Heterogeneity of phenotype and function reflects the multistage development of t follicular helper cells

T follicular helper cells (Tfh) provide crucial signals for germinal center (GC) formation, but Tfh populations are heterogeneous. While PD1^hi Tfh are important in the GC response, the function of the PD1^lo Tfh-like subset is unknown. We show that these cells, like the PD1^hi GC-Tfh, depend upon B cells; however, their entry to follicles is independent of CXCR5 or cognate interactions with B cells. The differentiation into PD1^hi Tfh is dependent on MHC class II interactions with B cells and requires CXCR5. Our data suggest a Tfh differentiation pathway that is initially B cell-independent, then dependent on non-cognate B cell interactions, and finally following cognate interaction with B cells and CXCR5-ligands allows the formation of GC-Tfh. The PD1^lo Tfh-like cells make early cytokine responses and may represent precursors of CD4 memory cells.