Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.