Expression of CD69 and CD70 differentiates B1 cells from activated memory and naive B cells. (A) CD20⁺CD27⁺CD43⁻ memory and CD20⁺CD27⁻CD43⁻ naive B cells were sort-purified from adult peripheral blood and were cultured with SAC (0.005%) plus IL-2 (5 ng/ml) for 4 d, after which surface expression of CD43, CD69, and CD70 was determined by immunofluorescent staining and flow cytometry. Unstimulated CD20⁺CD27⁺CD43⁺ B1 cells were immunofluorescently stained and analyzed for CD69 and CD70 at the same time. CD69 and CD70 expression is displayed for CD43⁺ B cells. Results are shown for one of three separate samples. Fluorescence minus one isotype control staining is shown in solid gray for each population. (B and C) Memory and naive B cells from adult blood were cultured for 4 d with SAC plus IL-2, PMA at 100 ng/ml plus ionomycin at 400 ng/ml (P + I), or anti-CD40 at 10 µg/ml plus anti-IgM at 7 µg/ml (αCD40 plus αIgM), and immunofluorescently stained, after which surface expression of CD43, CD69, and CD70 was determined. Unstimulated CD20⁺CD27⁺CD43⁺ B1 cells were immunofluorescently stained and analyzed at the same time. The proportion of CD43⁺ B cells that stained positively for CD69 (B) and for CD70 (C) is shown. Mean values for three separate adult blood samples for each population are shown along with error bars indicating SEM.