Characterization of murine polyreactive antigen-binding B cells: presentation of antigens to T cells

Monoclonal polyreactive antibodies (Ab) can bind, at low affinity, a variety of different self and non-self antigens (Ag). Recent studies in humans showed that polyreactive Ab are expressed on the surface of a subset of peripheral B lymphocytes and clonal analysis revealed that a variety of different Ag can bind to single cells expressing these Ab. To see if these polyreactive Ag-binding B (PAB) cells also are present in mice, fluorescein-conjugated Ag and FACS sorting were used to identify and separate PAB cells from non-polyreactive Ag-binding B cells. Depending on the Ag used for screening, up to one-third of mouse splenic B cells displayed polyreactive Ag-binding properties. Confirmation that the Ag actually bound to surface Ig came from treating PAB cells with anti-Ig which inhibited Ag binding by up to 80 %. Further studies showed that PAB cells could present Ag to Ag-specific T cells, but despite their Ag-presenting ability, PAB cells from normal mice failed to trigger Ag-specific T cells to proliferate. Analysis of the co-stimulatory molecules B7-1 and B7-2 showed that these molecules were not expressed on PAB cells from normal mice. These findings argue that the lack of co-stimulatory molecules on PAB cells is the most likely explanation for their failure to stimulate Ag-specific T cells. The ability of PAB cells from normal mice to bind and present Ag to Ag-specific T cells, without causing them to proliferate, suggests that PAB cells may contribute to the induction and / or maintenance of immunological tolerance.     

CD5+/Mac-1- peritoneal B cells: a novel B cell subset that exhibits characteristics of B-1 cells

The peritoneal cavity of mice is enriched for B-1 B cells, a lymphocyte subset that differs from conventional B-2 cells phenotypically, functionally, and developmentally. According to current paradigms, all peritoneal B-1 cells express Mac-1 whereas B-2 cells do not and thus these populations are often purified by FACS sorting or magnetic bead isolation based on B cell expression of Mac-1 or lack thereof. However, in the course of studying B220+/Mac-1- peritoneal B-2 cells, we discovered that this population is actually heterogeneous, with approximately 30-40% of these B220+/Mac-1- cells expressing the B-1 cell marker CD5. It was unclear whether this B220+/CD5+/Mac-1- peritoneal B cell population represented aberrantly CD5 expressing B-2 cells or Mac-1- B-1 cells. To address this issue we tested CD5+/Mac-1- peritoneal B cells for several traits that distinguish B-1 and B-2 cells. We found that CD5+/Mac-1- peritoneal B cells resembled CD5+ B-1 cells and not B-2 cells in terms of expression of several additional surface markers (IgM, IgD, CD23, CD43, and CD80). Further, CD5+/Mac-1- peritoneal B cells expressed high levels of V(H)11 and V(H)12, two Ig variable genes that are expressed mainly by B-1 but not B-2 cells. In addition, CD5+/Mac-1- peritoneal B cells responded to PMA, a mitogen that stimulates B-1 cells but not B-2 cells, and not to anti-Ig, that stimulates B-2 cells but not B-1 cells. ELISPOT analyses of freshly isolated CD5+/Mac-1- peritoneal B cells revealed that they secreted IgM constitutively, like B-1 cells and unlike B-2 cells. These results indicate that CD5+/Mac-1- peritoneal B cells are a new subset of B-1 cells, here termed B-1c, and stress the importance of using multiple surface markers to identify and purify specific B cell populations.     

In-vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. I. Effect of anti-Thy-1 antibody on the tissue distribution of lymphocytes.

A procedure is described for the in vivo removal of all detectable T lymphocytes from spleen and lymph nodes in mice. A single intraperitoneal injection of monoclonal anti-Thy-1 antibody into mice leads to rapid depletion of functional T cells from peripheral lymphoid organs, but not thymus. The extent of T-cell depletion is dependent on the cytotoxic titre of the anti-Thy-1 antibody used. Antibody with a median cytotoxic titre greater than 10(6) causes the complete removal of cells bearing Thy-1, Lyt-1 and Lyt-2 surface antigens from peripheral lymphoid populations in 3 days. Eight days after treatment Thy-1+, Lyt-1+ and Lyt-2+ cells begin to reappear in these organs. Splenic B cells, assayed by the expression of surface immunoglobulin (sIg) and by mitogenic responsiveness to bacterial lipopolysaccharide (LPS), are not affected by this treatment. The monoclonal anti-Thy-1 antibody does not appear to penetrate thymus tissue and bind to thymocytes. Anti-Thy-1 antibody, but not F(ab')2 is required for in-vivo T-cell depletion. These findings indicate that anti-Thy-1 antibody causes the removal of Thy-1+ cells from peripheral lymphoid tissue, and as the circulating levels of anti-Thy-1 antibody decrease, cells from the thymus repopulate the thymus-dependent areas of the depleted lymphoid organs.     

