DC-SIGN mediates low-level antigen-independent signaling in sIg-Mann⁺ cells. (A) The histograms show SYK phosphorylation at Y525/526 in a representative sIg-Mann⁺ primary sample (16-TB0014) and in the WSU-FSCCL cell line after stimulation with DC-SIGN (red), anti-Ig (blue), or no treatment (green) (top). Geometric mean fluorescence intensity (MFI) levels of pSYK are shown for each condition. Heat-map of DC-SIGN–mediated and anti-Ig–mediated signaling in all cell lines and primary samples analyzed at 1, 5, and 15 minutes. MFI of pSYK after stimulation was normalized with pSYK MFI of the untreated (NT) sample at the respective time point (NT sample was normalized to 1) (bottom). The statistical difference between DC-SIGN and anti-Ig stimulation was significant at each time point (P = .03; Wilcoxon signed-rank test). (B) Immunoblotting of AKT phosphorylation at S473 and ERK phosphorylation at T202/Y204 in the primary DLBCL sample 17-TB0084 after exposure to DC-SIGN or anti-IgM or left untreated (NT) for 15 minutes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) Soluble recombinant DC-SIGN was incubated with 500 nM hIgG1-D1 before treating NU-DHL1 cells. Phosphorylation of SYK at Y525/526 was measured by flow cytometry. Data are represented as the mean ± standard error of the mean (SEM) of 3 independent experiments. Supplemental Tables 6 and 7 provide sIg characteristics and DC-SIGN binding of the primary samples and cell lines.