Phenotypic characterization of rhesus macaque lymphocytes after in vitro stimulation. (A) Rhesus macaque PBMCs were stimulated with PWM + SAC + CpG, IL-2 + IL-10 + CpG + CD40L, or were nonstimulated (n-s) for 4 d and then stained for CD20, CD4, CD8, and intracellular IgG (IC-IgG). Nonviable cells were excluded via forward scatter and side scatter gating and B cells were defined as CD4⁻CD8⁻CD20^+/low cells. The numbers in the quadrants represent the percent cells of the total CD4⁻CD8⁻CD20^+/low cell population. The dot blots are representative of two donors. (B) Rhesus PBMCs were stained with CFSE and stimulated with PWM + SAC + CpG, IL-2 + IL-10 + CpG + CD40L, or CpG or nonstimulated for 6 d. Proliferation of CD20^+/low CD4⁻CD8⁻ B cells (bottom) and CD20^lowCD4⁺CD8⁺ T cells (middle) was analyzed. The numbers represent the percentage of cells in each indicated gate of total cells gated on side scatter/forward scatter. The dot blots and histograms are representative of two donors. (C) CD19⁺ or CD20⁺ B cells and CD4⁺ T cells were isolated from human (right) and rhesus macaque (left) PBMCs. The B cells were stimulated alone (white) or in combination with T cells (black) for 7 d and IgG production was evaluated by ELISA. The bar graph describing human data represents mean and standard deviation of triplicate samples. Stimulations on sorted human B cells were independently repeated three times with similar results. Data from rhesus macaque PBMCs represent mean and SEM of three donors.