Abnormal morphology of DOCK8-deficient lymphocytes when migrating in 3D collagen gel matrices. (A) Confocal imaging of T cells migrating in a collagen gel matrix (3.6 mg/ml) with nuclear staining. BF, brightfield. DAPI, blue. Representative of six experiments. (B) Confocal imaging after in-gel immunofluorescence staining of control and patient T cells for pericentrin (red, indicated by arrows), α-tubulin (green), and nuclei (blue). Stained gels were physically compressed before imaging. Left panels: migrating cells, each showing one centrosome. Right panels: dividing anaphase cells, each showing two centrosomes. Representative of three patients and three healthy controls from three experiments. (C) Proportions of resting T cells that were abnormally elongated. Less than 2% of cells were in S/G2/M phases after 6 wk of culture. Three controls and three patients were tested from three experiments. (D) Proportions of patient T cells that were abnormally elongated after cell cycle blockade. Similar analysis as for C, except that cells were from three patients that were treated with either aphidicolin (Aph) or vehicle (DMSO), tested in three experiments. (E) Nuclear elongation among abnormally elongated cells. Nuclei in cells from D were stained with Hoechst for analysis. (F) Percentage of time cells spent elongated in the collagen matrices. 6 patients and 12 healthy controls were tested from five experiments. (G) Similar analysis as for F, except performed with T cells transfected with DOCK8 or nonspecific (NS) siRNA, from four experiments. Representative immunoblot showing DOCK8 knockdown efficiency. (H and I) Proportions elongated of T cells (H) and NK cells (I) from 10 Dock8-deficient (KO) or control (WT) mice from three experiments, migrating in collagen gel matrices. Bars, 10 µm. C–I show means ± SD. Unpaired two-tailed Student’s t test were performed. Statistical significance indicated by *, P < 0.05; ****, P < 0.0001; ns, not significant.