Protective Roles of Natural IgM Antibodies

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

B lineage expression of the 67-kD pan–T-cell antigen CD5 was first detected on the surface of certain human and murine malignancies 30 yr ago, and was subsequently identified on a subset of normal B cells in both species (Kantor and Herzenberg, 1993; Morris and Rothstein, 1994; Hardy and Hayakawa, 2001). In mice, CD5 expression identifies a distinct B cell lineage, termed B1, which manifests unique ontological, anatomical, and functional characteristics. In contrast to conventional B2 cells, murine B1 cells derive from CD19+B220− progenitors, appear early in development, and preferentially locate to coelomic cavities (Herzenberg, 2000; Berland and Wortis, 2002; Rothstein, 2002; Dorshkind and Montecino-Rodriguez, 2007). Most importantly, B1 cells differ functionally from B2 cells by spontaneously secreting “natural” Ig that is generated in the absence of specific immunization and which accounts for most of the resting IgM, and a large portion of the resting IgA, found in normal serum (Sidman et al., 1986; Forster and Rajewsky, 1987; Ishida et al., 1992; Kroese et al., 1993). This B1 cell–derived natural Ig differs from B2 cell–derived antibody because it is more germline-like as a result of minimal N-region addition and somatic hypermutation—and is broadly reactive, autoreactive, and repertoire-selected (Forster et al., 1988; Hayakawa and Hardy, 1988; Hardy et al., 1989; Pennell et al., 1989; Gu et al., 1990). Natural Ig is vitally important in the early defense against bacterial and viral infections (Briles et al., 1981; Boes et al., 1998; Ochsenbein et al., 1999; Baumgarth et al., 2000; Haas et al., 2005), and may play a role in a wide variety of diseases through recognition of self-antigens and binding of cellular debris (Binder and Silverman, 2005). In addition, B1 cells differ functionally from B2 cells in efficiently presenting antigen to T cells (Zhong et al., 2007) and in displaying evidence of tonic signaling (Karras et al., 1997; Wong et al., 2002; Holodick et al., 2009b) in the “resting” state in the absence of specific stimulation. Somewhat akin to anergic B cells, B1 cells are relatively nonresponsive to B cell receptor (BCR) engagement (Morris and Rothstein, 1993; Wong et al., 2002). Whereas B1 cells have been considered to be self-renewing, and thus self-perpetuating, in adult animals (Hayakawa et al., 1986; Kantor et al., 1995), recent evidence suggests that new bone marrow emigrants are continually added to the B1 cell pool (Duber et al., 2009; Holodick et al., 2009a). A subpopulation of CD5-expressing B cells is found in various human tissues; these CD5+ B cells are capable of autoantibody production, and the number of such CD5+ B cells is expanded in some autoimmune diseases (Plater-Zyberk et al., 1985; Taniguchi et al., 1987; Burastero et al., 1988; Dauphinee et al., 1988). The significance of these findings vis a vis B1 cells is uncertain, however, because it is not clear that CD5 is a durable marker of the B1 cell population across species. Not only is CD5 expressed on B2 cell populations in the human system (including transitional, prenaive, and activated B cells), but in other mammals CD5 is nondiscriminatory (Freedman et al., 1989; Raman and Knight, 1992; Sims et al., 2005; Wilson and Wilkie, 2007; Lee et al., 2009). Further, both CD5− and CD5+ B cells can produce IgM autoantibodies (Casali and Notkins, 1989; Kasaian et al., 1992; Mackenzie et al., 1991). As a result, there has been much controversy regarding whether B1 cells exist at all in Homo sapiens, and if so, how human B1 cells might be characterized. Resolution of this problem is of great importance, because a full understanding of the relationship between B1 cells and diseases ranging from autoimmune dyscrasias to lymphoid malignancies, and the development of therapeutics to enhance natural Ig, depends on elucidating identifying features that will allow human B1 cells to be