Research On Cd4~+Cd25~+Foxp3~+Regulatery T Cells And Cd45Subsets In Patients Of Different Immune State Post-liver Transplantation

Objectives:The purpose of this research is investigate the expression, changes and influencing factors of CD4+CD25+Foxp3+regulatory T cells (Treg) and their CD45subsets of liver transplant patients in different immune status and then the role of CD4+CD25+Foxp3+Tregs in immune tolerance in liver transplantation.Experimental Design:The changes of CD4+CD25+Foxp3+Tregs and their CD45subsets in the peripheral blood of liver transplant patients were investigated. Immune status of patients who received liver transplantation was assessed by CD4+T lymphocyte ATP concentration in peripheral blood,and then Treg expression was investigated at different immune status according to different CD4+T lymphocyte ATP level in liver transplant patients.Methods:Part Ⅰ:A total91cases of patients with more than6months post liver transplant were randomly selected as post liver transplantation (LT) group and patients’clinical status were stable at the time of selection. A total23cases of patients who were diagnosed as end-stage liver disease were selected as surgery control group and all of them received liver transplantation in the observation period. Blood samples in surgery control group were collected at two weeks pre-LT and1st month,3rd month and6th month post-LT respectively. Following staining of specimens by fluorescent antibodies, CD4+CD25+Foxp3+Tregs and the expression of CD45RA, CD45RO and CD31were detected by flow cytometry. Part Ⅱ: A total189cases of patients post-LT were randomly selected in this research and CD4+T lymphocyte ATP concentration was evaluated by ImmuKnow. The effect of gender, age, postoperative time, rejection, infection and other factors on CD4+T lymphocyte ATP level was investigated. Based on CD4+T lymphocyte ATP levels, patients were divided into into low immune response group (<225ng/mL), normal immune response group (225-525ng/mL) and high immune response group (>525ng/mL), and then the incidences of rejection and infection were calculated and evaluated respectively. Part Ⅲ:Patients with more than six months after LT and are receiving tacrolimus (FK506) therapy were enrolled in the study. Patients were divided into infection group, rejection group and stable group according to their clinical status. Then the difference in CD4+CD25+Foxp3+Tregs proportions and total numbers were analyzed among these groups. These factors were also analyzed among groups that were divided according to different CD4+T lymphocytes ATP levels.Results:Part Ⅰ:The number of CD4+CD25+Foxp3+Tregs in patients post-LT was significantly lower than that of the healthy controls (2.79×107/L±2.02×107/L VS4.29×107/L±2.40×107/L, P=0.005). The types and concentrations of immunosuppressants could impact CD4+CD25+Foxp3+Tregs significantly. The proportion of CD4+CD25+Foxp3+Tregs in rapamycin (Rapa) group was significantly higher than that in FK506group (10.36%±2.52%VS6.97%±3.28%, P=0.004) and healthy controls (6.66%±2.41%, P=0.005). According to the FK506concentration at7.0ng/mL, patients could be divided into high concentration group and low concentration one. The proportion of Tregs in low concentration group (7.56%±3.31%) was significantly higher than that in the high concentration group (4.84%±2.11%, P=0.001). The proportion of CD4+CD25+Foxp3+Tregs in liver transplant recipients gradually decreased (P=0.003), whichwas higher at pre-LT (10.99%±5.49%) than that at1month (7.59%±4.20%),3months (6.26%±2.98%) and6months (5.65%±2.97%) post-LT and was significantly higher than the healthy controls (6.66%±2.41%)(P<0.05, respectively). The proportion of CD45RO+Treg in CD4+CD25+Foxp3+Tregs was significantly higher than CD45RA+Treg (71.77%±14.66%VS14.64%±11.08%, P=0.000). However, there was no difference between the proportions of CD31in these two types of cells (P=0.517) which could be expressed by both activated and naive Tregs. The proportion of Foxp3expression in CD45RA+Tregs was77.04%±20.69%, which was higher than that in CD45RO+Tregs (70.77%±19.08%, P=0.002). Importantly, the proportion of CD45RO+Treg was positively related with patients’age (r=0.526,P=0.000), however, the proportion of CD45RA+Treg was negatively correlated (r=-0.325, P=0.003). Part Ⅱ:CD4+T lymphocyte ATP levels were tested in a total of290cases of peripheral blood from189patients. The incidences of rejection were0.9%,3.4%and15.8%in low, normal and the high immune response group, respectively. There was difference among these groups (P=0.021). The incidences of infection were20.7%,13.7%and5.3%respectively, and no significant difference was detected(P=0.000). Part Ⅲ:The proportions of CD4+CD25+Foxp3+Tregs were different in different clinical status (P=0.010). The proportion in rejection group (4.38%±1.22%) was lower than that in the stable group (6.97%±3.28%, P=0.029) and infection (9.16%±4.12%, P=0.001) respectively. However, there was no difference compared with that in the healthy control group (6.66%±2.41%, P=0.088). The proportion in infection group was higher than that in the rejection group, stable group and healthy controls (P=0.001, P=0.022and P=0.028, respectively). Regardless of the proportions and absolute numbers of CD4+CD25+Foxp3+Tregs in peripheral blood, there was no difference among the low group, the normal and the high immune response group (P=0.355and P=0.342). There was no linear correlation between the proportion of CD4+CD25+Foxp3+Tregs and CD4+T lymphocytes ATP value (r=-0.130, P=0.188).Conclusions:(1) The proportion of CD4+CD25+Foxp3+Tregs pre-LT was higher than that of healthy controls and showed a decreasing trend at post-LT. The effect of immunosuppressants on Treg proportions was different that Rapa could promote an increase in Treg proportion, whereas FK506could inhibit Treg proliferation. In addition, Treg proportions were related to FK506concentration. The proportion of CD45RO+Treg (the activated Treg) was higher than CD45RA+Treg (the naive Treg) in all patients who received transplantation. The proportion of CD45RO+Treg was positively related with patients’age, whereas CD45RA+Treg was negatively correlated. However, the proportion of Foxp3expression was higher in CD45RA+Tregs than that in CD45RO+Treg.(2) CD4+T lymphocyte ATP level in peripheral blood could accurately reflect the immune status of patients post-LT. The incidence of infection is significantly higher in low immune response groups, whereas the incidence of rejection is significantly higher in high immune response group.(3) The differences in the proportion of CD4+CD25+Foxp3+T cells were significant in different clinical status. The ratio declined in the rejection group and increased in the infection group. But the proportion or number of CD4+CD25+Foxp3+T cells were not different within immune response groups divided by CD4+T lymphocyte ATP value. In addition, there were no linear relationships between the ratio or number of CD4+CD25+Foxp3+Tregs and CD4+T lymphocyte ATP.