Figure 4. Mutant Ezh2 centrocytes fail to re-enter the DZ.

A. YFP⁺IgD^- splenocytes sorted from 3 YFP;Ezh2^Y641F and 3 YFP;Cγ1-cre mice immunized with SRBC for 8 days were subjected to single cell RNA-seq. Dimensionality reduction with UMAP was performed on normalized gene expression values, using graph based clustering and K nearest neighbor analysis to assign cells to clusters with distinct expression profiles.

B. Mature B cell signatures were projected on clusters defined in A. MBC: memory B cell. DECP are positively selected GC B cells (Ersching et al., 2017).

C. Specific gene expressions were projected on clusters from A. D. Gene expression profiles were organized into a pseudotime vector by Slingshot.

E-F. After normalization for total number of analyzed cells, the abundance of Ezh2^Y641F and WT was calculated for CC (LZ) (E) and recycling cells (F), based on the projected signature score from (B); data are mean ± SE (n=3 mice), p values generalized linear model.

G. GFP-Myc mice were SRBC-immunized for 8 days and spleens analyzed by flow cytometry. CBs and CCs were gated from GC B cells.

H. Total GFP⁺ GC B cells were assigned to CBs and CCs, gated as shown in G. Each dot represents a mouse (n=4), mean ± SEM.

I. Flow cytometry plots showing the percentage of GFP⁺ cells among GC B splenocytes from GFP-Myc mice.

J. Quantification of GFP⁺ cells among GC B splenocytes from 4 GFP-Myc;Ezh2^Y641F and 4 GFP-Myc;Cγ1-cre mice; mean ± SEM; unpaired t test.

Results representative of 2 experiments.

See also Figure S4.