Optimal CXCR5 Expression during Tfh Maturation Involves Bhlhe40-Pou2af1 Axis

Bcl6/Blimp1 are well-known for Tfh cell determination, but they are dispensable in regulating the expression of CXCR5, which is critical for Tfh migration into germinal center (GC). In this study, we uncovered a novel pair of transcription factors, Bhlhe40/Pou2af1, that regulate Tfh maturation through regulation of CXCR5. Pou2af1 promotes Tfh generation by upregulating CXCR5, while Bhlhe40 represses Tfh differentiation by inhibiting Pou2af1. Pou2af1 was further verified being essential for GC Tfh cell localization, whereas Bhlhe40 inhibits pre-Tfh cells import into GC. Such migration behavior change can be explained by an alteration in the MFI of CXCR5. RNA-Seq analysis confirmed Bhlhe40 inhibiting Pou2af1 and the requirement of Pou2af1 upregulation in optimal CXCR5 expression. Thus, our study filled a long-standing gap in the regulation of optimal CXCR5 expression by revealing a key transcriptional regulatory circuit involving Bhlhe40/Pou2af1, which operates downstream of the Bcl6/Blimp-1 circuit that determines Tfh cell fate. RNA-Seq analysis of gene expression in T cell subsets (Th1, Th2, Th17 and iTreg), which are primed and differentiated in vitro from WT mice or Bhlhe40f/f-CD4Cre mice. RNA-Seq analysis of gene expression in antigen-specific Tfh cells and non-Tfh cells, which were sorted from the indicated bone marrow chimera mice (WT, Pou2af1-/-, Bhlhe40-/-). ChIP-Seq analysis of binding sites by Bhlhe40 in Th1, Th2, Th17 and iTreg. ChIP-Seq analysis of binding sites by Pou2af1 in splenic B cells. Overall design: RNA-Seq analysis of gene expression in T cell subsets (Th1, Th2, Th17 and iTreg), which are primed and differentiated in vitro from WT mice or Bhlhe40f/f-CD4Cre mice. RNA-Seq analysis of gene expression in antigen-specific Tfh cells and non-Tfh cells, which were sorted from the indicated bone marrow chimera mice (WT, Pou2af1-/-, Bhlhe40-/-). ChIP-Seq analysis of binding sites by Bhlhe40 in Th1, Th2, Th17 and iTreg, which are primed and differentiated in vitro from Bhlhe40-V5 tagged mice . ChIP-Seq analysis of binding sites by Pou2af1 in splenic B cells from WT or Pou2af1-HA-TG mice. AS15-specific cells from draining lymph nodes of two weeks AS15/CFA-immunized mice were identified by tetramer staining. All the cell subsets were harvested by cell sorting after staining.