5 per group; each sample is from an individual mouse.