Cutting Edge: A/WySnJ Transitional B Cells Overexpress the 15 Proapoptotic Blk and Succumb to Premature This information is current as of September 28, 2021. Ian J. Amanna, Karen Clise-Dwyer, Faye E. Nashold, Kathleen A. Hoag and Colleen E. Hayes J Immunol 2001; 167:6069-6072; ; doi: 10.4049/jimmunol.167.11.6069

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: A/WySnJ Transitional B Cells Overexpress the Chromosome 15 Proapoptotic Blk Gene and Succumb to Premature Apoptosis1

Ian J. Amanna, Karen Clise-Dwyer, Faye E. Nashold, Kathleen A. Hoag, and Colleen E. Hayes2

stage of peripheral B cell development (4, 5). These features make Better knowledge of peripheral B lymphocyte homeostasis is the A/WySnJ mouse a valuable genetic model for defining regu- needed to address the human hypogammaglobulinemia dis-

latory processes in peripheral B cell development. Downloaded from eases. A defect in the Bcmd gene shortens the B cell life span Our previous genetic analyses placed Bcmd on chromosome and causes B cell deficiency in A/WySnJ mice. Previous genetic (Chr) 15, between D15 Mit259 and D15 Mit118, an empirical dis- mapping placed Bcmd near Srebf2 on chromosome 15. Inspec- tance of ϳ0.8 centimorgans (cM) (6, 7). The human Chr 22 syn- tion of the human chromosome 22 syntenic region identified tenic region (8) contains the Bcl-2 interacting killer (Bik) gene that the proapoptotic Bik gene as a candidate. Two mapping meth- encodes a death-inducing BCL-2 family member (9, 10). In ods placed the homologous mouse gene, Blk, near Srebf2. The human B104 B lymphoma cells, surface IgM ligation increased Bik http://www.jimmunol.org/ Blk genomic structure was highly homologous to Bik. Sequence transcription and triggered apoptosis (11). These two reports sug- analysis ruled out coding region mutations, but Blk transcripts gested that a Bik homolog involved in excessive negative selection were overly abundant in sorted A/WySnJ T1 B cells. More- might be a Bcmd candidate gene. Investigating this hypothesis, we over, enriched transitional B cells showed a cell-autonomous mapped the putative Bik homolog, Bik-like killer (Blk), to Chr 15, defect leading to excessive apoptosis. Thus, Bcmd may be a demonstrated its overexpression in A/WySnJ transitional B cells, direct mutation in Blk, or in a gene involved in Blk regulation, and showed that these transitional B cells have a cell-autonomous such that excess expression pushes the A/WySnJ transitional B defect leading to excessive apoptosis. These results are discussed cells past the apoptosis checkpoint to cell death. The Journal in the context of a second suggested Bcmd candidate gene, provi- of Immunology, 2001, 167: 6069Ð6072. sionally designated Baffr (12). by guest on September 28, 2021 Materials and Methods nowledge of the factors that regulate peripheral B lym- Mice phocyte homeostasis is incomplete. Perturbations of B The production and simple sequence-length polymorphism (SSLP) geno- cell homeostasis can cause hypogammaglobulinemia or typing of (A/WySnJ ϫ CAST/Ei)F ϫ A/WySnJ mice for Chr 15 markers K 1 B cell lymphoma (1). Our discovery of a heritable B cell deficiency was described (7). The mice were housed in our pathogen-free mouse in A/WySnJ mice has afforded us the opportunity to investigate B colony; protocols were approved by the Institutional Animal Care and Use cell homeostasis (2). The A/WySnJ mice have 90% fewer periph- Committee of the University of Wisconsin (Madison, WI). eral B cells than normal and fail to make significant Ig memory Nucleic acid isolation and PCR responses (3). The deficiency is controlled by a single, autosomal, 3 The DNA isolation, PCR, and electrophoresis were described (6). The ex- incompletely dominant gene, B cell maturation defect (Bcmd), pressed region of Blk was cloned and sequenced, and transcript abundance that does not alter lymphopoiesis (3) but leads to premature B cell studies were performed with the PCR primers 5Ј-AGCTCAGCTTGGCAGAA apoptosis at the transitional type-1 (T1) to transitional type-2 (T2) CAC-3Ј and 5Ј-GGGGACAGTCAGAAAACACC-3Ј. The PCR primers for the intronic, Blk-associated SSLP were 5Ј-TGTAGGGACTTTCCAGTGAG-3Ј and Ј Ј 5 -CCATTGAAACAGGAGACACAC-3 . The Bcl-xL transcript abundance stud- ies were performed with the PCR primers 5Ј-TTTTTCTGAGTTACCGGCG-3Ј Department of Biochemistry, College of Agricultural and Life Sciences, University of Ј Ј Wisconsin, Madison, WI 53706 and 5 -GGGAGGTGAGAGGTGAGTGG-3 . The Bax and Bcl-2 specific prim- ers were described (13). Received for publication July 19, 2001. Accepted for publication October 1, 2001. The costs of publication of this article were defrayed in part by the payment of page RH mapping charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The DNA from each T31 radiation hybrid (RH) cell line (Research Ge- 1 netics, Huntsville, AL) and controls was amplified with Blk-specific PCR This research was supported by National Institutes of Health Grant AI42990. primers and analyzed for the unique 305-bp mouse gene product three 2 Address correspondence and reprint requests to Dr. Colleen E. Hayes, Department times. of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706. E-mail address: [email protected] B cell enrichment, analysis, and culture 3 Abbreviations used in this paper: Bcmd, B cell maturation defect; BAFF, B cell Flow-sorted splenic T1 B cells were obtained from adult mice 13 days after activating factor belonging to the TNF family; BAFF-R, BAFF receptor; BAX, Bcl- 500-rad irradiation (14). Splenocytes were stained following RBC lysis 2-associated X protein; BCR, B cell Ag receptor; BH, Bcl-2 homology; Bik, Bcl-2 ␮ interacting killer; Blk, Bik-like killer; Chr, chromosome; cM, centimorgan; PI, pro- with biotin-goat Ab to mouse (Fab; Jackson ImmunoResearch Labora- pidium iodide; RH, radiation hybrid; SAC, splenic adherent cell; SSLP, simple se- tories, West Grove, PA), and PE Ab to CD23 (clone B3B4), FITC Ab to quence-length polymorphism; T1, transitional type-1; T2, transitional type-2. CD21 (clone 7G6) and Cy5PE-streptavidin, all from BD PharMingen (San

