The Role Of T Cell Immune Checkpoint CD226/TIGIT In The Pathogenesis Of Primary Biliary Cholangitis

Objective Primary biliary cholangitis(PBC)is a kind of autoimmune liver disease characterized by non-suppurative injury of middle and small bile ducts.According to published studies,the overactive T cells are considered to play an important role in the pathogenesis of PBC,but the detail mechanism has not been elucidated.In recent years,immune checkpoints have attracted much attention as its intrinsic ability to control the activity of lymphocytes.The novel immune checkpoint of T cells,CD226/TIGIT is reported to be closely related with the occurrence and development of autoimmune diseases,and is expected to be a potential therapeutic target of these diseases.This is the first study to explore the role of CD226/TIGIT in the pathogenesis of PBC.Methods A total of 42 PBC patients and 55 controls containing disease control(DC)and health control(HC)were enrolled.The phenotype of peripheral lymphocytes and their effector function,immunoassay of involved tissue,the levels of soluble immune checkpoint molecules,intervene the immune checkpoint in vitro,and their clinical association were analyzed by flow cytometry and cell sorting,immunohistochemistry,cell culture in vitro,real-time PCR and statistical analysis etc.,so as to establish the role that the immune checkpoint CD226/TIGIT plays in PBC and its probable molecular mechanism.Results Significant proportional alteration of CD226 and TIGIT from the peripheral CD8+T cells and CD4+T cells of PBC patients could be observed:the percentages of CD8+CD226+T cells,CD8+TIGIT+T cells,CD4+CD226+T cells and CD4+TIGIT+T cells in PBC were remarkably higher than those of HC(71.8 ± 12.0 vs.52.0± 14.1,p<0.001;60.0±15.60vs.41.7± 12.9,p<0.001;63.0±13.3 vs.50.1±11.7,p<0.001;31.5±8.7 vs.26.2 ± 7.1,p=0.032).Comparisons between PBC and DC also presented similar results,while no statistical difference between DC and HC was found.In the correlation analysis,the frequency of CD8+ T cells expressing CD226/TIGIT have close clinical associations with biochemical indices:in PBC group,the proportion of CD8+TIGIT+T cells was negatively correlated with serum levels of alkaline phosphatase(r=-0.39,p=0.01),gamma glutamyl transpeptidase(r=-0.35,p=0.02),total bilirubin(r=-0.38,p=0.01),direct bilirubin(r=-0.43,p<0.01)and total bile acid(r=-0.35,p=0.03),and positively associated with platelet count(r=0.38,p=0.03).The frequency of CD8+CD226+T cells was positively correlated with alkaline phosphatase(r=0.38,p=0.03).Of note,the CD226/TIGIT ratio of CD8+T cells in PBC patients was positively associated with alkaline phosphatase(r=0.42,p<0.01),gamma glutamyl transpeptidase(r=0.31,p=0.04),total bilirubin(r=0.31,p=0.04),direct bilirubin(r=0.35,p=0.02)and total bile acid(r=0.47,p<0.01),and negatively correlated with platelet count(r=-0.34,p=0.04).Moreover,this ratio was positively associated with the prognosis marker,the ratio of aspartate aminotransferase to platelet count(r=0.35,p=0.04)and negatively correlated with the level of serum albumin(r=-0.40,p=0.02).In the model established by inter-group difference analysis,the phenotype data of CD226/TIGIT showed good accuracy in distinguishing PBC and DC(80%-89%),and also PBC and HC(86-92%).In this model,CD8+T cells related data was the most important features when distinguishing PBC from others.Further experiments show that the effector function of CD226+T cells was more robust than the CD226-T subsets:higher percentages of CD107a+(77.5±7.8 vs.67.8±9.8,p<0.001),IFN-?+(71.3 ± 13.9 vs.55.6±15.7,p=0.04),and TNF-?+(68.0± 10.7 vs.56.9 ± 14.4,p<0.001)cells were observed among CD226+T cells comparing with the CD226-counterparts.Regarding the TIGIT+T cells,they may be the product of feedback regulation owing to the abnormal activation of the immune system:the frequency of TIGIT+cells in HLA-DR+cells was significantly higher than that of HLA-DR-cells(63.2± 17.4 vs.42.6 ± 14.0,p<0.01);TIGIT+T cells could be generated from TIGIT-T cells after 72 hours of stimulation.the imbalance of CD226/TIGIT was also found by pathological analysis in the involve tissues:the liver slices from PBC showed no TIGIT staining cells,but a large number of CD226 expressing lymphocytes and positive staining of CD155.The positive rates of soluble CD226 and CD155 were low and no significant difference was found among groups.Blocking CD226 in vitro resulted in significantly decreased frequencies of CD 107a+(81.1 ± 5.1 vs.72.7±7.8,p=0.032),IFN-y+(69.2±6.7 vs.52.9 ± 15.6,p=0.024),and TNF-?+(76.4 ± 5.9 vs.58.6±14.7,p=0.021)cells among CD8+CD226+T cells from PBC patients.The addition of the ligand of CD226/TIGIT,recombinant CD 155 had no significant effect on the function of CD8+TIGIT+T cells in vitro.Conclusion The imbalance of CD226/TIGIT immune checkpoint may involve in the pathogenesis of PBC.Future studies should be carried out in both a more comprehensive cohort and animal model,so as to fully clarify the role that CD226/TIGIT immune checkpoint plays in the occurrence and development of PBC and to further evaluate its potential value for clinical application.