【摘要】 目的 探讨结直肠癌(colorectal cancer, CRC)患者外周血滤泡辅助性T细胞(T follicular helper cell, Tfh)中程序性死亡受体-1(programmed death-1,PD-1)和细胞毒性T淋巴细胞相关蛋白4(cytotoxic T-lymphocyte-associated protein 4,CTLA4)的共表达模式及临床意义。方法 纳入2020年1月至2020年12月在我院治疗的75例CRC根治术后的患者，30例结直肠息肉病理证实为腺瘤性息肉患者作为病例对照组和30名健康体检者作为健康对照组，留取空腹外周血标本，提取外周血单个核细胞(peripheral blood mononuclear cells, PBMC),流式细胞术检测Tfh细胞上PD-1和CTLA4的表达水平，并评估其与CRC临床特征的关系。采用Spearman等级相关分析Tfh细胞上PD-1和CTLA4的共表达模式与TNM分期的相关性。采用二元Logistic回归分析影响CRC发病的危险因素。结果 CRC组中Tfh细胞、PD-1~+CTLA4~-Tfh、PD-1~-CTLA4~+Tfh和PD-1~+CTLA4~+Tfh细胞比例显著高于病例对照组和健康对照组(P<0.001),病例对照组与健康对照组上述细胞亚群的差异无统计学意义(P>0.05)。CRC患者PD-1~+CTLA4~-Tfh、PD-1~-CTLA4~+Tfh、PD-1~+CTLA4~+Tfh细胞比例在TNM分期、淋巴结转移、肝转移方面存在显著差异(P<0.05),Spearman等级相关分析结果显示，TNM分期Ⅰ～Ⅱ期患者与Ⅲ～Ⅳ期患者相比，Tfh细胞比例差异无统计学意义(P>0.05),而PD-1~+CTLA4~-Tfh、PD-1~-CTLA4~+Tfh和PD-1~+CTLA4~+Tfh细胞比例与TNM分期呈显著正相关(r=0.815、0.860、0.872,P<0.001)。二元Logistic回归模型分析显示，PD-1~+CTLA4~-Tfh、PD-1~-CTLA4~+Tfh和PD-1~+CTLA4~+Tfh的高表达均是CRC发病的危险因素，其中以PD-1~+CTLA4~+Tfh最具临床效能。结论 CRC患者外周血Tfh细胞上PD-1和CTLA4的高表达及共表达与疾病的发生发展密切相关，两者在Tfh细胞上具有协同抑制作用，共同促进了T细胞耗竭，导致CRC发生免疫逃逸，PD-1和CTLA4有望成为CRC新的治疗靶点。更多还原

【Abstract】 Objective To investigate the co-expression pattern and clinical significance of programmed death-1(PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) on peripheral blood T follicular helper(Tfh) cells in patients with colorectal cancer(CRC). Methods Seventy-five patients with CRC who underwent radical resection in our hospital from Jan. 2020 to Dec. 2020 were enrolled in this study. Thirty patients with colorectal polyps pathologically confirmed as adenomatous polyps(disease control group) and 30 healthy subjects(health control group) were enrolled. Fasting peripheral blood samples were collected to extract peripheral blood mononuclear cells(PBMC). The expression levels of PD-1 and CTLA4 on Tfh cells were detected by flow cytometry, and their relationship with the clinical characteristics of CRC was evaluated. Spearman rank correlation was used to analyze the correlation between the co-expression patterns of PD-1 and CTLA4 on Tfh cells and TNM stage. Binary Logistic regression was used to analyze the risk factors affecting the incidence of CRC. Results The proportions of Tfh cells, PD-1~+CTLA4~-Tfh, PD-1~-CTLA4~+Tfh, and PD-1~+CTLA4~+Tfh cells in CRC group were significantly higher than those in disease control group and health control group(P<0.001), and there was no significant difference in the above cell subsets between disease control group and health control group(P>0.05). The proportions of PD-1~+CTLA4~-Tfh, PD-1~-CTLA4~+Tfh, and PD-1~+CTLA4~+Tfh cells in CRC patients were significantly different in TNM stage, lymph node metastasis, and liver metastasis(P<0.05), and Spearman rank correlation analysis showed that there was no significant difference in the proportion of Tfh cells between TNM stage Ⅰ-Ⅱ patients and Ⅲ-Ⅳ patients(P>0.05), while the proportions of PD-1~+CTLA4~-Tfh, PD-1~-CTLA4~+Tfh, and PD-1~+CTLA4~+Tfh cells were significantly positively correlated with TNM stage(r=0.815, 0.860, 0.872, P<0.001). Binary Logistic regression model analysis showed that high expression of PD-1~+CTLA4~-Tfh, PD-1~-CTLA4~+Tfh, and PD-1~+CTLA4~+Tfh were all risk factors for CRC, with PD-1~+CTLA4~+Tfh being the most clinically effective. Conclusion The high expression and co-expression of PD-1 and CTLA4 on peripheral blood Tfh cells in patients with CRC are closely related to the occurrence and development of the disease. Both of them have a synergistic inhibitory effect on Tfh cells, which promotes the depletion of T cells and leads to immune escape in CRC. PD-1 and CTLA4 are expected to be new therapeutic targets for CRC.更多还原