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

● 6070 CUTTING EDGE: A/WySnJ B CELLS OVEREXPRESS THE Chr 15 PROAPOPTOTIC Blk GENE

Diego, CA). Nonviable, 4Ј,6Ј-diamidino-2-phenylindole-stained cells (Mo- Structure of the Blk gene and identification of a Blk gene SSLP lecular Probes, Eugene, OR) were excluded. A FACSVantage (BD Bio- sciences, Mountain View, CA) sorted the T1 B cells (IgMhigh/CD23Ϫ/ To confirm the RH mapping data, we sought a Blk-associated poly- CD21Ϫ/low)toϾ95% purity. morphism between strains A/WySnJ and CAST/Ei that could be Enriched transitional B cells were obtained from the spleens of 1-wk old used to genotype our panel of Chr 15 recombinant (A/WySnJ ϫ mice as described (15). A/J B cells, labeled with CellTracker Orange and CAST/Ei)F ϫ A/WySnJ mice (7). To this end we examined a Blk A/WySnJ B cells labeled with CellTracker Green (Molecular Probes) were 1 mixed in equal proportions and cultured. After culture, cells were analyzed intron, anticipating polymorphisms between A/WySnJ and for CellTracker dyes and propidium iodide (PI) exclusion. Some cell cul- CAST/Ei due to less sequence conservation. To determine the tures also included splenic adherent cells (SAC). Splenocytes (107 cells in genomic organization of Blk, basic local alignment search tool 1.0 ml/well; 48-well plates) were cultured overnight and nonadherent cells searches were done by comparing the Blk mRNA sequence to the were removed before the purified B cells were added. publicly available High Throughput Genomic Sequence database For apoptosis assays, RBC-depleted splenocytes (viability Ͼ95%) from neonatal mice were cultured in triplicate (2 ϫ 106 cells in 1.0 ml/well; (www.ncbi.nlm.nih.gov/genome/seq/MmHome.html). This search 48-well plates) in IMDM supplemented with 5% FBS, 50 ␮M 2-ME, 2 mM yielded a Chr 15 mouse genomic clone (National Center for Bio- glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. Samples technology Information LocusID AL583887) encompassing the were analyzed by flow cytometry for apoptotic B cells after B220 staining, Blk coding region. Comparing the mRNA sequence (18) to the ethanol fixation, and PI staining. genomic sequence, and following the gt/ag rule for intron/exon Data analysis boundaries, yielded the gene structure (Fig. 1, B and C). The mouse Blk gene structure was similar to the human Bik gene, with The interval mapping data were analyzed with Map Manager QT software five exons and four introns of similar size and functional organi- (16) (http://mapmgr.roswellpark.org/mapmgr.html). The map positions in Downloaded from centimorgans were obtained from the 2000 Chromosome Committee Re- zation. Inspection revealed a putative SSLP in intron 2. When ports, Mouse Genome Database, Mouse Genome Informatics, The Jackson genomic A/WySnJ and CAST/Ei DNA were amplified with PCR Laboratory (Bar Harbor, ME) (17) (http://www.informatics.jax.org/ccr/). primer pairs designed to bracket this repeat, the two strains yielded distinct band sizes (CAST/Ei ϭ 150 bp and A/WySnJ ϭ 180 bp). Results RH mapping of the Blk gene SSLP mapping of the Blk gene using Chr 15 recombinant To find the Chr 15 syntenic region, we searched the annotated strains http://www.jimmunol.org/ human Chr 22 sequence (http://www.sanger.ac.uk/HGP/Chr22) For fine resolution mapping of Bcmd, we assembled a panel of 17 ϫ ϫ for loci mapped in the human and the mouse (8). Srebf2 has been (A/WySnJ CAST/Ei)F1 A/WySnJ N2 progeny with recom- mapped in both species (8, 17) and is very close to D15 Mit118, binations in the interval between D15 Mit144 and D15 Mit118, and the closest known SSLP to Bcmd (6). Near Srebf2 on human Chr measured splenic B cell phenotypes (7). The DNA from this re- 22 is the Bik locus (8, 9). The mouse Blk gene is believed to be the combinant panel was examined for the Blk-associated intronic Bik homolog, because mouse BLK protein is 43% homologous to SSLP. The genotyping results placed the Blk gene within the gray human BIK protein (18), but Blk has not been mapped. area shown in Fig. 2, consistent with the RH mapping data.

