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目的:观察脓毒症患者外周血Toll样受体（Toll-like receptor, TLR）表达谱变化，分析活化TLR7对脓毒症患者CD8+T细胞杀伤活性的影响。方法:采用横断面研究方法，纳入2020年6月至2021年6月在新乡医学院第一附属医院就诊的脓毒症患者和对照者，于就诊本院后1 h内采血，分离外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），逆转录实时定量PCR法检测PBMCs中TLR1~10 mRNA，流式细胞术检测CD8 +T细胞中TLR7和TLR9，分析TLR对预后的诊断价值。分选CD8 +T细胞，使用TLR7激动剂CL097刺激CD8 +T细胞，CD8 +T细胞与人脐静脉内皮细胞直接接触或间接接触共培养，检测免疫检查点受体表达、靶细胞死亡比例、毒性分子和细胞因子分泌。组间比较采用 t检验或Mann-Whitney检验，刺激前后比较采用配对 t检验或配对Wilcoxon秩和检验。 结果:纳入58例脓毒症患者和24例对照者。PBMCs中TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR8和TLR10 mRNA在脓毒症患者和对照者之间的差异无统计学意义（均 P>0.05）。脓毒症患者PBMCs中TLR9 mRNA低于对照者[0.94(0.81, 1.15) vs. 1.14(0.82, 2.12)， P=0.046]，但CD8 +T细胞中TLR9平均荧光强度与对照者差异无统计学意义[(23.83±7.44) vs. (24.95±7.97)， P=0.548]。脓毒症患者PBMCs中TLR7 mRNA[1.01 (0.88, 1.15) vs. 1.61(1.04, 2.63)， P<0.001]和CD8 +T细胞中TLR7平均荧光强度[(130.0±45.07) vs. (250.9±79.09)， P<0.001）均低于对照者。对脓毒症患者存活组和死亡组TLR7水平进行ROC曲线分析，TLR7 mRNA和TLR7 MFI曲线下面积分别为0.70和0.73（均 P<0.05），预测价值较好。脓毒症患者CD8 +T细胞中免疫检查点受体mRNA表达升高、直接接触杀伤功能及分泌毒性分子、细胞因子水平降低（均 P<0.05）。在直接接触共培养体系中，CL097刺激可增加脓毒症患者CD8 +T细胞诱导靶细胞死亡比例[(11.92±2.81)% vs. (9.28±2.03)%， P=0.008]，促进毒性分子和细胞因子分泌。在间接接触共培养体系中，CD8 +T细胞诱导靶细胞死亡比例和毒性分子分泌在经CL097刺激和无CL097刺激之间的差异无统计学意义。 结论:TLR7水平降低对脓毒症患者预后不佳有提示意义。脓毒症患者存在CD8+T细胞功能衰竭，TLR7激动剂可增强脓毒症患者CD8+T细胞杀伤功能。

Objective:To investigate the changes of Toll-like receptor (TLR) expression profile in peripheral blood of septic patients, and to analyze the influence of TLR7 activation to CD8 + T cells from septic patients. Methods:A cross-sectional study was conducted. Septic patients and controls admitted to the First Hospital of Xinxiang Medical University between June 2020 and June 2021 were enrolled in this study. Peripheral blood samples were collected within 1 h after admission. Peripheral blood mononuclear cells (PBMCs) were isolated. TLR1~10 mRNA expressions were semi-quantified by reverse transcription real-time PCR. TLR7 and TLR9 expressions in CD8 + T cells were measured by flow cytometry. The prognosis values of TLRs were analyzed. CD8 + T cells were purified, and stimulated with TLR7 agonist CL097. CD8 + T cells were co-cultured with human umbilical vein endothelial cells in direct contact or indirect contact manner. Immune checkpoint inhibitor expressions, target cell death percentage, cytotoxic molecules and cytokines secretions were assessed. Student’s t test or Mann-Whitney test was used for comparison between groups. Paired t test or Wilcoxon matched pair test was used for comparison between with and without stimulation. Results:Fifty-eight septic patients and twenty-four controls were included. There were no significant differences of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8, and TLR10 mRNA expressions in PBMCs between septic patients and controls (all P>0.05). TLR9 mRNA expression in PBMCs was notably down-regulated in septic patients compared with controls [0.94(0.81, 1.15) vs. 1.14(0.82, 2.12), P=0.046]. However, there was no remarkable difference of TLR9 mean fluorescence intensity (MFI) in CD8 + T cells between septic patients and controls [(23.83±7.44) vs. (24.95±7.97), P=0.548]. Both TLR7 mRNA expression in PBMCs [1.01 (0.88, 1.15) vs. 1.61(1.04, 2.63), P<0.001] and TLR7 MFI in CD8 + T cells [(130.0±45.07) vs. (250.9±79.09), P<0.001] were significantly reduced in septic patients compared with controls. TLR7 mRNA expression and MFI in the survival and death group was analyzed by receiver operating characteristic curve with area under curve of 0.70 and 0.73 (both P<0.05), indicating better prognosis value. CD8 + T cells from septic patients revealed the up-regulation of immune checkpoint inhibitor mRNA expressions, down-regulation of direct CD8 + T cell-induced target cell death, and reduced cytotoxic molecules and cytokines secretions (all P<0.05). In direct contact co-culture manner, CL097 stimulation enhanced CD8 + T cell-induced target cell death [(11.92±2.81)% vs. (9.28±2.03)%, P=0.008], and promoted cytotoxic molecules and cytokines secretion in septic patients. In indirect contact co-culture manner, there were no significant differences of either CD8 + T cell-induced target cell death percentage or cytotoxic molecules secretions between CD8 + T cells with and without CL097 stimulation. Conclusions:Down-regulation of TLR7 was an indicator for poor prognosis of septic patients. CD8 + T cells exhaustion was found in septic patients. TLR7 agonist enhanced the cytotoxicity of CD8 + T cells in septic patients.