FIG. 4.

EBV replicates in the IgD⁻ CD10⁻ CD20⁺ subset of tonsil B cells. (a) T-cell-depleted tonsil lymphocytes (CD3⁻) were fractionated by MACS into IgD⁺ and IgD⁻ fractions. The IgD⁻ subset was further fractionated by MACS into CD20⁺ and CD20⁻ fractions. The purified cells were analyzed by flow cytometry for purity (top panels). (The T-cell depletion was less efficient for this experiment than for the one shown in Fig. 2.) The CD20⁻ fraction probably consists of contaminating T cells and macrophages but could also include plasma cells that had lost their lineage markers. (b) Cells (10⁶) of each fraction were subjected to serial twofold dilutions, and the dilutions were tested by RT-PCR for expression of the immediate-early gene BZLF1. PCR products were identified by Southern blotting with a gene-specific probe. Tonsil 1 from Table 1 was used in this experiment. (c) CD10⁺ CD20⁺ and CD10⁻ CD20+ tonsil lymphocytes were separated by FACS sorting. (d) The samples were serially diluted, and then multiple replicates were made for each dilution. Each sample was analyzed by RT-PCR (upper panels) for expression of the immediate-early BZLF1 gene. PCR products were identified by Southern blotting with a gene-specific probe. Tonsil 2 from Table 1 was used in this experiment.