A Hard(y) Look at B-1 Cell Development and Function

This review focuses on a small population of B cells, termed “B-1 cells,” which exists in lymphoid tissues and body cavities of mice and is distinct in development, phenotype, and function from the majority B cell population, termed “conventional” or “B-2 cells.” B-1 cells were identified initially as “Ly-1” B cells, expressing the surface Ag Ly-1 (mice) or “Leu-1” (humans) now known as CD5. This discovery, nearly 35 y ago (1), was tightly linked to the development of mAbs and of multicolor flow cytometry and its application for the analysis of leukocytes. It was the team around Len and Lee Herzenberg at Stanford University, including K. Hayakawa, R.R. Hardy, and D.R. Parks (1), who first identified this B cell subset in mice.

The discovery of CD5⁺ B cells was not a chance finding, but rather was spurred by the discovery that CD5 was expressed on most human B-type chronic lymphatic leukemia (CLL) cells (B-CLL) (2, 3) and on many B cell tumors (4). The original goal had been to identify the normal CD5⁺ B cell counterparts of these tumors, although follow-up studies revealed a likely more complex picture on the precursor relationship between CD5⁺ B cells and CLL. CD5, whose ligand is still unknown, has been identified as a negative regulator of T and B cell Ag–receptor signaling. CD5 is expressed on all T cells, where it is required for normal thymocyte development. Its expression by B-1 cells has been linked to their inability to proliferate in response to IgM–BCR stimulation, whereas CD5 expression by conventional B cells has been linked to the maintenance of the anergic state (5–7).

Although the initial impetus was to find CD5-expressing B cells, it soon became clear that B-1 cells were distinct in many other ways from conventional B cell populations. Indeed in 1992, Kantor et al. (8) and Stall et al. (9) reported on a population of B-1 cells that lacked expression of CD5, but otherwise showed many similar characteristics. This included their 1) maintenance by self-renewal; 2) ability to survive long term and expand after adoptive transfer, in contrast to the rapid death seen after transfer of conventional B cells; 3) predominance in the pleural and peritoneal cavities of mice; and 4) and ability to secrete IgM without foreign Ag exposure (8, 9). Collectively, the data showed that CD5 expression was insufficient for delineating all cells with the characteristics of Ly-1 B cells. A new nomenclature was therefore adopted in 1991 (10), in which these early developing B-1 cells were distinguished from later developing, bone marrow–derived, conventional B-2 cells and in which B-1 cells were separated based on their expression or not of CD5 into B-1a and B-1b, respectively.

This review outlines the scientific milestones that have led to our current understanding of B-1a cell development and regulation. I will attempt to highlight major findings made by R.R. Hardy, who died recently and who together with K. Hayakawa made some of the most impactful discoveries about this still-enigmatic B cell subset.

The original studies on CD5⁺ B cells revealed what has turned out to be one of their most important characteristics, namely their relative abundance in young mice and their reduced frequencies as mice age (from 30% in the spleen on day 5 after birth to ∼1–2% by 8 wk) (1). Cell transfer experiments soon demonstrated that adult bone marrow transfer did not fully reconstitute the B-1a cell compartment of lethally irradiated mice, whereas transfers of fetal liver as well as newborn spleen and bone marrow were able to do so (11). The selective ability of early but not later developing precursors to fully replenish the B-1a compartment suggested that distinct B cell hematopoietic precursors in fetal and adult compartments give rise to B-1 and B-2 cells, respectively. In the 30-plus intervening years since these original studies, many subsequent data were published in support of and against the hypothesis of an independent lineage of B-1 cells. We refer to prior reviews on that topic for a more comprehensive discussion (12–15).

As outlined below, lineage tracing studies together with the recent identification of B-1 cell–restricted precursors in the fetal liver and neonatal bone marrow, which lose de novo B-1 cell development potential by ∼6 wk after birth, have provided support for a lineage distinction between B-1 and B-2 cells. Furthermore, recent studies showed that a newly discovered master regulator of fetal but not adult hematopoiesis, Lin28b, drives B-1 cell development (16, 17). Together the data support the early observations of B-1 cells as belonging to a wave of B cells that develop early in ontogeny and onto which later developing B-2 cells are “layered” (14).

