In BO cGVHD EZH2 is necessary for the GC reaction. (A-D) Transplants were performed as in Figure 1; groups are as defined in Figure 3. (A-B) Flow cytometry analysis of mouse splenocytes taken 49 days posttransplant. Cell populations for flow cytometry analysis are as defined in Figure 2. Results show that when EZH2 is knocked out in both the BM compartment (A) (n = 4/group) and T-cell compartment (B) (n = 14/BM-only, n = 13/cGVHD, n = 10/BM [WT] + T [EZH2 KO]) of the graft, there is a significant reduction in the TFH and GCB frequencies and a significant increase in the TFR/TFH ratio. (C) Representative images of cryopreserved spleen sections stained to show GCs from mice 49 days posttransplant. Sections are stained with DAPI (blue), PNA rhodamine (red), and CD4 FITC (green). Images are ×200 magnification. (D) Left panel shows the number of GCs observed in each spleen section normalized for the area of spleen in each section. Right panel is a quantification of the average size of GCs observed in each section. GCs were identified and quantified as in Figure 2. Quantification shows when EZH2 is knocked out in either the BM and T-cell compartments, there is a comparable reduction in both the GC count and average size. N = 8/BM-only, n = 6/cGVHD, n = 5/BM (EZH2 KO) only, n = 4/BM (EZH2 KO) + T (WT), n = 4/BM (WT) + T (EZH2 KO). For all panels, statistics shown are results of an unpaired t test with Bonferroni corrected P values, where appropriate. *P < .05, **P < .01, ***P < .001, ****P < .0001.