Allelic exclusion of membrane but not secreted immunoglobulin in a mature B cell line

Abstract

Although many B lymphocytes contain two completely rearranged immunoglobulin (Ig) heavy (H) chain genes, only one of the alleles specifies a protein product. This phenomenon is termed allelic exclusion and is controlled in part by the membrane portion of Ig H chains. We have identified a mature B cell line derived from murine bone marrow that expresses two H chain proteins, gamma 3 and gamma 2b. Proteins of appropriate size for the membrane and secreted forms of both H chains are produced and both IgG3 and IgG2b are secreted from the cells. However, only IgG3 is expressed on the membrane. Detailed restriction mapping of the H chain genes indicates that both are rearranged, one containing a VHJ558 gene linked to the gamma 3 constant region and the other a VHS107 gene linked to the gamma 2b constant region. Primer extension sequencing of the RNA reveals that the gamma 2b RNA is transcribed from the allele containing VHS107 sequences while the gamma 3 RNA is transcribed from the allele containing VHJ558 sequences. Thus, this mature B cell line secretes two distinct antibody molecules but is allelically excluded at the level of surface Ig expression. This cell line provides a model system to study factors, in addition to aberrant rearrangement, that influence allelic exclusion.

Expression of individual immunoglobulin genes occurs in an unusual system consisting of multiple independent loci

“…Dual expression of different IgNAR isoforms, a third class of Ig that does not associate with light chain, occurs in~4% of cells in shark [16]. The expression of multiple IgH transcripts in a small but significant number of individual cells is expected, as haplotypic exclusion of heavy-chain genes (such as in mammals), even at the level of translation and surface expression, is not an absolute characteristic of either B or T lymphocytes [17,18]. Co-expression of IgM and IgG has been described in transgenic mice [19], as well as in a murine B lymphoma cell line [2].…”

Moloney leukemia virus immortalizes B lymphocytes in vitro

“…Similar to the B-cell lines derived with Ab-MLV (15,18), these cell lines contained between three and eight copies of integrated Mo-MLV that could be detected with a 500-bp BstXI-BamHI probe from the Mo-MLV env region (bases 6032 to 6537) (data not shown). This probe does not hybridize to endogenous viruses under stringent conditions (15).…”

“…Similar to the B-cell lines derived with Ab-MLV (15,18), these cell lines contained between three and eight copies of integrated Mo-MLV that could be detected with a 500-bp BstXI-BamHI probe from the Mo-MLV env region (bases 6032 to 6537) (data not shown). This probe does not hybridize to endogenous viruses under stringent conditions (15). In addition, like the Ab-MLV-derived cell lines (18), all of the Mo-MLV-derived cell lines tested were LPS dependent for "Cultures containing 2 x 106 nucleated bone marrow cells were treated with 100 ,ug of LPS per ml as indicated, infected with Ab-MLV or Mo-MLV, and monitored for the appearance of lymphocyte cell lines as described previously (18).…”

“…Serial twofold dilutions of the RNA were applied to a nitrocellulose filter (Schleicher & Schuell, Keene, N.H.), and the filter was processed and hybridized with a v-abl probe according to the manufacturer's suggestions (9) (B). The filter was stripped and reprobed with a c-myb probe obtained from pgt- [11][12][13][14][15] (2) (a gift of T. Bender, University of Virginia) (A). RNA prepared from the nonadherent lymphocytes (lanes 2 and 5) and stromal cells (lanes 1 and 4) from two independent cultures of B cells, RNA from two other established B-cell lines (lanes 3 and 8), and RNA from the Ab-MLV-transformed pre-B-cell line 298-18 grown in the presence (lane 7) and absence (lane 6) of LPS were analyzed.…”