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

Abstract

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, <i>n</i> = 16) and poor prognosis (BA2, <i>n</i> = 14), with samples from choledochal cyst patients (<i>n</i> = 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^hiCD45RO^low, and preferentially expressed more CD73. Besides, RT-qPCR revealed elevated expression of <i>CD25, CD73, TGF-</i>β, and <i>BCL-2</i> 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 <i>in vitro</i>. 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.