Most B cells that have switched surface immunoglobulin isotypes generate clones of cells that do not secrete IgM

The order of genes coding for the constant regions of immunoglobulin heavy chains is 5'-C mu, C delta, C gamma 3, C gamma 1, C gamma 2b, C gamma 2a, C epsilon, C alpha-3'. To try to determine whether switching of the expression of heavy chain genes is irreversible and occurs in a direction from 5' to 3' within a cell line, we have isolated B cells bearing particular membrane isotypes, stimulated them to generate clones, and assayed daughter cells for secreted antibody. B cell with and without surface IgM and IgD, and B cells with and without surface IgG, were isolated by fluorescence-activated cell sorting. Fractionated cells were individually stimulated with phosphorylcholine in a splenic fragment assay, and the antibody secreted by clonal progeny was assayed for several heavy chain isotypes by radioimmunoassay. The results show that 1) B cells bearing IgM and IgD can produce individual clones of cells secreting 3 isotypes of anti-phosphorylcholine antibody--IgM, IgG, and IgA; 2) most B cells expressing surface IgG generate clones that secrete IgG but not IgM; and 3) most B cells from gut-associated lymphoid tissue that do not express surface IgM or IgD generate clones of cells that secrete IgA but not IgM. Therefore, IgM- and IgD-bearing cells can give rise to progeny that secrete IgM, IgG, and IgA. Once B cells switch to a different surface isotype, they appear restricted in isotype potential, since they frequently generate clones of cells that do not secrete IgM. These findings suggest that isotype switching is generally irreversible, and isotypes are expressed in a 5'- to -3' direction that is consistent with the germline gene order.