程序性死亡受体-1(PD-1)以及T细胞免疫球蛋白-3(Tim-3)等免疫检查点受体在肿瘤微环境当中发挥着调节免疫细胞功能抑制的重要作用。在长期肿瘤抗原刺激下，这些免疫检查点受体在肿瘤浸润性T淋巴细胞表面的表达导致免疫细胞的功能耗竭从而使肿瘤细胞逃脱免疫监管。在头颈鳞癌患者中PD-1抑制剂的治疗有效率在20％以下，因此研究肿瘤微环境中免疫检查点受体维持免疫细胞功能抑制状态相关的机制具有重要意义。在前期的研究当中，我们发现PD-1与Tim-3的共表达标记了肿瘤微环境中一组炎性细胞因子分泌减少的特异的耗竭T细胞。Tim-3通过Akt／S6相关信号通路介导免疫细胞的功能抑制。在这些有着PD-1以及Tim-3共表达的耗竭T细胞当中，PD-1通路的抑制导致Akt／S6的磷酸化水平出现明显下降，提示PD-1与Tim-3之间可能存在相互调控。我们观察到在体外抑制人类头颈鳞癌浸润性T细胞表面的PD-1受体后，Tim-3的表达水平明显升高。代偿性的Tim-3表达升高使PD-1通路的抑制不能有效的激活肿瘤免疫细胞，提示这种代偿调节可能是免疫细胞维持耗竭状态的机制。另外，这一现象也在小鼠头颈肿瘤模型当中得到验证，PD-1受体抑制剂治疗的小鼠肿瘤微环境中Tim-3在免疫细胞表面的表达水平明显升高。序贯Tim-3受体抑制剂治疗有效的抑制了小鼠头颈肿瘤的生长，加强了PD-1受体抑制剂的疗效。上述发现提示我们在PD-1抑制剂治疗后，免疫细胞对Tim-3的表达上调可能是通过T细胞受体下游的PI3K／Akt通路介导的。综上，我们认为PD-1受体抑制剂的获得性耐药机制是通过PI3K／ Akt信号通路上调免疫细胞表面的Tim-3受体表达，增强免疫细胞内抑制性信号并维持肿瘤微环境中的免疫耗竭状态。这一发现为肿瘤治疗中两种或两种以上免疫检查点受体抑制剂的联合应用提供了理论依据。 关键词：头颈肿瘤；免疫检查点受体；单克隆抗体；PD-1；Tim-3

 Immune checkpoint receptors such as Programmed Death 1 (PD-1) and T cell Immunoglobulin and mucin domain-3 protein (Tim-3) plays an important role in the tumor microenvironment. They are expressed on activated tumor-infiltrating lymphocytes (TIL) to mediate immune cell dysfunction. The response rate of anti-PD-1 therapy in head and neck squamous cell carcinoma (HNSCC) patients is less than 20%, thus it is of great importance to understand the mechanism of immune check point receptors in maintaining suppressed cellular immunity. We have previously reported that PD-1 and Tim-3 marks the exhausted phenotype of TIL with greatly reduced production of inflammatory cytokines upon activation. In these exhausted TIL with PD-1 and Tim-3 expression, dampened Akt/S6 phosphorylation were observed upon PD-1 activation suggesting a signaling cross-talk between these two receptors. Indeed, upon PD-1 blockade, Tim-3 expression was upregulated in human HNSCC TIL, suggesting a compensatory signaling circuit in mediating potential adaptive resistance to anti-PD-1 therapy in the tumor micro-environment. In addition, this adaptive resistant mechanism exists in a murine head and neck cancer (HNC) model that showed modest responsiveness to anti-PD-1 therapy with significant upregulation of Tim-3 expression in TIL. Addition of Sequential anti-Tim-3 mAb to PD-1 blockade showed significant increase in antitumor activity possibly by overcoming the adaptive resistance. We proved that the Tim-3 mediated resistant mechanism to anti-PD-1 therapy in cancer treatment was mediated by the downstream signaling of TCR through phospho-inositol-3 kinase (PI3K)/Akt complex instead of the cytokine-mediated pathways. In summary, we conclude that the adaptive resistant mechanism in anti-PD-1 immunotherapy is mediated by Tim-3 upregulation dependent on the PI3K/Akt pathway, suggesting potential benefit of dual targeting of immune check point receptors by permitting more effective anti-tumor immune response in cancer immunotherapy. Keywords: Head and neck cancer; immunotherapy; monoclonal antibody; PD-1; Tim-3