Inhibition of B-cell activation by heme is through the DOCK8/STAT3 signaling pathway. (A) Purified HD naïve B cells were stimulated with B-cell activation cocktail, and phosphorylation of Pyk2 (pPyk2), pSyk, pSRC, and pSTAT3 was analyzed by intracellular flow cytometry after 15 minutes. Histogram overlays depict the extent of phosphorylation without (gray) or with stimulation (blue) with the phosphorylated signal gate placed by reference to the baseline prestimulation histogram. (B) Purified HD naïve B cells were stimulated for the same length of time as in panel A in the absence or presence of heme (10 μM), and the frequency of cells positive for pPyk2, pSyk, pSRC, and pSTAT3 is shown. (C) Purified HD naïve B cells were stimulated with B-cell activation cocktail for 7 days. Contour plots depict gating strategy for DOCK8 Low and DOCK8 High in proliferated B cells as well as plasma B cells (CD27^hiBlimp1⁺ cells) within DOCK8^low or DOCK8^high cells. (D) Fold change in plasma B-cell frequencies in 7-day purified HD naïve B-cell–stimulated cultures in the presence of 2.5 μM or 5 μM heme relative to no heme stimulated B cells. (E) DOCK8^high (left) and DOCK8^low (right) cell numbers in stimulated B-cell cultures treated with 2.5 μM or 5 μM heme. Fold change in DOCK8^high or DOCK8^low cell numbers with heme treatment relative to no treatment. (F) Representative histogram overlay showing DOCK8 expression (blue) in peripheral B cells relative to isotype control (gray). Relative DOCK8 expression (mean fluorescence intensity [MFI]) in circulating B cells from HD and allo-negative and allo-positive patients is shown. (G) Fold change in DOCK8^high B-cell and CD27^hi plasma cell frequency in 7-day heme-treated cultures of stimulated naïve B cells from allo-negative and allo-positive patients. Data represent means ± standard error of the mean. SSC, side scatter (light).