Indications for the presence of distinct B-1 and B-2 precursors were first provided by studies with embryonic tissues such as the para-aortic splanchnopleura (18) and the fetal liver (11) which, when taken from early embryos, were shown to contain precursors that give rise exclusively to B-1 cells. In contrast, transfer of bone marrow fully reconstituted the B-2 but not the B-1a cell compartment. Two important technological advances eventually led to the identification of distinct precursor populations for B-1 and B-2 cells.

First, the establishment of culture systems that recapitulated B cell development from precursors in tissue culture (19). In support of those efforts, Hardy et al. (20) established a fetal liver-derived stromal cell line (ST-2) that is now widely used for these purposes. Second, using the ability to generate distinct stages of B cell development in vitro, and assessing Ig gene rearrangement in the cultured cells by PCR, Hardy and his team developed a flow cytometry–based scheme to distinguish distinct B cell developmental stages. This now widely adopted “Hardy scheme” uses a set of seven surface markers, AA4.1, CD24, CD43, CD45R (B220), BP-1, IgM, and IgD, to distinguish the stages of B cell development from early pre–pro–B cells (“Hardy fraction” A) to mature B cells (“Hardy fraction” F). Each step of the B cell developmental path identified by phenotype is reflective of a step in the successive rearrangement of Ig H and L chain genes in that cell (20, 21).

Montechino-Rodriquez et al. (22) then used this approach to identify distinct precursor populations for B-1 and B-2 cells in fetal liver and bone marrow. B-1 cell precursors were identified as lacking expression of CD45R (B220) among AA4.1⁺ CD19⁺ CD43⁺ pro–B cell precursors, whereas early B-2 cell precursors expressed CD45R but lacked CD19. Consistent with previous studies (23, 24), follow-up studies then demonstrated that B-1a cell output from bone marrow precursors strongly diminished over time, such that by ∼6–8 wk of life they no longer contribute significantly to the B-1 cell pool (25). The data were consistent with reports that total bone marrow transfer into irradiated adult mice results in only limited B-1a cell reconstitution. However, the extent of B-1 cell reconstitution observed following bone marrow transfer seems to vary greatly from laboratory to laboratory, with some researchers finding very poor reconstitution (23, 24) and others finding reconstitution to be extensive (26, 27). In our hands, and consistent with studies by Lalor et al. (23), B-1a cell reconstitution after bone marrow transfer is slow and incomplete, resulting in higher frequencies of B-1b than B-1a in the peritoneal cavity of reconstituted mice. The data by Dorshkind et al. (25) would support an overall poor B-1a cell reconstitution potential of bone marrow precursors. The discrepancies may indicate the presence of some unknown stimuli that could reactivate the reconstitution potential of B-1 precursors in the bone marrow. However, such a signal(s), if it exists, has not been identified (23, 26, 27).

The above outlined experiments provided significant evidence for the existence of B-1 cell–restricted precursors in embryonic and fetal tissues. Already in 1991, Hardy and Hayakawa (28) argued that the appearance of CD5⁺ B cells from fetal but not adult pro–B cells represent a “developmental switch in B lymphopoiesis,” similar to that observed for erythropoiesis, and that these cells are then carried over into the adult long-lived B cell pool. They observed that such a developmental switch was consistent with existing data on the preponderance of B-1 cells early, but not later, after birth and the adoptive transfer data summarized above, and it supported the hypothesis of the layering of immune cell development (14) observed for γδ T cells (29, 30) and macrophages (31).

