Journal: Nature Communications
Article Title: Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf
Figure Lengend Snippet: Ezh2 Ser21 phosphorylation is necessary for Bcl6 induction but dispensable for p19Arf repression. a Ezh2 phosphorylation status in polyclonal CD4 + T cell responses. CXCR5 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 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 + SLAM lo T FH cells and CXCR5 – SLAM hi T H 1 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 T FH 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 T FH 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 + SLAM lo T FH cells ( e ). For a more accurate detection of the CXCR5 + Bcl6 + T FH 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 – T FH 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
Article Snippet: For detection of pS21-Ezh2 (rabbit polyclonal, Bethyl Laboratories) or pT487-Ezh2 (rabbit polyclonal, abbexa, UK), the surface-stained and fixed cells were first stained with the primary antibody, followed by sequential staining with biotinylated goat anti-rabbit IgG (Cat. No. 111-066-144, Jackson ImmunoResearch Laboratories) and fluorochrome-conjugated streptavidin.
Techniques: Infection, Mouse Assay, Staining, Derivative Assay, Mutagenesis, In Vitro, Transduction, Expressing, Adoptive Transfer Assay, Quantitative RT-PCR