Ova323 339

Conjugate formation assays were performed as previously described (Ise et al., 2018 (link)). Sorted splenic centrocytes (B220⁺CD38^-Fas⁺CXCR4^loCD86^hi) from NP-CGG immunized Cγ1-cre mice were incubated with Fc block and then stained with 1 µM cell tracer CFSE (Life Technologies) and Ezh2^Y641F centrocytes with 1 µM cell tracer Violet (Life Technologies). After washing with PBS, centrocytes were incubated with 5 µM OVA323–339 (MBL International, TS-M703) for 2 hours at 37°C. Ten thousand centrocytes (10,000 WT or 5,000 WT + 5,000 Ezh2^Y641F) were mixed with 5,000 splenic Tfh (CD4⁺B220^-PD1^hiCXCR5^hi) sorted from OVA immunized OT-II mice, and cultured in 96-well U-bottom plates for 45 minutes at 37°C. Cells were next stained with anti-B220 (PE) and anti-CD4 (APC). Cells were vigorously pipetted to disrupt nonspecific conjugates and CD4 expression on B220⁺ cells was analyzed by flow cytometry.