Cytotoxic ability and IFN-γ production of NK cells from MM patients. (A) NK cells isolated from PB of MM patients or HDs were cocultured with allogeneic primary MM cells or U266B1myeloma cell line at an E/T ratio 1:1 for 6 hours. (Left) Degranulation activity was assessed by evaluating the percentage of CD107a⁺ NK cells. One representative experiment of 8 is shown. Data from all the experiments are summarized in the bars as mean ± SEM of CD107a⁺ cells. (Right) IFN-γ production by NK cells was analyzed by flow cytometry. One representative experiment of 8 is shown. Results of all experiments performed are summarized in the bars as mean ± SEM of IFN-γ⁺ cells. (B) NK cells isolated from PB of MM patients or HDs were cocultured with U266B1 at an E/T ratio 1:1 for 3 hours. Killing capability of NK cells was assessed by evaluating target cell death on coculture. U266B1 target cells are gated as PKH26⁺ events and, following coculture with NK cells, dead cells were identified as positive for the viability dye 7AAD. One representative experiment out of 5 is shown. Results of all experiments performed are summarized in the bars as mean ± SEM of target cell death. (C) Degranulation ability of patient-derived NK cells against autologous or allogeneic MM cells. Bars show data from 10 independent experiments and represent mean ± SEM of CD107a⁺ cells. MM cells used for the degranulation assay were further analyzed for the expression of total HLA class I (clone W632) by flow cytometry. Histograms show data from a MM representative patient compared with healthy B plasma cells isolated from human tonsil. (D, top) Degranulation ability of patient-derived NK cells was analyzed against autologous MM cells in the presence either of an anti-HLA-I blocking antibody (clone A6-136, IgM) or an isotype-matched irrelevant IgM (IgM ctrl). One representative experiment is shown and numbers in each gate represent the percentage of CD107a⁺ cells. Data from 6 independent experiments are summarized in the bars and presented as mean ± SEM of CD107a⁺ NK cells, Student t test. (D, bottom) The same experiment but in relation to CD94 expression on NK cells. *P < .05, **P < .01, ***P < .001; Student t test.