The H2 haplotype regulates the distribution of B cells into B-1a,B-1b and B-2 subsets

The size of B-cell subsets appears to be under genetic control, but the mechanism of this regulation is unknown. By analyzing five congenic strains of mice that differ only in their H2 haplotype, we addressed the issue of whether the MHC genes are involved in the relative proportions of B-1a, B-1b and B-2 cells. Not only were there considerable differences in the percentages of B-1 in B cells between H2s mice which were the highest [78.5+/-0.8% in the peritoneal cavity (PerC), and 26.3+/-0.5% in the spleen] and H2d mice, which were the lowest (15.2+/-0.6% in the PerC, and 10.9+/-0.6% in the spleen), but the percentages of B-1a cells varied inversely to those of B-1. Crosses between H2s and H2d strains showed that the highest B-1 frequencies occurred in F2 progeny expressing the homozygous H2s (70.8+/-2.1% in the PerC, and 30.0+/-0.5 in the spleen), and the lowest in that expressing the homozygous H2d haplotype (8.9+/-0.6% in the PerC, and 8.6+/-0.4% in the spleen). A dose effect of H2 was established in heterozygous F1 and F2 mice. As mice aged, there was a reduction of B-1 cells in the PerC, at the expense of B-1b in the H2s, but not in the H2d mice. Hence, the H2 genes appear to participate in regulating the proportions of B-1a, B-1b and B-2 cells.     

IL-9-induced expansion of B-1b cells restores numbers but not function of B-1 lymphocytes in xid mice

Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton's tyrosine kinase and were shown to be specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1 lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature peritoneal B-1 cells that all belonged to the CD5(-) B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5(+) B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally distinct from CD5(+) B-1a lymphocytes.     

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ^null engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ^null mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D_H-J_H pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.     

B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures.

Analyses of VHDJH rearrangements isolated from murine peritoneal B-1a cells (CD5+, IgMhi, B220lo), peritoneal B-1b cells (CD5-, IgMhi, B220lo), and conventional splenic B cells provide evidence that a unique repertoire of VH regions is displayed by each of these B-cell subsets. The B-1a subset is characterized by a low N-region diversity, by a high frequency of sequence homologies in the VH-D and D-JH junctions, and by a limited exonuclease nibbling of the terminals of the joining gene segments. Through expansion in ageing mice, B-1a clones with these properties are favoured. B-1b cells are similar to conventional B-2 cells with respect to N-region diversity, but are unique in terms of D gene expression. Thus, while most murine pre-B and B cells preferentially use DSP and DFL gene segments in a given reading frame (RF1), B-1b cells frequently express D genes in another reading frame (RF2). Together, these findings provide structural evidence for a model where B-1a, B-1b and B-2 cells are produced by separate progenitors that are active at different stages of ontogeny.     

B-1 B cell development

CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.     

Maintenance of peritoneal B-1a lymphocytes in the absence of the spleen

Positive selection by autoantigens is believed to play an important role in the generation/maintenance of B-1a cells. Recently, it has been described that splenectomy results in the loss of an already established B-1a cell pool. To elucidate whether the spleen influences the peritoneal B-1a repertoire, we have analyzed the consequences of splenectomy in the recently established IgL-transgenic L2 mouse model. L2 mice are characterized by a severe block of B-2 development and predominance of B-1a cells, which exhibit a pronounced IgH oligoclonality, presumably due to positive selection by autoantigens. In this study, we show that, in striking contrast to splenectomized normal mice, L2 mice exhibit unchanged frequencies of peritoneal B-1a cells. The IgH repertoire of these B-1a cells, however, was severely perturbed in that the previously described predominant B-1a H chains were no longer present. The repertoire changes were partial since phosphatidylcholine-specific B-1a cells were present in similar numbers before and after splenectomy. Thus, splenic Ags appear to act as "survival factors" for major subsets of peritoneal B cells. The loss of B-1a cells in the absence of such factors is compensated by repertoire changes among B-1a cells in B cell lymphopenic L2 but not normal mice.     

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.     

Association of B-1 B cells with follicular dendritic cells in spleen

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.     

Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.     

