Antibodies from donor B cells perpetuate cutaneous chronic graft-versus-host disease in mice.

Cutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4(+) T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)H(µÎ³1) DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgH(µÎ³1) grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgH(µÎ³1) grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgH(µÎ³1) grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells.

B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis.

RATIONALE: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

Protective Role for B-1b B Cells and IgM in Obesity-Associated Inflammation, Glucose Intolerance, and Insulin Resistance.

OBJECTIVE: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell-specific deletion of Id3 (Id3(Bcell KO)) to identify B-cell functions involved in the metabolic consequences of obesity. APPROACH AND RESULTS: Diet-induced obese Id3(Bcell KO) mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3(Bcell KO) mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1(-/-) VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1(-/-) hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. CONCLUSIONS: NAb-producing B-1b B cells are increased in Id3(Bcell KO) mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.

Antitumor effector B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10.

We have previously reported that adoptive transfer of tumor-draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL-10-producing cells within these B cells, and found that IL-10 removal, either by using IL-10(-/-) TDLN B cells or by systemic neutralization of IL-10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL-10 in B-cell adoptive transfers significantly increased CTLs and B-cell activity of PBMCs and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by coculture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen specific and Fas ligand-dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.

ABCG1 is required for pulmonary B-1 B cell and natural antibody homeostasis.

Many metabolic diseases, including atherosclerosis, type 2 diabetes, pulmonary alveolar proteinosis, and obesity, have a chronic inflammatory component involving both innate and adaptive immunity. Mice lacking the ATP-binding cassette transporter G1 (ABCG1) develop chronic inflammation in the lungs, which is associated with the lipid accumulation (cholesterol, cholesterol ester, and phospholipid) and cholesterol crystal deposition that are characteristic of atherosclerotic lesions and pulmonary alveolar proteinosis. In this article, we demonstrate that specific lipids, likely oxidized phospholipids and/or sterols, elicit a lung-specific immune response in Abcg1(-/-) mice. Loss of ABCG1 results in increased levels of specific oxysterols, phosphatidylcholines, and oxidized phospholipids, including 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine, in the lungs. Further, we identify a niche-specific increase in natural Ab (NAb)-secreting B-1 B cells in response to this lipid accumulation that is paralleled by increased titers of IgM, IgA, and IgG against oxidation-specific epitopes, such as those on oxidized low-density lipoprotein and malondialdehyde-modified low-density lipoprotein. Finally, we identify a cytokine/chemokine signature that is reflective of increased B cell activation, Ab secretion, and homing. Collectively, these data demonstrate that the accumulation of lipids in Abcg1(-/-) mice induces the specific expansion and localization of B-1 B cells, which secrete NAbs that may help to protect against the development of atherosclerosis. Indeed, despite chronic lipid accumulation and inflammation, hyperlipidemic mice lacking ABCG1 develop smaller atherosclerotic lesions compared with controls. These data also suggest that Abcg1(-/-) mice may represent a new model in which to study the protective functions of B-1 B cells/NAbs and suggest novel targets for pharmacologic intervention and treatment of disease.

Myeloid cells limit production of antibody-secreting cells after immunization in the lymph node.

Ab-secreting cell (ASC) expansion and survival are important processes in optimizing vaccines and controlling autoimmunity. The microenvironment of the medullary cords is positioned to control these key processes. Previously, we imaged and characterized ASC differentiation and migration by intravital microscopy in the lymph node (LN) by transferring and activating B cells expressing yellow fluorescent protein only in the ASC compartment. In this study, we observed that yellow fluorescent protein(+) ASCs in the medullary cords migrated along myelomonocytic cells and arrested in contact with them. Acute ablation of myeloid cells using the human diphtheria receptor system (diphtheria toxin receptor [DTR]) expressed in Lysmd1-cre-positive cells increased ASC and Ab production by 2-fold. Increases in ASC numbers were associated with cell proliferation based on Ki-67 staining, rather than reduced apoptosis, or changes in egress from the LN. Using DTR-mediated ablation targeted to Ccr2-expressing myeloid cells also generated increases in ASCs. In contrast, neither the depletion of Gr-1-positive cells with an Ab nor the ablation of cells using a cd11c-DTR resulted in any change in ASCs. IL-6 cytokine signaling can enhance ASC production and has been implicated in dampening ASCs in lupus mouse models through myeloid cells. Using mixed bone marrow chimeras, we observed that IL-6 enhances ASC production, but IL-6 production was not required by myeloid cells to dampen ASCs in the LN. Inhibition of ASCs by these myeloid cells in the LN provides a new regulatory mechanism with implications for tuning Ab responses.

