Fig. 3

Assessment of B-cell activation upon IgM Fab stimulation and B-cell replication history in p.P522R-carriers and non-carriers. A Expression of phosphorylated PLCγ2 (pPLCγ2) in B cells before and after stimulation of the B-cell receptor with IgM Fabs. Detection of pPLCγ2 was used as a direct measure of B-cell receptor activation after IgM Fab stimulation. N = 14. % increase in PLCγ2 phosphorylation in carriers vs non-carriers is indicated behind each population in the legend. B-D Measurement of calcium release (‘flux’) after B-cell stimulation with IgM Fabs of pre-GC B cells (CD27-IgG-IgA-) B, unswitched memory B cells (CD27+IgG-IgA-) C, or class-switched memory B cells (CD27+IgG+ or CD27+IgA+) D in cohort II. Differences between carriers and non-carriers were evaluated by comparing the area under the curve (AUC) of the total Fab stimulation (from stimulation until the moment ionomycin was added, ~ 10 min, flux intensity and duration), the peak of the response after Fab stimulation (the 5 highest points after the Fabs were added to the cells; flux intensity), and after ionomycin was added (to determine the maximum flux). AUC was calculated only for points that were higher than baseline value (unstimulated sample). N = 13 (one sample was lost due to technical failure). % increase or decrease in AUC in carriers vs non-carriers is indicated behind each population in the legend. E + F Measurement of the average number of undergone cell divisions by total pre-GC B cells (CD27-IgM + IgG-IgA-) or unswitched memory B cells (CD27+IgM+IgG-IgA-) by means of qPCR-based KREC assay in the total cohort E or separated into carriers and non-carriers F. For pre-GC B cells; n = 14. Due to low DNA concentration, for unswitched MBC; n = 8. Differences were evaluated by Mann-Whitney U test. Pre-GC; pre-Germinal Center, MBC; memory B cells, iMFI; integrated mean fluorescence intensity (for calculation, see Methods) Unsw. MBC; unswitched memory B cells, Cs. MBC; class-switched memory B cells. ** p < 0.01