readily enumerated and functionally evaluated in clinical situations. To address this issue, we screened various phenotypically defined populations from umbilical cord and adult peripheral blood for key characteristics of the well-studied murine B1 cell population, specifically, spontaneous IgM secretion, efficient T cell stimulation, and tonic intracellular signaling. Using these functional criteria, we identified the phenotypic profile for human B1 cells as CD20+CD27+CD43+CD70−. We determined that some characteristics attributed to human memory B cells identified on the basis of CD27 expression are actually produced by (CD27+) B1 cells. RESULTS In the murine system, B1 cell progenitors are more abundant in fetal hematopoietic tissues than in adult hematopoietic tissues, and B1 cells emerge before the bulk of B2 cell production occurs (Montecino-Rodriguez et al., 2006). Following this paradigm, we first examined umbilical cord blood specimens for spontaneous IgM secretion by ELISPOT assay, which we used for initial screening, and then looked for efficient T cell stimulation and tonic intracellular signaling. We isolated B cells that were CD20+ to avoid CD19+CD20− plasmablasts that might secrete IgM. Umbilical cord blood contains CD20+CD27+CD43+ B cells Initially, we sort-purified CD20+ B cells from umbilical cord blood samples, and then tested for IgM secretion in 3-h ELISPOT assays. We found that unstimulated cord blood B cells spontaneously generated IgM-containing ELISPOTs (unpublished data). To phenotypically characterize the Ig-secreting population, we examined various CD20+ cord blood populations defined by known B cell surface antigens. Among these populations, we unexpectedly identified a small subset of CD27+ cells, ranging from 3 to 11% of CD20+ B cells. Similar numbers of CD27+ cord blood B cells were detected by a variety of different anti-CD27 immunofluorescent reagents (unpublished data), and expression of CD27 mRNA coincident with expression of CD27 surface antigen was verified by real-time PCR conducted on sort-purified CD20+CD27+ and CD20+CD27− cord blood cells (unpublished data). These CD20+CD27+ cord blood B cells uniquely express CD43, a well-described marker for murine B1 cells (Wells et al., 1994), whereas CD20+CD27− cord blood B cells are CD43− (Fig. 1 A). Thus, CD20+ cord blood B cells segregate into two populations, CD27+CD43+ (amounting to 6.1 ± 1.1%; mean ± SEM; n = 13) and CD27−CD43− (amounting to 93.9 ± 1.1%). Figure 1. Umbilical cord blood CD20+CD27+CD43+ B cells spontaneously secrete IgM and efficiently stimulate T cells. (A) Umbilical cord blood mononuclear cells were stained with immunofluorescent antibodies and evaluated by flow cytometry. The contour plot displays expression of CD27 and CD43 by gated CD20+ cells. Results shown represent 1 of 13 separate cord blood samples. (B) Sort-purified CD20+CD27−CD43− (27−43−) and CD20+CD27+CD43+ (27+43+) cord blood B cells were plated at 1 × 104 cells per well, incubated for 3 h at 37°C, and analyzed for IgM secretion by ELISPOT. Images shown are representative of three separate experiments on three different cord blood samples each done in triplicate. (C) Enumeration of ELISPOT results displayed as mean values for triplicate wells, with error bars indicating the SEM. Each bar graph indicates an individual experiment on a separate cord blood sample. (D) Sort-purified CD20+CD27−CD43− and CD20+CD27+CD43+ cord blood B cells were cultured for 5 d, after which supernatants were evaluated for secreted IgM by ELISA. Each bar graph indicates an individual experiment on a separate cord blood sample. (E) Sort purified and irradiated CD20+CD27−CD43− (27−43−) and CD20+CD27+CD43+ (27+43+) cord blood B cells were evaluated for the ability to drive allogeneic T cell proliferation as measured by tritiated thymidine incorporation for 8 h at the end of 5 d co-cultures. Data shown are representative of three separate experiments on three different cord blood samples each done in triplicate. Mean cpm