Elongation when migrating through confined spaces leads to cytothripsis. (A) Time-lapse microscopy of a T cell from a DOCK8-deficient patient migrating in collagen matrix. PI (orange) added when indicated. Shown is a representative of three patients and three healthy controls, from three experiments. (B) Flow cytometric quantification of dead and dying cells by Annexin V and PI staining, after collagenase treatment to recover unstained cells. Normal human T cells were transfected with either DOCK8 (open symbols) or nonspecific (NS) siRNA (filled symbols), and allowed to migrate in collagen matrices (solid line) or medium (dotted line) for the indicated times. Shown is a representative of three experiments. (C) Similar analysis as for B, after migration in medium or collagen for 24 h, of NK cells from six Dock8-deficient (KO) or control (WT) mice from three experiments. (D) Percentages of time spent elongated, for those Dock8-deficient T cells that died or remained alive after migrating in collagen matrix for 16 h. (E) Similar to D, except that duration of elongation episodes was measured for each single episode of elongation that immediately preceded the cell fragmenting (F), or for all other elongation episodes (NF). Bar, 20 µm. Three patients were tested in D and E from three experiments. C–E show means ± SD. Two-way ANOVA was performed for C. Unpaired Student’s t test was performed for D and E. Statistical significance indicated by *, P < 0.05; ***, P < 0.001; ns, nonsignificant.