Differentiation of macaque memory B cells into ASC in vitro. (A) PBMCs isolated from rhesus macaques inoculated with Env trimers in adjuvant were stimulated 4 d in vitro for differentiation of memory B cells into ASC. Stimulations were performed in 48-well plates with combinations of CpG, IL-2, IL-10, CD40L, anti-CD40, SAC, and PWM as indicated. PWM (underlined) was tested at concentrations ranging from 10 µg/ml to 1 ng/ml. Stimulated cells were added to ELISpot plates for detection of total and Env-specific IgG-producing cells. Bar graphs represent mean values + SEM of two to three monkeys normalized to 10⁶ cultured PBMCs. (B) The correlation between Env-specific and total IgG-producing ASCs was determined for each stimulation after log₁₀ transforming the values. (C) The correlation between Env-specific ASC quantified by B cell ELISpot assay and secreted Env-specific Abs quantified by ELISA for each stimulation condition was determined. Pearson’s correlation (r²) and the p-value for the correlation efficiency are shown. The experiment was repeated three times.