TABLE 1.

Immunoglobulin Class Produced by Surface Isotype-Selected and EBV-Transformed B Lymphocytes

Surface markers for B cell selection	Percentage of B cells producing			Percentage of B cells immortalized
	IgG	IgA	IgM
B1 (CD20)	5.4 ± 1.5	5.4 ± 1.0	86.3 ± 4.1	2.1 ± 0.3
γ heavy chains	99.6 ± 0.7	0.1 + 0.2	0.3 ± 0.3	3.3 ± 0.4
α heavy chains	0.5 ± 0.3	98.6 + 0.9	0.9 ± 0.4	2.8 ± 0.3
μ heavy chains	0.5 ± 0.2	0.3 + 0.2	99.6 ± 0.8	4.0 ± 0.5

Note. Mononuclear cells were obtained from healthy subjects (NIH Blood Bank, Leukopheresis Research Program, NIH, Bethesda, MD) and depleted of monocytes and T lymphocytes. The residual enriched B lymphocyte fraction contained approximately 60% B cells. 2 × 10⁷ enriched B cells (5 × 10⁶/ml) from three different subjects were incubated for 1 h in ice-chilled sterile Hanks’ balanced salt solution without Ca²⁺ and Mg²⁺, without phenol red, with 1% bovine serum albumin (BSA–HBSS), and containing appropriate amounts FITC mouse monoclonal antibody to CD20 or of FITC goat F(ab′)₂ fragment to human γ, α, or μ Ig heavy chains. A negative control sample of similar B cells (10⁶) from each subject was simultaneously incubated with BSA–HBSS containing FITC goat F(ab′)₂ fragment with irrelevant binding activity and allowed to react under similar conditions. After washing with cold BSA–HBSS, cells from all samples were resuspended at 2×10⁶/ml in the same medium and applied at different times to a Model 440 FACS equipped with an argon laser (Becton and Dickinson Immunocytometry Systems, Mountain View, CA). Cells that had been sorted for γ, α, or μ Ig were then infected with EBV and cultured at 1000, 500,100, 50, 20, 10, 5, and 2.5 cells/well in 96-well plates in the presence of 10⁵ irradiated (1800 rad) peripheral blood mononuclear cells as feeders. After 4 weeks, culture fluids were assayed for total Ig content and specific classes (IgG, IgA, and IgM) of Ig using ELISA. Standard reference curves were constructed using purified IgG, IgA, or IgM of known concentration. Wells containing more than 10 ng/ml of IgM, IgG, or IgA were scored as positive. Twenty-five to thirty percent of B cells that had been sorted for surface CD20 or specific Ig heavy chain isotype produced Ig after EBV infection. The proportion of lymphocytes producing Ig of various classes is expressed as a percentage of total Ig-producing B cells. Numbers are mean values ± standard deviation of three experiments using lymphocytes from different donors. Fraction of B lymphocytes producing Ig was calculated by limiting dilution methodology and analysis according to Poisson distribution. Forty-eight microculture wells were used for each cell dose. The frequency of immortalized B lymphocytes was determined by limiting dilution and analysis based on Poisson distribution after 6 weeks of culture.