Indeed, studies published since have demonstrated the presence of a developmental switch in B cell lymphopoiesis and established that fetal hematopoietic stem cells (HSC) differ significantly from adult HSC. The transcription factor Sox 17 was the first molecular factor identified as being required selectively for the maintenance of fetal but not adult HSC (32). Subsequently, Yuan et al. (16) reported in 2012 on the expression of Lin28b in fetal but not adult HSC and common lymphoid progenitors in both humans and mice. Lin28b is known as a posttranscriptional inhibitor of Let-7 microRNAs (miRNAs), a family of miRNAs expressed in adult but not fetal B cell precursors. This important study showed that ectopic expression of Lin28b in adult HSC instructed a “fetal HSC” transcriptional program, leading to their preferential reconstitution of various innate-like lymphocytes, including the B-1a cell compartment (16). Hardy et al. also identified Lin28b as a regulator of fetal hematopoiesis and confirmed that the regulation of Let-7 by Lin28b was sufficient to instruct fetal/adult B cell development. Furthermore, his group showed that the major target of Let-7 miRNA–mediated transcriptional control is the transcription factor Arid3a, which modulates BCR signaling (17). Importantly, the study by Hardy et al. (17) also suggested that ectopic expression of Lin28b, although sufficient to drive extensive development of B-1a cells from adult bone marrow precursors, was insufficient to reconstitute the normal BCR repertoire of B-1a cells. Thus additional differences between fetal and adult B cell development must exist that are independent of Lin28b/Let-7 and that mediate development of B-1 cells and affect the B-1 cell repertoire.

A fetal versus adult developmental switch integrates known differences in the development and selection of B-1 and B-2 cells. However, additional complexities in the developmental paths of fetal/neonatal B-1 cells appear to exist. Early B cell precursors are present in multiple tissues, including the embryonic yolk sac, para-aortic splanchnopleura, fetal liver, and neonatal bone marrow. In keeping with the hypothesis of a layered immune system, Montecino-Rodriquez and Dorshkind (33) suggested that each of these tissues, at different times in ontogeny, give rise to waves of B-1a cells: A first wave that is derived from nonhematopoietic precursors, potentially developing from hemogenic endothelium in the embryonic yolk sac (34–37); a previously identified wave of B-1 cell development from the aorta–gonad–mesonephric region (18) and the fetal liver; and finally an early wave of bone marrow–derived B-1 cells in the neonate (38–40) (Fig. 1). A question emerging from these findings is, which of these precursors contribute to the B-1 cell pool that persists into adulthood?

Earlier studies showed that fetal-derived HSC had long-term reconstitution potential after adoptive transfer into lethally irradiated adult recipients (41). Using the receptor tyrosine kinase Flk2, previously shown to be expressed on fetal, but not adult, HSC with long-term multilineage reconstitution potential (41); Forsberg and colleagues (42) generated a reporter mouse in which expression of Flk2 would lead to deletion of expression of one fluorochrome and expression of another. Using this system, they could then follow the fate of adoptively transferred HSC. The results demonstrated the continued presence of B-1 cells from both Flk2-expressing and -nonexpressing precursors, not all of which persisted into adulthood. Furthermore, they demonstrated the preferential generation of peritoneal cavity B-1 cells from fetal-derived, transient, Flk2-expressing HCS (43). Thus, at least two distinct B cell precursors contribute to B-1 cell development (Fig. 1).

The repertoire of B-1 cells in body cavities and the spleen reflects their developmental paths. A relatively large fraction of B-1 cells lack nontemplate-derived nucleotide (N-region) insertions at the VDJ joining ends, consistent with a lack of TdT expression in the fetus (44, 45). TdT induction is controlled by IL-7αR signaling, which is expressed at lower levels in fetal B cell precursors (46) and is downregulated following expression of a rearranged μ-H chain as part of the pre-BCR complex (47). Increased frequencies of N-region–containing sequences of peritoneal cavity B-1 cells were reported for aging mice (48).

Hardy and colleagues identified the skewing of the B-1 cell repertoire to mostly autoreactive specificities early in their studies. One to two percent of B-1 cells in the spleen (49) and closer to 10% in the peritoneal cavity were found to bind to the head group of phosphatidylcholine (PtC), a cell surface phospholipid expressed on senescent RBCs. V-gene usage by these PtC binders was found to be highly restricted, but not monoclonal. In C57BL/6 mice, PtC-binding BCR are predominantly encoded by VH11 or VH12 in conjunction with JH1 (49–51); whereas in BALB/c mice, PtC binders are encoded predominantly by VHQ52/JH4 (52). Self-reactivity, predominance, and clonal relationship of anti-PtC–specific B-1 cells in the body cavities suggested an Ag-specific expansion process (49, 50, 53). Consistent with that, repertoire studies demonstrated continued expansion of B-1a cells with that specificity over the first few months of life, including in mice kept germ free (45).

