Figure 1. Development of a VRC01^gHL B Cell Transfer Model to Assess Precursor Frequency Effects on Immune Responses.

(A) pERK induction in VRC01^gHL B cells upon stimulation with eOD-GT8 60-mer. n = 3 mice.

(B) 10⁶ CFSE-labeled heterozygous VRC01^gHL B cells were transferred into CD45.1⁺ hosts. Host mice were immunized with eOD-GT8 60-mer. Splenocytes were harvested 3 days later and VRC01^gHL B cell division was analyzed by flow cytometry. Gated on scatter/singlet/live (SSL), B220⁺, CD4⁻, CD45.2⁺, CD45.1⁻. n = 3 mice.

(C) Schematic of the VRC01^gHL B cell transfer system used for (D)–(J).

(D) Frequency of total VRC01^gHL B cells (SSL, CD4⁻, B220⁺, CD45.2⁺, CD45.1⁻) on day 8 in spleens of mice immunized as in (B), seeded with different VRC01^gHL cell precursor frequencies. n = 4 mice/group.

(E) Representative plot of (red) eOD-GT8 60-mer binding to VRC01^gHL GC B cells (SSL, CD4⁻, B220⁺, CD45.2⁺, CD45.1⁻, GL7⁺, BCL6⁺) or (blue) naive host B cells (SSL, CD4⁻, B220⁺, CD45.2⁻, CD45.1⁺, GL7⁻, BCL6⁻) on day 8 post-immunization. Host mice were seeded with a 1:10⁶ VRC01^gHL B cell precursor frequency prior to immunization. n = 4 mice/group.

(F) Representative day 8 serum IgG ELISA data. Blue = WT eOD-GT8 60-mer; Red = CD4bs-KO eOD-GT8 60-mer. n = 4 mice/group.

(G–I) Day 8 GC B cells in mice immunized as in (D). n = 4 mice/group. Representative flow cytometry (G) and quantification (H) of GC B cells. VRC01^gHL B cells (CD45.2⁺) and endogenous B cells (CD45.1⁺) are indicated within the GC compartment. Pre-gated on SSL, B220⁺, CD4⁻. (I) Quantitation of VRC01^gHL B cells among GC B cells from G. * = p < 0.05. Full gating shown in Figure S1P.

(J) Day 8 spleen GC histology from a host mouse seeded with a 1:10⁶ VRC01^gHL B cell precursor frequency. Green, B220; red, CD45.2; white, GL7. Independent Experiments = E. (A) E = 3; (C) E = 2; (D) E = 2; (E) E = 2; (F) E = 2; (G) E = 2; (H) E = 2; (I) E = 2; (J) E = 2. Data from one experiment are shown in each panel. See also Figures S1 and S2. Error bars are SEM.