Anti-Sm B cell differentiation in Ig transgenic MRL/Mp-lpr/lpr mice: altered differentiation and an accelerated response

To determine the regulation of B cells specific for the ribonucleoprotein Sm, a target of the immune system in human and mouse lupus, we have generated mice carrying an anti-Sm H chain transgene (2-12H). Anti-Sm B cells in nonautoimmune 2-12H-transgenic (Tg) mice are functional, but, in the absence of immunization, circulating anti-Sm Ab levels are not different from those of non-Tg mice. In this report, we compare the regulation of anti-Sm B cells in nonautoimmune and autoimmune MRL/Mp-lpr/lpr (MRL/lpr) and bcl-2-22-Tg mice. Activation markers are elevated on splenic and peritoneal anti-Sm B cells of both nonautoimmune and autoimmune genetic backgrounds indicating Ag encounter. Although tolerance to Sm is maintained in 2-12H/bcl-2-22-Tg mice, it is lost in 2-12H-Tg MRL/lpr mice, as the transgene accelerates and increases the prevalence of the anti-Sm response. The 2-12H-Tg MRL/lpr mice have transitional anti-Sm B cells in the spleen similar to nonautoimmune mice. However, in contrast to nonautoimmune mice, there are few if any peritoneal anti-Sm B-1 cells. These data suggest that a defect in B-1 differentiation may be a factor in the loss of tolerance to Sm and provide insight into the low prevalence of the anti-Sm response in lupus.     

Low-affinity anti-Smith antigen B cells are regulated by anergy as opposed to developmental arrest or differentiation to B-1.

Understanding the regulation of B lymphocytes specific for self-Ags targeted in human and murine systemic lupus erythematosus, such as the ribonucleoprotein Smith Ag (Sm), is crucial to understanding the etiology of this autoimmune disease. To address the role of B cell receptor affinity in the regulation of anti-Sm B cells, we generated low-affinity anti-Sm transgenic mice by combining the anti-Sm 2-12H transgene with a V(kappa)8 transgene. In contrast to 2-12H transgenic mice, in which anti-Sm B cells are predominantly splenic transitional, and peritoneal B-1, low-affinity anti-Sm B cells are long-lived B-2 cells and are found in the spleen, lymph nodes, and peritoneum. However, they are unresponsive to LPS in vitro, indicating that they are anergic, although they do not down-regulate IgM and are not excluded from follicles even in the presence of nonautoreactive B cells. Thus, low-affinity anti-Sm B cells appear to have a partial form of anergy. Interestingly, these cells have elevated levels of MHC class II and CD95, but not CD40, CD80, or CD86, suggesting that they are poised to undergo deletion rather than activation upon T cell encounter. These data identify anergy as a mechanism involved in anti-Sm B cell regulation.     

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.     

EBV latent membrane protein 2A induces autoreactive B cell activation and TLR hypersensitivity

EBV is associated with systemic lupus erythematosus (SLE), but how it might contribute to the etiology is not clear. Since EBV-encoded latent membrane protein 2A (LMP2A) interferes with normal B cell differentiation and function, we sought to determine its effect on B cell tolerance. Mice transgenic for both LMP2A and the Ig transgene 2-12H specific for the ribonucleoprotein Smith (Sm), a target of the immune system in SLE, develop a spontaneous anti-Sm response. LMP2A allows anti-Sm B cells to overcome the regulatory checkpoint at the early preplasma cell stage by a self-Ag-dependent mechanism. LMP2A induces a heightened sensitivity to TLR ligand stimulation, resulting in increased proliferation or Ab-secreting cell differentiation or both. Thus, we propose a model whereby LMP2A induces hypersensitivity to TLR stimulation, leading to activation of anti-Sm B cells through the BCR/TLR pathway. These data further implicate TLRs in the etiology of SLE and suggest a mechanistic link between EBV infection and SLE.     

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

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

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.     

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.     