We mapped Blk using the T31 RH mapping panel (Research by guest on September 28, 2021 Genetics). The mouse Blk gene retention pattern was analyzed by Overexpression of the Blk gene in A/WySnJ transitional B cells PCR and entered into a server at the Whitehead Center for Genome We next sought evidence for a Blk coding sequence mutation. The Research (Cambridge, MA) (http://www-genome.wi.mit.edu). The cDNA from A/J and A/WySnJ B cells was PCR-amplified, cloned, RHMAPPER software (Whitehead Center for Genome Research) and sequenced (data not shown). There were no sequence differ- determined that the Blk gene cosegregated with the Chr 15 markers ences between these strains and the published Blk coding sequence D15 Mit198 and D15 Mit33 at 48.6 cM on the Mouse Genome (18). Therefore, we investigated the level of Blk transcript expres- Database map (Fig. 1A). This position is very near to D15 Mit118, sion in sorted T1 B lymphocytes through the use of RT-PCR. within the interval defined by our most current analysis (7). Serially diluted cDNA was examined for expression of Blk and a

FIGURE 1. RH mapping of the Blk gene and Blk genomic organization. A, The map position of Blk in relation to high-resolution genetic mapping markers and some that map in this region (MGD). B, The National Center for Biotechnology Information LocusID genomic clone AL583887. The filled box indicates the unspliced Blk transcript (17.8 kb). C, The intron-exon structure of the Blk gene (GenBank ID NM-007546). D, A schematic of the BLK protein; the conserved BH3 domain and the transmembrane region are indicated (18). The Journal of Immunology 6071