Another binding specificity of B-1a cells, extensively studied by Hayakawa et al. (54), is a carbohydrate epitope on the thymocyte glycoprotein “Thy-1” that is recognized by a natural IgM. Elegant studies by that group showed that mice lacking this self-antigen also lacked B-1 cells of that specificity (55, 56). This led them to propose that B-1 cells, similar to T cells, are positively selected based on their ability to bind to self-antigens. Further supporting strong self-antigen binding as a driver of B-1a cell development were studies that showed the lack of B-1a cell development in CD19-deficient mice (57), as well as in mice lacking either other costimulatory molecules (reviewed in Ref. 13) or components of the NF-κB signaling cascade (58); all defects that reduce BCR signaling. Thus, heavy usage of germline-encoded BCR and the presence of relatively large clones of distinct, self-reactive B-1a cells develop in all mouse strains to shape the B-1 cell repertoire, possibly following BCR stimulation by self-antigens.

Hardy et al. (59) further aimed to understand the mechanisms for the repertoire skewing and distinct VH usage of B-1 compared with B-2 cells, which led them to study pre-BCR signaling. In pro/pre–B cells, the surrogate L chains, V-preB and λ5, are expressed as binding partners for the rearranged μ-H chain, with which they form the pre-BCR (60). Pairing of the rearranged μ-H chain with surrogate L chains was shown to be critical for conventional B cell development in the bone marrow. In contrast, by using a B-1a cell–restricted VH11 transgene, the dominant VH gene associated with PtC binding, Hardy’s group (59) demonstrated that VH11 μ-H chains did not pair with surrogate L chains, yet they still enabled B-1 cell development. The data thus suggested that B-1 and B-2 cell development rely on distinct pre-BCR signals. Altered pre-BCR signaling was also suggested to play a role in the development of B-1 cells expressing a BCR specific for the hapten arsonate in A/J mice (61). Such differential requirement for pre-BCR signaling provides a potential further distinction between fetal and adult-developing B cells that could lead to strong differences in VH gene usage among B-1 and B-2 cells and explain their repertoire differences.

Together, the existing data suggest that self-reactivity acts as a positive selection step for B-1a cell development and/or expansion. Yet, positive selection for self-reactivity is anathema to our understanding of adaptive T and B cell development, a process that is expected to result in the development of a broadly reactive repertoire of Ag receptors devoid of, or at least greatly curtailed for, self-reactivity. Nevertheless, the evidence for positive selection of B-1 cells is overwhelming, as is the finding of B-1 cell–derived spontaneous natural IgM production that contributes broadly self-reactive natural Abs. This points not only to a distinction between fetal and adult B cell development but also to fundamental differences between B-1 and B-2 cell function.

Despite the restricted de novo development of B-1 cells in adulthood, dramatic and dynamic changes of the B-1a cell repertoire in ontogeny have been documented for B-1a cells from peritoneal cavities and spleens via next generation sequencing (45). Importantly, those studies confirmed that the de novo appearance of B-1 cell clones is restricted to the early phase of ontogeny, but also provided evidence that strong ongoing selection and/or clonal expansion shapes the repertoire of the adult B-1 cell pool. Similar repertoire changes among B-1a cells seem to develop over time even when mice were reared in the absence of microbiota, as the B-1a cell repertoire of mice housed in specific pathogen-free and germ-free conditions were indistinguishable (45).

In apparent contrast to the above studies with gnotobiotic mice, evidence for direct effects of foreign Ag exposure on the B-1 cell repertoire came from studies by Kearney and colleagues. They showed that injection of α1-3 dextran permanently altered the repertoire of carbohydrate-reactive B cells when given within a short window after birth, but not later (summarized in Ref. 62). This Ag is known to activate B-1 cells (63), and is expressed on Aspergillus fumigatus as well as house dust mites. Although immunization did not change anti–α1-3 dextran serum IgM levels, it expanded the repertoire of polysaccharide-specific B cells and protected the mice from increased airway reactivity and signs of TH2-mediated airway disease (64). In addition, recent studies suggested that a portion of mainly B-1 cell–derived maternal IgG3 Abs binds to gut microbiota and is induced in a TLR-dependent manner (65). Although this could be explained by cross-reactivity of certain B-1 cells induced originally to self-antigens or damage-associated molecular pattern molecules; significant changes are reported in the B-1a cell repertoire of mice around the time of weaning, when food Ags and the gut microbiota undergo extensive changes (45).

