RA potentiates CpG-mediated DNA synthesis and proliferation of human memory B cells. (A) Peripheral-blood B cells (0.5 × 10⁶/mL) were cultured in triplicates with medium alone or with CpG (1 μg/mL) or SAC (0.005%) in the presence or absence of RA (100 nM). [³H]thymidine was added for the last 20 hours of a 72-hour period, and thymidine incorporation was determined. Data represent mean cpm ± SEM of the median of triplicates (**P < .01, paired sample t test; n = 7). (B) B cells were cultured with CpG (1 μg/mL) in the presence or absence of increasing concentrations of RA, and thymidine incorporation was determined. The results are presented as mean cpm ± SD of triplicates from 1 representative experiment of 3. (C) Peripheral-blood B cells were fractionated into CD27⁺ or CD27⁻ cells as described in “Materials and methods,” and cells of the fractionated and unfractionated populations were analyzed for the expression of CD27, IgD, and IgM as indicated. Isotype-matched control antibodies were included as negative controls (dotted lines), and the flow cytometry histograms represent 1 of 3 reproducible experiments. (D) CD27⁺ and CD27⁻ B cells were cultured in medium alone or with CpG or SAC in the presence or absence of RA as described in panel A, and thymidine incorporation was determined. The results are presented as mean cpm ± SEM of the median of triplicates (*P < .05, Wilcoxon signed-rank test; **P = .01, paired sample t test; n = 7). (E) CD27⁺ B cells (0.5 × 10⁶/mL) were labeled with CFSE, and cultured in medium alone or with CpG (1 μg/mL) with or without RA (100 nM). Cell division was analyzed by flow cytometry on successive days as indicated. During the acquisition, events were obtained for a fixed time so that the amplitude of the peak is proportional to the cell yield. The data were gated for living cells based on forward- and side-scatter distribution, and the results are from 1 representative experiment of 3.