Figure EV4. NEF interferes with antibody affinity maturation, while GC B‐cell numbers or B‐cell follicle size is not altered by NEF.

• A–C
  Mice were immunized with HEL‐OVA/CFA following adoptive transfer of B_HEL and CD4_OT‐II cells expressing WT or mutant NEF. Draining lymph nodes (dLN) were harvested at d7 post‐immunization, cryosectioned, and stained with fluorophore‐coupled antibodies to visualize GCs (GL‐7), follicular dendritic cells (CD21/CD35), and nuclei (Hoechst). Quantification of dLN size (A), B‐cell follicle size (B), and GC numbers (C) per section are quantified from confocal micrographs as shown in Fig 4A. Shown are mean values with SD from three mice in two independent experiments. P‐values were calculated by one‐way ANOVA with Tukey’s post‐test. ***< 0.001, **< 0.01.
• D–O
  Representative flow cytometry histogram plots showing surface expression of PD‐L1 (D), GL‐7 (F) and CD95 (H) on B cells and CXCR5 (J), ICOS (L), and OX40 (N) on T cells harvested from dLN of immunized mice. Receptor expression was quantified on cells that were positive for CD45.2 (donor cell) and either B220 (for B cells) or CD3 (for T cells). Panels (E, G, I) and (K, M, O) show mean values of the data shown in (D, F, H) and (J–N, respectively. Data shown are derived from 2‐3 independent experiments. Shown are mean values with (E, G, I) and without (K, M, O) SD, each dot represents one animal.