Figure 4.

Highly Multimerized Low-Affinity Antigen Effectively Induces Early B Cell Responses

(A and B) Intravital imaging of iLNs 2–3.5 h after immunization with eOD-GT2 60-mer. (A) Normalized speed of VRC01^gHL B cells (^∗∗∗∗p < 0.0001 versus unimmunized). (B) Frequency of stationery VRC01^gHL B cells (speed <3 μm/min; ^∗p < 0.05 versus unimmunized). Data in (A) and (B) are from three experiments with n = 4 mice. Bars represent the mean. See also Figures 1C and 1D.

(C and D) VRC01^gHL B cell activation at 24 h. (C) VRC01^gHL B cell size (mean FSC-A). (D) Activation markers on VRC01^gHL B cells (normalized geometric MFI). Data in (C) and (D) are from three experiments with n = 6–9 mice. Bars represent the mean.

(E) Distribution of VRC01^gHL B cells at 24 h. Data are from two experiments with n = 6 mice and 580 cells.

(F)–(H) Intravital imaging showing T and B cell interactions at 24 h. (F) Schematic. (G) Representative movie frames. (H) VRC01^gHL B cells (rows) were classified as “in contact” (red) or “not in contact” (blue) with SMARTA CD4⁺ T cells. Randomly selected 71 VRC01^gHL B cells are shown.

(I) Percentage of time VRC01^gHL B cells were in contact.

Data in (G)–(I) are from two experiments with n = 3 mice.

(J–P) Proliferation and differentiation of VRC01^gHL B cell 3 days after immunization. (J) Division profiles. The gate identifies undivided and divided cells. (K) CTV (geometric MFI) on divided VRC01^gHL B cells, normalized to eOD-GT5_gp61 60-mer. (L) Divided VRC01^gHL B cell number. (M) Undivided VRC01^gHL B cells as a frequency of total B cells, normalized to unimmunized. (N) Bcl6 and IRF4 expression in divided VRC01^gHL B cells. Percentages are indicated in quadrants. (O) Divided VRC01^gHL B cells that are Bcl6^hi IRF4^lo GC B cells. (P) Divided VRC01^gHL B cells that are IRF4^hi Bcl6^lo PCs. Data in (J)–(P) are from two experiments with n = 6 mice. Bars represent the mean.

^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Videos S2 and S3 and Figure S4.