Frequency of B-lymphocyte transformation by Epstein-Barr virus decreases with entry into the cell cycle

The relationship between in vitro B-cell activation and transformation by Epstein-Barr virus (EBV) was studied. B cells were fractionated using discontinuous Percoll gradients to purify cells with resting morphology. Activation of resting cells for 24 hr with anti-Ig (mu chain specific) or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G1 phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B-cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr with high concentrations of SAC or anti-mu or using BCGF. SAC-activated cells entered S phase on Day 2 and anti-mu treated cells on Day 3. Control (G0) cells and cell activated for varying lengths of time (G0/G1, G1/S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a transformation frequency of 1-2%. With continued activation with SAC or anti-mu, however, transformation frequency decreased at a rate paralleling the entry of the population into S phase. Treating cells with low concentrations of anti-mu or SAC in combination with BCGF decreased the transformation frequency to levels lower than anti-mu or SAC alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation, and that with in vitro activation into the cell cycle B cells become resistant to EBV transformation.