BAFF costimulation of Toll-like receptor-activated B-1 cells

B cell-activating factor belonging to the TNF family (BAFF) and its receptor BAFF-R play critical roles in the maturation and survival of conventional peripheral B cells. However, they appeared to be dispensable for the generation and maintenance of CD5(+) B-1 cells as BAFF(-/-) and BAFF-R(-/-) mice have normal B-1 cell populations. Hence, it is presently unclear if B-1 cells are responsive to BAFF and if BAFF regulates some aspects of B-1 cell function. We show here that BAFF-R and transmembrane activator and CAML interactor (TACI) are the major receptors expressed by B-1 cells. Specifically, we show that BAFF treatment of B-1 cells leads to increased NF-kappaB p100 processing and CD21/CD35 expression. Interestingly, toll-like receptor (TLR) engagement of B-1 cells augmented the surface expression of BAFF receptors and rendered them responsive to BAFF costimulation, as evidenced by their increased proliferation, expression of cell surface activation markers and secretion of the pro-inflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10. This costimulatory effect is achieved primarily through BAFF-R as BAFF failed to costimulate B-1 cells obtained from A/WySnJ mice which have defective BAFF-R signaling. Thus, as TLR are innate immune receptors and B-1 cells are "innate-like" lymphocytes, our data provide evidence that BAFF plays a role in innate immunity.     

The ontogeny and functional characteristics of human B-1 (CD5+ B) cells.

We demonstrate that, on average, greater than 90% of B lymphocytes in fetal spleen express CD5 at gestational ages of 17-23 weeks. Similarly, CD5+ B cells (B-1 cells) are the major B cell subset in umbilical cord blood. These findings depend on the optimization of fluorochrome conjugated anti-CD5 reagents for multiparameter fluorescent-activated cell sorter (FACS) analysis. From infancy through childhood the percentage of B-1 cells gradually diminishes in both spleen and peripheral blood. Stable adult levels, 25-35% of the total B cell population, are reached in late adolescence. The decrease in the percentage of B-1 cells in spleen is accompanied by an increase in conventional (CD5-) B cells, keeping the percentage of total B cells per mononuclear cells relatively constant. In contrast, in peripheral blood, the concentration of both B-1 cells and total B cells decreases, while T cells increase. At the functional level, we show that polyreactive IgM autoantibodies are produced by FACS-sorted CD5high B cells, but not by CD5- B cells from adolescent spleen. In contrast, fetal splenic CD5high and CD5- B cells appear functionally uniform, both producing IgM autoantibodies that are typical of B-1 cells. The apparent level of CD5- B cells in fetal spleen, on average 10% of total B cells, may still result from limitations of our reagent. The prominence of B-1 cells in fetal spleen and cord blood, the gradual reduction of B-1 cells with increasing age, and its characteristic repertoire, all suggest a role for this cell type in immunologically immature hosts.     

Regulation of BCR signal transduction in B-1 cells requires the expression of the Src family kinase Lck

Although found predominantly in the peritoneal and pleural cavities, B-1 cells are also present in other peripheral tissues such as spleen and lung. While similar in surface phenotypes, such as CD5, all B-1 cells are not equivalent in their response to stimuli. Here, we report that the src family kinase Lck is required to confer the BCR hyporesponsiveness typical of CD5+ B-1 cells and appears involved in the maintenance of their unique function. Splenic B-1 cells express CD5 but not Lck and are not hyporesponsive; however, within the peritoneum, these B-1 cells are induced to express Lck and acquire a hyporesponsive phenotype. Peritoneal B-1 cells from lck-deficient mice, while CD5+, no longer exhibit attenuated BCR signaling. Interestingly, lck-null mice exhibited increased natural antibody levels characteristic of B-1 cells. Taken together, these results demonstrate a key role for Lck in modulating the signaling and cellular fate of B-1 B cells.     

