An intronic silencer regulates B lymphocyte cell- and stage-specific expression of the human complement receptor type 2 (CR2, CD21) gene.

Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from -5 kb to +75 bp (-5 kb/+75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (-5-kb/+75) construct resulted in appropriate reporter gene expression, defined as the silencing of the (-5-kb/+75) promoter activity only in non-CR2-expressing cells. Interestingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity.

Defective B cell clonal regulation and autoantibody production in New Zealand black mice.

By using the splenic fragment assay in a KLH-primed host, we have evaluated the clonal anergy model of tolerance in DBA/2 and spontaneously autoimmune NZB mice. Unlike immature B cells from DBA/2 mice (which are tolerized by encounter with TNP-OVA), SIg- B cells from NZB mice respond to TNP-KLH with equal precursor frequency in TNP-OVA-tolerized or control fragments. In additional experiments, SIg- bone marrow or mature spleen cells of DBA/2 or NZB origin were adoptively transferred into irradiated (DBA/2 X NZB) F1 X xid hosts, and host-derived splenic fragments were stimulated in vitro with LPS and growth factors. These experiments revealed a substantial anti-ssDNA precursor frequency in NZB marrow and spleen (2.5 and 5.1, respectively, per 10(7) transferred cells). In DBA/2 SIg- marrow cells, there was an anti-ssDNA precursor frequency of 1.3 to 3.5/10(7) transferred cells; however, anti-ssDNA-producing clones were reduced in fragments derived from recipients of DBA/2 as compared with NZB spleen cells (0.2 to 1.9/10(7) transferred cells). By using a replica plate technique, we evaluated fragments from recipients of DBA/2 SIg- marrow cells or mature spleen cells for anti-TNP reactivity. In fragments derived from recipients of DBA/2 SIg- marrow cells, 92% of anti-TNP-producing fragments also bound ssDNA. In fragments derived from recipients of DBA/2 spleen cells, only 43% of anti-TNP-producing fragments also bound ssDNA. Our findings document that NZB marrow-derived immature B cells abnormally resist tolerance induction, and that clonal anergy/selection operates in directing the B cell repertoire away from autoantibody formation.