V(D)J recombination in peritoneal B cells of leaky scid mice.

Developing lymphocytes in immune-deficient severe combined immunodeficient (scid) mice express a defective recombinase activity and rarely succeed in making an antigen receptor; those cells that do succeed account for the known B and T cell leakiness in this mutant mouse strain. To gain more insight into the nature of the scid defect, we assessed the status of heavy (H) and light (L)k, chain genes in immunoglobulin (Ig)Mk-secreting B cells from the peritoneal cavity of old leaky scid mice, the only lymphoid site where scid B cells have been routinely detected. We found these cells to be unusual in that their nonexpressed H chain alleles were either abnormally rearranged or in germline configuration (wild-type B cells generally show normal rearrangements at both H chain alleles).

Scid mouse Pre-B cells with intracellular mu chains: analysis of recombinase activity and IgH gene rearrangements.

Four Pre-B cell clones with intracellular mu chains were recovered from individual leaky scid mice by transformation of bone marrow or peritoneal cells with Abelson murine leukemia virus. Three clones were derived from independent bone marrow cell cultures. These express the defective scid recombinase activity and contain truncated mu chains resulting from abnormal and/or incomplete (D to J only) gene rearrangements.

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching.

Carrier-specific T cells sufficient for the expression of multiple isotypes in B cell cultures.

A modified splenic fragment assay was used to assess the role of antigen-specific helper T cells in B cell isotype expression. Limiting numbers of carrier-specific helper T cells from lines or clones were injected along with a source of B cells into lethally irradiated unprimed recipients. The incidence of lodging of the T cell lines in recipient spleens at 18 h was determined by autoradiography to be 1.

CH isotype switching in B cell differentiation.

We have functionally defined a number of B cell subsets that likely represent B cells at different stages of development, based on the pattern of CH isotypes expressed by their clones in splenic fragment or microcultures and on those factors necessary in culture to support the growth of a clone displaying a particular isotype or set of isotypes. Our observations are consistent with isotype switching being a stochastic process which results in the occurrence of progressive isotype restriction in members of a diversifying clone. The surface marker best predictive of the pattern of isotypes a clone may secrete is the sIg isotype of its B cell precursor.