Figure 3. Boosting with B11 or B16 protein trimers increases GC-resident BG18^gH B cells.

(A) Mutations in B11 and B16 relative to BG505 are highlighted with the equivalent regions on GT5 shown for comparison. Orange indicates mutations that are only present in B16 that were selected for reduced binding to WT mouse serum that was immunized with GT2 or GT5. (B) SPR dissociation constants (KD) for GT2, GT5 and B11 trimer binding to epitope specific (GT2⁺KO⁻) Fabs derived from WT GC B cells 10 dpi of GT2 trimer protein; n=6 Fabs. Dashed line indicates limit of detection (LOD). (C) SPR dissociation constants (KD) for GT5, B16 and B11 trimer binding to epitope-specific (GT5⁺KO⁻) Fabs derived from GC CD45.2⁺ B cells 42 days after GT5 protein immunization of BG18^gH B cell adoptive transfer recipient mice. Each symbol corresponds to a different Fab and represents one or two measurements. Bars indicate geometric mean and geometric SEM; n=10 Fabs. GT5 prime-only KD values are those presented in Fig. 2J. (D) Schematic showing BG18^gH B cell adoptive transfer recipients primed with GT5 trimer protein on day 0, followed by boosting with B11 and B16 trimer protein on day 56. Samples from all treatments were collected on day 65 for analysis. (E) Representative flow cytometry plots of GC B cells, CD45.2⁺ cells in GC, B11 binders of GC CD45.2 cells and epitope binding specificity on day 65. (F) GC and B11-specific CD45.2⁺ GC B cells as a percentage of total B cells on day 65. Each symbol represents a different mouse: BG18^gH (GT5 prime), BG18^gH (B11 boost), n=11. Bars indicate mean + SD. Analyzed using Welch’s t test. *p <0.05, ****p < 0.0001. We have pooled data from two independent experiments. (G) Representative flow cytometry plots of GC B cells, CD45.2⁺ cells in GC, B16 binders of GC CD45.2 cells and epitope binding specificity on day 65. (H) GC and B16-specific CD45.2⁺ GC B cells as a percentage of total B cells on day 65. Each symbol represents a different mouse: BG18^gH (GT5 prime), n=11; BG18^gH (B16 boost), n=10; analysis otherwise as in (F). *p <0.05, ****p < 0.0001. We have pooled data from two independent experiments.