values are displayed with error bars indicating SEM. Umbilical cord blood CD20+CD27+CD43+ B cell Igs express few mutations In the human system, CD27 expression is generally considered to mark memory B cells (Agematsu et al., 2000), which suggests incongruity in the identification of CD27+ B cells in cord blood that is obtained at birth before exogenous antigen exposure. To clarify the nature of umbilical cord blood CD20+CD27+CD43+ B cells, we examined the mutational status of Ig amplified from single cells, as described in Materials and methods. In contrast to the large number of somatic mutations present in adult CD27+ B cell–derived antibodies (Klein et al., 1998), we found that all cord blood B cells express antibodies with very low levels of somatic mutation, both CD20+CD27+CD43+ and CD20+CD27−CD43− B cells (Table I, Fig. S1 and Tables S1 and S2). All cord blood CD27+CD43+ B cell Ig sequences contained N region additions at the V–D and D–J junctions, much like CD27−CD43− B cell Ig sequences (Table I) reflecting the presence of terminal deoxynucleotidyl transferase throughout ontogeny in the human system (Asma et al., 1986). Among cord blood B cells, CD27 expression does not correlate with increased Ig mutational status and does not correspond to memory B cell status, and thus is presumably indicative of another B cell population. Table I. Umbilical cord blood CD20+CD27+CD43+ B cell immunoglobulins express few mutations B cell populations Total mutations (no. of bases) VH mutations (no. of bases) N1 additions (no. of bases) N2 additions (no. of bases) CDR3 length (no. of bases) B1: CD20+CD27+CD43+ 2.0 ± 0.3 0.14 ± 0.05 6.8 ± 1.2 3.8 ± 0.7 14.5 ± 0.7 B2: CD20+CD27 − CD43 − 1.9 ± 0.4 0.08 ± 0.06 6.4 ± 0.8 4.6 ± 0.8 14.8 ± 0.7 Number of cells analyzed: B1, n = 43; B2, n = 25. Mean number ± SEM. Umbilical cord blood CD20+CD27+CD43+ B cells spontaneously secrete IgM We sort-purified cord blood CD20+ B cells and found that CD27 expression (and synonymous CD43+ expression) neatly and completely separated cord blood B cells that spontaneously secrete IgM from those that do not, as shown by ELISPOT (Fig. 1 B,C) and ELISA (Fig. 1 D) analyses. Thus, a small population of CD20+CD27+CD43+ B cells, amounting on average to 50-fold in the ability to stimulate T cell proliferation. Thus, in comparison with the bulk of adult peripheral B cells that do not express CD43, only the small population of CD20+CD27+CD43+ B cells efficiently stimulates T cells. Adult peripheral blood CD20+CD27+CD43+ B cells exhibit tonic intracellular signaling To further evaluate IgM-secreting, T cell–stimulating adult peripheral blood B cells, we examined tonic intracellular signaling by phosphoflow analysis of phosphorylated PLC-γ2 and Syk, as described earlier. Untreated B cells showed little evidence of phosphorylated PLC-γ2 or phosphorylated Syk. As with cord blood B cells, within minutes of phosphatase inhibition we found that CD20+CD27+CD43+ B cells expressed substantial levels of phosphorylated PLC-γ2 and phosphorylated Syk, whereas the levels of phosphorylated PLC-γ2 and phosphorylated Syk in CD20+CD27+CD43− and CD20+CD27−CD43− B cells changed little (Fig. 4). Thus, the small population of CD20+CD27+CD43+ adult blood B cells, and only that population, displays tonic intracellular signaling. Figure 4. Adult peripheral blood CD20+CD27+CD43+ B cells exhibit tonic intracellular signaling. (A) Adult blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, or 12 min, and then fixed, permeabilized, and stained for surface antigens and intracellular phosphorylated PLC-γ2 with specific immunofluorescent antibodies. Because isotype control antibody staining by “fluorescence minus one” did not vary between the three cell populations under study, only a single control tracing is shown. Results for one of three comparable experiments are shown. (B) Mean values of MFI are shown (with error bars indicating the SEM) for intracellular phosphorylated PLC-γ2 staining at various time points from three