Activation of Toll-like receptor 7 enhances cytotoxicity of CD8+T cells in septic patients
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.
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