Figure 1.

Previously elicited high-affinity antibodies restrict naive BG18^gH B cell responses

(A) Schematic of evaluation of naive BH18^gH B cell responses to immunization by N332-GT2 NP in primed and unprimed recipients.

(B) Representative FACS plots of gating strategy to quantify FAS⁺CD38⁻ GC B cells and CD45.2⁺ BG18^gH GC B cell responses versus endogenous CD45.1⁺ GC B cell responses in unprimed (left) and primed (right) recipients.

(C and D) (C) GC cells as the percentage of total B cells and (D) CD45.2⁺ BG18^gH cells as the percentage of total GC B cells at experiment day 52 (10 days post-immunization [dpi]) in previously unprimed (gray) or primed (red) recipients.

(E) Experimental design to evaluate naive BH18^gH B cell responses in primed and unprimed MD4 recipients.

(F and G) (F) GC cells as the percentage of total B cells and (G) GFP⁺ BG18^gH cells as the percentage of total GC B cells at experiment day 52 (10 dpi) in previously unprimed (gray) or primed (red) MD4 BG18^gH recipients.

(H) N332-GT2 trimer binding serum IgG of unprimed (gray) and primed (red) MD4 BG18^gH recipients at experiment day 42 (prior to immunization) and experiment day 52 (10 dpi). AUC = area under the ELISA binding curve.

(I) Schematic of evaluation of the effect of serum IgG from previously immunized mice on naive BG18^gH recipients consisting of (1) isolation of serum from N332-GT2 immunized cohort and (2) administration of purified IgG to the naive BG18^gH recipients.

(J and K) (J) GC cells as the percentage of total B cells and (K) CD45.2⁺ BG18^gH cells as the percentage of total GC B cells at 10 dpi in BG18^gH recipients receiving 200 μg of IgG isolated from 7- and 28-dpi serum.

(L and M) (L) GC cells as the percentage of total B cells and (M) CD45.2⁺ BG18^gH cells as the percentage of total GC B cells at 10 dpi in BG18^gH recipients receiving escalating doses of IgG isolated from 28-dpi serum.

p values were calculated by unpaired Student’s t test (C, D, F, and G) or ordinary one-way ANOVA with Dunnett’s multiple comparisons (K and M) (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001; ns, not significant). Figures represent data from one of at least two experiments with 3–5 mice per condition, with data presented as mean ± SD.

See also Figures S1–S3.