Development of HEL-specific GC and EFPBs is impaired in the absence of OVA-specific T cells expressing Bcl-6. Naive OT-II Bcl6^+/+ or OT-II Bcl6^−/− with or without SW_HEL B cells were transferred i.v. 4 h apart into Cd28^−/− CD45.1 recipient mice, which were then immunized with HEL-OVA and/or OVA in alum i.p. (A and B) CXCR5 versus PD-1 phenotype of OT-II Bcl6^+/+ cells on day 2 or 4 after transfer into mice immunized with OVA in alum. Gating strategy to identify naive versus activated donor OT-II cells on day 2 or 4 after transfer (A) and representative flow cytometric plots, histograms, and bar graphs showing PD-1/ CXCR5 expression on naive (CD44^lo) and activated (CD44^hi) OT-II cells on either day 2 or 4 after adoptive cell transfer and immunization, analyzed on the same day (B). (C) Representative flow cytometric plots showing the gating strategy to identify donor-origin SW_HEL GC B cells (CD45.2 B220^hi HEL-binding^hi CXCR5^hi small forward scatter [FSC]) and EFPBs (CD45.2 B220^lo HEL-binding^lo CXCR5^lo large forward scatter) in the spleens of Cd28^−/− CD45.1 recipient mice 5.5 d after adoptive transfer. (D–F) Flow cytometric profiles (D) and quantification of SW_HEL EFPBs (E) and GC B cells (F) in the same recipient mice 5.5 d after immunization with HEL-OVA. (G and H) Representative flow cytometric plot showing gating strategy (G) and quantification (H) of donor-origin Tfh cells identified as CXCR5^hi PD-1^hi CD45.2 (gated on CD4⁺ cells) after transfer of OT-II Bcl6^+/+ or OT-II Bcl6^−/− T cells. These data are representative of two independent experiments with three to six mice per group.