Primary B cell repertoire remodeling to achieve humoral transplantation tolerance.

The current mainstay of immunotherapy in clinical transplantation is T lymphocyte directed. However, it has long been appreciated that the emergence of an alloimmune response mounted by the B lymphocyte compartment and detectable as donor-specific antibodies is a critical challenge to long-term graft survival. Thus, achieving robust transplantation tolerance will require induction of tolerance in both the T- and B-cell compartments. Here we propose that the natural developmental propensity of the B-lymphocyte compartment acquisition of tolerance to self-antigens can be recapitulated to achieve humoral transplantation tolerance. It is our contention B-lymphocyte directed induction immunotherapy would be an important component of emerging strategies for induction of Transplantation tolerance.

Role of B cells in host defense against primary Coxiella burnetii infection.

Despite Coxiella burnetii being an obligate intracellular bacterial pathogen, our recent study demonstrated that B cells play a critical role in vaccine-induced immunity to C. burnetii infection by producing protective antibodies. However, the role of B cells in host defense against primary C. burnetii infection remains unclear. In this study, we investigated whether B cells play an important role in host defense against primary C. burnetii infection. The results showed that peritoneal B cells were able to phagocytose virulent C. burnetii bacteria and form Coxiella-containing vacuoles (CCVs) and that C. burnetii can infect and replicate in peritoneal B1a subset B cells in vitro, demonstrating a potential role for peritoneal B cells in host defense against C. burnetii infection in vivo. In addition, the results showing that B1a cells secreted a high level of interleukin-10 (IL-10) in response to C. burnetii infection in vitro suggest that B1a cells may play an important role in inhibiting the C. burnetii infection-induced inflammatory response. The observation that adoptive transfer of peritoneal B cells did not significantly affect the severity of C. burnetii infection-induced diseases in both severe combined immunity-deficient (SCID) and µMT mice indicates that peritoneal B cells alone may not be able to control C. burnetii infection. In contrast, our finding that C. burnetii infection induced more-severe splenomegaly and a higher bacterial burden in the spleens of B1a cell-deficient Bruton's tyrosine kinase x-linked immunity-deficient (BTK(xid)) mice than in their wild-type counterparts further suggests that B1a cells play an important role in host defense against primary C. burnetii infection.

Quorum sensing contributes to activated IgM-secreting B cell homeostasis.

Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity.

B1a cells enhance susceptibility to infection with virulent Francisella tularensis via modulation of NK/NKT cell responses.

B1a cells are an important source of natural Abs, Abs directed against T-independent Ags, and are a primary source of IL-10. Bruton's tyrosine kinase (btk) is a cytoplasmic kinase that is essential for mediating signals from the BCR and is critical for development of B1a cells. Consequentially, animals lacking btk have few B1a cells, minimal Ab responses, and can preferentially generate Th1-type immune responses following infection. B1a cells have been shown to aid in protection against infection with attenuated Francisella tularensis, but their role in infection mediated by fully virulent F. tularensis is not known. Therefore, we used mice with defective btk (CBA/CaHN-Btk(XID)/J [XID mice]) to determine the contribution of B1a cells in defense against the virulent F. tularensis ssp. tularensis strain SchuS4. Surprisingly, XID mice displayed increased resistance to pulmonary infection with F. tularensis. Specifically, XID mice had enhanced clearance of bacteria from the lung and spleen and significantly greater survival of infection compared with wild-type controls. We revealed that resistance to infection in XID mice was associated with decreased numbers of IL-10-producing B1a cells and concomitant increased numbers of IL-12-producing macrophages and IFN-γ-producing NK/NKT cells. Adoptive transfer of wild-type B1a cells into XID mice reversed the control of bacterial replication. Similarly, depletion of NK/NKT cells also increased bacterial burdens in XID mice. Together, our data suggest B cell-NK/NKT cell cross-talk is a critical pivot controlling survival of infection with virulent F. tularensis.