separate adult blood samples. (C) Adult blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, or 12 min, and then fixed, permeabilized, and stained for surface antigens and intracellular phosphorylated Syk with specific immunofluorescent antibodies. Results for one of three comparable experiments are shown. (D) Mean values of MFI are shown (with error bars indicating the SEM) for intracellular phosphorylated Syk staining at various time points from three separate adult peripheral blood samples. In sum, because CD20+CD27+CD43+ adult blood B cells reproduce the characteristics of CD20+CD27+CD43+ cord blood B cells in manifesting three key B1 cell functional characteristics, CD27 and CD43 identify a B cell population throughout ontogeny that represents the human equivalent of murine B1 cells. Additional surface antigens displayed by CD20+CD27+CD43+ B cells (as single peak expression with >90% positive) are as follows: IgD, CD19, CD21, CD44, CD45RB, and HLA-DR (Fig. S2). CD20+CD27+CD43+ B1 cells display two typical B1 cell specificities The production of particular antimicrobial and autoantibody specificities is a well-described characteristic of murine B1 cells (Hayakawa et al., 1984) and it has been reported that autoreactive Ig is preferentially produced by human CD5+ B cells (Casali and Notkins, 1989), although it has also been reported that human CD5− B cells produce autoantibodies (Mackenzie et al., 1991; Kasaian et al., 1992). Because the repertoire of human B1 cells might well differ from murine B1 cells in terms of pathogenic microbes and self-antigens in view of the tens of millions of years that have passed since murine and human evolution diverged, we did not initially use specificity criteria based on the mouse system to identify human B1 cells. However, having determined the phenotype of human B1 cells, it was of interest to examine whether, as in murine B1 cells, the repertoire of CD20+CD27+CD43+ B1 cells is weighted in favor of antimicrobial and autoantibody specificities. To address this issue, we exposed human adult peripheral blood mononuclear cells to phosphorylcholine (PC; Lalor and Morahan, 1990) and to aspartate-tryptophan-glutamate-tyrosine-serine (DWEYS) tetramer DNA mimetope (Shirai et al., 1991; Jacobi et al., 2009) and determined the level of binding to CD20+CD27+CD43+ (B1), CD20+CD27+CD43− (memory), and CD20+CD27−CD43− (naive) B cells, as described in Materials and methods. We found that only B1 cells, and not memory or naive B cells, contained a substantial number of PC-binding and mimetope-binding antigen receptors (Fig. 5). Thus, human B1 cells are similar to murine B1 cells in displaying a skewed antigen receptor repertoire as indicated by preferential expression of anti-PC and anti-DNA specificities. Figure 5. CD20+CD27+CD43+ B1 cells display two typical B1 cell specificities. Adult peripheral blood mononuclear cells were immunofluorescently stained with specific antibodies and with either PC-BSA-fluorescein (A and B) or biotinylated DWEYS tetramer plus PE-streptavidin (C and D), as described in the Materials and methods. Representative results are shown in A and C for three different B cell populations (CD20+CD27+CD43+ B1 cells, CD20+CD27+CD43− memory B cells, and CD20+CD27−CD43− naive B cells). (top) Gated CD20+CD43− cells; (bottom) gated CD20+CD27+CD43+ cells. Aggregate results are shown in B and D, with the proportion of B cells that bound antigen displayed on the Y axis as mean ± SEM, n = 4. CD20+CD5+ B cells are not the same as CD20+CD27+CD43+ B1 cells Inasmuch as human CD5+ B cells have been reported to express autoreactive specificities, our results raise the question of the relationship between CD5+ B cells and CD27+CD43+ B1 cells. To address this issue, we immunofluorescently stained and analyzed adult peripheral blood CD20+ B cells for CD5, CD27, and CD43. We found that CD20+CD27+CD43+ B cells were largely CD5+, with ∼75 ± 2.5% (mean ± SEM; n = 46) expressing CD5 (Fig. 6 and Fig. S2 B). However, CD20+CD27+CD43+ B1 