Competition for BLyS-mediated signaling through Bcmd/BR3 regulates peripheral B lymphocyte numbers

Striking cell losses occur during late B lymphocyte maturation, reflecting BcR-mediated selection coupled with requisites for viability promoting signals. How selection and survival cues are integrated remains unclear, but a key role for B lymphocyte stimulator (BLyS(TM); trademark of Human Genome Sciences, Inc.) is suggested by its marked effects on B cell numbers and autoantibody formation as well as the B lineage-specific expression of BLyS receptors. Our analyses of the B cell-deficient A/WySnJ mouse have established Bcmd as a gene controlling follicular B cell life span, and recent reports show Bcmd encodes a novel BLyS receptor. Here we show that A/WySnJ B cells are unresponsive to BLyS, affording interrogation of how Bcmd influences B cell homeostasis. Mixed marrow chimeras indicate A/WySnJ peripheral B cells compete poorly for peripheral survival. Moreover, in vivo BrdU labeling shows that (A/WySnJ x BALB/c)F(1) B cells have an intermediate but uniform life span, indicating viability requires continuous signaling via this pathway. Together, these findings establish the BLyS/Bcmd pathway as a dominant mediator of B cell survival, suggesting competition for BLyS/Bcmd signals regulates follicular B cell numbers.     

A quantitative-trait locus controlling peripheral B-cell deficiency maps to mouse Chromosome 15

Peripheral B-lymphocyte homeostasis is determined through incompletely defined positive and negative regulatory processes. The A/WySnJ mouse, but not the related A/J strain, has disturbed homeostasis leading to peripheral B-lymphocyte deficiency. B lymphopoeisis is normal in A/WySnJ mice, but the B cells apoptose rapidly in the periphery. This B cell-intrinsic defect segregated as a single locus, Bcmd, in (A/WySnJxA/J)F2 mice. Here we mapped a quantitative-trait locus (QTL) that contributes to the A/WySnJ B-cell deficiency by examining the F2 progeny of a cross between strains A/WySnJ and CAST/Ei. In this cross, minimally 1.9 QTLs controlling peripheral B lymphocyte deficiency segregated. The (A/WySnJxCAST/Ei)F2 mice were phenotyped for splenic B-cell percentage and the DNA from progeny with extreme phenotypes was used to map the QTL by the simple-sequence length polymorphism method. A genome scan showed linkage between peripheral B-cell deficiency and Chromosome (Chr) 15 markers. When closely spaced Chr 15 markers were analyzed, the 99% confidence interval for the QTL map position extended along the entire chromosome length. The peak lod scores >17 occurred between 30 and 45 cM. We conclude that a significant QTL segregating in (A/WySnJxCAST/Ei)F2 mice resides in this middle region of Chr 15.     

Cutting edge: B cell receptor signals regulate BLyS receptor levels in mature B cells and their immediate progenitors

This study examines how B lymphocyte stimulator (BLyS) receptor expression and responsiveness are influenced by B cell receptor (BcR) signaling. Our results show that resting and BcR-stimulated B cells are dependent on BLyS for survival and that B cells remain BLyS responsive during BcR-induced activation. Further, BcR ligation up-regulates expression of the BLySR B cell maturation defect/BLySR3 (Bcmd/BR3), but not other known BLySRs. Finally, the coupling of BcR signaling with Bcmd/BR3 expression is limited to late transitional and mature B cells. Together, these findings establish the coupling of BcR signaling with Bcmd/BR3 expression as a fundamental aspect of follicular B cell selection, survival, and activation.     

Enforced bcl-xL gene expression restored splenic B lymphocyte development in BAFF-R mutant mice

The TNFR family member BAFF-R facilitates peripheral B cell development, although it is unclear whether it promotes survival of B cells, or also initiates a differentiation program. We show that disruption of the BAFF-R encoding gene Tnfrsf13c in strain A/WySnJ mice causes a progressive decline in peripheral B cell numbers, beginning at the transitional 1 developmental stage and continuing through the mature peripheral B cell stage. Bcl-x(L) overexpression in A/WySnJ B cells decreased the turnover of transitional B cells, as determined by 5-bromo-2'-deoxyuridine labeling, and restored follicular B cell development. We conclude that the mutant A/WySnJ allele of Tnfrsf13c can be complemented through the survival signal provided by Bcl-x(L).     

