Figure 2.

Human Mϕs induce terminal differentiation of PCs from naive and memory B cells in vitro. (A) Sorted naive or memory B cells were co-cultured with autologous in vitro generated Mϕs or medium for 6 d and analyzed by flow cytometry for the expression of CD138, CD38, CD20, and CFSE. Both the percentage of CD138⁺CD38⁺ PCs and the absolute cell counts are shown. Each dot represents one independent experiment using a different donor. P-values were determined by two-sample Student’s t test. (B) Naive B cells co-cultured with autologous Mϕs for 6 d were stained intracellularly with Abs against IgA and IgG. (C) Percentage of CD138⁺ PCs per cell division was calculated based on FlowJo cell proliferation analysis. (D) IgM, IgA, and IgG production in the supernatants of co-culture of naive B cells and Mϕs, as measured by ELISA. Data are presented as mean ± SD from triplicate cultures and represent >10 different experiments using cells from different donors. (E and F) Naive B cells cultured with or without Mϕs were harvested on day 4 for isolation of mRNA and synthesis of cDNA. qPCR was performed to measure the expression of germline transcripts Iγ3-Cγ3, Iα1-Cα1, and Iα2-Cα2 (E) and switch circle transcripts Iγ-Cμ and Iα-Cμ (F). Expression was normalized to the amount of β-actin mRNA (for germline transcripts) or Im-Cm (for switch circles). B cells cultured alone were used as control for the relative expression. Data are mean ± SEM of three experiments using cells from different donors. (G) Sorted plasmablasts (CFSE^lowCD38⁺) were co-cultured with Mϕs for various days and analyzed for PC phenotype and counts. Mean ± SD of duplicate cultures is shown. (H) qPCR was performed to measure the expression of Blimp-1 on naive B cells co-cultured with Mϕs. P = 0.029, paired Student’s t test.