Fig. 7
Ezh2 Ser21 phosphorylation is necessary for Bcl6 induction but dispensable for p19Arf repression. a Ezh2 phosphorylation status in polyclonal CD4+ T cell responses. CXCR5+SLAMlo TFH cells and CXCR5–SLAMhi TH1 cells were sorted from activated CD4+ T cells in spleens of LCMV-Arm-infected WT C57BL/6 mice on 8 dpi, and immunoblotted with antibodies specific for pS21-Ezh2, pT487-Ezh2, and total Ezh2. Data are representative from 2 independent experiments with similar results. b Ezh2 phosphorylation status in monoclonal CD4+ T cell responses. CD45.2+ WT Smarta CD4+ T cells were adoptively transferred into congenic recipients followed by LCMV-Arm infection. On 4 dpi, CXCR5+SLAMlo TFH cells and CXCR5–SLAMhi TH1 cells were analyzed for total Ezh2, pT487-Ezh2, or pS21-Ezh2 with intracellular staining. Values denote gMFI, and data are representative of ≥3 experiments. c–d Predominant association of pS21-Ezh2 with TFH lineage cells. Blimp1-YFP+ Smarta CD4+ T cells were adoptively transferred into WT congenic recipients followed by LCMV infection. On 4 dpi, donor-derived CD4+ T cells were analyzed for Blimp1-YFP, CXCR5, T-bet, Tcf1 and pS21-Ezh2 at indicated combinations. e–g Effect of WT or mutant Ezh2 on rectifying TFH defects. Ezh2–/– Smarta CD4+ T cells were primed in vitro and transduced with EV-GFP retrovirus or that expressing WT or mutant forms of Ezh2, followed by adoptive transfer and LCMV-Arm infection. WT Smarta CD4+ cells infected with EV-GFP retrovirus were used as a control. On 4 dpi, equivalent to day 7 after initial CD4+ T cell priming, GFP+ Smarta CD4+ T cells were analyzed for frequency of CXCR5+SLAMlo TFH cells (e). For a more accurate detection of the CXCR5+Bcl6+ TFH subset in retrovirally transduced cells, CD45.2+CD4+ T cells were intracellularly stained for Ezh2, and Ezh2– cells in the EV-GFP group and Ezh2+ cells in other groups were analyzed (f). In (g), GFP+CD45.2+CXCR5+SLAM– TFH cells were sorted for analysis of Bcl6, Arf, and Ink4a transcripts by quantitative RT-PCR. Cumulative data are means ± s.d. from ≥2 independent experiments. ns, not statistically significant; *p < 0.05; **p < 0.01; ***p < 0.001 for indicated pairwise comparison by Student’s t-test, coupled with one-way ANOVA for multi-group comparisons