cells comprised only a minority (34 ± 3.0%) of CD20+CD5+ B cells, so that approximately 2/3 of CD20+CD5+ B cells were outside the CD20+CD27+CD43+ B1 cell pool. Thus, B cell positivity for CD5 captures approximately 3/4, but misses approximately 1/4, of human CD20+CD27+CD43+ B1 cells, and includes many B cells that are not B1 cells, consistent with reports that CD5 marks prenaive, transitional, and activated B cell populations. Figure 6. CD20+CD5+ B cells are not the same as CD20+CD27+CD43+ B1 cells. Adult peripheral blood mononuclear cells were immunofluorescently stained with specific antibodies. The proportion of CD20+CD27+CD43+ B1 cells that express CD5 (75 ± 2.5%; n = 46) is shown in A; the proportions of CD20+CD5+ B cells that phenotype as CD20+CD27+CD43+ (34 ± 3.0%; n = 46), CD20+CD27+CD43− (4.8 ± 0.41%), and CD20+CD27−CD43− (61 ± 3.1%) are shown in B. Representative flow cytometry data are shown in Fig. S2. CD20+CD27+CD43+ B1 cells decline with age in normal individuals To evaluate the variation in B cell populations among normal individuals and with advancing age, we screened 6 umbilical cord and 43 adult peripheral blood samples obtained from normal volunteers. As expected, the fraction of B cells expressing CD27 that lack CD43 (CD20+CD27+CD43−) increased from very few in cord blood to >50% in the sixth through eighth decades as (true) memory B cells accumulated. We found, conversely, that the fraction of CD27+ B cells expressing CD43 (CD20+CD27+CD43+) declined from nearly 100% in cord blood to 99% positive for CD69 and >97% positive for CD70, whereas B1 cells (n = 3) were 90% of CD20+CD27+CD43+ adult peripheral blood B1 cells (Fig. S2) and 100% of umbilical cord blood B1 cells (unpublished data) express IgD. It remains possible that the circulating pool of human B1 cells differs from B1 cells that are tissue-resident, and in that respect ELISPOT assays have not been performed on murine peripheral blood B1 cells. Still, results on tonic intracellular signaling reflect a single-cell population, and no other CD20+ B cell population showed even minimal secretion, so despite the small proportion of B1 cells that are positive on ELISPOT assay we suggest that this most likely represents a single type of B cell in which the test for Ig secretion is imperfectly sensitive. As defined by CD20, CD27, and CD43, human B1 cells are for the most part CD5+, although some are CD5 negative. In preliminary experiments we found these two subpopulations to be equivalent in IgM secretion, T cell stimulation, and tonic signaling (unpublished data) and the existence of CD20+CD27+CD43+CD5− B1 cells may explain previous reports that CD5− B cells are capable of producing autoreactive and polyreactive antibodies. Importantly, CD5 positivity encompasses many B cells that are not B1 as defined herein, consistent with previous reports that follicular B cells at various stages of development and activated B cells express CD5. Thus, isolating B1 cells as CD20+CD27+CD43+ provides a much more refined and discrete population as compared with the very heterogeneous nature of CD5+ B cells. The phenotypic composition of B1 cells has already led to additional insights regarding human B cell populations. First, whereas peripheral (cord) blood at birth would seem to be necessarily devoid of memory B cells, as has been previously reported (Maurer et al., 1990; Agematsu et al., 1997), our results indicate that a population of CD27+ B1 cells is, in fact, present, as has been suggested (Shi et al., 2005), furthering the notion that CD27 is not an immutable indicator of memory B cell status, as also suggested by its presence on developing B cells (Nilsson et al., 2005). Second, whereas adult peripheral blood memory B cells have been reported to efficiently stimulate T cells (Good et al., 2009), our results indicate that this function is actually contributed by B1 cells contained within the CD27+ population and not by memory B cells, per se. In other words, CD20+CD27+ B cells represent a heterogeneous