E-box binding proteins (E-proteins) have recently been demonstrated to regulate follicular helper T (TFH) cell differentiation, but the mechanisms underlying E-protein-mediated TFH differentiation remain elusive. To evaluate T cell-intrinsic effects of E-proteins, we generated OT-II TCR-transgenic mice with peripheral T cell-specific deletion of either E2A (E2AcKO) or HEB (HEBcKO). Naïve CD4+ OT-II T cells from mutant or control mice were transferred into congenic C57BL/6 mice, and the recipients were immunized with OVA in alum. Seven days after immunization, E2AcKO donor cells differentiated into pre-TFH and germinal center (GC) TFH cells comparably to control cells, whereas HEBcKO donor cells underwent pre-TFH cell differentiation but failed to mature into GC TFH cells. HEBcKO pre-TFH cells exhibited normal TFH signature gene expression but a substantial increase in the Gpr183 gene encoding Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2), which recognizes 7α,25-dihydroxycholesterol secreted by stromal cells in the outer T cell zone that affects T cell localization at the T/B border. EBI2 expression on HEBcKO donor cells was induced normally at day 2 and downregulated at day 4 post-immunization. However, at day 5 post-immunization and later, HEBcKO pre-TFH cells re-expressed high levels of EBI2, and accumulated at the T/B border. The failure of HEBcKO pre-TFH cells to enter B cell follicles and undergo subsequent GC TFH maturation was completely rescued by further conditional deletion of Eomes, which was highly expressed in HEBcKO donor cells during the first 2 days of priming. Our results suggest that HEB fine-tunes the localization of pre-TFH cells by preventing EBI2 re-expression through the suppression of Eomes, allowing for their entry to the follicles to promote subsequent maturation into GC TFH cells.
This work was supported by the Intramural Research Program of NIAID/NIH.