CD5 expression by B lymphocytes and its regulation upon Epstein-Barr virus transformation

Abstract

Dim expression of CD5 on human B lymphocytes has been used to delineate B1 and B2 subsets. Nevertheless, others have suggested that the molecule is an activation marker and does not predicate a subset distinction. We have used enzymatic amplification staining, a technology that enhances the resolution of flow cytometric analysis of cell surface molecules by as much as 100-fold, to determine that essentially all human B cells express CD5. Furthermore, we show that this expression is regulated during Epstein-Barr virus transformation.

Figures

Figure 1

Human B lymphocytic CD5 expression assessed by EAS and a standard amplification procedure. Peripheral blood cells from five healthy donors were simultaneously stained with anti-CD19 antibodies conjugated to phycoerythrin and with 100 ng of biotinylated anti-CD5 monoclonal antibody for conventional amplification (indirect staining) or with 10 ng of the same antibody for EAS. EAS was performed with a kit from Flow-Amp Systems. Equivalent amounts of isotype control biotinylated murine IgG1 (open histograms) were assessed in parallel with biotinylated anti-CD5 (shaded histograms). Fluorescein isothiocyanate was used as the detecting fluorochrome for both the standard and EAS procedures. CD5 expression on the B cells was assessed in the FL1 channel after gating on the CD19-expressing cells determined in the FL2 channel. Fluorescence in the FL1 channel due to phycoerythrin was eliminated by setting compensation using cells stained for CD19 alone. Similar results were obtained with cells from nine additional healthy donors.

Figure 2

Comparison between CD5 expression by B lymphocytes and neutrophils. Peripheral blood mononuclear cells and peripheral blood neutrophils were obtained by gradient centrifugation. The mononuclear cells were stained with anti-CD19PE to identify B lymphocytes, and the cells were stained simultaneously with either 10 ng of biotinylated control IgG1 (open histogram) or 10 ng of biotinylated anti-CD5 IgG1 (shaded histogram). The B lymphocytes are shown by gating on the lymphocytic population and the CD19-phycoerythrin-positive population. The neutrophils were stained with either 10 ng of biotinylated control IgG1 (open histogram) or 10 ng of biotinylated anti-CD5 IgG1 (shaded histogram). Binding of the biotinylated antibody was detected by EAS with a kit from Flow-Amp Systems.

Figure 3

Absent CD5 expression on EBV-transformed human B lymphocytes. CEM, a human T cell tumor line, two EBV-transformed human B cell lines, JY(LCL) and DR(LCL), and JY(LCL)-CD5, JY(LCL) cells transfected with a recombinant retrovirus encoding CD5 expression, were stained by EAS for CD5 expression by using 5 ng of biotinylated anti-CD5 monoclonal antibody for CEM, 100 ng for JY(LCL)-CD5, 500 ng for JY(LCL) and DR(LCL) (shaded histograms), or equivalent amounts of biotinylated control IgG1 (open histograms). For panels that appear to contain only one histogram, the shaded and open histograms precisely overlap. EAS was performed with a kit from Flow-Amp Systems. Results with equivalent amounts of biotinylated isotype control murine IgG1 are also shown.

Figure 4

Loss of CD5 expression by human B lymphocytes on EBV transformation. Peripheral blood mononuclear cells were obtained from a healthy volunteer, and T lymphocytes and natural killer cells were removed by sheep erythrocyte rosetting. The remaining cells, B lymphocytes and monocytes, were exposed to EBV-containing medium from the B95-8 cell line and assessed serially for both cell surface CD5 expression by EAS, gating on CD19 expressing cells, and intracellular EBV LMP-1 expression. The intracellular antigen expression was assessed by an EAS kit from Flow-Amp Systems. The anti-LMP-1 monoclonal antibody was obtained from Dako, and the validity of this technique was ascertained by staining EBV-positive and EBV-negative cell lines. The top panels show control staining for CD5 on CEM cells and for LMP-1 on JY(LCL) cells. Staining with isotype control antibodies is shown as open histograms, and staining with specific antibodies is shown as shaded histograms. These results are representative of three experiments.