Excluding the contribution of food Ags to the observed repertoire changes at various times after birth is experimentally very challenging. Even sterile food can contain pathogen-associated molecular pattern molecules or other structural components that may influence B-1 cells in a TLR-dependent manner, without the presence of actual microbiota. This would be consistent with the findings by Koch et al. (65), who showed that maternal IgG2b and IgG3 Ab development to gut microbiota requires TLR signaling. In contrast, weaning is also the time that the Flk2⁺ HSC, which give rise preferentially to B-1 cells, seem to be lost (43). Their disappearance could alter the B-1 cell pools. Future studies will be required to understand more fully the extent to which self-antigens and foreign Ags shape the B-1 cell repertoire during and after development and selection, and to understand what triggers B-1 cell clonal expansion.

Once established, B-1 cell numbers seem to be maintained through self-renewal, i.e., slow turnover. This was first suggested by studies demonstrating that mature IgM⁺ CD5⁺ B-1 cells from the peritoneal cavity were able to fully reconstitute all B-1 cell compartments after adoptive transfer, a stem cell–like behavior (11, 23, 24, 66). The findings by Beaudin et al. (43), which provided evidence for a developmentally restricted HSC with preferential B-1 cell reconstitution potential, seem to support those early data by also suggesting that once established, the maintenance of the peripheral B-1 cell pool is achieved at the level of self-renewal rather than de novo development. If correct, then the continued presence of B-1 cells from those time-restricted HSC precursors would not require the continued presence of the HSC, but rather signals that trigger self-renewal of the mature B-1 cells. Identification of the signals and the biological context that cause B-1 cell expansion and/or maintenance should be an important future research goal for the field.

In this context it is interesting to note that the transfer of peritoneal cavity B-1 cells into neonatal mice, rendered otherwise temporarily B cell deficient, caused a near complete arrest of subsequent B-1 cell reconstitution from the bone marrow (23). Thus, B-1 cells or their products, particularly natural IgM Abs, might be considered candidates for providing feedback regulation. Indeed, mice genetically engineered to lack secreted IgM (sIgM), but not membrane-bound IgM, were originally reported to harbor increased frequencies of peritoneal cavity B-1a cells (67, 68), suggesting that the presence of sIgM might control B-1 cell development. Similarly, a recent study suggested that natural Ab production regulates the size of the B-1 cell compartment (69). However, with regards to the former study, multiple lines of evidence demonstrated that the accumulating CD5⁺ B cells in the body cavities of sIgM^−/− mice, previously identified as B-1a, were not B-1 cells, but anergic B cells: Phenotypic analysis showed that they lacked surface expression of CD43, a hallmark of many albeit not all B-1 cells (70); they did not express high levels of CD19, characteristic of B-1a cells; and they nearly completely lacked cells binding to liposomes containing PtC, which is predominantly B-1a cell specific and encoded by VH11 or VH12, genes that were not expressed by peritoneal cavity B cells in sIgM^−/− mice (71). Functional analyses confirmed that most CD5⁺ B cells in the sIgM^−/− mice were anergic B-2 cells (71). Conclusions from the earlier studies that reported expansion of B-1a cell populations in the absence of sIgM (67, 68) were thus likely based on the somewhat subtle phenotypic differences between B-1a cells and anergic B cells. B-1 cell frequencies were either unaffected or even enhanced in mice that lacked the FcμR (72–74), although FcμR^−/− mice had increased B-1 cell–derived serum IgM levels. Therefore, sIgM does not appear to provide a direct negative feedback signal for B-1 cell development and/or expansion.