IL-21 promotes lupus-like disease in chronic graft-versus-host disease through both CD4 T cell- and B cell-intrinsic mechanisms

T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus erythematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and function, promotes T follicular helper (T(FH)) cell and Th17 cell differentiation, and downregulates the induction of T regulatory cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4(+) T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-versus-host disease by using wild-type or IL-21R(-/-) mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6 model. We show that IL-21R expression on donor CD4(+) T cells is essential for sustaining T(FH) cell number and subsequent help for B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17 cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4(+) T cell- and B cell-intrinsic mechanisms and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant T(FH) cell pathway that contributes to lupus pathogenesis.     

B cell autophagy mediates TLR7-dependent autoimmunity and inflammation

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5^f/f). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.     

B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.     

Delineation of the human systemic lupus erythematosus anti-Smith antibody response using phage-display combinatorial libraries

The anti-Smith (Sm) autoantibody response is highly specific for systemic lupus erythematosus and is predominantly targeted to the Sm-B/B' and -D1 polypeptides. In all animal species thus far studied, anti-Sm Abs initially recognize proline-rich epitopes in the carboxyl terminus of the Sm-B/B' protein and subsequently to multiple other epitopes in B/B' and D. The absence of appropriate mAbs has limited our understanding of the genetic and structural basis of this autoimmune response. Using phage-display technology and lymphocytes from a systemic lupus erythematosus patient we have generated the first and only panel of human IgG anti-Sm mAbs thus far available. These Abs reproduced to a remarkable extent the serological reactivity of the patient. Epitope mapping and genetic studies revealed that the anti-Sm response is produced by distinct B cell clones with restricted epitope reactivity. All of the Abs in our study were exclusively encoded by different members of the V(H)4 gene family. On the aggregate, our results demonstrate that combinatorial libraries can recapitulate the immune repertoire of peripheral blood B memory cells and that epitope spreading appears to occur through the sequential recruitment of nonclonally related autoreactive B cell clones.     

Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes

Loss of tolerance is considered to be an early event that is essential for the development of autoimmune disease. In contrast to this expectation, autoimmune (type 1) diabetes develops in NOD mice that harbor an anti-insulin Ig transgene (125Tg), even though anti-insulin B cells are tolerant. Tolerance is maintained in a similar manner in both normal C57BL/6 and autoimmune NOD mice, as evidenced by B cell anergy to stimulation through their Ag receptor (anti-IgM), TLR4 (LPS), and CD40 (anti-CD40). Unlike B cells in other models of tolerance, anergic 125Tg B cells are not arrested in development, and they enter mature subsets of follicular and marginal zone B cells. In addition, 125Tg B cells remain competent to increase CD86 expression in response to both T cell-dependent (anti-CD40) and T cell-independent (anti-IgM or LPS) signals. Thus, for anti-insulin B cells, tolerance is characterized by defective B cell proliferation uncoupled from signals that promote maturation and costimulator function. In diabetes-prone NOD mice, anti-insulin B cells in this novel state of tolerance provide the essential B cell contribution required for autoimmune beta cell destruction. These findings suggest that the degree of functional impairment, rather than an overt breach of tolerance, is a critical feature that governs B cell contribution to T cell-mediated autoimmune disease.     

A VH11V kappa 9 B cell antigen receptor drives generation of CD5+ B cells both in vivo and in vitro

B lymphocytes can be divided into different subpopulations, some with distinctive activation requirements and probably mediating specialized functions, based on surface phenotype and/or anatomical location, but the origins of most of these populations remain poorly understood. B cells constrained by transgenesis to produce an Ag receptor derived from a conventional (B-2) type cell develop a B-2 phenotype, whereas cells from mice carrying a B-1-derived receptor acquire the B-1 phenotype. In this study transgenic enforced expression of a B cell receptor (mu/kappa) originally isolated from a CD5+ (B-1a) B cell generates B-1 phenotype cells in bone marrow cultures that show a distinctive B-1 function, survival in culture. Despite their autoreactivity, we find no evidence for receptor editing or that the paucity of B-2 cells is the result of tolerance-induced selection. Finally, Ca2+ mobilization studies reveal a difference between transgenic B-1 cells in spleen and peritoneal cavity, with cells in spleen much more responsive to anti-B cell receptor cross-linking. We discuss these results in terms of specificity vs lineage models for generation of distinctive B cell subpopulations.     

