研究背景及目的: 外周 T 细胞淋巴瘤（Peripheral T-cell lymphoma，PTCL）是一组起源于成熟 T 细胞、具有高度异质性的非霍奇金淋巴瘤。在我国的发病率明显高于西方国家。T细胞淋巴瘤的预后明显差于B细胞淋巴瘤，标准化疗方案有效率尚可，但存在缓解时间短、复发率较高的问题。因此，进一步研究阐明PTCL的发病机制，对寻找新的治疗方法及改善 PTCL 患者的预后具有重要的意义。临床上，PD-1/PD-L1 单抗用于肿瘤治疗有一定疗效，但存在反应率低及治疗有效后复发的问题。PD-1/PD-L1单抗用于治疗外周T细胞淋巴瘤的临床研究目前仍然较少。因此，探究如何抑制PD-1/PD-L1 通路介导的肿瘤免疫逃逸具有重要的临床意义。HDAC（Histone deacetylase，组蛋白去乙酰化酶）抑制剂西达本胺通过表观遗传修饰调控宿主免疫 系统的抗肿瘤作用，对肿瘤尤其是血液系统肿瘤具有一定的疗效。临床上，西达本胺与 PD-1/PD-L1 单抗联用对淋巴瘤的治疗表现出协同作用，提示西达本胺可能具有调控 PD-1/PD-L1 免疫逃逸信号通路的作用。本研究从表观遗传学及免疫检查点联合用药的角度出发，探究西达本胺对 PD-1/PD-L1 免疫逃逸的调控机制，为今后临床治疗上的联合用药提供新的理论基础。研究方法: 使用不同浓度的西达本胺处理 Jurkat 细胞系，通过荧光定量 PCR 检测 PD-L1、 JAK/STAT 通路基因以及趋化因子 CXCL3、CXCL9 的 RNA 表达水平；通过流式细胞术检测细胞表面PD-L1蛋白表达。从人外周血中提取淋巴细胞，体外刺激增殖。设置西达本胺单独处理淋巴细胞组、与西达本胺处理的 Jurkat 细胞共培养的淋巴细胞 组。通过荧光定量 PCR 检测各组淋巴细胞 CXCL3、CXCL9 的 RNA 表达水平，通过流式细胞术检测其CD8+T细胞的比例。研究结果: 1. 西达本胺上调 Jurkat 细胞系 PD-L1 的 RNA 表达，上调水平呈剂量依赖。 2. Jurkat 细胞系 PD-L1 阳性的细胞比例较低。低剂量西达本胺（0.5-1μmol/L）上 调 Jurkat 细胞系中 PD-L1 阳性细胞比例，高剂量西达本胺（2-5μmol/L）则无此 作用。3. 西达本胺上调 Jurkat细胞表面 PD-L1 蛋白的平均水平，不同剂量作用相近。4. 西达本胺上调 Jurkat 细胞系 JAK2、STAT1、STAT3 的 RNA 表达，高浓度组（5μmol/L 组）上调水平明显；同时导致 JAK/STAT 通路上游负调控基因 SOCS1、SOCS3 的 RNA 表达上调。5. 在 Jurkat 细胞系中，西达本胺可能通过上调 STAT1，上调 T 细胞趋化因子，增 强 PD-1 单抗疗效。6. 西达本胺上调 Jurkat 细胞系 CXCL9 的 RNA 表达，上调水平呈剂量依赖，可能具 有增强PD-1单抗疗效的作用。7. 西达本胺上调淋巴细胞CXCL9 的RNA表达，上调水平明显低于同浓度西达本胺处理的 Jurkat 细胞系。 8. 与西达本胺处理的Jurkat细胞共培养，可以上调淋巴细胞中CD8+T 细胞的比例， 可能机制为西达本胺通过上调 Jurkat 细胞 STAT1 的表达上调 CXCL9 表达，增加 环境中 CXCL9 的浓度，导致 CD8+T 细胞数量的增加。结论: 西达本胺调控 PD-1/PD-L1 免疫逃逸的可能机制：1. 西达本胺上调 Jurkat 细胞 系 PD-L1 的 RNA 表达，增加 PD-1 单抗的作用靶点。2. Jurkat 细胞系中，西达本胺 通过 STAT1 上调 CXCL9，增加环境中 CXCL9 的浓度，导致 CD8+T 细胞数量的增加，克服免疫逃逸，与PD-1单抗发挥协同作用。

Background and Objectives Peripheral T-cell lymphoma (PTCL) is a group of highly heterogeneous non-Hodgkin lymphoma originated from mature T cells. The incidence rate in China is obviously higher than western countries. The prognosis of T-cell lymphoma is significantly worse than B-cell lymphoma. The standard chemotherapy regimen is effective, but there are some problems such as short remission time and high recurrence rate. Therefore, further study on the pathogenesis of PTCL is of great significance to find new treatment methods and improve the prognosis of patients with PTCL. In clinical, PD-1/PD-L1 monoclonal antibody has a certain effect on tumor treatment, but there are low response rate and recurrence after effective treatment. At present, there are few clinical studies of PD-1/PD-L1 monoclonal antibody in the treatment of PTCL. Therefore, it is of great clinical significance to explore how to inhibit tumor immune escape mediated by PD-1/PD-L1 pathway. Chidamide, an HDAC inhibitor, regulates the antitumor effect of host immune system by epigenetic modification, which has certain effect on tumor, especially hematological malignancies. In clinical, Chidamide and PD-1 monoclonal antibody have synergistic effect on the treatment of lymphoma, suggesting that Chidamide may play a role in regulating the signaling pathway of PD-1/PD-L1 