Fig 3. EBV-specific Ab-dependent removal of B cell-bound VP by NK cells is an active process independent of perforin and proteases.

(A, C-G) B cells were pretreated with AKBM VP (A, C, E) or with B95.8 VP (D, F, G), stained after washing with anti-gp350 and cultured with NK cells together with EBV S- or EBV S+ sera for 4h. (A) NK cells were pre-incubated for 2h with 50nM Dasatinib or vehicle (DMSO), or for 30 min with EDTA (20mM) or EGTA (10mM) and the same concentrations were maintained during the co-culture. The proportions of gp350⁺ B cells detected in three (Dasatinib, EDTA) or two experiments (EGTA) are shown. (B-D) NK cells were pre-treated or not for 2h with 1μM of Concanamycin A, 50μM of Chloroquine or vehicle (DMSO) and co-cultured with 721.221 with or without 100ng/ml of Rituximab (B), or with AKBM VP- (C) or B95.8 VP-coated (D) B cells; the same concentrations of inhibitors were maintained during co-cultures. Proportions of DAPI+ 721.221 cells (B) or of gp350⁺ B cells (C, D) from four (B) or three experiments (C, D) are shown. (E) Proportions of CD16^low-neg cells detected among CD56^dim NK cells from six experiments are shown. Statistical analysis was performed with Wilcoxon test. (F-G) NK cells were pre-treated or not for 2h with 25μM of GM6001, protease inhibitor cocktail of 200μM Leupeptin, 8μM Aprotinin, 15μM Pepstatin, 25μM GM6001 and 10μM Amastatin or the equivalent volume of vehicle (DMSO or H₂O plus DMSO). B and NK cells were co-cultured for 4h in the presence of EBV S- or S+ sera, with or without fresh inhibitor or vehicle at the same concentration used for pre-treatment. Proportions of CD16^low-neg cells within CD56^dim NK cells (F) and of gp350⁺ B cells (G) are shown. Representative plots of the indicated conditions (left) and results of four experiments (right) are shown. (H) B cells were incubated with B95.8 VP, stained for gp350 and cultured alone or with NK cells in the presence of EBV S-, S+ or Rituximab (25ng/ml) for 4h. The proportions of gp350⁺ B cells were analyzed. Representative plots (left) and results of four experiments are shown. For statistical analysis Friedman test with Dunn’s post-test was applied to the last 3 columns.