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.     

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.     

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.     

Heterogeneous antibody repertoire of marginal zone B cells specific for virus-like particles

Marginal zone (MZ) B cells differ from follicular (FO) B cells in their functional, phenotypic and localization properties. It is still unclear whether B cells from the MZ compartment also have distinct or biased BCR specificities, recognizing only a limited number of conserved antigenic structures. To address the complexity of the immune response mounted by marginal zone B cells, we compared the antibody repertoire of murine MZ and FO B cells induced by immunization with two different virus-like particles (VLPs). Antibody sequences isolated from sorted VLP-specific MZ and FO B cells were similar in heavy chain V, D and J gene segment usage. Sequence analysis of CDR3 regions of antibodies from MZ and FO B cells also revealed no consistent difference in N nucleotide additions or CDR3 length. In contrast, somatic hypermutations were reduced in CDR regions of antibodies from MZ B cells compared to those from FO B cells. These results indicate that the response of MZ B cells to VLPs is clonotypically heterogeneous and suggest that the MZ B cell compartment is capable of generating variable and diverse antibody responses.     

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.     

Marginal zone B cells regulate antigen capture by marginal zone macrophages

The marginal zone (MZ) of the mouse spleen contains macrophages that express receptors that trap pathogens, including the scavenger receptor macrophage receptor with a collagenous structure and the C-type lectin specific intracellular adhesion molecule-grabbing nonintegrin receptor 1 (SIGN-R1). We previously reported that expression of SIGN-R1 was decreased in CD19-deficient mice. In this study, we demonstrate that SIGN-R1 is expressed on a subset of macrophage receptor with a collagenous structure (MARCO)(+) macrophages. This subset is diminished when MZ B cells are absent due to either genetic developmental defects or following transient migration of B cells out of the MZ. When B cells return to the MZ, there is a delay in recovery of SIGN-R1-expressing macrophages. During this period, capture of Ficoll, which for the macrophages requires SIGN-R1, remains defective not only by the macrophages, but also by the B cells. Thus, MZ B cells regulate expression of molecules on macrophages that are important for trapping Ag, which, in turn, is required for Ag capture by the B cells.     

TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses

Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-alphabeta receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.     

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.     

Alterations in marginal zone macrophages and marginal zone B cells in old mice

Marginal zones (MZs) are architecturally organized for clearance of and rapid response against blood-borne Ags entering the spleen. MZ macrophages (MZMs) and MZ B cells are particularly important in host defense against T-independent pathogens and may be crucial for the prevention of diseases, such as streptococcal pneumonia, that are devastating in older patients. Our objective was to determine whether there are changes in the cellular components of the MZ between old and young mice. Using immunocytochemistry and a blinded scoring system, we observed gross architectural changes in the MZs of old mice, including reduction in the abundance of MZMs surrounding the MZ sinus as well as disruptions in positioning of mucosal addressin cell adhesion molecule 1 (MAdCAM-1)(+) sinus lining cells and metallophilic macrophages. Loss of frequency of MZMs was corroborated by flow cytometry. A majority of old mice also showed reduced frequency of MZ B cells, which correlated with decreased abundance of MZM in individual old mice. The spleens of old mice showed less deposition of intravenously injected dextran particles within the MZ, likely because of the decreased frequency in MZMs, because SIGN-R1 expression was not reduced on MZM from old mice. The phagocytic ability of individual MZMs was examined using Staphylococcus aureus bioparticles, and no differences in phagocytosis were found between macrophages from young or old spleens. In summary, an anatomical breakdown of the MZ occurs in advanced age, and a reduction in frequency of MZM may affect the ability of the MZM compartment to clear blood-borne Ags and mount proper T-independent immune responses.     

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.     

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.     

The Yaa mutation promoting murine lupus causes defective development of marginal zone B cells

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.     

Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen

The early responses of follicular (Fo) and marginal zone (MZ) B cells to T cell-dependent Ag were compared using anti-hen egg lysozyme (HEL+) B cells capable of class switch recombination and somatic hypermutation (SHM). Purified CD21/(35int)CD23high Fo and CD21/35(high)CD23low MZ splenic B cells from SW(HEL) Ig-transgenic mice were transferred into wild-type recipients and challenged with HEL-sheep RBC. Responding HEL+ B cells from both populations switched efficiently to IgG1, generated syndecan-1+ Ab-secreting cells, and exhibited equivalent rates of proliferation. However, the expansion of HEL+ MZ B cells lagged significantly behind that of HEL+ Fo B cells due to less efficient homing to the outer periarteriolar lymphatic sheath and reduced recruitment into the proliferative response. Despite the equivalent rates of class switch recombination, the onset of SHM was delayed in the MZ subset, indicating that these two activation-induced cytidine deaminase-dependent events are uncoupled in the early response of MZ B cells. Migration of HEL+ B cells into germinal centers coincided with the onset of SHM, occurring more rapidly with Fo vs MZ responders. These results are consistent with the concept that Fo and MZ B cells have evolved to specialize in T cell-dependent and T-independent responses respectively.     

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.     

Activation of marginal zone B cells from lupus mice with type A(D) CpG-oligodeoxynucleotides

Several types of CpG-oligodeoxynucleotides (ODN) have been recently characterized. In mice, type A(D) CpG-ODNs primarily stimulate macrophages and dendritic cells, but fail to stimulate B cells. On the contrary, type B(K) CpG-ODNs are excellent B cell activators. Type C CpG-ODNs combine features of both types A(D) and B(K) CpG-ODNs. Despite cell type preferences, all CpG-ODNs require the presence of TLR9 for activation. In this study, we show that a subset of B cells from lupus mice responds to type A(D) CpG-ODN stimulation vigorously and directly with increased CD25 and CD86 expression and IL-10 secretion. Furthermore, these CpG-ODNs induce high surface IgM expression and promote 50- to 100-fold higher IgM and IgG3 secretion in lupus B cells than in controls. This response is similar to that seen with bacterial DNA stimulation of B cells. Type A(D)-responsive cells are enriched within lupus B cells with the marginal zone (MZ) phenotype. These cells are at least twice more numerous in lupus mice than in controls. The ability of lupus B cells to respond to type A(D) CpG-ODN stimulation is not due to differential TLR9 expression. Therefore, type A(D) CpG-ODNs may contribute to the lupus pathogenesis by inducing MZ-B cell activation, costimulatory molecule expression, and polyclonal Ig secretion. Through increased IL-10 secretion, MZ-B cells may also modify the activity of other cell types, particularly dendritic cells and macrophages.     

Fc receptor homolog 3 is a novel immunoregulatory marker of marginal zone and B1 B cells

Two members of the recently identified FcR homolog (FcRH) family in mice demonstrate preferential B cell expression. One of these, FcRH3, encodes a type I transmembrane protein with five extracellular Ig domains and a cytoplasmic tail with a consensus ITIM and a noncanonical ITAM. Analysis of full-length cDNAs from five different mouse strains defines two FcRH3 alleles. A panel of FcRH3-specific mAbs was generated to define its expression pattern and functional potential on B lineage cells. Although poorly detected on the majority of bone marrow or peripheral blood cells, FcRH3 was readily identified on splenic marginal zone (MZ) and MZ precursor B cells, but not on the bulk of newly formed B cells, follicular B cells, germinal center B cells, and plasma cells. In the peritoneal cavity, FcRH3 was found on B1 cells, and not on the majority of B2 cells. Consistent with its possession of an ITIM and ITAM-like sequence, FcRH3 was tyrosine phosphorylated following pervanadate treatment, and its coligation with the BCR inhibited calcium mobilization. These results suggest FcRH3 is a novel immunoregulatory marker of MZ and B1 B lineage cells.     

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.