immune escape. From the perspective of epigenetics and immune checkpoint combination, this study aims to explore the regulatory mechanism of Chidamide on PD-1/PD-L1 immune escape and to provide a new theoretical basis for future clinical treatment. Methods Jurkat cells were treated with different concentrations of Chidamide. The mRNA expression levels of PD-L1, JAK/STAT pathway genes and chemokines CXCL3 and CXCL9 were detected by fluorescence quantitative PCR. Cell surface PD-L1 protein expression was detected by flow cytometry. Lymphocytes were extracted from human peripheral blood and stimulated to proliferate in vitro. Lymphocytes treated with Chidamide alone and lymphocytes co-cultured with Chidamide treated Jurkat cells were set. The RNA expression levels of CXCL3 and CXCL9 in lymphocytes of each group were detected by fluorescence quantitative PCR. The proportion of CD8+T cells was detected by flow cytometry. Results 1. Chidamide upregulated PD-L1 mRNA expression in Jurkat cell line in a dose- dependent manner.2. The proportion of PD-L1 positive cells in Jurkat cell line was low. Low dose of Chidamide (0.5-1μmol/L) increased the proportion of PD-L1 positive cells in Jurkat cell line, but high dose of Chidamide (2-5μmol/L) did not. 3. Chidamide upregulated the average level of PD-L1 protein on Jurkat cell surface. The effects of different doses were similar. 4. Chidamide upregulated the RNA expression of JAK2, STAT1 and STAT3 in Jurkat cell line. The level of up-regulation was obvious in high concentration group (5μmol/L group). At the same time, the mRNA expression of SOCS1 and SOCS3, the negative regulatory genes upstream of the JAK/STAT pathway, were up-regulated. 5. In Jurkat cell line, Chidamide may enhance the efficacy of PD-1 monoclonal antibody by upregulating STAT1 and T cell chemokines. 6. Chidamide upregulated CXCL9 RNA expression in Jurkat cell line in a dose-dependent manner, which may enhance the efficacy of PD-1 monoclonal antibody. 7. Chidamide upregulated CXCL9 mRNA expression in lymphocytes. The up-regulated level was significantly lower than that in Jurkat cell line treated with the same concentration of Chidamide. 8. Co-culture with Chidamide treated Jurkat cells upregulated the proportion of CD8+T cells in lymphocytes. The possible mechanism is that Chidamide can upregulate CXCL9 expression by upregulating STAT1 expression of Jurkat cells, leading to the increase of the CXCL9 concentration and the number of CD8+T cells in the environment. Conclusion Possible mechanisms of how Chidamide regulates PD-1/PD-L1 immune escape: 1.Chidamide upregulates the RNA expression of PD-L1 in Jurkat cells and increases the action target of PD-1 monoclonal antibody. 2. In Jurkat cell line, Chidamide upregulates CXCL9 through STAT1, increasing the CXCL9 concentration and the number of CD8+T cells in the environment, overcoming immune escape and playing a synergistic role with PD-1 monoclonal antibody.