Correlation Between Expression Of Programmed Cell Death Receptor-1 By T Cells And Sepsis In Elderly Patients

Backgroud and ObjectiveSepsis is a life-threatening organ dysfunctional disease caused by dysregulated host responses to severe infection.This disease displays high morbidity and elevated mortality in the elderly.Immunosuppression has recently been demonstrated to be a key element causing the death of patients with sepsis.Cell exhaustion plays an important role among various mechanisms facilitating immunosuppression.T cell exhaustion possesses the typical features of an immunosuppression mechanism,which is characterized by the upregulation of negative immune checkpoint molecules such as programmed death protein-1(PD-1)and programmed death-ligand 1(PD-L1).Patients with sepsis have shown similarly selective upregulation of negative immune checkpoint expression to those suffering malignant tumors.As the currently targeted drugs in clinical trials can block the PD-1/PD-L1 pathway,delay tumor progression,and extend survival time,it has triggered great interest in studying the PD-1/PD-L1 pathway in sepsis.High expression of PD-1 in CD4+T cells and/or CD8+T cells has been observed in both patients with sepsis and animal models.Targeted interference of the PD-1/PD-L1 pathway can enhance the function of innate and adaptive immune cells,decrease the apoptosis of lymphocytes,and thus reduce the mortality rate in sepsis models.However,little information is known about the timing of targeted interference and the relationship of PD-1 expression among T cell subpopulations,including CD4+T cells,CD8+T cells,and regulatory T cells(Tregs).The effect of PD-1 on CD4+T cells and inhibitory Tregs in the establishment of the immunosuppressive network is also unknown.The PD-1 gene is named for its activation and involvement in a typical programmed cell death process.It is potentially linked to programmed cell death,apoptosis.Therefore,the purposes of this study include the following:1.Exploring the expression patterns of PD-1 among T cells along with subpopulation CD4+T cells,CD8+T cells,and Tregs in the acute phase of sepsis in elderly patients;determining the relationship between PD-1 expression,apoptosis and prognosis;investigating the expression correlation among three T cell subpopulations2.Determining the impact of PD-1 expression on the proliferation of CD4+T cells and the secretory function of Tregs in elderly patients with sepsis3.Uncovering the correlation of PD-1 expression and ratio changes in T cells along with subpopulation CD4+T cells,CD8+T cells,and Tregs in SD rats with severe sepsis,the apoptosis of lymphocytes/T cells,and the severity and prognosis of sepsis.Methods1.Participants(?65 years of age)enrolled in this study were divided into three groups.The number of patients in sepsis group(S Group),mild to moderate community acquired pneumonia group(CAP Group),and health group(H Group)was 22,19,and 25,respectively.The specimens of the first day included peripheral blood samples from S Group and CAP Group within 24 hours of diagnosis and peripheral blood samples from H Group on the sampling day.Flow cytometry was utilized to compare the levels of PD-1 expression and apoptosis rates on the first day in T cells with subpopulation CD4+T cells,CD8+T cells,and Tregs from all three different groups.Peripheral blood from S Group on the 3th,5th,7th,and 10th days was sampled at 24-hour intervals.The PD-1 expression patterns in CD4+T cells,CD8+T cells,and Tregs within 10 days were analyzed using a last observation carried forward analysis(LOCF).And apoptotic rate on the first day and apoptotic rate at the last observation point were also analyzed by LOCF.2.The clinical data related to infection and severity of the disease,including first lymphocyte count,SOFA score,APACHE ? score,Prognosis within 60 days and last lymphocyte count in sepsis group before discharge,were collected.Further analysis was conducted to determine the correlation of PD-1 expression among Tregs,CD4+T cells,and CD8+T cells from S Group and the correlation of PD-1 expression with the severity and prognosis of sepsis.3.The difference of SOFA score and APACHEE ? score in different prognostic groups of sepsis group,the change of first lymphocyte number and last lymphocyte number before discharge,and the correlation between last lymphocyte number before discharge and SOFA score and APACHEE ? score were compared.4.In vitro cultivated and stimulated Tregs from S Group were sorted by magnetic beads.Those cells were further divided into control,stimulation,and interference groups.Next,2?M of monension,0.081 ?M of PMA,1.338 ?M of Ionomycin and 167?g/ml of PD-1 antibody were supplied to the interference group.The stimulation group was treated with the same reagents except for the PD-1 antibody,while the control group only contained the cell suspension without any addition.All cells were cultivated for 6 hours before the detection of IL-10 expression level changes in Tregs by flow cytometry.5.In vitro-stimulated PMBCs from S Group and H Group were divided into control,stimulation,and interference groups.Next,10 ?g/ml of phytohemagglutinin(PHA)and 167?g/ml of PD-1 antibody were added to the interference group.The stimulation group was treated with the same amount of PHA,while the control group contained the cell suspension without any addition.All three groups of cells were cultivated for 48 hours before the analysis of the average fluorescence intensity changes in CD4+T cells.6.The cecal ligation puncture procedure was conducted with rats in S Group to