Fig. 3. BCR antigenicity and signaling within Public Clonal Lineage 1 (Lin1).

A sHsL and gHgL BCRs in Lineage 1, along with VRC01 BCRs were expressed in our B cell reporter system⁷⁸ and evaluated by flow cytometry for binding to fluorescent versions of WT Env (red lines) vs D368R Env (blue lines; D368R + A281R + G366R + P369R in the case of YU2¹²) for each antigen used in the immunization regimen. Gray histograms represent binding to BCR isotype control or binding to surface BCR negative for LC expression. B Acquisition of D368R sensitivity, as further resolved by gHgL BCR vs sHsL BCR triggering in response to bivalent Env-Fc, Env-D368R-Fc or anti-IgM as a positive control. All Env variants used in the heterologous immunization regimen were tested. Presented is the Ca²⁺ flux activity, measured kinetically by the ratiometric Ca²⁺ sensing dye fura red and normalized to total flux capacity, as defined by the ionophore ionomycin⁷⁸. (A) vs (B) represents two independent experiments to evaluate BCR antigen recognition via the two orthogonal methods (binding to membrane presented BCR and BCR triggering following antigen exposure). Antigenicity of sHsL vs gHgL BCR was independently confirmed by reversing the fluorescent label on Env vs Env-D368R (Supplementary Fig. 9), and LC vs HC contribution to the acquisition of D368R sensitivity was evaluated by independently comparing antigenicity of sHgL vs sHsL BCR (Supplementary Fig. 10). BCR triggering was first established in response to 122E Env-Fc vs 122E Env-D368R-Fc (Supplementary Fig. 11), and then independently evaluated for Fc-presentation of all the Env strains used in the heterologous immunization regimen, as shown in this Figure (B).