B-1 cells: the lineage question revisited

Summary: The origins and functions of B-1 cells have sparked a good deal of controversy, largely centered on whether these B cells are developmentally distinct from the principal B cell populations (B-2) found in peripheral lymphoid organs. However, the prime criteria for assigning B-1 and B-2 cells to separate developmental lineages are satisfied by studies published some time ago that 1) identify distinct sources of progenitors for B-1 and B-2 cells; 2) show that these progenitors express their inherent commitment developing under the same conditions in co-transfer recipients; and, 3) have distinctive developmental patterns revealed by analysis of cells at various stages along the B-cell development pathway. I review these developmental studies here both to clarify the issue and to set the stage for presentation of evidence from more recent studies, which further define the functional differences between B-1 and B-2 cells and reveal intriguing complexities in the selective and other mechanisms that control the VH composition of the B-1 antibody repertoire.     

B-1-like cells exist in sheep. Characterization of their phenotype and behaviour.

Two populations of B lymphocytes, B-1 (CD5+ and/or CD11b+) and B-2 (CD5- and CD11b-) cells have been described. In mice, which is the species of reference for B-1 and B-2 cell studies, these two subsets present different developmental schemes, phenotypes, antibody repertoires, localization and behaviours. Interestingly, in sheep, B cells rearrange their immunoglobulin (Ig) loci around the neonatal period, similarly to murine B-1 cells. However, the phenotype of the sheep B cells has not been characterized with regards to their developmental pathway. In this report, we show that two sheep B-cell subsets can be distinguished on the basis of CD11b expression. Relative to CD11b- B cells, the CD11b+ B cells frequently co-express CD5, CD11c, higher levels of surface IgM (sIgM), show larger cell size and higher cell-cycling activity, and thus present a B-1-like phenotype. However, unlike murine B-1 cells, sheep B-1 like cells mainly localize in blood, display a higher propensity to spontaneous apoptosis relative to B-2-like cells, and proliferate after sIgM stimulation. Our data show that despite neonatal immunoglobulin loci rearrangements, sheep B cells do not all express a B-1-like phenotype. However, B-1-and B-2-like cells co-exist and present phenotypic and behavioural specificities. Nevertheless, sheep B-1-and B-2-like cells differ from the murine B-1 and B-2 cells in their cell behaviour. These subsets can thus not be considered as true homologues among species.     

Peritoneal B-2 cells comprise a distinct B-2 cell population with B-1b-like characteristics

B-1 and B-2 cells are lymphocyte populations that differ in development, surface marker expression, tissue localization, and function. Though mainly found in the spleen, lymph nodes, and circulation of mice, small numbers of B-2 cells are found in the peritoneal cavity, a site predominantly populated by B-1 cells. Here, we characterized peritoneal B-2 cells, and determined their relationship to B-1 cells. We found that peritoneal B-2 cells appear to be intermediate between splenic B-2 and peritoneal B-1 cells in terms of surface marker expression of B220, CD80, and CD43, expression of several marker genes, and in vitro viability and IgM secretion. Adoptive transfer of peritoneal B-2 cells into severe combined immunodeficiency mice resulted in the acquisition of a phenotype reminiscent of B-1b cells, as shown by up-regulation of Mac-1 and CD43, and down-regulation of CD23. Moreover, adoptively transferred peritoneal B-2 cells recapitulated B-1 cell function by producing natural IgM in recipient mice. These data suggest that peritoneal B-2 cells express some characteristics of B-1b cells and that this similarity increases with additional time in the peritoneal cavity.     

Cell cycle control mechanisms in B-1 and B-2 lymphoid subsets

An effective humoral response requires that a given B lymphocyte population express a repertoire of receptors capable of recognizing a distinct array of antigens, while at the same time disregarding self-antigens. Mature B cells interacting with antigen via their B cell antigen receptors (BCRs) enter G(1) phase of the cell cycle and, depending on the strength of the signal, can commit to S phase entry. Input from co-receptors, which may function to either enhance or inhibit BCR signals, also influence the decision to proliferate. We review herein recent advances in the biochemistry of G(1)-cyclin holoenzymes that function to integrate BCR-coupled signaling pathways to the phosphorylation (and inactivation) of the retinoblastoma gene product (pRb) in splenic B lymphocytes (B-2 cells). We also highlight differences in the control of G(1)-to-S phase progression between B-2 cells and peritoneal CD5+ B cells (B-1 cells).     

CD5+ B lymphocytes are the main source of antibodies reactive with non-parasite antigens in Trypanosoma congolense-infected cattle.

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non-trypanosome antigens such as trinitrophenol and single-strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as beta-galactosidase, ovalbumin and ferritin. The parasite non-specific IgM antibodies appear around the same time as the parasite-specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B-1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non-trypanosome antigens. Splenic CD5+ and CD5- B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin-secreting cells were similar in both B-cell populations. However, antibodies with reactivity for non-trypanosome antigens were significantly more prevalent in the CD5+ B-cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non-specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B-1 cells are up-regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non-specific antibodies.     

