Cytotoxic activity of NK cells from cases #1 and #2. (A) Case #1 (4 years after HSCT, showing 8% donor chimerism), his parents and unrelated healthy donor PBMCs were cultured overnight in the absence (left) or presence of 100 IU/ml of recombinant human (rh) IL-2 (right). The cytotoxic activity of NK cells was assessed against K562 target cells using ⁵¹Cr-release assay. Patients’ NK cells show similar activity to his parents and a healthy control despite the loss of donor chimerism. The assay was standardized with respect to the NK-cell percentage in PBMCs (CD3-/CD16+/CD56dim). (B) Case #1 NK cells show normal levels of degranulation as measured by LAMP-1/CD107a externalization in the presence of K562 target cells. (C) Case #2 PBMCs were incubated overnight in the absence or in the presence of 100 IU/mL of rh IL-2, and NK-cell cytotoxicity was assessed by ⁵¹Cr-release assay. The assay was standardized as in panel A. (D) Case #2 NK-cell degranulation was measured by LAMP-1/CD107a externalization using K562 as target cells. (E) Western immunoblotting, performed under reducing conditions, shows reduced MUNC13-4 expression in the isolated T cells from case #2, compared to a healthy donor control. PBMCs, peripheral blood mononuclear cells.