Fig. 8

Ezh2 is necessary for T_FH lineage specification during the early activation stage. a Rapid induction of Ezh2 and Ser21 phosphorylation upon CD4⁺ T cell activation in vivo. CTV-labeled WT Smarta CD4⁺ T cells were adoptively transferred into B6.SJL recipients, either uninfected or infected with LCMV-Arm. Thirty-six hours later, undivided donor cells (Div0) or those in the first division (Div1) were analyzed for expression of CD25, total Ezh2 and pS21-Ezh2. Values denote geometric MFI, and data are representative from at least 2 independent experiments. b–c Predominant association of pS21-Ezh2 with nascent T_FH cells at the fate-bifurcation stage. CTV-labeled Blimp1-YFP⁺ Smarta CD4⁺ T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. Sixty hours later, donor cells in the 5th division were analyzed for Blimp1-YFP in combination with surface staining of CXCR5 or CD25 or intracellular staining of T-bet and Tcf1 (b), or with total Ezh2 and pS21-Ezh2 (c). d–e Impact of Ezh2 deficiency on early T_FH cells. CTV-labeled WT, Ezh2^–/–, or Ezh2^–/–Arf^–/– Smarta CD4⁺ T cells were adoptively transferred, and recipients infected with LCMV-Arm as in (b). Cells in the 5th division at 60 h post-infection were analyzed for nascent T_FH and T_H1 subsets based on CD25 and CXCR5 expression. The nascent T_FH and T_H1 cells were then analyzed for expression of T_H1-associated Irf4 and T_FH-associated Bcl6 (d), with values denoting geometric MFI and dotted lines marking histogram peaks in isotype control staining. Caspase-3/7 activation was determined in nascent T_FH cells (lower panels in e). Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; *p < 0.05; **p < 0.01 for indicated pairwise comparison by Student’s t-test, coupled with one-way ANOVA for multi-group comparisons