EBV S+ induces VP transfer from B to NK cells.

(A) B cells were coated with B95.8 VP, stained for gp350 and incubated for 4h with NK cells in the presence of EBV S-, S+ serum or 25ng/ml of Rituximab. Proportions of gp350+ NK cells detected in representative plots (left) or four experiments (right) are shown. Statistical analysis with Friedman test and Dunn’s post-test was applied. (B) B cells were incubated with AKBM SN, stained for gp350, co-cultured for 4h with NK cells in the presence of sera from various EBV S- (n = 5) or S+ (n = 6) donors, and analyzed for the proportions of gp350+ NK cells. (C-D) B cells were treated and co-cultured with NK cells as in (A), in the presence of EBV S- or S+ sera. (C) A gp350 fluorescence index was calculated for B and NK cells multiplying the percentage of the population by the corresponding gp350 MFI; the absolute variation (|Δ|) of the index comparing conditions with EBV S- and S+ was calculated. For each experiment, the absolute variation of the index in NK cells was expressed as a percentage of that calculated for B cells. (D) For each experiment, MFI of all live cells in the co-culture with EBV S+ was expressed as a percentage of that calculated for EBV S- co-cultures. In C-D results of eight experiments are shown. (E-G) B cells were incubated with B95.8 VP (E, F) or AKBM VP (G), stained for gp350 and co-cultured with NK cells, in the presence of EBV S- or S+ for 4h (E) or during the indicated times (F, G). The proportions of gp350+ B (F, G; left) and NK cells (E-G, right) corresponding respectively to four (E) and three (F, G) experiments are shown. Statistical analysis was performed with Friedman test with Dunn’s post-test. (H-I) B cells coated with B95.8 VP were cultured alone or with NK cells in the presence of EBV S- or S+ serum for 4h, followed by separation of B and NK cells. DNA was extracted from both cell fractions and analyzed by qPCR for the EBV gene EBNA1 and the endogenous gene 36B4. Relative quantification of EBNA1 in relation to 36B4 for B (H) and NK cells (I) was calculated and normalized to the first condition of each graph. Data correspond to three experiments.