grouping that includes CD27+CD43+ B1 cells and CD27+CD43− “true” memory B cells. Thus, the features ascribed to memory B cells must now be reevaluated, and functional studies of CD27+ B cells should proceed only after depletion of CD27+CD43+ B1 cells to ensure a true memory B cell population. The confusion between CD27+CD43+ B1 cells and CD27+ memory B cells may be particularly acute with respect to so-called “IgM memory” B cells. Like B1 cells, IgM memory B cells express both IgM and CD27; however, as noted above in respect to all memory B cells, IgM memory B cells and B1 cells are two distinct populations separable by CD43 expression. IgM memory B cells lack the functional features of B1 cells; this is clear from the complete failure of CD27+CD43− B cells to secrete IgM, stimulate T cells, or exhibit tonic signaling, even though more than half of these B cells are IgM+. Thus, IgM memory B cells, constituting a substantial portion of CD27+ non-B1 cells, lack the characteristics of B1 cells and are completely separable from them. Notably, it has been reported that IgM memory B cells are responsible for controlling infections produced by Streptococcus pneumoniae and other encapsulated organisms (Kruetzmann et al., 2003); however, the population of IgM memory B cells studied in previous work presumably included B1 cells because prior investigations used CD27 to identify memory B cells. Instead, it may be that (CD27+CD43+) B1 cells are responsible for producing anti-PC antibody in the human system as they are in the murine system, a notion supported by our finding that CD20+CD27+CD43+ B1 cells are repertoire-skewed to preferentially recognize PC in comparison to CD20+CD27+CD43− memory B cells and CD20+CD27−CD43− naive B cells (Fig. 5). This again emphasizes the importance of distinguishing B1 cells from memory B cells so that characteristics of the former not be attributed to the latter. The number of human CD20+CD27+CD43+ B1 cells found in the peripheral circulation was small, and this raises the possibility that human B1 cells may be primarily located in a reservoir other than the peripheral circulation. This is, in fact, the situation with mouse B1 cells, which are located primarily in coelomic cavities and the spleen. The report that anti-Streptococcus pneumoniae IgM memory B cells are generated in the spleen (Kruetzmann et al., 2003) is reminiscent of previous reports on mouse B1 cells indicating they also require the spleen for development (Wardemann et al., 2002), and again raises the possibility that at least some B cells previously described as IgM memory B cells may actually be human B1 cells. Regardless of the relationship between human B1 cells and IgM memory B cells, the number of CD27+ B cells declines with advancing age (Shi et al., 2005), and our results indicate that the fraction of CD43+ B1 cells within the CD20+CD27+ population declines even more precipitously, particularly in the very old age group. Still, preliminary results suggest that the proportion of IgD+ B1 cells changes little with age and remains above 90%. Collectively, however, it is likely that B1 cell protective IgM natural antibody also declines with age, and this may explain, at least in part, the susceptibility of aged individuals to overwhelming infection by encapsulated organisms. It has been conjectured for many years that the normal counterpart for malignant CD5-expressing chronic lymphocytic leukemia cells lies in human B1 cells; however, the identity of such cells has not been known up until now. With the characteristics of human B1 cells in hand, several similarities between normal human B1 cells and malignant chronic lymphocytic leukemia (CLL) cells, at least of the poor prognosis type, are evident. For example, both are CD20+CD27+CD43+CD70−; most normal B1 cells express CD5, as do malignant CLL cells; and, both express relatively nonmutated Ig (Damle et al., 1999; Hamblin et al., 1999; Jung et al., 2003). In addition, we have found that normal human B1 cells