CD9 is a unique marker for marginal zone B cells,B1 cells,and plasma cells in mice

Marginal zone (MZ), follicular (FO), and B1 B cells form the long-lived naive B cell compartment. To identify surface markers that define MZ B cells in mice, we generated a panel of mAbs reactive with MZ but not FO B cells. One of these mAbs, MZ3, was found to recognize the tetraspanin CD9. CD9 expression not only distinguishes MZ B cells from FO B cells but also divided peritoneal cavity B1 cells into smaller subsets. After short-term in vitro stimulation with various mitogens, FO B cells failed to induce CD9 protein, while MZ B cells up-regulated the level of CD9 protein. However, after prolonged culture of FO B cells with LPS, surface CD9 was induced, together with syndecan 1, indicative of plasma cell differentiation. Following immunization with a T-independent-2 Ag, R36A, or a T-dependent Ag, SRBC, we found that CD9 is not expressed by germinal center B cells but is eventually expressed on plasma cells in response to both T-independent-2 and T-dependent Ags. Collectively, these results suggest that MZ B cells and B1 cell subsets are the immediate precursors of plasma cells in the primary response and that CD9 is acquired by T-dependent plasma cells.     

Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells

The early involvement of marginal zone (MZ) B lymphocytes in T-independent immune responses is well established. In this study we compared the abilities of MZ and follicular (FO) B cells to collaborate with T cells. After immunization with soluble hen egg lysozyme, both MZ and FO B cells captured Ag and migrated to T cell areas in the response to hen egg lysozyme. MZ B cells were far superior to FO B cells in inducing CD4+ T cell expansion both in vitro and in vivo. MZ, but not FO, B cells, after interaction with T cells, differentiated into plasma cells, and in addition they stimulated Ag-specific CD4+ T cells to produce high levels of Th1-like cytokines upon primary stimulation in vitro. These results indicate that MZ B cells rapidly and effectively capture soluble Ag and activate CD4+ T cells to become effector T cells. The enhanced capacity of MZ B cells to prime T cells in this study appeared to be intrinsic to MZ B cells, as both MZ and FO B cell populations express an identical Ag receptor.     

Rapid response of marginal zone B cells to viral particles

Marginal zone (MZ) B cells are thought to be responsible for the first wave of Abs against bacterial Ags. In this study, we assessed the in vivo response of MZ B cells in mice immunized with viral particles derived from the RNA phage Qbeta. We found that both follicular (FO) and MZ B cells responded to immunization with viral particles. MZ B cells responded with slightly faster kinetics, but numerically, FO B cells dominated the response. B1 B cells responded similarly to MZ B cells. Both MZ and FO B cells underwent isotype switching, with MZ B cells again exhibiting faster kinetics. In fact, almost all Qbeta-specific MZ B cells expressed surface IgG by day 5. Histological analysis demonstrated that a population of activated B cells remain associated with the MZ, probably due to the elevated integrin levels expressed by these cells. Thus, both MZ and FO B cells respond with rapid proliferation to viral infection and both populations undergo isotype switching, but MZ B cells remain in the MZ and may be responsible for local Ab production, opsonizing pathogens entering the spleen.     

DNA microarray gene expression profile of marginal zone versus follicular B cells and idiotype positive marginal zone B cells before and after immunization with Streptococcus pneumoniae

Marginal zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and follicular (FO) B cells were sort purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, Id-positive and -negative MZ B cells were sort purified before (0 h) or after (1 h) i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up-regulated or down-regulated at 1 h following immunization in the Id-positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs FO B cells and the specific regulation of gene expression in Ag-specific MZ B cells following interaction with Ag.     

Antigen receptor proximal signaling in splenic B-2 cell subsets

Splenic marginal zone (MZ) and follicular mantle (FO) B cells differ in their responses to stimuli in vitro and in vivo. We have previously shown that MZ cells exhibit greater calcium responses after ligation of membrane IgM (mIgM). We have now investigated the molecular mechanism underlying the difference in calcium responses following ligation of mIgM and studied the response to total B cell receptor ligation in these two subsets. We compared key cellular proteins involved in calcium signaling in MZ and FO cells. Tyrosine phosphorylation and activity of phospholipase C-gamma 2 and Syk protein tyrosine kinase were significantly higher in MZ cells than in FO cells after mIgM engagement, providing a likely explanation for our previous findings. Tyrosine phosphorylation of CD22 and expression of Src homology 2-containing inositol phosphatase and Src homology 2-containing protein tyrosine phosphatase-1 were also higher in the MZ cells. Expression and tyrosine phosphorylation of Btk, BLNK, Vav, or phosphatidylinositol 3-kinase were equivalent. In contrast, stimulation with anti-kappa induced equivalent increases in calcium and activation of Syk in the two subsets. These signals were also equivalent in cells from IgM transgenic, J(H) knockout mice, which have equivalent levels of IgM in both subsets. With total spleen B cells, Btk was maximally phosphorylated at a lower concentration of anti-kappa than Syk. Thus, calcium signaling in the subsets of mature B cells reflects the amount of Ig ligated more than the isotype or the subset and this correlates with the relative tyrosine phosphorylation of Syk.     

