Figure 4.

High affinity GT5 immunogen activates PCT64 precursors at physiological frequencies

(A) Schematic of adoptive transfer model to calibrate PCT64^LMCA B cell frequencies. Experiments were performed in duplicate with one presented; n = 5.

(B) Gating strategy for titration of cell transfer model.

(C) Precursor frequencies (y axis) corresponding to number of B cells transferred (x axis). Bars are mean ± SD.

(D) Analysis of linearity of CD45.2 PCT64^LMCA B cells recovered 24 h post transfer.

(E) Schematic of immunizations performed at precursor frequencies of 100, 20, and 10 per 10⁶. Experiments were performed in triplicate with one presented; n = 4.

(F) Representative FACS plots at 8 dpi with GT, showing CD45.2 GC B cell responses at precursor frequency of 100, 20, and 10 per 10⁶ of B cells.

(G) Quantification of GC B cells and CD45.2⁺ PCT64^LMCA cells present in GC at 8 dpi with GT2. Bars are mean ± SD.

(H) SPR of PCT64^LMCA for GT2 and GT5 trimers.

(I) Representative FACS plots 8 dpi with GT5, showing CD45.2 GC B cell responses at precursor frequency of 100, 20, and 10 per 10⁶ of B cells.

(J) Quantification of GC cells and CD45.2⁺ PCT64^LMCA cells present in GC at 8 dpi with GT5 at decreasing precursor frequencies (n = 4 and n = 5 for 10:10⁶). Bars are mean ± SD. See also Figures S5F and S5G.

(K) Representative FACS plots at 8, 16, and 42 dpi with GT5, showing CD45.2 GC B cell responses at precursor frequency of 100, 10, and 1 per 10⁶ B cells (n = 4).

(L) Quantification of K. Data are represented as mean ± SD.

(M) ELISA quantification of GT2-/GT5-binding (top) and GT2-/GT5-KO-binding (bottom) serum IgG from PCT64^LMCA recipient mice compared to WT C57BL/6J mice (n = 4). AUC was assessed prior to immunization by GT2 or GT5 and at 7, 14, 21, and 42 dpi. Points are mean ± SD.