As outlined above, early work on B-1 cells, including that by Hardy and colleagues, recognized that a major function of B-1 cells is the generation of natural Abs, mainly IgM and IgG3, that react with both self-antigens (anti–Thy-1 and anti-PtC) as well as foreign Ags (Ags on Streptococcus pneumonia, influenza, Borrelia hermsii, Salmonella, among others, reviewed in Ref. 12). However, the exact mechanisms that induce and control natural Ab production remain incompletely understood. In contrast to B-2 cells, which require foreign Ag and often CD4 T cell help for activation to Ab secretion, B-1 cells apparently “spontaneously” differentiate into natural Ab-forming cells (AFC). Given the observed dynamic changes to the repertoire outlined above, it seems that BCR-engagement to self-antigens and/or environmental Ags drives these processes. This is supported by studies with gene-targeted mice that show enhanced BCR signaling and increased serum IgM levels in mice with deletions of a repressor of BCR signaling, such as the two members of the sialic acid–binding Ig-like lectin family, CD22 and Siglec G (75), as well as CD72 (13). Furthermore, B cell–specific deletion of the FcμR, which was shown to increase BCR expression and BCR tonic signaling due to enhanced BCR transport from the trans-Golgi (72), resulted in increased numbers of CD138⁺ B-1 plasma cells in the spleen and higher concentrations of serum IgM (72). In all cases, however, both B-1 cell development and B-1 cell differentiation were enhanced. In contrast, genetic ablation of IL-5Rα, or the lack of IL-5, resulted in selective reductions in natural serum IgM and IgG3 levels as well as reduced B-1 cell frequencies (76, 77). Given that injection of IL-5 induces strong IgM production by B-1 cells in vivo (76–78) and drives plasma cell differentiation of B-2 cells in vitro (79), it is tempting to speculate that IL-5 enhances B-1 cell differentiation to Ab-secreting cells. It will be important to identify the cellular source for this IL-5 and the biological processes by which IL-5 production regulates natural Ab production in vivo.

To better understand the regulation of natural Ab production and development of B-1 cell AFC we studied the phenotype and developmental paths of IgM- and IgG3-secreting B-1 cells. Consistent with previous studies (reviewed in Ref. 80), we found spontaneous IgM production to be restricted to bone marrow and spleen in both specific pathogen-free–housed as well as germ-free mice (81, 82). The study identified two distinct populations of natural AFC: B-1 cell–derived CD19⁻ CD43⁺ IgM⁺ plasma cells (B-1PC) and CD19⁺ CD43⁺ IgM⁺ B-1 cells (82). Both cell populations contributed significantly to natural Ab production. Although B-1PC was the dominant IgM-AFC population in the bone marrow (67% of IgM-AFC), they contributed only ∼25% of IgM-AFC in the spleen. Intriguingly, a subset of B-1 cells expressed J chain and intracellular IgM, but neither expressed nor required Blimp-1 for maximal IgM or IgG3 secretion.

Further analysis suggested that these B-1 cells develop into AFC without terminal differentiation (82), which is consistent with reduced but not absent serum IgM levels and normal serum levels of IgG3 in mice with a B cell–specific deletion of prdm1, the gene encoding Blimp-1 and shown to be a master regulator of B cell differentiation. The above-described effects of IL-5 on natural IgM and IgG3 may only affect the Blimp-1–dependent arm of natural Ab production, as IL-5 induces Blimp-1 expression (79), which could explain why natural IgM and IgG3 production is reduced but not gone in the absence of IL-5. The data may also explain previous contradictory results regarding the need for Blimp-1 in natural Ab secretion (83–85). Interestingly, a similar lack of Blimp-1 expression was noted also for a subset of IgM-secreting AFC in the shark (86). Lack of terminal differentiation might allow B-1 cells to rapidly adjust to changing needs for natural Ab production and/or it may enable continued self-renewal.

B-1 cells were shown to regulate immunity by cytokine secretion. A subset of body cavity B-1 cells appears to constitutively express IL-10, which may imbue them with regulatory functions (87). B-1 cell–derived IL-10 production has been associated with attenuated responses to infection with Leishmania (88, 89) and artherosclerosis (90), and it appears to enhance B-1 cell expansion via induction of proliferation (91). The term “innate response activator (IRA) B cell” was coined by Swirski and colleagues for B-1a cells from the body cavities that migrated in response to inflammatory cues and accumulated in the spleen and lung early in sepsis. These cells generated not only IgM, but also GM-CSF and IL-3, importantly affecting the inflammatory responses (92–94).

