Single-cell profiling of tumor infiltrating T cells

Immune checkpoint blockade has shown tremendous anti-tumor potential in the clinic. However, these therapies are only effective in a subset of patients, so identification of additional immunomodulatory molecules that enhance the anti-tumor activity of these treatments may expand their clinical utility. In particular, identifying small molecules that complement existing immunotherapies has been relatively unexplored, so we performed a small molecule screen to identify compounds that can enhance co-inhibitory molecule blockade, to improve the anti-tumor adaptive immune response. Our unbiased screen identified inhibitors of cyclin-dependent kinase 4 and 6 (CDK4/6), including the FDA-approved palbociclib, as a class of small molecule compounds that exhibited significant immunostimulatory activity in vitro. In accordance with our in vitro finding of enhanced NFAT signaling, single-cell RNA-sequencing confirmed that in vivo exposure to CDK4/6 inhibitors enhanced NFAT signaling in tumor infiltrating T cells. Moreover, our results revealed that CDK4/6 inhibition up-regulated activation molecules and down-regulated suppressive molecules in these cells. CDK4/6 inhibition also increased the number of T cells with activated TCR (T cell receptor) signaling, as well as factors that are important for signal transduction downstream of TCR signaling. In summary, the impact of CDK4/6i on cell cycle progression and T cell proliferation are balanced favorably towards increased T cell recruitment and enhanced effector cell function, mediated in part by activation of the NFAT family of transcription factors. Further, our results demonstrate that CDK4/6i enhances PD-1 blockade through increased T-cell effector function and inhibition of immune suppressive cytokine production. While prolonged CDK4/6i treatment could be immunosuppressive due to adverse effects on lymphocyte proliferation, properly timed/sequenced CDK4/6i may potentiate the clinical impact of anti-PD-1/PD-L1 antibodies. As palbociclib is FDA-approved and multiple other CDK4/6 inhibitors are in clinical trials, we expect that this hypothesis will undergo rapid testing in humans. Overall design: Single-cell comparison of control and CDK4/6 inhibitor treated tumor infiltrating T cells免疫检查点阻断疗法在临床中已展现出卓越的抗肿瘤潜力。然而此类疗法仅对部分患者有效，因此发掘可增强此类疗法抗肿瘤活性的新型免疫调节分子，有望拓展其临床应用边界。尤为值得关注的是，目前针对可协同现有免疫疗法的小分子化合物的研究仍相对匮乏，为此本研究开展了小分子筛选实验，以筛选能够增强共抑制分子阻断效果、改善抗肿瘤适应性免疫应答的活性化合物。本研究的无偏筛选实验发现，细胞周期蛋白依赖性激酶4/6（cyclin-dependent kinase 4 and 6, CDK4/6）抑制剂类小分子（包括获美国食品药品监督管理局（Food and Drug Administration, FDA）批准的帕博西尼（palbociclib））在体外实验中展现出显著的免疫激活活性。结合我们体外实验中观察到的活化T细胞核因子（Nuclear Factor of Activated T cells, NFAT）信号通路增强的结果，单细胞RNA测序（single-cell RNA-sequencing）进一步证实，体内给予CDK4/6抑制剂可提升肿瘤浸润T细胞中的NFAT信号通路活性。此外，研究结果显示，CDK4/6抑制可上调此类细胞中的激活相关分子，并下调抑制性分子的表达。CDK4/6抑制还可增加携带激活型T细胞受体（T cell receptor, TCR）信号的T细胞数量，并上调TCR信号通路下游关键信号转导相关因子的表达。综上，CDK4/6抑制剂（CDK4/6i）对细胞周期进程与T细胞增殖的调控作用整体偏向于促进T细胞招募并增强效应细胞功能，该效应部分由NFAT家族转录因子的激活所介导。此外，本研究结果证实，CDK4/6抑制剂可通过增强T细胞效应功能并抑制免疫抑制性细胞因子的产生，来强化PD-1（programmed cell death protein 1）阻断疗法的效果。尽管长期使用CDK4/6抑制剂可能因对淋巴细胞增殖的不良影响而产生免疫抑制效果，但适时给药或序贯给药的CDK4/6抑制剂疗法，或可增强抗PD-1/PD-L1（programmed cell death ligand 1）抗体的临床疗效。鉴于帕博西尼已获FDA批准，且多款其他CDK4/6抑制剂正处于临床试验阶段，我们预计该假说将快速开展人体实验验证。实验整体设计：对对照组与CDK4/6抑制剂处理组的肿瘤浸润T细胞进行单细胞对比分析。