B G Van Ness

Initiation and processing of two kappa immunoglobulin germ line transcripts in mouse B cells.

The splicing patterns and sequences of two processed kappa immunoglobulin germ line mRNAs are presented. A 1.1-kilobase (kb) mRNA appeared to be derived from splicing of the previously characterized 8.4-kb germ line transcript, while a 0.8-kb mRNA was the splice product of a second 4.7-kb germ line transcript that initiated 50 base pairs upstream of J kappa 1. The interaction of the two kappa germ line promoters with nuclear binding factors is also examined. The potential role of these germ line transcripts in establishing the rearrangement potential of the locus is discussed.

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed.

Identification of a germ line transcript from the unrearranged kappa gene in human B cells

A novel kappa immunoglobulin-hybridizing mRNA in cell lines derived from human B cells arrested at several stages of development has been identified. Hybridization studies demonstrate that this 1.5-kilobase mRNA species is the spliced product of a precursor germ line transcript initiating upstream of the unrearranged JKappa locus.

Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3 J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

In vitro splicing of kappa immunoglobulin precursor mRNA

The in vitro splicing of kappa immunoglobulin precursor mRNA was studied as an example of a naturally occurring mRNA possessing multiple 5 splice sites. Several kappa mRNAs were generated in vitro by using an SP6 transcription system and were spliced in nuclear extracts derived from HeLa cells. Products and intermediates resulting from in vitro splicing were identified and characterized. In contrast to the in vivo situation, in which apparently only the 5-most splice donor site is used, all of the 5 splice sites were used in vitro with equal frequency. Neither the presence or absence of variable region coding sequences nor the deletion of intron sequences had an effect on in vitro splice site selection.

Fc-specific suppressor T cells.

BALB/c mice bearing IgA-secreting plasmacytomas exhibit large numbers of Thy-1+, Ly-2+ lymphocytes with surface membrane receptors specific for IgA (Ta cells) (1). These lymphocytes have several characteristics of immunoregulatory T cells (2) and develop in response to the high circulating levels of polymeric IgA as evidenced by: a) their failure to develop in mice bearing IgA non-secreting variant plasmacytomas (1) and (b) their occurrence in normal mice infused daily with high concentrations of polymeric IgA (3). When suppressor Ta cells were transferred to orallyimmunized mice the recipient mice exhibited a marked suppression of the IgA component, but not the IgM or IgG components, of an immune response to an orally administered antigen (4). These findings identify an immunoregulatory circuit in which IgA secreting cells are simultaneously effector cells and regulatory cells. Secreted IgA antibody, in addition to its antigen-specific effector function, can provide an inductive signal for the IgA-binding suppressor T cells which in turn can inhibit further IgA secretion. Enhanced expression of IgA receptors on suppressor T cells requires polymeric forms of IgA which suggests that IgA-Fc receptor cross-linking is a necessary step in the induction process. Under physiological conditions it may be that receptor crosslinking is mediated by the polyvalent arrays of Fc determinants that occur in immune complexes.