BCR⁺ but not BCR⁻ germinal center B cells infected with LMP2A⁻ 2525 EBV enter the S phase of the cell cycle and continue to proliferate in vitro. (A) Primary lymphocytes were isolated from nasal adenoids. The BCR^+/− fraction consisted of approximately 30% immunoglobulin surface-negative cells and more than 60% immunoglobulin light chain–positive B cells. The BCR⁻ fraction consisted of more than 90% CD19⁺, light chain–negative B cells. (B) Both fractions were infected with virus stocks of LMP2A⁺ 2190 EBV or LMP2A⁻ 2525 mutant virus and analyzed for forward and sideward scatter characteristics to identify cells in the lymphocyte gate R1 4 days after infection. (C) BCR^+/− B cells readily proliferated when infected with either virus (top 2 panels), but cell cycle entry of BCR⁻ B cells was observed with LMP2A⁺ but not with LMP2A⁻ mutant EBV (bottom 2 panels). Total cells were analyzed for cell-cycle distribution 4 days after infection by dual parameter flow cytometry with an APC-conjugated α-BrdU antibody to detect incorporation of the nucleotide analog BrdU and with 7-AAD to reveal the cellular DNA content as described.¹⁹  (D) Cell surface expression of λ and κ light chains of cells as in panel A. Ten days after infection, EBV growth-transformed BCR^+/− B cells infected with LMP2A⁺ 2190 EBV (top panel) maintained the typical initial distribution of BCR⁺ (>60% of λ or κ light chain staining) and BCR⁻ (approximately 30%) primary B cells as in panel A. In contrast, BCR⁻ B cells were absent 10 days after infection when the same initially heterogeneous BCR^+/− fraction was infected with the LMP2A⁻ 2525 virus mutant as indicated by the loss of λ- and κ-positive cells in the lower left quadrant (middle panel). The majority of BCR⁻ cells infected with LMP2⁺ 2190 EBV maintained their immunoglobulin surface-negative status (bottom panel). No cells survived when BCR⁻ B cells were infected with LMP2A⁻ 2525 mutant EBV. (E) Cell-cycle distribution and immunoglobulin light chain surface expression of cells were analyzed.