generate the D rat model with severe sepsis.To establish a self-control study,the tail vein blood samples were withdrawn before modeling,6 hours/24 hours/48 hours/7 days after modeling,and after the death of rats.The PD-1 expression patterns in T cells with subpopulation CD4+T cells,CD8+T cells,and Tregs and the changes in the apoptosis of T cells and lymphocytes were compared before and after modeling.The changes in the proportion of T cells and their related subpopulations in the survival group(survived more than 7 days)and the death group(survived less than 7 days)were also monitored.Results1.The CD4+T cells,CD8+T cells,and Tregs from S Group with the acute phase of sepsis showed continuously high PD-1 expression levels.The changes in the expression rates at five time points within 10 days displayed no statistical differences.The PD-1 expression among Tregs,CD4+T cells,and CD8+ T cells was positively correlated.The levels of PD-1 expression in T cells,Tregs,and CD4+T cells from S Group were higher than those from CAP Group and H Group.No significant difference was observed for the PD-1 expression in T cells H and their subpopulations between CAP Group and H Group.2.CD4+T cells,CD8+T,and Tregs in the survival group and the death group within S Group showed no significant difference in the levels of PD-1 expression.The PD-1 expression in Tregs from S Group and CAP Group was positively correlated to the SOFA score and the APACHE ? score..The PD-1 expression in CD8+T cells and CD4+T cells from S Group were not correlated to either the SOFA score or the APACHE ? score.3.The apoptotic rates of lymphocytes and T cells and their subsets were significantly higher in S Group and CAP Group than in health examination group.There was no significant difference in apoptotic rate of CD4+T cells and Treg cells between S Group and CAP Group,but the apoptotic rate of lymphocytes,T cells and CD8+T cells in CAP group was higher than that in S Group.4.No significant difference in the number of lymphocytes between the survival group and the death group from S Group was observed on the first day.Before patients discharge from micu,the number of lymphocytes in the death group was lower than that in the survival group.Before patients discharge from micu,the number of the last lymphocytes in the death group displayed a decreasing trend compared to the first day;however,this was not statistically significant.The number of the last lymphocytes in the survival group before patients discharge from micu was increased significantly compared to the first day.5.The SOFA score and APACHE ? score in the sepsis death group were statistically significantly higher than those in the survival group.The SOFA score and the APACHE ? score from S Group were negatively correlated with the number of lymphocytes measured before patients left the hospital.6.After the in vitro interference of PD-1 expression in Tregs from S Group,IL-10 expression levels in both the interference group and control group declined compared to the stimulation group,while the difference between the interference group and the control was not statistically significant.7.An increased proliferation rate was detected for the PBMCs in the surviving patients from S Group after the stimulation.The average fluorescence intensity of CD4+T cells showed a decreasing trend in the stimulation group compared to the control group.The intensity values in the interference group and stimulation group displayed no statistical significance.CD4+T cells died upon stimulation of PBMCs in patients dying shortly after the experiment.The proliferation of CD4+T cells in the interference group and the stimulation group was higher than that in the control group within the H Group.The proliferation of CD4+T cells in the interference group was higher than that in the stimulation group,while the average fluorescence intensity of CD4+T cells declined.8.The levels of PD-1 expression in T cells along with their subpopulations and the apoptosis of lymphocytes and T cells in S Group of D rats suffering severe sepsis increased compared to the values before the sepsis rat model establishment.Before the death of the rats,the proportion of T cells with their subpopulations in the death S Groupignificantly decreased at the last observation point compared to the observations before the sepsis model establishment.The proportion of T cells with their subpopulations on the 7th day and the PD-1 expression in the survival S Grouphowed no significant differences upon the establishment of the sepsis model.The apoptosis of lymphocytes and T cells in the surviving group first increased and then decreased.On the 7th day,the apoptosis of lymphocytes and T cells displayed no significant difference before and after sepsis model establishment.ConclusionT cells,CD4+T cells,and Tregs showed consistently high PD-1 expression levels in elderly patients with acute sepsis.The interference of PD-1 expression in Tregs reduced the secretion of inhibitory cytokines,but could not reverse the limited proliferation or death of CD4+T cells.The persistently high levels of PD-1 expression in T cells were accompanied by a consistent increase of apoptosis in lymphocytes and T cells.It might explain the decline in circulating T cells along with their subpopulations and the decreased number of lymphocytes,which are associated with the poor prognosis.Therefore,PD-1 overexpression on Treg and persistent decrease of lymphocyte number and T cell subsets ratio can be used as feasible markers for indicating severe infection and predicting death,respectively.