Differentially regulated expression and function of CD22 in activated B-1 and B-2 lymphocytes

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5(+) B-1 cells, consistent with a weak Ca(2+) response in anti-IgM-treated CD5(+) B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca(2+) influx in CD5(+) B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

The selection of marginal zone B cells differs from that of B-1a cells

Transgenic (Tg) L2 mice expressing high levels of the lambda2 (315) L chain contain only B cell populations involved in the first line of defense, i.e., B-1 and marginal zone (MZ) B cells. The strongly oligoclonal IgH chain repertoire of Tg B-1a cells in such mice was attributed to strong positive selection by autoantigens. In this study, we show that the MZ B cells of L2 mice correspond very closely to MZ B cells of normal mice, as revealed by surface marker expression and gene expression profiling. We demonstrate that the IgH chain repertoire of these Tg MZ B cells is extremely heterogeneous. This is in sharp contrast to the oligoclonality found in B-1a cells of the same mice, which was attributed to strong positive selection mediated by autoantigens. Therefore, the strong positive selection of the IgH chain repertoire in L2 mice is B-1a specific. Thus, our data demonstrate that despite common functional properties, MZ B and B-1a cells exhibit striking differences in their selection and/or maintenance requirements.     

Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese diabetic mice and lupus-prone MRL-Fas(lpr) mice

The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Fas(lpr) mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT(-/-) mice, and reduced glomerulonephritis and increased life span in MRL-Fas(lpr) TdT(-/-) mice. Using tetramer staining, TdT(-/-) and TdT(+/+) NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4(+) T cells. We found no increase in CD4(+)CD25(+) regulatory T cells in NOD TdT(-/-) mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.     

Fetal B-cell lymphopoiesis and the emergence of B-1-cell potential

Most B cells in peripheral lymphoid tissues are produced in adult bone marrow and are referred to as B-2 cells. A minor B-cell population, known as the B-1-cell population, that is mainly involved in innate immune responses has been identified in mice. In contrast to B-2 cells, B-1-cell progenitors are produced most efficiently during fetal life. This Review focuses on the emergence of B-1-cell potential during embryogenesis, summarizes recent advances in the delineation of a fetal B-1-cell-specified progenitor, and discusses the possibility that distinct fetal and adult B-cell developmental programmes might be operative in humans.     

Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures

Immunoglobulin junctional diversity is concentrated in the third complementarity-determining region of the heavy chain (CDR-H3), which often plays a dominant role in antigen binding. The range of CDR-H3 lengths in mouse is shorter than in human, and thus the murine repertoire could be presumed to be a subset of the human one. To test this presumption, we analyzed 4751 human and 2170 murine unique, functional, published CDR-H3 intervals. Although tyrosine, glycine, and serine were found to predominate in both species, the human sequences contained fewer tyrosine residues, more proline residues, and more hydrophobic residues (p<0.001, respectively). While changes in amino acid utilization as a function of CDR-H3 length followed similar trends in both species, murine and human CDR-H3 intervals of identical length were found to differ from each other. These differences reflect both divergence of germline diversity and joining gene sequence and somatic selection. Together, these factors promote the production of a rather uniform repertoire in mice of tyrosine-enriched CDR-H3 loops with stabilized hydrogen bond-ladders versus a much more diverse repertoire in human that contains CDR-H3 loops sculpted by the presence of intra-chain disulfide bonds due to germline-encoded cysteine residues as well as the enhanced presence of somatically generated proline residues that preclude hydrogen bond ladder formation. Thus, despite the presumed need to recognize a similar range of antigen epitopes, the murine CDR-H3 repertoire is clearly distinct from its human counterpart in its amino acid composition and its predicted range of structures. These findings represent a benchmark to which CDR-H3 repertoires can be compared to better characterize and understand the shaping of the CDR-H3 repertoire over evolution and during immune responses. This information may also be useful for the design of species-specific CDR-H3 sequences in synthetic antibody libraries.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

Antibody repertoire and gene expression profile: implications for different developmental and functional traits of splenic and peritoneal B-1 lymphocytes

In L2 mice, a high expression level of the transgenic lambda2(315) L chain results in nearly complete exclusion of endogenous L chains and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum. The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in functional properties.