Unraveling B-1 progenitors

B-1 cells comprise a small percentage of the B lymphocytes that reside in multiple tissues in the mouse, including the peritoneal and pleural cavities. Functionally, B-1 cells participate in innate immunity by producing the majority of the natural IgM in serum, which protects against invading pathogens before the onset of the adaptive immune response. B-1 cells arise from fetal and neonatal progenitors and are distinct from the adult bone marrow progenitors that give rise to follicular and marginal zone B-2 cells. Recent studies have attempted to delineate the progenitors of B-1 cells from those of B-2 cells. Notably, the identification of CD45R(-/lo)CD19(+) B-1 progenitors and expression of two surface determinants, CD138 and major histocompatibility class II antigens, distinguish developing B-1 cells from B-2 cells.     

Morphological characterization of mouse B-1 cells

At least three B cell subsets, B-1a, B-1b and B-2 are present circulating peripherally in the mouse. In these animals, B-1 cells constitute a minor fraction of B cells in spleen and are absent in lymph nodes although they represent the main B cell population in peritoneal and pleural cavities. Currently these cells are identified by a surface phenotypic repertoire; they express Mac-1, IgM(high), and B220(low). B-1a cells express CDS. The aim of this work emerged from the fact that the morphology of B-1 cells is not fully characterized. Here we identified B-1 cells using colloidal gold immunocytochemical assays and purified B-1 cells from supernatants of adherent peritoneal cell cultures by a magnetic bead technique. These techniques lead us to demonstrate that, in mice, either B-1a or B-1b cells have a unique morphology distinct from that of B-2 cells.     

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

Up-regulation of CD80-CD86 and IgA on mouse peritoneal B-1 cells by porin of Shigella dysenteriae is Toll-like receptors 2 and 6 dependent

Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-κB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in robust NF-κB activation for signaling. Porin treatment of B-1 cell populations of C57BL/6 mice, and C3H/HeJ mice in particular, selectively up-regulated the expression of the costimulatory molecules. CD80 expression got enhanced on the B-1a cells whereas CD86 got solely expressed on B-1b cells. Porin-induced cell surface expression of IgM and IgA on B-1 cell populations from C57BL/6 mice. The IgA-generating capacity, hallmark of mucosal immune response, was confirmed with B-1 cells of C3H/HeJ, the lipopolysaccharide non-responder mouse, in response to the protein. The porin-mediated induction of IgA was augmented by interleukin-6 on B-1a and B-1b cells, by 2.4- and 2.6-fold, respectively. The IgA expressed on both B-1a and B-1b cell surfaces after 72 h of culture was found to bind to the 38 kDa monomer of porin confirming it to be anti-porin IgA antibody.     

Evidence that intestinal IgA plasma cells in {micro},{varkappa} transgenic mice are derived from B-1 (Ly-1 B) cells

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived,Igh-C^a allotype) µ. heavy chain and light chain transgenes,specific for trinitrophenyl, on a C57BL background (Igh-C^b allotype).FACS analyses show that the majority of B cells in peripherallymphoid organs and bone marrow(BM) express transgenic IgM exclusively.A small proportion of the B cells, however, express endogenousIgM, usually concomitant with transgenic IgM. Three criteriaestablish that the endogenous IgM expressing B cells belongto the B-1 cell lineage. (I) Endogenous IgM expressing B cellsin B6-Sp6 mice have the same localization pattern as B-1 cellsfrom normal animals: they are enriched in the peritoneal cavity.(II) The endogenous IgM⁺ B cells have the phenotype of B-1 cells:the endogenous IgM⁺ peritoneal B cells express Mac-1 (CD11b)and low levels of IgD, and most also express CD5 (L-1). (III)B6-Sp6 BM poorly reconstitutes endogenous IgM⁺ B cells, justas adult BM from normal mice poorly reconstitutes B-1 cells.In contrast, B cells which only express the transgene are readilyreconstituted by B6-Sp6 BM. The few endogenous IgM⁺ cells inthe B6-Sp6 BM recipients are located in the peritoneal cavityand have the phenotype of B-1b cells (previously the Ly-1 Bsister population), which are known to be reconstituted by adultBM.Two-color immunofluorescence staining of tissue sectionsfrom the gut and from isolated gut lamina propria cells showsthe presence of many IgA containing cells, about one-third ofwhich simultaneously express cytoplasmic (transgenic) IgM. TheC-region of this IgA is produced by endogenous C a genes, becausethe transgene encodes only for C_µ. Furthermore, the majorityof gut IgA containing cells do not express the Idiotype of thetransgene, indicating that most of the gut IgA cells are encodedby endogenous V_H genes and thus the result of an isotype switchfrom endogenous IgM expressing B cells. Since the endogenousIgM⁺ cells are B-1 cells (both B-1a and B-1b), the data stronglyindicate that the intestinal IgA plasma cells also belong tothe B-1 cell lineage.     