The capsular polysaccharide Vi from Salmonella typhi is a B1b antigen.

Vaccination with purified capsular polysaccharide Vi Ag from Salmonella typhi can protect against typhoid fever, although the mechanism for its efficacy is not clearly established. In this study, we have characterized the B cell response to this vaccine in wild-type and T cell-deficient mice. We show that immunization with typhoid Vi polysaccharide vaccine rapidly induces proliferation in B1b peritoneal cells, but not in B1a cells or marginal zone B cells. This induction of B1b proliferation is concomitant with the detection of splenic Vi-specific Ab-secreting cells and protective Ab in Rag1-deficient B1b cell chimeras generated by adoptive transfer-induced specific Ab after Vi immunization. Furthermore, Ab derived from peritoneal B cells is sufficient to confer protection against Salmonella that express Vi Ag. Expression of Vi by Salmonella during infection did not inhibit the development of early Ab responses to non-Vi Ags. Despite this, the protection conferred by immunization of mice with porin proteins from Salmonella, which induce Ab-mediated protection, was reduced postinfection with Vi-expressing Salmonella, although protection was not totally abrogated. This work therefore suggests that, in mice, B1b cells contribute to the protection induced by Vi Ag, and targeting non-Vi Ags as subunit vaccines may offer an attractive strategy to augment current Vi-based vaccine strategies.

Selective blockade of herpesvirus entry mediator-B and T lymphocyte attenuator pathway ameliorates acute graft-versus-host reaction.

The cosignaling network mediated by the herpesvirus entry mediator (HVEM; TNFRSF14) functions as a dual directional system that involves proinflammatory ligand, lymphotoxin that exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT; TNFSF14), and the inhibitory Ig family member B and T lymphocyte attenuator (BTLA). To dissect the differential contributions of HVEM/BTLA and HVEM/LIGHT interactions, topographically-specific, competitive, and nonblocking anti-HVEM Abs that inhibit BTLA binding, but not LIGHT, were developed. We demonstrate that a BTLA-specific competitor attenuated the course of acute graft-versus-host reaction in a murine F(1) transfer semiallogeneic model. Selective HVEM/BTLA blockade did not inhibit donor T cell infiltration into graft-versus-host reaction target organs, but decreased the functional activity of the alloreactive T cells. These results highlight the critical role of HVEM/BTLA pathway in the control of the allogeneic immune response and identify a new therapeutic target for transplantation and autoimmune diseases.

Donor B cells in transplants augment clonal expansion and survival of pathogenic CD4+ T cells that mediate autoimmune-like chronic graft-versus-host disease.

We reported that both donor CD4(+) T and B cells in transplants were required for induction of an autoimmune-like chronic graft-versus-host disease (cGVHD) in a murine model of DBA/2 donor to BALB/c recipient, but mechanisms whereby donor B cells augment cGVHD pathogenesis remain unknown. In this study, we report that, although donor B cells have little impact on acute GVHD severity, they play an important role in augmenting the persistence of tissue damage in the acute and chronic GVHD overlapping target organs (i.e., skin and lung); they also markedly augment damage in a prototypical cGVHD target organ, the salivary gland. During cGVHD pathogenesis, donor B cells are activated by donor CD4(+) T cells to upregulate MHC II and costimulatory molecules. Acting as efficient APCs, donor B cells augment donor CD4(+) T clonal expansion, autoreactivity, IL-7Rα expression, and survival. These qualitative changes markedly augment donor CD4(+) T cells' capacity in mediating autoimmune-like cGVHD, so that they mediate disease in the absence of donor B cells in secondary recipients. Therefore, a major mechanism whereby donor B cells augment cGVHD is through augmenting the clonal expansion, differentiation, and survival of pathogenic CD4(+) T cells.

