Figure 6.

Dock8 deficiency disrupts the activation and transcription of CD19 as well as the early activation of memory B cells. Human B cells from HCs and Dock8 patients were incubated with AF546–mB-Fab′–anti-Ig without or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. (A-E) After fixation and permeabilization, the cells were stained for pCD19 and CD19 and analyzed using CFm (A-D) or flow cytometry (E). (C) The Pearson’s correlation coefficients between BCR and pCD19 staining in sAg-stimulated cells were determined using NIS-Elements AR 3.2 software. (D) The MFI of CD19 in B cells from HC and Dock8 patients was measured by NIS-Elements AR 3.2 software. (E) The MFI of pCD19 in sAg-stimulated cells were determined by flow cytometry. (F) Two hundred ninety-three cells in 24-well plates were transfected with 0.45 μg pGL3-CD19, 0.45 μg pcDNA3.1, or pcDNA3.1-Dock8 and 0.045 μg pRL-TKB (internal control) for 24 hours, followed by luceferase reporter assay; pGL3-promoter and pGL3-basic were used as positive and negative controls. TIRFM and interference reflection microscopy analysis of pY and pBtk staining in the contact zone of naïve and memory B cells from HCs and Dock8 patients incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (G-J) and the B-cell contact area (K), the MFI of Fab′–anti-Ig in the B-cell contact zone (L), MFI (±SD) of pY (M), and pBtk (N) in the B-cell contact zone from 3 independent experiments. All 3 patients were included, and each patient was an independent experiment. Scale bars, 2.5 µm. *P < .01; **P < .001, in comparison with naïve B cells from Dock8 patients. M, memory B cell; N, naïve B cell.