Premature apoptosis is a cell-autonomous trait of the A/WySnJ transitional B cells Increased Blk gene transcription in A/WySnJ transitional B cells suggested that these cells may have a defect leading to premature apoptosis. To test this hypothesis, splenocytes from neonatal mice were cultured and, at various times, collected and analyzed. At 1 wk of age the majority of splenic B cells are transitional B cells (19). Analysis of B220ϩ cells with subdiploid amounts of DNA revealed a rapid B cell apoptosis rate for A/WySnJ compared with A/J cells (Fig. 4A). We next isolated the transitional B cells from 1-wk old mice as described (15). These B cells were cultured with FIGURE 2. Genetic mapping of the Blk-associated, intronic SSLP using ϫ ϫ or without SAC, recovered 1 day later, and analyzed by flow cy- the (A/WySnJ CAST/Ei)F1 A/WySnJ recombinant N2 mapping panel. A Blk-associated SSLP was identified and the N2 mapping panel tometry. Cell viability was determined by enumeration of B cells was genotyped for this SSLP. Interval mapping data analysis for this SSLP that excluded PI and appeared in the live lymphocyte scatter gate was accomplished by calculating the likelihood ratio statistic (16). The map (Fig. 4B). The A/J B cells had more than twice the viability of the positions are from the Mouse Genome Database (17). A/WySnJ B cells, in very good agreement with the apoptosis data. Adding A/J or A/WySnJ SAC had no effect on B cell survival for either A strain. Neither A/J nor A/WySnJ transitional B cells ex- Downloaded from pressed Fas, ruling out Fas-mediated apoptosis as a mechanism for second proapoptotic gene, Bax, as well as the two antiapoptotic premature cell death (data not shown). genes that interact with Blk, Bcl-2, and Bcl-xL (18) (Fig. 3). Blk transcripts were ϳ3-fold more abundant in the A/WySnJ T1 B Discussion cells than in the A/J B cells. This was in contrast to Bax and Bcl-2, The evidence presented in this work establishes that the Blk gene which were not different, and Bcl-xL, which was increased fits the Bcmd candidate gene profile by placement, expression http://www.jimmunol.org/ ϳ2-fold. polymorphism, and function. Two methods mapped Blk within the region with the highest probability of including Bcmd. Moreover, the A/WySnJ T1 B cells overexpressed Blk ϳ3-fold compared with A/J and showed a cell-autonomous defect leading to prema- ture apoptosis, consistent with increased expression of a proapop- totic gene. The molecular basis for this expression polymorphism is not yet known. The Bik gene encodes a death-inducing member of the BCL-2

protein family (10). The BCL-2 protein family members share one by guest on September 28, 2021 or more of the BCL-2 homology (BH) domains, BH1, BH2, BH3,

