Effect of IL-6 on antigen-specific CD8+ T cell activation in vitro

Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. Here we performed transcriptional profiling of OT-I CD8+ T cells activated in the presence or absence of IL-6. In this study, splenocytes from wild type C57BL/6J mice were incubated with SIINFEKL peptide for two days to activate OT-I CD8+ T cells. SIINFEKL was then withdrawn, T cells were rested in medium with IL-2 for three days, and CD8+ T cells were restimulated with anti-CD3 + anti-CD28 antibodies and purified by FACS for RNA sequencing analysis on day 7. Throughout this culture period, some cells were treated with anti-IL6R antibody (clone MR16-1, 5 ug/ml), isotype control antibody (5 ug/ml), recombinant IL-6 (10 ng/ml), or recombinant hyper-IL-6 (IL-6 linked to IL6R; 20 ng/ml). Experimental conditions were run in triplicate, for a total of 15 samples. Conclusion: Neutralization of endogenous IL-6 signaling (with anti-IL6R antibody) enhances effector differentiation of CD8+ T cells, while addition of exogenous IL-6 or hyper-IL-6 suppresses effector differentiation. Overall design: Activation of TCR-transgenic T cells in vitro with cognate peptide in the presence or absence of IL-6 signaling inhibitors or recombinant IL-6.

白细胞介素6（Interleukin 6, IL-6）是一种多效性细胞因子，在机体稳态、炎症反应与肿瘤发生发展中发挥多种调控功能。本研究针对存在或缺失IL-6条件下活化的OT-I型CD8+T细胞开展转录谱分析。
本实验以野生型C57BL/6J小鼠的脾细胞为材料，用SIINFEKL肽段孵育两天以活化OT-I型CD8+T细胞；随后移除SIINFEKL肽段，将T细胞置于含IL-2的培养基中静置培养三天；于第7天采用抗CD3抗体联合抗CD28抗体再次刺激CD8+T细胞，并通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）纯化细胞以进行RNA测序分析。
整个培养周期内，部分细胞分别接受如下处理：抗IL-6受体抗体（克隆号MR16-1，浓度5 μg/ml）、同型对照抗体（浓度5 μg/ml）、重组IL-6（浓度10 ng/ml），或重组超IL-6（即IL-6与IL-6R融合蛋白，浓度20 ng/ml）。所有实验条件均设置三次生物学重复，共计15个样本。
研究结论：阻断内源性IL-6信号通路（采用抗IL-6受体抗体）可增强CD8+T细胞的效应分化过程，而添加外源性IL-6或重组超IL-6则会抑制CD8+T细胞的效应分化。
实验整体设计：本实验于体外采用同源肽段活化T细胞受体转基因T细胞，并设置是否添加IL-6信号通路抑制剂或重组IL-6的不同实验条件。