CD4(+)T Cell Subset Profiling in Biliary Atresia Reveals ICOS(−) Regulatory T Cells as a Favorable Prognostic Factor

Biliary atresia (BA) is a destructive pediatric liver disease and CD4(+)T cell activation is demonstrated to play an important role in BA. However, a comprehensive scenario regarding the involvement of CD4(+)T cell subsets to the development of BA remains unclear. Here, we aim to explore the infiltration of CD4(+)T cell subsets and their clinical significance in BA. In the present study, thirty BA liver samples were collected during surgery and were divided into good (BA1, n = 16) and poor prognosis (BA2, n = 14), with samples from choledochal cyst patients (n = 8) as control. By using multiplex immunohistochemistry, we evaluated the infiltration level of CD4(+)T cell subsets in the portal areas. RT-qPCR and flow cytometry were further applied to explore detailed features of Treg subsets. We revealed that hepatic infiltrating Th1, Th2, Th17, and ICOS(+)Treg cells were significantly increased in BA patients compared to controls and were negatively associated with prognosis, while high infiltrating ICOS(−)Tregs showed a favorable outcome. Phenotypic analysis indicated that, in contrast to ICOS(+)Tregs, ICOS(−)Tregs were mainly CD45RA(hi)CD45RO(low), and preferentially expressed more CD73. Besides, RT-qPCR revealed elevated expression of CD25, CD73, TGF-β, and BCL-2 genes in ICOS(−)Tregs. Finally, functional assay confirmed that ICOS(−)Tregs had a higher suppressive capacity to cytokine secretion and were more resistant to apoptosis in vitro. Collectively, we demonstrate that a mixed immune response is involved in BA pathogenesis, and the globally enhanced effector CD4(+)T cell response is associated with unfavorable prognosis, highly suppressive ICOS(−)Tregs is a protective factor and may serve an important reference to predict prognosis.