Identification of a WIP-deficient patient and of the role of WIP in lymphocyte chemotaxis. (A) Immune reconstitution and plasma CMV load in the patient after sequential infusion of peripheral blood lymphocytes and stem cells from the HLA-identical mother (sequential DLI/HSCT). Doses were calculated for a target number of T cells (from 1 × 10⁶/kg CD3⁺ to maximum 1 × 10⁷/kg CD3⁺ cells; white triangles) and stem cells (2 × 10⁷/kg CD34⁺ plus 2 × 10⁷/kg CD3⁺ cells; black triangles). Plasma concentration of CMV nucleic acid is shown as copies per milliliter (dotted line, left y-axis). Chimerism of maternal CD3⁺ T cells is plotted as absolute number (solid line, left y-axis) and proportion (gray-shaded area, right y-axis). (B) Electropherogram of Sanger sequencing depicting the homozygous c.373C>T mutation in WIPF1. (C) Western blot analysis of WIP and WASP in EBV–immortalized B cell lysates from 2 healthy donors, the WIP patient and a Wiskott-Aldrich syndrome (WAS) patient. Data were confirmed in 3 independent experiments. (D) Transwell migration of expanded T cells in response to the indicated concentrations of CCL19 and CXCL12. Histograms correspond to the mean ± standard deviation of triplicate values of 1 representative experiment out of 3. (E) Representative pictures of expanded T cells from the patient and a control, following stimulation with CCL19 or CXCL12 and staining for WIP and F-actin. (F) Roundness and aspect ratio values were extracted with ImageJ analysis from 45 to 106 cells per condition. (G) Representative pictures of T cells recovered post-DLI/HSCT, stimulated with CCL19 or CXCL12 and stained for WIP and F-actin. (H) Roundness and aspect ratio values were extracted with ImageJ analysis from 42 to 72 cells per condition. (I) Speed and forward migration index were extracted from the trajectory records of the indicated cells using the Ibidi chemotaxis tool. Data represent 1 representative experiment out of 3. (J) Plots showing correlation between cell speed and forward migration index along the y-axis (chemokine gradient) for the indicated cells. Data represent 1 representative experiment out of 3 performed. (K) Time-lapse video recording of the LifeAct-GFP expressing wt or WIP-KD B cells exposed to a CCL19 gradient, with color plot showing time evolution of cell contour. The volatility values pertain to the representative cells shown in the time-lapse series. (L) Plots representing the cell polarization angle frequency. The CCL19 source is on top. The orientation of the major cell axis was calculated for 400 cell snapshots per condition. (M) Values of actin polarization vector, calculated as the distance between cell and actin centers of mass. The length of the vector was projected along the y-axis to combine actin polarization with cell orientation along the chemokine gradient. Data stem from 400 cell snapshots per condition. DLI, donor lymphocyte infusion; ns, not significant. Pt, patient. *P < .05, **P < .01, ***P < .001.