Antigen-specific suppression of humoral immunity by anergic Ars/A1 B cells.

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Marginal zone B cell is a major source of Il-10 in Listeria monocytogenes susceptibility.

Rag-1-knockout (KO) mice are highly resistant to Listeria monocytogenes infection. The role played by the many Rag-1-dependent lymphocyte lineages was studied using a genetic approach in which each Rag-1-dependent lymphocyte lineage was eliminated one at a time. Only B cell-deficient Igh-KO mice displayed reduced bacterial load and improved survival upon Listeria infection. Listeria infection of Rag-1-KO and Il-10-KO hosts that had been adoptively transferred with wild-type marginal zone B (MZB) cells, but not follicular B cells, resulted in heightened bacterial load and increased Il-10 production in the spleen, but not the liver. This MZB cell-dependent increase in bacterial load was eliminated by anti-Il-10 mAb. In addition, Listeria infection of MZB cell-deficient Rbpj-cKO mice showed decreased bacterial load and increased survival. Whereas multiple cell types have been shown to be capable of Il-10 production, our results indicate that the MZB cell is the most dominant and relevant Il-10 source in the context of Listeria susceptibility. In marked contrast to the generally protective nature of MZB cells in defending against pathogenic infection, our results demonstrate that MZB cells play a detrimental role in Listeria infection and possibly other infections as well.

Treatment of experimental stroke with IL-10-producing B-cells reduces infarct size and peripheral and CNS inflammation in wild-type B-cell-sufficient mice.

Clinical stroke induces inflammatory processes leading to cerebral and splenic injury and profound peripheral immunosuppression. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B-cells led to larger infarct volumes and CNS damage after middle cerebral artery occlusion (MCAO) that could be prevented by transfer of IL-10(+) B-cells. The purpose of this study was to determine if the beneficial immunoregulatory effects on MCAO of the IL-10(+) B-cell subpopulation also extends to B-cell-sufficient mice that would better represent stroke subjects. CNS inflammation and infarct volumes were evaluated in male C57BL/6J (WT) mice that received either RPMI or IL-10(+) B-cells and underwent 60 min of middle cerebral artery occlusion (MCAO) followed by 96 h of reperfusion. Transfer of IL-10(+) B-cells markedly reduced infarct volume in WT recipient mice when given 24 h prior to or 4 h after MCAO. B-cell protected (24 h pre-MCAO) mice had increased regulatory subpopulations in the periphery, reduced numbers of activated, inflammatory T-cells, decreased infiltration of T-cells and a less inflammatory milieu in the ischemic hemispheres of the IL-10(+) B-cell-treated group. Moreover, transfer of IL-10(+) B-cells 24 h before MCAO led to a significant preservation of regulatory immune subsets in the IL-10(+) B-cell protected group presumably indicating their role in immunomodulatory mechanisms, post-stroke. Our studies are the first to demonstrate a major immunoregulatory role for IL-10(+) regulatory B-cells in preventing and treating MCAO in WT mice and also implicating their potential role in attenuating complications due to post-stroke immunosuppression.

Post-transplantation B cell function in different molecular types of SCID.

PURPOSE: Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B and sometimes NK cell function. Non-ablative HLA-identical or rigorously T cell-depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T cell development in the recipients, leading to a high rate of long-term survival. However, the development of B cell function has been more problematic. We report here results of analyses of B cell function in 125 SCID recipients prior to and long-term after non-ablative BMT, according to their molecular type. METHODS: Studies included blood immunoglobulin measurements; antibody titers to standard vaccines, blood group antigens and bacteriophage Φ X 174; flow cytometry to examine for markers of immaturity, memory, switched memory B cells and BAFF receptor expression; B cell chimerism; B cell spectratyping; and B cell proliferation. RESULTS: The results showed that B cell chimerism was not required for normal B cell function in IL7Rα-Def, ADA-Def and CD3-Def SCIDs. In X-linked-SCID, Jak3-Def SCID and those with V-D-J recombination defects, donor B cell chimerism was necessary for B cell function to develop. CONCLUSION: The most important factor determining whether B cell function develops in SCID T cell chimeras is the underlying molecular defect. In some types, host B cells function normally. In those molecular types where host B cell function did not develop, donor B cell chimerism was necessary to achieve B cell function. 236 words.

IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation.

IL-10-producing CD1d(hi)CD5(+) B cells, also known as B10 cells, have been shown to possess a regulatory function in the inhibition of immune responses, but whether and how B10 cells suppress the development of autoimmune arthritis remain largely unclear. In this study, we detected significantly decreased numbers of IL-10-producing B cells, but increased IL-17-producing CD4(+) T (Th17) cells in both spleen and draining lymph nodes of mice during the acute stage of collagen-induced arthritis (CIA) when compared with adjuvant-treated control mice. On adoptive transfer of in vitro expanded B10 cells, collagen-immunized mice showed a marked delay of arthritis onset with reduced severity of both clinical symptoms and joint damage, accompanied by a substantial reduction in the number of Th17 cells. To determine whether B10 cells directly inhibit the generation of Th17 cells in culture, naive CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were co-cultured with B10 cells. These B10 cells suppressed Th17 cell differentiation via the reduction of STAT3 phosphorylation and retinoid-related orphan receptor γt (RORγt) expression. Moreover, Th17 cells showed significantly decreased proliferation when co-cultured with B10 cells. Although adoptive transfer of Th17 cells triggered the development of collagen-induced arthritis in IL-17(-/-)DBA/1J mice, co-transfer of B10 cells with Th17 cells profoundly delayed the onset of arthritis. Thus, our findings suggest a novel regulatory role of B10 cells in arthritic progression via the suppression of Th17 cell generation.

B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions.

RATIONALE: Aggravated atherosclerosis in B lymphocyte-deficient chimeric mice and reduced atherosclerosis after transfer of unfractionated spleen B lymphocytes into splenectomized mice have led to the widely held notion that B lymphocytes are atheroprotective. However, B lymphocytes can be pathogenic, because their depletion by anti-CD20 antibody ameliorated atherosclerosis, and transfer of B2 lymphocytes aggravated atherosclerosis. These observations raise the question of the identity of the atheroprotective B-lymphocyte population. OBJECTIVE: The purpose of the study was to identify an atheroprotective B-lymphocyte subset and mechanisms by which they confer atheroprotection. METHODS AND RESULTS: Splenectomy of apolipoprotein E-deficient mice selectively reduced peritoneal B1a lymphocytes, plasma IgM, and oxidized low-density lipoprotein IgM levels and lesion IgM deposits. These reductions were accompanied by increased oil red O-stained atherosclerotic lesions and increased necrotic cores, oxidized low-density lipoproteins, and apoptotic cells in lesions. Plasma lipids, body weight, collagen, and smooth muscle content were unaffected. Transfer of B1a lymphocytes into splenectomized mice increased peritoneal B1a lymphocytes; restored plasma IgM, oxidized low-density lipoprotein IgM levels, and lesion IgM deposits; and potently attenuated atherosclerotic lesions, with reduced lesion necrotic cores, oxidized low-density lipoprotein, and apoptotic cells. In contrast, transfer of B1a lymphocytes that cannot secrete IgM failed to protect against atherosclerosis development in splenectomized mice despite reconstitution in the peritoneum. CONCLUSIONS: B1a lymphocytes are an atheroprotective B-lymphocyte population. Our data suggest that natural IgM secreted by these lymphocytes offers protection by depositing IgM in atherosclerotic lesions, which reduces the necrotic cores of lesions.

Acquisition of humoral transplantation tolerance upon de novo emergence of B lymphocytes.

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant--despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire "remodeling" to a humoral tolerant state.