抗PD-1通过IFN-γ抑制Tim-3介导的Treg细胞功能调控治疗头颈部鳞癌的作用及机制

Treg cells play an important role in tumor immune escape. Anti-PD-1 has been shown to stimulate CD8+ T cells to secrete IFN-γ, and the efficacy of anti-PD-1 was positively correlated with increased IFN-γ secretion and maintenance of Treg cell function. However, the inhibiting mechanism of anti-PD-1 on Treg cells is not clear. The applicant previously found that Tim-3 inhibited the proliferation of CD8+T cells in the tumor infiltrating Treg cells of patients with head and neck cancer. Meanwhile, anti-PD-1 or IFN-γ inhibited the Tim-3 expression and function in Treg cells. Surprisingly, IFN-γ capture antibody could partially reverse the function of anti-PD-1 on Tregs. Furthermore, the expression of Tim-3 in Treg cells was decreased after inhibiting STAT3 in vitro. Based on previous studies, we hypothesize that anti-PD-1 stimulates IFN-γ secretion of CD8+ T cells, IFN-γ acts on the JAK2-STAT3 pathway in Treg cells, thereby inhibiting the Tim-3 expression and function of Treg cells. Thus we intend to study the molecular mechanism of anti-PD-1 affecting the phenotype and function of Treg cells by flow cytometry in vivo and in vitro using animal models and head and neck cancer samples, with a view to proposing that Tim-3 expression is a molecular target to predict the efficacy of anti-PD-1.

Treg细胞在肿瘤免疫逃逸中发挥重要作用，抗PD-1能刺激CD8+T细胞分泌IFN-γ，且抗PD-1疗效与IFN-γ分泌增加及Treg细胞功能维持正相关，但抗PD-1抑制Treg细胞作用机制不明确。申请者前期发现Tim-3在头颈部肿瘤患者浸润性Treg细胞中抑制CD8+T细胞增殖，同时抗PD-1或IFN-γ抑制Treg细胞Tim-3表达及功能，而捕获性IFN-γ抗体逆转抗PD-1对Treg细胞的调控。进一步体外抑制STAT3后Treg细胞Tim-3表达下降。据此我们推测抗PD-1通过刺激CD8+T细胞分泌IFN-γ，作用于Treg细胞JAK2-STAT3通路，从而抑制Treg细胞Tim-3表达及功能。我们拟用头颈部肿瘤动物模型及患者样本，体内及体外流式细胞术研究抗PD-1抑制Tim-3表达进而影响Treg细胞功能的分子机制，以期阐明Treg细胞Tim-3表达是预测抗PD-1疗效的分子靶标。

部分恶性黑色素瘤患者对抗PD-1治疗无应答，而Treg细胞在肿瘤免疫逃逸中起到重要作用，抗PD-1能刺激CD8+T细胞分泌IFN-γ，且抗PD-1疗效与IFN-γ分泌增加及Treg细胞功能维持正相关。本研究发现在皮肤黑色素瘤发生发展过程中，肿瘤组织浸润的免疫细胞百分比具有显著性差异，其中包括Treg细胞。在抗PD-1治疗抵抗的皮肤黑色素瘤患者组织中，组织的Tim-3表达增加。Treg细胞内IL-10和TGF-β分泌增加。在抗PD-1治疗后，黑色素瘤肿瘤组织和人外周血中Treg细胞中p-STAT3表达增加，激活了JAK-STAT3通路。使用STAT3抑制剂Stattic治疗人皮肤黑色素瘤和人外周血中的Treg细胞，Treg细胞上的Tim-3表达下降，并且分泌的TGF-β也减少。小鼠实验中，联合STAT3抑制剂和抗PD-1治疗后，黑色素瘤增长减慢，肿瘤浸润的Treg细胞减少，并且Treg细胞上Tim-3的表达以及IL-10和TGF-β分泌也相应减少。本研究阐明了调控Treg细胞功能的机制，为提高抗PD-1治疗疗效提供新思路。