FIGURE 3. Expression of proapoptotic and antiapoptotic transcripts in A/WySnJ and A/J T1 B cells. The T1 B cell total RNA was quantified, and equal amounts were reverse transcribed. A quantitative competitive PCR was done for G3PDH transcripts as described (24). The cDNA was nor- malized to G3PDH transcripts, serially diluted, amplified by PCR, and electrophoresed (A). The PCR products were imaged and quantified with TotalLab software, v. 1.10 (Nonlinear Dynamics, Durham, NC). The ratio of A/WySnJ:A/J transcript abundance was determined at each dilution and the mean for these ratios was calculated for one dilution series. This mea- surement was performed in triplicate on individual cDNA sample sets; a mean of means Ϯ SEM was calculated from the triplicate measurements and graphed (B). The significance of the differences was calculated by a paired, two-tailed Student’s t test. For the Student t test all data points from the triplicate measurements of one cDNA sample were used to compare FIGURE 4. The A/WySnJ transitional B cells rapidly succumb to apo- corresponding A/WySnJ and A/J band intensities. The Blk and Bcl-xL dif- ptosis in vitro. A, splenocytes from 2-wk old mice were cultured, and the ferences were significant (p Ͻ 0.05). The Bax and Bcl-2 differences were cells were analyzed on the flow cytometer for subdiploid, B220ϩ cells; one not significant. The values shown and the statistical analyses performed are experiment of three. B, enriched transitional B cells from 1-wk old mice from one representative experiment of two, each using separately derived were cultured, and the cells were analyzed on the flow cytometer for viable T1 B cell samples. cells by PI exclusion; one experiment of two. 6072 CUTTING EDGE: A/WySnJ B CELLS OVEREXPRESS THE Chr 15 PROAPOPTOTIC Blk GENE and BH4 (20). The Bcl-2 prosurvival gene was discovered at the will no doubt test these hypotheses and provide a detailed model of characteristic chromosomal translocation breakpoint t(14;18) of this most interesting B cell developmental transition. follicular B cell lymphoma (21). The Bax pro-death gene was dis- covered due to interactions between Bcl-2-associated X protein Acknowledgments (BAX) and BCL-2 (22). The BCL-2:BAX ratio determined a cell’s We thank K. Schell (University of Wisconsin Comprehensive Cancer Cen- susceptibility to apoptosis, leading to the concept that the ratio of ter, Madison, WI) for assistance with flow cytometry and Dr. A. Attie proapoptotic and antiapoptotic BCL-2 family serves as a (Department of Biochemistry, University of Wisconsin, Madison, WI) for the gift of the T31 RH panel DNA. checkpoint or rheostat regulating apoptosis (20). Bik was discovered due to interactions between BIK and pro- References survival BCL-2 family members (10). Bik promoted cell death in 1. Conley, M. E. 2000. Genetics of primary immunodeficiency diseases. Rev. Im- munogenet. 2:231. transient transfection assays, and Bcl-2 and Bcl-xL suppressed this 2. Miller, D. J., and C. E. Hayes. 1991. Phenotypic and genetic characterization of function. Thus, BIK is a proapoptotic protein and lacks all BH a unique B lymphocyte deficiency in strain A/WySnJ mice. Eur. J. Immunol. domains except BH3. The BH3-only proteins (BIK, BAD, BID, 21:1123. and HRK) are particularly potent death agonists (20). The mouse 3. Miller, D. J., K. D. Hanson, J. A. Carman, and C. E. Hayes. 1992. A single autosomal gene defect severely limits IgG but not IgM responses in B lympho- homolog of Bik, Blk, was discovered by searching GenBank for cyte-deficient A/WySnJ mice. Eur. J. Immunol. 22:373. sequences encoding conserved BH3 domains (18). The BLK BH3 4. Lentz, V. M., M. P. Cancro, F. E. Nashold, and C. E. Hayes. 1996. Bcmd governs domain interacts directly with BCL-X protein (18). In transfec- recruitment of new B cells into the stable peripheral B cell pool in the A/WySnJ L mouse. J. Immunol. 157:598. tion experiments, Blk was a more potent death effector than Bik or 5. Clise-Dwyer, K., M. P. Cancro, and C. E. Hayes. 2001. The Bcmd-controlled Bax, and the apoptotic cell percentage doubled with each doubling defects in A/WySnJ mice are limited to follicular B lymphocytes. In B Cell Downloaded from Immunobiology and Disease, Vol. E8. J. R. Parnes, A. L. DeFranco, and of the Blk to Bcl-xL ratio, indicating that this ratio is a very sen- K. L. Calame, eds. Keystone Symposia, April 17Ð22, Snowbird, UT, p. 105. sitive apoptosis rheostat (18). In the present experiments, Blk was 6. Hoag, K. A., K. Clise-Dwyer, Y. H. Lim, F. E. Nashold, J. Gestwicki, overexpressed 3-fold and Bcl-x was overexpressed 2-fold, so the M. P. Cancro, and C. E. Hayes. 2000. A quantitative-trait locus controlling pe- L ripheral B-cell deficiency maps to mouse chromosome 15. Immunogenetics 51: Blk:Bcl-xL ratio was higher in the A/WySnJ T1 B cells than in the 924. control cells. 7. Clise-Dwyer, K., I. J. Amanna, J. L. Duzeski, F. E. Nashold, and C. E. Hayes.

Little is known about regulation of Blk transcription (18). Jiang 2001. Genetic studies of B lymphocyte deficiency and mastocytosis in strain http://www.jimmunol.org/ A/WySnJ mice. Immunogenetics. In press. and Clark (11) showed that surface IgM but not surface IgD liga- 8. Dunham, I., N. Shimizu, B. A. Roe, S. Chissoe, A. R. Hunt, J. E. Collins, tion initiated Bik transcription and apoptosis in B104 B lymphoma R. Bruskiewich, D. M. Beare, M. Clamp, L. J. Smink, et al. 1999. The DNA sequence of human chromosome 22. Nature 402:489. cells, a model for B cell Ag receptor (BCR)-mediated negative 9. Verma, S., M. L. Budarf, B. S. Emanuel, and G. Chinnadurai. 2000. Structural selection. We reported that excessive apoptosis occurs at the analysis of the human pro-apoptotic gene Bik: chromosomal localization, T13T2 transition of B cell maturation in A/WySnJ mice (5). The genomic organization and localization of promoter sequences. Gene 254:157. ϩ Ϫ 10. Boyd, J. M., G. J. Gallo, B. Elangovan, A. B. Houghton, S. Malstrom, T1 B cells are IgM IgD and retain their sensitivity to negative B. J. Avery, R. G. Ebb, T. Subramanian, T. Chittenden, R. J. Lutz, et al. 1995. selection via a BCR-mediated apoptotic signal, whereas the T2 B Bik, a novel death-inducing protein, shares a distinct sequence motif with Bcl-2 cells are IgMϩIgDϩ and apparently insensitive to negative selec- family proteins and interacts with viral and cellular survival-promoting proteins.

