Heparinized samples of PB and BM aspirates (10 ml each) were collected, mononuclear leucocytes were separated and submitted to flow cytometric analyses and functional tests as described previously.[13, 43-45] The Y-27632 presence of EBV DNA was evaluated from the whole blood and BM aspirates using real-time PCR at the Virology Laboratory at Sahlgrenska University Hospital, Gothenburg, Sweden, as previously described.[25] Detection of 10 EBV-DNA copies was sufficient

to stratify a patient as EBV+. The BM and PB cells were prepared and stained for the FACS analysis as previously described.[43, 44] To avoid non-specific binding, cells were pre-incubated with 0·1% rabbit serum for 15 min at room temperature, where after cells were stained with the following monoclonal antibodies used in different combinations: Peridinin Chlorophyll-conjugated anti-CD3 (SK7), eFluor450-conjugated anti-CD19 (HIB19), phycoerythrin-conjugated or FITC-conjugated anti-CD25 (2A3), phycoerythrin- or allophycocyanin-conjugated

anti-CD27 Antiinfection Compound Library cell assay (LI28), allophycocyanin-conjugated CD95 (DX2). All the antibodies were produced in mice and purchased from BD-Bioscience (BD-Bioscience, Erebodegem, Belgium) except for anti-CD19, which was purchased from eBioscience (San Diego, CA). For the immunoglobulin analyses we used FITC-conjugated rabbit anti-IgA (F0057), rabbit anti-IgD (F0059), rabbit anti-IgG (F0056) and rabbit anti-IgM (F0058) antibodies (DakoCytomation, Glostrup, Denmark). Polyclonal rabbit F(ab’)2 anti-human immunoglobulin was used as isotype control. Between 3 × 105 and 1·5 × 106 events were collected using a FACSCanto II equipped with FACS Diva software (BD-Bioscience). Cells were gated based on fluorochrome

minus one setting when needed,[46] and a representative gating strategy is shown in Fig. 2(f). A minimum of 50 cells per gate was used as an inclusion criterion. All analyses were performed using FlowJo software (Three Star Inc., Ashland, OR). B cells were defined as CD19+ CD3−. PtdIns(3,4)P2 CD27 was used as a memory B-cell marker, alone or in combination with IgA, IgD, IgG and IgM. Combination of CD27 and IgD gave four different populations: IgD− CD27− (immature B cells), IgD+ CD27− (naive B cells), IgD+ CD27+ (unswitched memory B cells) and finally, IgD− CD27+ (switched memory B cells and plasmablasts).[47, 48] Mononuclear leucocytes of the PB were stained with Peridinin Chlorophyll-conjugated anti-CD3, eFluor450-conjugated CD19 and phycoerythrin-conjugated CD25 and sorted into CD19+ CD25+ and CD19+ CD25− populations using the FACS-Aria II (BD-Bioscience, San José, CA) as described previously.[49] The purity of these sorted cells was > 97·5%. The viability of the cells was assessed using trypan blue. The sorted cell populations were stimulated for 96 hr with EBV-rich medium (3·6 × 106 copies/culture, kindly provided by the Immunology Laboratory, Sahlgrenska University Hospital, Göteborg, Sweden).