Chromosomal localization of phospholipase A2 activating protein, an Ets2 target gene, to 9p21

A murine Ets2 target gene isolated by differential display cloning was identified as the phospholipase A2 activating protein (PLAA) gene. A 2.7-kb human cDNA demonstrating high homology to mouse and rat Plaa genes was then isolated and characterized. Human PLAA contains six WD-40 repeat motifs and three different protein kinase consensus domains. Fluorescence in situ hybridization (FISH) mapping placed PLAA on chromosome 9p21, a region frequently deleted in various cancers. A comprehensive mapping strategy was employed to define further the chromosomal localization of PLAA relative to CDKN2A within the 9p21 locus. Radiation hybrid mapping placed the gene 7.69 cR from WI-5735 (LOD >3.0), a marker in close proximity to CDKN2A and CDKN2B. Yeast artificial chromosome (YAC) mapping localized PLAA proximal to the CDKN2A/CDKN2B genes and to a region flanked by D9S171 and INFA commonly deleted in many neoplasms. Two YACs contained both PLAA and D9S259, a marker present in a second more proximal minimal deleted region observed in cutaneous melanoma and squamous cell lung carcinoma. Double-color fiber FISH mapping confirmed the location of PLAA centromeric to D9S171 and CDKN2A/CDKN2B. The mapping data suggest a possible tumor suppressor role for this gene.     

Isolation and characterization of a novel human gene (NESH) which encodes a putative signaling molecule similar to e3B1 protein

Using a conventional cloning technique, a novel full-length cDNA was isolated and sequenced from a human placental cDNA library. This cDNA consists of 2129 bp and has a predicted open reading frame encoding 366 amino acids. It possesses a Src homology 3 (SH3) motif, proline-rich region, serine-rich region and no catalytic domain, suggesting that it seems to be a signaling protein most similar to e3B1, an eps8 SH3 binding protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 17q21.3 near the marker D17S1795.     

Localization of human SAA gene(s) to chromosome 11 and detection of DNA polymorphisms

A human serum amyloid A (SAA) cDNA was used as a probe in chromosome mapping studies to detect human SAA gene sequences in DNA isolated from human/mouse somatic cell hybrids. Southern analysis of DNA from 20 hybrid cell lines, including some with translocations of human chromosomes, placed the SAA gene(s) in the p11----pter region of chromosome 11. Screening of human DNA from unrelated individuals by Southern analysis using the SAA cDNA probe revealed restriction fragment polymorphisms for HindIII and PstI. An analysis of the segregation of these polymorphisms with other markers on the short arm of chromosome 11 should more precisely map the SAA gene(s).     

Genetic mapping of the gene for a novel tyrosine kinase, Blk, to mouse chromosome 14

The gene Blk, which encodes a novel tyrosine kinase expressed preferentially in B-lymphoid cells, was mapped by Southern blot analysis of DNA from the progeny of an intersubspecific backcross. Blk maps to the proximal region of chromosome 14 with the gene order centromere--(Np-1,Tcra)-Blk-sys-Es-10.     

Characterization and expression of the human leukocyte-common antigen (CD45) gene contained in yeast artificial chromosomes.

The leukocyte-common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed uniquely by cells of hematopoietic origin. There are multiple isoforms of CD45 that are generated by the variable use of three exons (exons 4-6). The use of the variable exons results in changes near the amino-terminus of the mature glycoprotein. The gene is located on chromosome 1 for both human and mouse in a region that is homologous between these two species. This conserved linkage group contains a number of genes of immunological interest, such as the genes for complement regulatory proteins and the FCG2 receptor. Yeast artificial chromosomes provide a vector system in which large fragments of foreign DNA can be isolated and are suited to long-range physical mapping. To this end, three yeast artificial chromosomes containing the human CD45 gene have been isolated and characterized. They overlap to span 475 kb, establishing the largest physical map for DNA within the conserved linkage group. The CD45 gene is entirely encoded within one yeast artificial chromosome clone as determined by mapping with cDNA probes. A mouse B cell line transfected with this YAC clone expressed the low-molecular-weight isoform of the protein into the cell surface. The size of the human CD45 gene was determined to be approximately 120 +/- 10 kb.     

TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r(-/-) mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r(-/-) mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r(-/-) B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r(-/-) mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Human chromosome 3 mediates growth arrest and suppression of apoptosis in microcell hybrids.

Chemotherapeutic treatment of tumor cells leads either to tumor cell death (usually by apoptosis) or to the formation of drug-resistant subpopulations. Known mechanisms of cancer cell drug resistance include gene amplification and increased expression of drug transporters. On the other hand, normal cells survive many forms of chemotherapy with minimal damage probably because of their capacity for growth arrest and stringent control of apoptosis. Microcell hybrids between B78 (murine melanoma) and HSF5 (normal human fibroblasts) were analyzed to identify a new human chromosomal region involved in the promotion of drug-induced growth arrest and suppression of apoptosis. In these hybrids, the presence of human chromosome 3 was strongly associated with suppression of apoptosis via G1 and G2 growth arrest during exposure to the antimetabolite N-phosphonoacetyl-L-aspartate (PALA), suggesting that a gene(s) on chromosome 3 serves an antiproliferative role in a drug-responsive growth arrest pathway.     

Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic β-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.     

Regional assignment of the human acid sphingomyelinase gene (SMPD1) by PCR analysis of somatic cell hybrids and in situ hybridization to 11p15.1----p15.4

Human acid sphingomyelinase (SMPD1) is the lysosomal phosphodiesterase that cleaves sphingomyelin to ceramide and phosphocholine. The deficient activity of SMPD1 is the enzymatic defect in Types A and B Niemann-Pick disease. Previously, the gene encoding human SMPD1 was assigned to chromosome 17 by the differential thermostability of human and hamster SMPD1 in somatic cell hybrids. The recent isolation of the human SMPD1 cDNA (L. E. Quintern, E. H. Schuchman, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, 1989, EMBO J. 8: 2469-2473) permitted the mapping of this gene by molecular techniques. Oligonucleotide primers were synthesized to PCR amplify the human, but not murine, SMPD1 sequences in man-mouse somatic cell hybrids. In a panel of 15 hybrid cell lines, amplification of the human SMPD1 sequence was 100% concordant with the presence of human chromosome 11. For each of the other human chromosomes there were at least 6 discordant hybrid lines. Further analysis of somatic cell hybrids containing only chromosome 11 or chromosome 11 rearrangements localized the human SMPD1 gene to the region 11p15.1----p15.4. To provide an independent regional gene assignment, in situ hybridization was performed using the radiolabeled human SMPD1 cDNA. In the 58 metaphase cells examined, 34% of the 122 hybridization sites scored were located in the distal end of chromosome 11 with the major peak of hybridization at band 11p15. The absence of any other in situ hybridization site indicated the absence of pseudogenes or homologous sequences elsewhere in the genome. In contrast to the previous provisional localization to chromosome 17, these results assign a single locus for human SMPD1 to 11p15.1----p15.4.     

Human zinc finger gene ZNF23 (Kox16) maps to a zinc finger gene cluster on chromosome 16q22, and ZNF32 (Kox30) to chromosome region 10q23-–q24

Two members of the KOX gene family, ZNF23 (KOX16) and ZNF32 (KOX30), have been mapped by in situ hybridization to chromosome regions 16q22 and 10q23-q24, respectively. The map location of ZNF23 and ZNF32 placed these zinc finger protein genes near to chromosome loci that, under certain in vitro conditions, are expressed as fragile sites (FRA16B, FRA16C) and (FRA10D, FRA10A, FRA10B and FRA10E). Human zinc finger gene ZNF32 maps to a chromosome region on 10q23-24 in which deletions have been observed associated with malignant lymphoma on 10q22-23 and with carcinoma of the prostate on 10q24. ZNF23 is located on 16q22 in a chromosomal region that has been involved in chromosome alterations characteristic of acute myeloid leukemia. A second Kox zinc finger gene (ZNF19/KOX12) was recently mapped to the same chromosome region on human chromosome 16q22. In the analogous murine position, the murine zinc finger genes Zfp-1 and Zfp-4 are found in the syntenic 16q region of mouse chromosome 8. Thus, ZNF19 and ZNF23 might be members of an evolutionarily conserved zinc finger gene cluster located on human chromosome 16q22.     

B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF(-/-) and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.     

Characterization and mapping of the Bacillus subtilis prtR gene.

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.