Oncogene 11:1921. by guest on September 28, 2021 tion (23). Thus, our evidence is consistent with a model wherein 11. Jiang, A., and E. A. Clark. 2001. Involvement of Bik, a proapoptotic member of the A/WySnJ B cells have difficulty making the T13T2 transition the Bcl-2 family, in surface IgM-mediated B cell apoptosis. J. Immunol. 166: 6025. because they express inappropriately high levels of a BCR-induced 12. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, death gene, and excessive apoptosis is the result. C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al. 2001. Baff-r, a newly Intriguingly, Thompson et al. (12) very recently identified a new identified tnf receptor that specifically interacts with baff. Science 293:2108. 13. Tomayko, M. M., and M. P. Cancro. 1998. Long-lived B cells are distinguished receptor for B cell activating factor belonging to the TNF family by elevated expression of A1. J. Immunol. 160:107. (BAFF), and showed that the gene encoding this receptor, provi- 14. Allman, D. M., S. E. Ferguson, and M. P. Cancro. 1992. Peripheral B cell mat- sionally termed Baffr, maps to human Chr 22q13.1, which is syn- uration. I. Immature peripheral B cells in adults are heat-stable, antigen-high, and exhibit unique signaling characteristics. J. Immunol. 149:2533. tenic to the mouse Chr 15 Bcmd region. Also, they showed by 15. Norvell, A., and J. G. Monroe. 1996. Acquisition of surface IgD fails to protect Southern blotting that A/WySnJ and A/J genomic DNA are poly- from tolerance-induction: both surface IgM- and surface IgD-mediated signals induce apoptosis of immature murine B lymphocytes. J. Immunol. 156:1328. morphic at this locus, and that the A/WySnJ Baffr transcript was 16. Manly, K. F., and J. M. Olson. 1999. Overview of QTL mapping software and shortened and exon 3 was not intact. The exact nature of the Baffr introduction to map manager QT. Mamm. Genome 10:327. mutation in A/WySnJ is not known, and whether the A/WySnJ 17. Huppi, K., D. Siwarski, J. Kim, and V. Letts. 1999. Mouse chromosome 15. Mamm. Genome 10:956. BAFF receptor (BAFF-R) retains any signaling function is also 18. Hegde, R., S. M. Srinivasula, M. Ahmad, T. Fernandes-Alnemri, and unclear. Nevertheless, their studies together with ours and those of E. S. Alnemri. 1998. Blk, a BH3-containing mouse protein that interacts with Jiang and Clark (11) suggest a very interesting model, wherein Bcl-2 and Bcl-xL, is a potent death agonist. J. Biol. Chem. 273:7783. 19. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, membrane IgM and BAFF-R may compete for control of Blk tran- M. C. Lamers, and R. Carsetti. 1999. B cell development in the spleen takes place scription and thus the maturation or apoptosis fate of the transi- in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190:75. tional B cells. The IgM may enhance Blk transcription and 20. Chao, D. T., and S. J. Korsmeyer. 1998. BCL-2 family: regulators of cell death. BAFF-R may inhibit it. The outcome of the competition might Annu. Rev. Immunol. 16:395. determine peripheral B cell homeostasis. The A/WySnJ T1 B cells 21. Tsujimoto, Y., J. Gorham, J. Cossman, E. Jaffe, and C. M. Croce. 1985. The t(14;18) chromosome translocations involved in B-cell neoplasms result from may be unable to inhibit Blk transcription due to a BAFF-R defect, mistakes in VDJ joining. Science 229:1390. explaining their premature apoptosis phenotype. It is also interest- 22. Oltvai, Z. N., C. L. Milliman, and S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes ing to note that Baffr and Bik are only ϳ1.3 Mbp apart in the in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609. , raising the possibility that chromatin remodeling 23. Monroe, J. G. 2000. B-cell antigen receptor signaling in immature-stage B cells: during peripheral B cell development may lead to coordinated ex- integrating intrinsic and extrinsic signals. Curr. Top. Microbiol. Immunol. 245:1. 24. Nashold, F. E., K. A. Hoag, J. Goverman, and C. E. Hayes. 2001. Rag-1-depen- pression of these two genes, which may play critical roles in the dent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experi- life or death fate of the transitional B cell. Additional experiments mental autoimmune encephalomyelitis. J. Neuroimmunol. 119:16.