ZAP-70 and ILT3 (unpublished data) like CLL cells (Best et al., 2006; Colovai et al., 2007). And, in respect to pathophysiology, the chronically activated phenotype of normal B1 cells may predispose to malignant transformation. Thus, in regard to CLL, in some respects we may be back to the future that was postulated 30 yr ago; to wit, that the normal counterpart cell for, and the target for malignant tranformation by, CLL is the human B1 cell, only now the true identity of the normal counterpart is known. This will, in turn, provide the means to carry out informative experiments to elucidate the nature of CLL neoplasia by comparing malignant CLL cells with the proper corresponding non-transformed B1 cells. In summary, we have found that a small subset of human peripheral CD20+ B cells, specifically expressing CD27 and CD43 and present in both umbilical cord and adult peripheral blood, recapitulates key functional characteristics of murine B1 cells and for this reason is here denoted as the human B1 cell population. Identification of this population carries important implications for studying the normal behavior of B1 cells, for probing the production of natural Ig, for clarifying the functions previously ascribed to memory B cells, for determining the origin of chronic lymphocytic leukemia, and for evaluating the pathophysiology of autoimmune dyscrasias. MATERIALS AND METHODS Donors and samples. Adult peripheral blood samples were obtained by venipuncture of adult volunteers after obtaining informed consent in accordance with the Declaration of Helsinki. Additional samples in the form of leukopacks were obtained from the New York Blood Center on the day of donation. Anonymous umbilical cord blood samples were obtained from the Tissue Donation Program at The Feinstein Institute for Medical Research. This study was approved by, and all samples were obtained in accordance with, the Institutional Review Board of the North Shore-LIJ Health System. Processing. All blood samples were treated in a similar manner and processed promptly upon receipt. Mononuclear cells were obtained by density gradient separation using lymphocyte separation medium (Cellgro). Except as otherwise noted, mononuclear cells were then washed and resuspended in RPMI 1640 (Cellgro) containing 10% fetal calf serum plus 2 mM l-glutamine, 10 mM Hepes, pH 7.25, 100 U/ml penicillin, and 100 µg/ml streptomycin. B cell enrichment. For some experiments B cells were enriched by CD19+ selection using the EasySep Human CD19+ B Cell magnetic bead selection kit (StemCell Technologies) according to the manufacturer’s instructions. Flow cytometry analysis and cell sorting. Enriched B cells and mononuclear cells were sort-purified on an Influx instrument (BD) after immunofluorescent staining, as described in the Results. In all experiments displayed, except in Fig. 9, CD20+ cells were studied. Flow cytometric analysis of immunofluorescently stained cells was performed on a LSR-II instrument (BD). BCR specificity. Adult peripheral blood mononuclear cells were immunofluorescently stained and exposed to PC-BSA-fluorescein and biotinylated DWEYS tetramer. The biotinylation of the DWEYS tetramer was performed using the Miltenyi Biotec one step biotinylation kit according to the manufacturer’s instructions. After 30 min in the dark and on ice, the cells were washed and labeled with PE-streptavidin. Cells were then analyzed by flow cytometry. ELISPOT. Ig secretion was determined by ELISPOT assay as previously described (Tumang et al., 2005), using MultiScreen-IP plates (Millipore) coated with goat anti–human IgM (SouthernBiotech), and blocked with 5% bovine serum albumin (Sigma-Aldrich). In brief, 10,000 sort-purified B cells were cultured in 100 µl RPMI medium (supplemented with 10% FCS for 3 h at 37°C, after which plates were treated with alkaline phosphatase-conjugated anti–human IgM antibody (SouthernBiotech) and developed with 5-bromo-4-chloro-3-indoyl phosphate/p-NBT chloride substrate (KPL). Ig secreting