WIP controls CD8⁺T lymphocyte cytotoxicity and actin meshwork ultrastructure at the IS. (A) Flow cytometry analysis of CD4, CD8, and perforin expression in purified CD8⁺ T cells expanded from a healthy donor and the WIP-deficient patient. (B) LAMP-1 expression at the surface of CD8⁺ T cells upon incubation with P815 cells coated with the indicated concentrations of αCD3 antibody. (C) CD8⁺ T-cell cytotoxic activity assessed by counting the residual alive P815 target cells after 4 or 24 hours. (D) Representative pictures of healthy donor CD8⁺ T cells and patient CD8⁺ T cells forming conjugates with P815 cells coated or not with αCD3 antibody. Fixed conjugates were stained for WIP, F-actin, and perforin. (E) The proportion of T cells forming a synaptic contact with P815 cells was assessed from confocal images. At least 300 cells were analyzed per condition. (F) The distance from lytic granules to the T cell–P815 cell interface was calculated from confocal images. (G) Representative pictures of conjugates formed between T cells and DCs precoated or not with a cocktail of superantigens. Cells were isolated from the blood of the mother or the patient post-DLI/HSCT, as indicated. Fixed conjugates were stained for WIP, F-actin, and 4′,6-diamidino-2-phenylindole (DAPI). (H) The distribution of the number of T cells captured per DC is represented for each indicated condition. (I) Reconstructed structured illumination microscopy images of healthy donor and patient CD8⁺ T cells spreading over ICAM-1/αCD3. Staining for WIP, F-actin, and DAPI is shown for a ventral section of 110-nm thickness. Zoomed areas reveal actin meshwork architecture and WIP distribution. (J) The quantification of actin fiber coherency corresponds to the analysis of 20 zoomed areas per condition. DC, dendritic cell. HD, healthy donor. SAg, super-antigens. *P < .05, **P < .01, ***P < .001.