CUTTING EDGE IMMUNOLOGY THE OF JOURNAL Cutting Edge: Spontaneously Ig-Secreting B-1 Cells Violate the Accepted Paradigm for Expression of Differentiation-Associated Transcription Factors 1 Joseph R. Tumang, 2 * Rube ´n France ´s, 2 * Seung Geun Yeo, 3 * and Thomas L. Rothstein 4 * †‡ B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major short- fall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we demonstrate that secreting B-1 cells use some aspects of the recently recognized plasmacytic dif- ferentiation program but deviate from it in important ways. Specifically, we show that key repressors of the plas- macytic program, B cell leukemia/lymphoma-6 and paired box gene 5, are reduced in spontaneously secreting B-1 B cells, as in stimulated differentiated B-2 cells. Sur- prisingly, we find that key promoters of the plasmacytic program, B lymphocyte inducer of maturation program 1 and X-box binding protein 1, are not up-regulated in se- creting B-1 cells, in contrast to secreting B-2 cells. These data demonstrate that B-1 cells operate under a differen- tiation program that is unique and differs from the par- adigm associated with Ig-secreting B-2 cells. The Journal of Immunology, 2005, 174: 3173–3177. B -1 cells spontaneously secrete natural Ig, which plays a critical role in host immune defense. Natural Ig con- tains specificities for pathogens and pathogen-associ- ated epitopes (1– 4); these specificities have been shown to be protective in vivo (4 –7). Although the importance of these cru- cial effector molecules is well understood, the mechanisms reg- ulating natural Ab production are not. One accepted paradigm for Ig secretion derived from studies of LPS-stimulated B-2 cell plasma cell differentiation focuses on a central cascade con- trolled by four transcription factors, B cell leukemia/lympho- ma-6 (BCL-6), 5 B lymphocyte inducer of maturation program 1 (BLIMP-1), paired box gene 5 (PAX-5), and X-box binding protein 1 (XBP-1) (reviewed in Refs. 8 and 9). According to this model, BCL-6 and PAX-5a repress, and BLIMP-1 and XBP-1 stimulate, genes associated with differentiation to Ig secretion. These transcription factors are thought to act in cascade fash- ion, such that naive, nonsecreting B-2 cells express elevated BCL-6 that represses BLIMP-1 and elevated PAX-5 that re- presses XBP-1, whereas coincident with plasmacytic differenti- ation the level of BCL-6 falls, releasing repression of BLIMP-1 which rises, thereby suppressing PAX-5a, which in turn releases XBP-1 which also rises. Thus, the current paradigm holds that differentiated Ig-secreting B cells express low levels of BCL-6 and PAX-5a, but high levels of BLIMP-1 and XBP-1. BLIMP-1 in particular is currently cast as the master regula- tor of plasmacytic differentiation on the basis of several key observations. Normal plasma cells express BLIMP-1 (10), and ectopic expression of BLIMP-1 induces plasma cell differenti- ation (11). Conversely, BLIMP-1-deficient mice are deficient in serum Ig and B cells from these mice fail to mature to plasma cells and to up-regulate the plasma cell associated genes CD138 (syndecan-1) and J-chain (12). Final commitment to the plasma cell differentiation pathway appears to be reinforced through BLIMP-1 back-repression of BCL-6 (13). The studies described herein were designed to bridge the gap between the physiology of natural Ig secretion and the known molecular mechanisms governing plasmacytic differentiation. Materials and Methods Animals Male BALB/cByJ mice at 8 –14 wk of age were obtained from The Jackson Laboratory. Mice were cared for and handled in accordance with National In- stitutes of Health and institutional guidelines. B cell purification and culture Sorted B cell populations were obtained on the basis of CD5 and B220 staining as previously described (14), and reanalyzed for purity by immunofluorescent staining with Abs directed against Mac-1, CD43, and CD23. Peritoneal B-1a cells and splenic B-2 cells were found to be 96% pure (B220  /CD43  / CD23  /Mac-1  or B220  /CD43  /CD23  /Mac-1  , respectively). FACS- sorted B cells were cultured in RPMI 1640 medium as previously described (14). Where indicated, cells were cultured in the presence of 25 g/ml LPS. ELISPOT assay FACS-sorted, naive B cells, or B cells cultured for 48 h, were distributed at various dilutions onto MultiScreen*-IP Plates (Millipore) precoated with goat anti-mouse Ig (HL) and then incubated for 3 h at 37°C and 5% CO 2 . Plates Departments of *Medicine and † Microbiology, Boston University School of Medicine, and ‡ Immunobiology Unit, Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston, MA 02118 Received for publication November 15, 2004. Accepted for publication January 21, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Public Health Service Grants AI29690 and AI60896 awarded by the National Institutes of Health. 2 J.R.T. and R.F. contributed equally to this work. 3 Current address: Department of Otolaryngology, College of Medicine, Kyung Hee Uni- versity #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea. 4 Address correspondence and reprint requests to Dr. Thomas L. Rothstein, Immunobi- ology Unit, Evans Biomedical Research Center, Room 437, Boston Medical Center, 650 Albany Street, Boston, MA 02118. E-mail address: tr@bumc.bu.edu 5 Abbreviations used in this paper: BCL-6, B cell leukemia/lymphoma-6; qPCR, quanti- tative-PCR; BLIMP-1, B lymphocyte inducer of maturation program 1; PAX-5, paired box gene 5; XBP-1, X-box binding protein 1; XBP-1s, spliced XBP-1. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00