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.     

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.     

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.     

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.     

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.     

Homeostatic regulation of marginal zone B cells by invariant natural killer T cells

Marginal zone B cells (MZB) mount a rapid antibody response, potently activate naïve T cells, and are enriched in autoreactive B cells. MZBs express high levels of CD1d, the restriction element for invariant natural killer T cells (iNKT). Here, we examined the effect of iNKT cells on MZB cell activation and numbers in vitro and in vivo in normal and autoimmune mice. Results show that iNKT cells activate MZBs, but restrict their numbers in vitro and in vivo in normal BALB/c and C57/BL6 mice. iNKT cells do so by increasing the activation-induced cell death and curtailing proliferation of MZB cells, whereas they promote the proliferation of follicular B cells. Sorted iNKT cells can directly execute this function, without help from other immune cells. Such MZB regulation by iNKTs is mediated, at least in part, via CD1d on B cells in a contact-dependent manner, whereas iNKT-induced proliferation of follicular B cells occurs in a contact- and CD1d-independent manner. Finally, we show that iNKT cells reduce ‘autoreactive’ MZB cells in an anti-DNA transgenic model, and limit MZB cell numbers in autoimmune-prone (NZB×NZW)F1 and non-obese diabetic mice, suggesting a potentially new mechanism whereby iNKT cells might regulate pathologic autoimmunity. Differential regulation of follicular B cells versus potentially autoreactive MZBs by iNKT cells has important implications for autoimmune diseases as well as for conditions that require a rapid innate B cell response.     

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.     

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.     

Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.     

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.     

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.     

TLR stimulation modifies BLyS receptor expression in follicular and marginal zone B cells

Through their differential interactions with B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), the three BLyS family receptors play central roles in B cell survival and differentiation. Recent evidence indicates BLyS receptor levels shift following BCR ligation, suggesting that activation cues can alter overall BLyS receptor profiles and thus ligand sensitivity. In this study, we show that TLR stimuli also alter BLyS receptor expression, but in contrast to BCR ligation, TLR9 and TLR4 signals, preferentially increase transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) expression. Although both of these TLRs act through MyD88-dependent mechanisms to increase TACI expression, they differ in terms of their downstream mediators and the B cell subset affected. Surprisingly, only TLR4 relies on c-Rel and p50 to augment TACI expression, whereas TLR9 does not. Furthermore, although all follicular and marginal zone B cells up-regulate TACI in response to TLR9 stimulation, only marginal zone B cells and a subset of follicular B cells respond to TLR4. Finally, we find that both BLyS and APRIL enhance viability among quiescent and BCR-stimulated B cells. However, although BLyS enhances viability among TLR stimulated B cells, APRIL does not, suggesting that TACI but not BLyS receptor 3 may share survival promoting pathways with TLRs.     

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.     

B cells are crucial for both development and maintenance of the splenic marginal zone

The splenic marginal zone is a unique compartment that separates the lymphoid white pulp from the surrounding red pulp. Due to the orchestration of specialized macrophages and B cells flanking a marginal sinus, this compartment plays an important role in uptake of blood-borne Ags and it gives the spleen its specialized function in antibacterial immunity. In this study, we demonstrate that both development and maintenance of this marginal zone is highly dependent on the presence of B cells. Spleens from B cell-deficient mice were found to lack both metallophilic and marginal zone macrophages as well as mucosal addressin cellular adhesion molecule-1+ sinus lining cells. Using an inducible Cre/loxP-driven mouse model in which mature B cells could be partially depleted by removal of the B cell receptor subunit Igalpha, we could show that the integrity and function of an established marginal zone was also dependent on the presence of B cells. This was confirmed in a transgenic model in which all B cells were gradually depleted due to overexpression of the TNF family member CD70. The loss of all cellular subsets from the marginal zone in these CD70 transgenic mice was effectively prevented by crossing these mice on a CD27(-/-) or TCRalpha(-/-) background, because this prohibited the ongoing B cell depletion. Therefore, we conclude that B cells are not only important for the development, but also for maintenance, of the marginal zone. This direct correlation between circulating B cells and the function of the spleen implies an increased risk for B cell lymphopenic patients with bacterial infections.     

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.     

The early marginal zone B cell-initiated T-independent type 2 response resists the proteasome inhibitor bortezomib

The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll-stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.     

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.     

Impaired clearance of apoptotic cells induces the activation of autoreactive anti-Sm marginal zone and B-1 B cells

Since apoptotic cell Ags are thought to be a source of self-Ag in systemic lupus erythematosus, we have examined the role of apoptotic cells in the regulation and activation of B cells specific for Sm, a ribonucleoprotein targeted in human and murine lupus. Using Ig-transgenic mice that have a high frequency of anti-Sm B cells, we find that apoptotic cell injection induces a transient splenic B cell response, while simultaneously causing extensive splenic and peritoneal anti-Sm B cell death. In contrast, mice deficient in the clearance of apoptotic cells develop a chronic anti-Sm response beginning at 1-2 mo of age. These mice have expanded marginal zone and B-1 B cell populations and anti-Sm B cells of both types are activated to form Ab-secreting cells. This activation appears to be Ag-specific, suggesting that activation is due to increased availability of apoptotic cell Ags. Since marginal zone and B-1 cells are positively selected, these data suggest a loss of ignorance rather than a loss of tolerance.