FcRL4 expression enhances TLR9 signaling. (A) FcRL4⁻ and FcRL4⁺ B-cell subclones were stimulated with 0.5-1.0 μM control or CpG type B 2006 ODN for 18-24 hours and analyzed for the surface expression of CD23 by flow cytometry. Left panel: Representative data for CD23 expression for FcRL4⁻ and FcRL4⁺ cells after treatment with control ODN (Con) or CpG (CpG). Given are the percentages of CD23-positive cells with mean values in parentheses. Right panel: Data shown are expressed as the fold increase in NMFIs in CpG-treated cells relative to cells treated with control ODN. P values were calculated using unpaired, 2-tailed Student t test (FcRL4⁻; n = 5; FcRL4⁺; n = 10). (B) FcRL4⁻ B cells and B cells expressing either high (FcRL4^high) or low (FcRL4^low) levels of FcRL4 were analyzed for CD23 expression after CpG stimulation as above. Left panel: Representative histogram for CD23 expression after CpG stimulation in FcRL4^−, FcRL4^low or FcRL4^high cells. Right panel: NMFI of CD23 was calculated and P values obtained from unpaired, 2-tailed Student t tests either between FcRL4⁻ and FcRL4^low (*P = .0358) or between FcRL4^low and FcRL4^high (**P = .0017; n = 5). (C) B cells expressing either wild-type FcRL4, transfected with an empty vector (Vec), or expressing FcRL4 with individual tyrosine to phenylalanine mutations or all 3 tyrosine to phenylalanine mutations (Figure 5) were stimulated with CpG for 24 hours and analyzed for CD23 expression by flow cytometry. The results are expressed as NMFI. Shown is mean ± SD from 5 independent experiments. All FcRL4⁻ and FcRL4⁺ cell lines expressed similar levels of TLR9 (supplemental Figure 4). (D) Purified human B cells were nucleofected with either YFP (FcRL4⁻) or FcRL4-YFP (FcRL4⁺) plasmids and stimulated with 1.0μM CpG for 24-48 hours. Cells were analyzed for surface expression of CD23. Left panel: Representative FACS profiles for CD23 for FcRL4⁻ (YFP⁺) or FcRL4⁺ (FcRL4-YFP⁺) cells. The percentages of CD23-positive cells with the mean values in parentheses were given from the FcRL4⁻ and FcRL4⁺ populations. Right panel: The net increase of percentage of CD23-positive cells after CpG stimulation compared with the medium alone—percentage CD23⁺ cells (Δ)—was calculated within the FcRL4⁻ or FcRL4⁺ populations and shown are the means ± SD from repeated experiments with multiple donors (n = 7). P values were obtained from paired, 2-tailed Student t test. (E) FcRL4⁺ cells were stimulated with 3μM CpG-Cy3 in chambered coverglasses at 0°C or 37°C for 30 minutes, fixed, and imaged using a confocal laser scanning microscope ZEISS 510 META equipped with a 1.4 oil plan-apochromat ×63 objective lens. Two-color confocal images were acquired as described in “Preparation of PLBs and live cell imaging” and shown are pseudo-color images of FcRL4-YFP (green), CpG (red), DIC, and merged green and red images. Yellow scale bars represent 10 μm. Shown in the bottom panel are pixel intensity graphs for the FcRL4 (green) and CpG (Red) in the direction shown by the white arrows in 2 representative cells. (F) Co-localization analyses between the FcRL4 and CpG in FcRL4⁺ cells after CpG stimulation in panel E as described in “Preparation of PLBs and live cell imaging.” Shown is the mean ± SD of Rr taken in single cells. P values were calculated using paired Student t test from multiple cells treated at 0°C (n = 20, ●) or at 37°C (n = 30, ■).