cells were enumerated with Phoretix Expression software (NonLinear Dynamics) after plates were scanned. ELISA. Ig secretion was determined by ELISA assay, as previously described (Hastings et al., 2006). In brief, sort-purified B cells were cultured for 5 d at 1 × 106 per ml in RPMI medium (supplemented as above). Supernatants were evaluated using anti-IgM coated plates (Bethyl Laboratories) and concentrations were determined with a standard curve. Allogeneic stimulation. Naive CD4+ T cells were negatively selected from PBMC using the EasySep human naive CD4+ T cell magnetic bead selection kit (StemCell Technologies) according to the manufacturer’s instructions and were co-cultured at a ratio of 2:1 with sort-purified, irradiated (4,000 rads) B cells (50,000) in 0.2 ml in RPMI medium in triplicate wells of 96-well round bottom plates. Cultures were pulsed with 0.75 µCi [3H]thymidine for the last 8 h of 5-d cultures, and counts per minute (cpm) were determined by scintillation counting. Phosphoflow analysis. Mononuclear cells were analyzed after phosphatase inhibition, as previously described (Holodick et al., 2009b). In brief, cells were treated with sodium pervanadate for varying periods of time, after which they were fixed with paraformaldehyde, permeabilized with methanol, and stained for surface antigens and intracellular phosphorylated proteins with specific immunofluorescent antibodies. Flow cytometric analysis was performed using a LSR-II instrument. Single cell Ig sequencing. Individual cells were sort-purified onto a 48-well Ampligrid (Beckman Coulter) and Ig sequences were PCR-amplified in a semi-nested approach as previously described (Holodick et al., 2009a), using primers designed for human Ig gene transcripts (Wang and Stollar, 2000). Products were sequenced (GENEWIZ), and sequences were analyzed using the International ImMunoGeneTics Information System. Reagents. Sodium orthovanadate was obtained from MP Biomedicals; one-step pcr and gel extraction kits were obtained from Qiagen; LPS and PMA were obtained from Sigma-Aldrich; CpG was obtained from Invitrogen; ionomycin and SAC were obtained from Calbiochem; anti-IgM was obtained from Southern Biotech; recombinant IL-2 and anti-CD40 were obtained from R&D Systems; recombinant IL-6 was obtained from BD; and fluorescently labeled antibodies (anti–CD20-APC-Cy7, anti–CD27-V450, anti–CD43-FITC, anti–CD70-PE, anti–CD5-PE-Cy7, anti–-IgD-PE, anti–CD19-PE, anti–CD21-PE, anti–CD44-PE, anti–HLA-DR-PE, anti-CD45RB, anti–phospho-PLC-gamma-2-A647, and anti–phospho-Syk-A647) were obtained from BD. Anti–CD69-PE was obtained from Beckman Coulter. Anti–CD43-APC was obtained from eBioscience. PC-BSA-fluorescein was obtained from Biosearch Technologies. DWEYS tetramer protein was a generous gift from B. Diamond (The Feinstein Institute for Medical Research, Manhasset, NY). PE-Streptavidin was obtained from Biomeda. Online supplemental material. Fig. S1 shows that umbilical cord blood B1 cells display preferential heavy chain variable gene usage. Fig. S2 shows that adult peripheral blood B1 cells express CD19, CD21, IgD, CD44, and CD45RB, but not all B1 cells express CD5. Table S1 shows actual sequences of Ig genes from umbilical cord B1 cells. Table S2 shows actual sequences of Ig genes from umbilical cord naive B cells. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20101499/DC1.

Recent advances in genomics and proteomics, combined with the facilitated generation and analysis of transgenic and gene-knockout animals, have revealed new complexities in classical biological systems, including the B-cell compartment. Studies on an 'old', but poorly characterized, B-cell subset--the naive, marginal-zone (MZ) B-cell subset--over the past two years have spawned an avalanche of data that encompass the generation and function of these cells. Now that the initial 'infatuation' is over, it is time to reconsider these data and generate some conclusions that can be incorporated into a working model of the B-cell system.