Figure 3. EZH2 is required for H3K27me3 deposition and antiviral CD8⁺ T cell clonal expansion.

A) Naïve CD8⁺ T cells from Ezh2^f/f mice and CD8⁺ T cells from Ezh2^f/f and Ezh2^f/f CD4Cre⁺ mice activated in vitro with αCD3 and αCD28 for 3 day were purified using FACS and the amounts of EZH2, β-Actin, H3K27me3 and total H3 were measured by western blot (data from 2 different experiments). Note, H3K27me3 is virtually undetectable in activated Ezh2^f/f CD4Cre⁺ CD8⁺ T cells.

B) Ezh2^f/f and Ezh2^f/f GzmBCre⁺ mice were infected with LCMV Armstrong and the number of splenic D^bGP_33-41 and D^bNP_396-404 MHC class I tetramer⁺ CD8⁺ T cells combined were enumerated at d8 p.i.

C) Bar graph shows viral titer in the serum of Ezh2^f/f and Ezh2^f/f GzmBCre⁺ mice at d8 p.i.

D) Naïve P14 Ezh2^f/f (red) and Ezh2^f/f CD4Cre⁺ (black line) CD8⁺ T cells were labeled with CellTrace Violet and stimulated for 72 hours in vitro with GP_33-41 peptide. Unstimulated naïve P14 CD8 T cells are shown in gray.

E) Congenically mismatched naïve P14⁺ Thy1.1⁺Ly5.2⁺ Ezh2^f/f (red) and Thy1.2⁺Ly5.2⁺ Ezh2^f/f CD4Cre⁺ (black line) CD8⁺ T Cells were pulsed with CellTrace Violet and adoptively co-transferred into Thy1.2⁺ Ly5.1⁺ WT recipient mice that were subsequently infected with LCMV-Armstrong and analyzed for cell division 60 hours later. P14⁺ CD8⁺ T cells from an uninfected recipient are shown in gray.

F) Bar graphs show IFNγ, TNFα, and IL2 production in GP_33-41 peptide-stimulated Ezh2^f/f and Ezh2^f/f GzmBCre⁺ CD8⁺ T cells at d8 p.i.

Data shown are representative of two (C–E), three (A), or five (F) independent experiments (n=4–10 mice/group/experiment for C and F), or cumulative of five independent experiments (n=21 mice/group) (B). Data are expressed as mean ± SD. *p=0.02, ****p<0.0001