Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

Removal of the activating agents before immortalization significantly improves growth and viability of CD22 + Blymphocytes allowing for a higher proportion of cells to be immortalized. Indeed, when stimulation and EBV infection are performed separately rather than simultaneously, or with no stimulation at all, the cells exhibit increased proliferation potential and improved viability. In contrast, the combined presence of activating agents and EBV has adverse effects on both cell viability and EBV infectivity (Figure 2A , B). The negative effects of the COM-BINED method on viability are surprising, given that CpG2006 induces robust polyclonal activation and proliferation of B-lymphocytes [24, 25] and that these effects are significantly enhanced by IL-2 [26] . It has been reported that TLR-9 triggering resulted in higher transformation rates of B cells infected with EBV [12] but a comparison between EBV transformation in the presence of CpG2006 and EBV and after sequential exposure of B cells to CpG2006 and separately to EBV, was not investigated. The results obtained using different experimental setting on the same cell population clearly demonstrated that CpG2006 and IL-2 used in combination with EBV exert negative effects on cell transformation. It is conceivable that the detrimental effects are related to the anti-proliferative activity of anti-viral cytokines induced by CpG2006 [27, 28] . Indeed, the significant levels of TLR9 expressed by resting B-lymphocytes are further up-regulated by CpG2006 itself. Increased TLR9 expression correlates with increased responsiveness to the ligand, indicating a positive feedback loop in which CpG2006 enhances its own effects [29] . Kinetic studies demonstrated that optimal concentrations of CpG2006 combined with IL-2 provide maximal cell stimulation after 2-5 days (data not shown). These observations are in line with cell-cycle analyses demonstrating that, in the same time frame, more than Quantitative and qualitative comparison of different EBV transformation methods Figure 2 Quantitative and qualitative comparison of different EBV transformation methods. A) The B-lymphocyte subsets were prepared using the SEQUENTIAL, COMBINED or BASIC methods, as outlined in Fig. 1 . Ten days after exposure to EBV, samples of each population were compared for the total cell number (black bars), and for the number of viable cells (white bars) measured microscopically by trypan blue dye exclusion. Data are expressed as % of initial B cells exposed to EBV and represent the means ± s.d. of 5 cell counts for each condition. Results are representative of two independent experiments. P < 0.001: SEQUENTIAL vs. COMBINED; BASIC vs. COMBINED (white bars); P < 0.05: SEQUENTIAL vs. BASIC (white bars) and SEQUENTIAL vs. COMBINED; and BASIC vs. COMBINED (black bars). B) Ten days after infection with EBV, the cells prepared by the SEQUENTIAL, COMBINED, or BASIC method were analyzed to identify viable lymphoblasts by propodium iodide (PI) exclusion (top panels) and CD23 expression (bottom panels) by flow cytometry. For each population of cells, the viable lymphoblasts (gated in regions R2, top panels) were defined as those with relatively high forward scatter (horizontal axes) and negative for PI fluorescence (vertical axes). R2 gated cells were then analyzed for CD23 expression. Fluorescence was analyzed with a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson). Background fluorescence was determined with a