FcRL4 inhibits BCR-induced CD69 expression. (A) Several FcRL4⁻ and FcRL4⁺ subclones, n = 4 and n = 12, respectively, were incubated with F(ab′)₂ control Ig or F(ab′)₂ anti-IgM to cross-link the BCRs for 18-24 hours. Cells were stained with PE-coupled mAb specific for CD69 and analyzed by flow cytometry. Left panel: Representative histograms of CD69 expression on FcRL4⁻ and FcRL4⁺cells after treatment with control F(ab′)₂ or F(ab′)₂ anti-IgM are given. Right panel: Data shown are normalized MFI (NMFI) calculated as (MFI of positive cells × percentage of positive cells)/100 from 1 of 3 independent experiments. P values were obtained from unpaired, 2-tailed Student t test. (B) FcRL4⁺ cells were sorted into 2 populations based on the level of FcRL4 surface expression resulting in 2-fold differences in MFI between the low- (MFI = 30) and high- (MFI = 60) expressing cells. Cells were analyzed for CD69 expression after BCR cross-linking. Left panel: Representative histogram of CD69 expression for FcRL4^−, FcRL4^low or FcRL4^high cells after BCR cross-linking. Right panel: NMFI of CD69 was calculated and P value obtained from unpaired, 2-tailed Student t tests either between FcRL4⁻ and FcRL4^low (*P = .0134) or between FcRL4^low and FcRL4^high (**P = .0012; n = 4). (C) Purified peripheral B cells from healthy donors were nucleofected with either YFP-containing (FcRL4⁻) or FcRL4-YFP–containing plasmids (FcRL4⁺). After a 2-hour rest in culture, the cells were further incubated in medium alone or medium containing F(ab′)₂ anti-IgM + IgG for 18 hours. Cells were analyzed for CD69 expression. Left panel: Given are the representative FACS profiles of YFP and CD69 for FcRL4⁻ or FcRL4⁺ cells. Given are the percentages of YFP-positive or CD69-positive cells in the FcRL4⁻ and FcRL4⁺ populations. Right panel: NMFI of CD69 was calculated and shown by mean ± SD (n = 4). P values were obtained from paired, 2-tailed Student t tests (*P = .0124; **P = 0053).