Indeed, migration of B-1 cells from the body cavities to secondary lymphoid tissues and their activation to IgM-secreting cells is a common outcome of activation of peritoneal and pleural cavity B-1 cells in response to cytokines and various pathogen-associated molecular pattern molecules (95–99) (Fig. 2). Whether all B-1a cells can respond to inflammatory cues with cytokine production remains to be determined. Together the data indicate the functional heterogeneous nature of the peripheral B-1 cell pool, which serve to both attenuate inflammatory responses and control infections by generating broadly reactive natural Abs as well as cytokines.

Continuing from the earliest studies that aimed to identify the CD5⁺ nonmalignant counterparts to the CD5⁺ B-CLL, Hardy and Hayakawa continued to investigate the potential link between B-1 cells and the development of this common leukemia emerging in aging individuals. B-CLL is characterized not only by CD5 expression but also by a skewed BCR repertoire encoding auto- and poly-reactive Abs (100), all features they have in common with B-1 cells. In their most recent studies, the group identified a nonmutated BCR CDR3 region in B-1 cells of mice that encoded binding to myosin IIA, a specificity recurrent among human B-CLL (101). Interestingly, they observed that B-1 cells with that specificity could expand greatly with age and eventually develop into CLL. The data suggest that the inherent ability of B-1 cells for self-renewal, and a constant activation trigger provided by self-antigen recognition, may predispose B-1 cells and their potential homologs in humans toward malignant transformation. In addition to the studies outlined above that demonstrate the importance in BCR signaling for B-1 cell activation and natural Ab production, the data further suggest that the presence of self-antigens provide potent and continued stimulation for B-1 cells, including after development and selection, driving clonal expansion, differentiation, and potentially, in rare instances, malignant transformation.

Early studies demonstrated that similar to mice, human cord blood and fetal spleens also contain higher frequencies of CD5⁺ B cells than adult spleen and blood (102). In 1987, Hardy et al. (103) and Casali et al. (104) simultaneously reported on the presence of CD5⁺ B cells in human blood that dominated the cord blood B cell population of neonates, and they reported an increase of CD5⁺ cells in patients with autoimmune diseases. Furthermore, we now understand that in humans, as in mice, a developmental switch from fetal to adult-like B cell hematopoiesis occurs that is controlled by Lin28b/Let-7 (16), thus strongly suggesting that human fetal-derived B-1 cell orthologs exist. However, given the much higher frequencies of CD5⁺ B cells in adult humans (17% of PBMC) versus mice (0.5% of PBMC), and the fact that not all B-1 cells in mice express CD5, it is clear that CD5 alone does not mark B-1 cells. Rather, CD5 expression seems to mark B cells having a certain degree of autoreactivity, both among fetal-derived B-1 cells and postnatal-derived (anergic) B-2 cells (6).

The exact nature and the phenotype of the human B-1 cell orthologs, however, has remained a topic of much study and controversy. This includes the most recent efforts by the group of Rothstein et al. (105) that led them to propose that a population of PBMC with the phenotype CD20⁺ CD27⁺ CD43⁺ and an ability to spontaneously secrete self-antigen reactive IgM is the human B-1 cell. However, the results of that study have been vigorously questioned (106–108) and further studies are required. The discovery of the Lin28b dependency of human fetal B cell development (16) might facilitate such studies.

The work by Hardy et al. has provided multiple seminal contributions to the field of B cell and B-1 cell biology. Their original findings of a fetal-derived B cell population that persists into adulthood and their continued quest to understand and define these cells in the context of their development has moved the field of B-1 cell biology in a way few others have done. They remained steadfast in their quest, even at times when the sheer existence of these cells was questioned. Despite the continued advances made, much remains to be explored about the mechanisms and signals that control B-1 cell activation and differentiation and to more fully understand the breadths of their functions in health and disease.

This review is dedicated to the memory of R.R. Hardy. I apologize for not being able to include all relevant work, due to space limitations.

The authors have no financial conflicts of interest.