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 (CD8ΔIL6R mice). Compared to WT littermate controls, we found that CD8ΔIL6R mice had stronger respones to anti-PD-L1 therapy in terms of improved CD8 T cell function (e.g. increased production of IFNγ 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. CD8ΔIL6R 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 CD8ΔIL6R 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. 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.

白细胞介素6（Interleukin 6, IL-6）是一种多效性细胞因子（pleiotropic cytokine），在机体稳态、炎症反应及肿瘤发生进程中发挥多种生物学功能。在多种同基因小鼠肿瘤模型（syngeneic mouse tumor models）中，我们发现采用IL6R阻断性抗体阻断IL-6信号通路，可与抗PD-L1治疗产生协同效应，诱导强效的抗肿瘤CD8+ T细胞应答并促成肿瘤消退。为更深入表征抗PD-L1治疗期间，IL-6信号通路在肿瘤反应性CD8+ T细胞中的细胞固有效应，我们通过将IL6R.loxp小鼠与E8i.CD8.Cre小鼠杂交，构建了CD8 T细胞特异性IL6R基因敲除小鼠（CD8ΔIL6R小鼠）。与野生型（WT）同窝对照小鼠相比，CD8ΔIL6R小鼠在接受抗PD-L1治疗后表现出更强的应答：包括CD8+ T细胞功能改善（例如经流式细胞术（flow cytometry）检测的IFNγ与TNF分泌增加）以及肿瘤控制能力增强，这表明CD8+ T细胞内直接的IL-6信号通路足以削弱抗肿瘤免疫。本研究旨在通过全转录组测序分析（whole-transcriptome profiling）更详细地表征IL6R敲除CD8+ T细胞的表型。我们将CD8ΔIL6R小鼠及其野生型同窝对照小鼠的右侧背部植入MC38肿瘤；当肿瘤体积达到约150mm³时，将动物随机分配至同型对照（isotype control）组或抗PD-L1治疗组。7天后从肿瘤组织中经荧光激活细胞分选（FACS）纯化CD8+ T细胞，并通过批量RNA测序（bulk RNAseq）进行转录组分析。与野生型小鼠来源的CD8+ T细胞相比，CD8ΔIL6R小鼠来源的CD8+ T细胞表现出干扰素诱导基因特征的表达上调、细胞周期相关基因的表达升高，以及氧化磷酸化关键基因的表达增强。与之相反，野生型细胞则更高表达与初始T细胞和记忆前体细胞相关的基因。由此可见，肿瘤反应性CD8+ T细胞中的IL-6信号通路会限制其分化为强效抗肿瘤效应细胞的能力。我们从经酶解消化的MC38肿瘤组织中通过FACS分选获取总CD8+ T细胞，所用小鼠的基因型分为两类：IL6R.wt/wt_E8i.CD8.Cre+（野生型，WT）以及IL6R.loxp/loxp_E8i.CD8.Cre+（IL6R敲除型，IL6R.ko）。分别给予同型对照抗体或抗PD-L1（克隆号6E11）抗体治疗，给药剂量为10mg/kg，于第0天和第4天给药。给药1周后收获肿瘤组织。每组设置5只生物学重复，每个样本均来自单只小鼠。