Figure 6

Proliferating CD23^hiCD58⁺ cells and non-proliferating CD23^loCD58⁺ cells express EBV latency genes. A. EBV-exposed B cells were FACS-sorted on day 4 into regions R1 (CD23^loCD58^-), R2 (CD23^loCD58⁺), and R3 (CD23^hiCD58⁺). Sorting strategy and purity of each population are shown. Regions were drawn with spaces in between to prevent contamination between regions. B. The relative transcript levels of latency genes EBNA1, LMP1, and EBNA2 in each of the three sorted sub-populations were determined by real-time reverse transcription-PCR (qRT-PCR) with gene-specific primers. RNA from already immortalized cells (LCL) from the same subject was used as positive control while RNA from un-infected PBMC from the same subject was used as negative control. C. EBV-exposed B cells were harvested on day 3 followed by staining for CD23 (PE), CD58 (PE-Cy7), and intracellular LMP1 (FITC) and flow cytometry. Expression of LMP1 in cells gated on R1, R2, and R3 is shown. Percentages represent fractions of gated cells expressing LMP1 or detected by the corresponding isotype control antibody. D. Sorted CD27⁺ memory and CD27^- naïve B cells as in the experiment shown in Fig. 5B were exposed to EBV, harvested on day 5, and examined for expression of intracellular IL6 (APC) and LMP1 (PE). Numbers represent percentages of CD27⁺ memory or CD27^- naïve B cells.