Presence of Activated B-1 Cells in Chronic Inflamed Gingival Tissue

B-1 cells are physically and functionally unique B cells that produce polyreactive natural antibody. This study examined the activation of B-1 cells in inflamed gingival tissue. Serum IgG antibodies to phosphorylcholine, E. coli LPS, DNA, and some commensal bacteria were examined in adult periodontitis patients and healthy subjects. In addition, the proportion of B-1a (CD20⁺ CD5⁺) cells and the amount of IL-6 and IL-10 in the inflamed gingival tissues were examined. The serum levels of IgG antibodies to phosphorylcholine, E. coli LPS, and commensal bacteria were significantly higher in the adult periodontitis patients than the healthy subjects. The proportion of B-1a cells and the amount of IL-6 and IL-10 were significantly higher in the inflamed gingival tissues than in peripheral blood from the healthy subjects. These results suggest the activation of B-1 cells in the inflamed gingival tissue of adult periodontitis patients, and that B-1 cells may serve as the first line of defense by producing polyreactive antibodies to phosphorylcholine, LPS, and commensal bacteria.     

B-1a cell origin of the murine B lymphoma line BCL1 characterized by surface markers and bacterial reactivity of its surface IgM

B cells are divided into two categories: conventional or B-2B cells and B-1B cells, the latter of which are distinguished by their different ontogeny. B cell lymphoma 1 (BCL1), the first-reported case of a spontaneously developed mouse B-lymphoma, expresses CD5, surface IgM, Mac-1, CD43 and low level of B220, and is likely to have B-1a cell origin. However, antigens recognized by IgM produced by the BCL1 cells (BCL1-IgM) have not been identified. Here, we demonstrate that BCL1-IgM reacts with Escherichia coli (E. coli). Our initial finding that several recombinant proteins expressed in E. coli bound to BCL1-B20 prompted us to examine the possibility that BCL1 cells may bind E. coli. Indeed, BCL1 cells bound fluorescein-labeled E. coli. To elucidate the structure on the BCL1 cells responsible for E. coli-binding, we produced a monoclonal antibody capable of inhibiting BCL1 binding to E. coli. The antibody recognizes an idiotypic epitope on the BCL1-IgM. Moreover, polyclonal antibody against IgM and secreted BCL1-IgM purified from the supernatants inhibited BCL1 binding to E. coli. Finally, transfection of non-lymphoid cells with cDNA of heavy and light chains of BCL1-IgM conferred the cells ability to bind E. coli. These results clearly indicate that BCL1-IgM bind E. coli and suggest that BCL1 lymphoma is a typical B-1 cell-derived lymphoma, characterized not only by the surface phenotype, but also by the reactivity of its IgM with commensal bacteria.     

Normal B-1 cell development but defective BCR signaling in Lck-/- mice

Mature B cells are grouped into two major subsets, B-1 and B-2, believed to derive from separate lineages. We have recently shown that B-1 cells, which are characterized by CD5 surface expression, specifically exhibit significant levels of the tyrosine kinase Lck in man. Here we show that also in mice Lck expression is restricted to B-1 cells and address the potential role of Lck in B-1 cell development and activation. Using as a model an Lck-/- mouse, we show that, while dispensable for B-1 cell development, Lck is required for full and sustained activation of the tyrosine phosphorylation and MAP kinase cascades triggered by the BCR in CD5+, B-1 cells. The data suggest a potential role for Lck in the achievement of the higher activation threshold required for productive BCR signaling in B-1 as compared to B-2 cells.     

B-1 cells in the bone marrow are a significant source of natural IgM

Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies identified BM and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B-cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM-secreting cells in spleen and, for the first time, in the BM as IgM(+) IgD(lo/-) CD19(hi) CD43(+) CD5(+/-) B-1 cells. The newly identified population of BM B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and BM B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Overall, these data identify populations of non-terminally differentiated B-1 cells in spleen and BM as the most significant producers of natural IgM.