Mechanisms governing B cell developmental defects in invariant chain-deficient mice

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Abeta doubly deficient mice, revealing that Ii-free I-Abeta chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Abeta. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.     

A hypomorphic IgH-chain allele affects development of B-cell subsets and favours receptor editing

The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene-targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin μ heavy chain (μHC) mRNA levels and hence lower pre-BCR and BCR levels. Hypomorphic expression of μHC leads to augmented selection processes at all stages of B-cell development, noticeably at the expansion of pre-B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B-lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B-cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre-BCR and BCR receptor levels for the normal development of B-lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.     

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.     

B cell receptor affinity and B cell subset identity integrate to define the effectiveness, affinity threshold, and mechanism of anergy

In this study we show that BCR affinity and subset identity make unique contributions to anergy. Analysis of anti-Smith (Sm) B cells of different affinities indicates that increasing affinity improves anergy's effectiveness while paradoxically increasing the likelihood of marginal zone (MZ) and B-1 B cell differentiation rather than just follicular (FO) B cell differentiation. Subset identity in turn determines the affinity threshold and mechanism of anergy. Subset-specific affinity thresholds for anergy induction allow discordant regulation of low-affinity anti-Sm FO and MZ B cells and could account for the higher frequency of autoreactive MZ B cells than that of FO B cells in normal mice. The mechanism of anergy changes during differentiation and differs between subsets. This is strikingly illustrated by the observation that blockade of BCR-mediated activation of FO and MZ B cells occurs at different levels in the signaling cascade. Thus, attributes unique to B cells of each subset integrate with signals from the BCR to determine the effectiveness, affinity threshold, and mechanism of anergy.     

Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic β-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.     

Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

Marginal zone (MZ) B cells, identified as surface (s)IgM^highsIgD^lowCD23^low/−CD21⁺CD38⁻ B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27⁺ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.     

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.     

Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax?23

Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax^®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA⁺ but not IgM⁺ or IgG⁺ memory B cells and a predominant generation of PnPS-specific IgA⁺ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax^®23. Our results demonstrate that a characteristic TI response induced by Pneumovax^®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.     

Marginal Zone Macrophage Receptor MARCO Is Trapped in Conduits Formed by Follicular Dendritic Cells in the Spleen

The marginal zone (MZ) region of the spleen plays an important role in leukocyte traffic and the removal of blood-borne pathogens by resident macrophages. Macrophage receptor with a collagenous structure (MARCO), expressed by MZ macrophages, recognizes several microbial ligands and is also involved in the retention of MZ B cells. Here, we report that MARCO is also associated with follicular dendritic cells (FDCs) in the spleen. In its FDC-associated form MARCO is arranged in 0.3–0.5-μm diameter granular-fibrillar structures with an appearance similar to the white pulp conduit system formed by fibroblastic reticular cells (FRCs), but with different compartment preference. The follicular display of MARCO resists irradiation and requires the presence of both MZ macrophages and differentiated FDCs. The follicular delivery of MARCO is independent from the shuffling of marginal zone B cells, and it persists after clodronate liposome-mediated depletion of MZ macrophages. Our findings thus indicate that MARCO is distributed to both MZ and follicles within the spleen into conduit-like structures, where FDC-bound MARCO may mediate communication between the stromal microenvironments of MZ and follicles.     

The role of B cell CD22 expression in Staphylococcus aureus arthritis and sepsis

Severe Staphylococcus aureus infections give rise to a pronounced antigen-specific and polyclonal B cell response with elevated serum immunoglobulin levels. However, it has been difficult to correlate the antibody levels with the clinical outcome of sepsis and/or arthritis concerning both protection and pathogenic aspects. Earlier studies have shown that macrophages and neutrophils are of great importance for bacterial clearance. However, deletion of the complete B cell compartment affected neither S. aureus-induced arthritis nor survival. MZ B cells are believed to be of importance for clearance of blood-borne antigens and have been implicated in protection against S. aureus infections. CD22 is a B-cell-specific inhibitory receptor binding to alpha2,6-linked sialic acids, and deficiency in CD22 leads to a 75% reduction of the MZ B cell compartment. CD22-/- mice and congeneic controls were inoculated intravenously with an arthritogenic dose of live S. aureus. No differences between the groups were detected regarding frequency and severity of arthritis, survival, bacterial clearance, or induction of inflammatory response. This study shows explicitly that a reduced MZ B cell compartment in the absence of CD22 expression does not interfere with the inflammatory response during S. aureus infection.     

Defining the Recognition Elements of Lewis Y-Reactive Antibodies

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1–4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.     

Notch2 haploinsufficiency results in diminished B1 B cells and a severe reduction in marginal zone B cells

Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele of Notch2 (Notch2(+/-)). Notch2(+/-) mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B cells.     

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self-interaction and polyspecificity compared to non-phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non-phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation-prone natures of antibodies which should be avoided in early development phases.