B cell depletion with anti-CD79 mAbs ameliorates autoimmune disease in MRL/lpr mice

MRL/lpr mice develop a spontaneous systemic lupus erythematosus-like autoimmune syndrome due to a dysfunctional Fas receptor, with contributions from other less well-defined genetic loci. The removal of B cells by genetic manipulation not only prevents autoantibody formation, but it also results in substantially reduced T cell activation and kidney inflammation. To determine whether B cell depletion by administration of Abs is effective in lupus mice with an intact immune system and established disease, we screened several B cell-specific mAbs and found that a combination of anti-CD79alpha and anti-CD79beta Abs was most effective at depleting B cells in vivo. Anti-CD79 therapy started at 4-5 mo of age in MRL/lpr mice significantly decreased B cells (B220(+)CD19(+)) in peripheral blood, bone marrow, and spleens. Treated mice also had a significant increase in the number of both double-negative T cells and naive CD4(+) T cells, and a decreased relative abundance of CD4(+) memory cells. Serum anti-chromatin IgG levels were significantly decreased compared with controls, whereas serum anti-dsDNA IgG, total IgG, or total IgM were unaffected. Overall, survival was improved with lower mean skin scores and significantly fewer focal inflammatory infiltrates in submandibular salivary glands and kidneys. Anti-CD79 mAbs show promise as a potential treatment for systemic lupus erythematosus and as a model for B cell depletion in vivo.     

Modification of ligand-independent B cell receptor tonic signals activates receptor editing in immature B lymphocytes

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.     

VISA is required for B cell expression of TLR7

B cells play a critical role in the initialization and development of the systemic lupus erythematosus that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the type I IFN secreted by plasmacytoid dendritic cells. In this article, we report that VISA, also known as MAVS, IPS-1, and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from a VISA(-/-) mouse express reduced TLR7 but normal basal levels of type I IFN. We also show that although IFN-β and TLR7 agonists synergize to promote TLR7 expression in VISA(-/-) B cells, they do not fully complement the defect seen in VISA(-/-) cells. Cell transfer experiments revealed that the observed effects of VISA(-/-) are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced upregulation of activation markers CD69 and CD86, cell proliferation, production of IFN-α, TNF, and IL-12, and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA(-/-) mice, because VISA(-/-) B cells differ in CD23 and TLR7 expression when on C57BL/6 versus 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity.     

A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells

V(H)12 B cells undergo stringent selection at multiple checkpoints to favor development of B-1 cells that bind phosphatidylcholine. Selection begins with the V(H) third complementarity-determining region (CDR3) at the pre-B cell stage, in which most V(H)12 pre-B cells are selectively eliminated, enriching for those with V(H)CDR3s of 10 aa and a fourth position Gly (designated 10/G4). To understand this selection, we compared B cell differentiation in mice of two V(H)12 transgenic lines, one with the favored 10/G4 V(H)CDR3 and one with a non-10/G4 V(H)CDR3 of 8 aa and no Gly (8/G0). Both H chains drive B cell differentiation to the small pre-BII cell stage, and induce allelic exclusion and L chain gene rearrangement. However, unlike 10/G4 pre-B cells, 8/G0 pre-B cells are deficient in cell division and unable to differentiate to B cells. We suggest that this is due to poor 8/G0 pre-B cell receptor expression and to an inability to form an 8/G0 B cell receptor. Our findings also suggest that V(H)12 H chains have evolved such that association with surrogate and conventional L chains is most efficient with a 10/G4 CDR3. Thus, selection for phosphatidylcholine-binding B-1 cells is most likely the underlying evolutionary basis for the loss of non-10/G4 pre-B cells.     

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19⁺ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19⁺ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.