REGULATIONS OF BCL6 EXPRESSION BY NOTCH SIGNALING IN HUMAN FOLLICULAR HELPER T CELL

Abstract

The highly conserved Notch signaling pathway operates in multi-cellular organisms. It plays important roles in gene expression regulation that controls many cellular processes, including differentiation, proliferation and activation of T helper cells. Follicular helper T cell (TFH), identified as CXCR5hiICOShi CD4+ subset T cell, is one of the CD4+ T helper cells which express BCL6 as a critical transcription regulator. This cell subset mainly localizes in the germinal center (GC) of secondary lymphoid tissues such as tonsils, lymph nodes and spleen. Their functions are to help maintain the formation of GC, and to regulate differentiation of B cells and production of antibodies. Previous study reported the unique transcriptomic patterns of genes related to the Notch signaling pathway in human TFH. Moreover, in mice with Notch1/Notch2 double-deficient CD4+ T cells immunized with Schistosoma mansoni eggs, numbers of TFH decreased and TFH development was impaired. These previous studies suggesting a role of Notch signaling in differentiation and/or functions of TFH. However, how Notch signaling regulates TFH differentiation was not well documented. Therefore, this study proposed to investigate the involvement of Notch signaling pathway in the regulations of BCL6 expression in human TFH. TFH from freshly dissected tonsils were sorted using CD4+CXCR5hiICOShi as marker. The expression of BCL6 and IL21 mRNA were confirmed by quantitative PCR (qPCR) to be higher in these sorted cell population. Moreover, cleaved Notch1 was found in freshly isolated TFH. The expression profiles of mRNA of Notch receptors and their ligands were higher in freshly isolated TFH, compared with nonTFH (CD4+CXCR5loICOSlo). However, expression of Notch receptors were down-regulated after in vitro stimulation using anti-CD3/CD28 antibodies, possibly by negative feedback of IL-2. Promoter analysis of BCL6 gene identified a putative RBP-Jϰ recognition site at -849 bp upstream of transcription starting site. Therefore, the direct association of Notch1 on the BCL6 promoter of TFH was investigated by Chromatin immunoprecipitation (ChIP) using anti-Notch1 antibody. ChIP analysis revealed specific association of Notch1 with BCL6 promoter at position 849 base pairs upstream of transcription starting site. Taken together, this result reveals a critical role of Notch signaling in direct regulation of BCL6 expression.