The regulation of autoreactive B cells during innate immune responses

Systemic lupus erythematosus (SLE) highlights the dangers of dysregulated B cells and the importance of initiating and maintaining tolerance. In addition to central deletion, receptor editing, peripheral deletion, receptor revision, anergy, and indifference, we have described a new mechanism of B cell tolerance wherein dendritic cells (DCs) and macrophages (MΦs) regulate autoreactive B cells during innate immune responses. In part, DCs and MΦs repress autoreactive B cells by releasing IL-6 and soluble CD40L (sCD40L). This mechanism is selective in that IL-6 and sCD40L do not affect Ig secretion by naïve cells during innate immune responses, allowing immunity in the absence of autoimmunity. In lupus-prone mice, DCs and MΦs are defective in secretion of IL-6 and sCD40L and cannot effectively repress autoantibody secretion suggesting that defects in DC/MΦ-mediated tolerance may contribute to the autoimmune phenotype. Further, these studies suggest that reconstituting DCs and MΦs in SLE patients might restore regulation of autoreactive B cells and provide an alternative to immunosuppressive therapies

Structural constraints within a trimeric transcriptional regulatory region. Constitutive and interferon-gamma-inducible expression of the HLA-DRA gene.

Constitutive and inducible transcription of the major histocompatibility class II HLA-DRA gene involves the upstream S element and the conserved X and Y elements. In this report we have addressed the roles of spatial constraints and stereospecific alignment between the upstream S and X elements, and the X and Y elements, in both constitutive and interferon-gamma (gamma-IFN)-induced expression. Analysis of the constitutive expression in B cell lines (B-LCL) has previously shown that the X and Y elements must be stereoaligned. Further study reveals that any spacing changes between S and X, regardless of the helical alignment of these two elements, is not tolerated. These same restraints are involved in an inducible system, because the response to gamma-IFN treatment requires both stereo alignment between the X and Y elements and precise spacing between the S and X elements. Neither constitutive nor inducible expression can be restored by correcting the distance and spacing between only the S and Y elements with misalignment of X. These results reveal a common pathway for constitutive and inducible expression that may require either direct or indirect protein complex formation among proteins bound to three highly conserved regulatory elements. We have also evaluated the role of the A/T-rich sequence located immediately 5' of the Y element and show that it exerts little effect on constitutive and gamma-IFN induced DRA expression

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

Activation of innate sensors by self-antigen contributes to autoimmunity, although how intracellular sensors are chronically exposed to self-antigen has remained unknown. Here, we identify a previously unidentified defect in which lupus-prone macrophages fail to mature the lysosome, promoting the accumulation of apoptotic debris-containing IgG–immune complexes (IgG-ICs). Interestingly, macrophages from other autoimmune diseases accumulate IgG-ICs, indicating that lysosomal defects may underlie multiple autoimmune diseases. Furthermore, the prolonged intracellular residency chronically activates Toll-like receptors and permeabilizes the phagolysosomal membrane, allowing activation of cytosolic sensors. These findings identify lysosomal maturation as a unique defect in MRL/lpr mice that impacts multiple events known to underlie SLE, including pathogenic cytokine secretion

B-cell anergy: from transgenic models to naturally occurring anergic B cells?

Anergy, a condition in which cells persist in the periphery but are unresponsive to antigen, is responsible for silencing many self-reactive B cells. Loss of anergy is known to contribute to the development of autoimmune diseases, including systemic lupus erythematosus and type 1 diabetes. Multiple transgenic mouse models have enabled the dissection of mechanisms that underlie anergy, and recently, anergic B cells have been identified in the periphery of wild-type mice. Heterogeneity of mechanistic concepts developed using model systems has complicated our understanding of anergy and its biological features. In this Review, we compare and contrast the salient features of anergic B cells with a view to developing unifying mechanistic hypotheses that explain their lifestyles

Independent Trafficking of Ig- /Ig- and -Heavy Chain Is Facilitated by Dissociation of the B Cell Antigen Receptor Complex

The BCR relays extracellular signals and internalizes Ag for processing and presentation. We have previously demonstrated that ligation of the BCR destabilizes Ig-α/Ig-β (Ig-αβ) from μ-H chain (μm). In this study we report that receptor destabilization represents a physical separation of μm from Ig-αβ. Sucrose gradient fractionation localized Ig-αβ to GM1-containing lipid microdomains in the absence of μm. Confocal and electron microscopy studies revealed the colocalization of unsheathed μm with clathrin-coated vesicles. Furthermore, μm failed to associate with clathrin-coated vesicles when receptor destabilization was inhibited, suggesting that unsheathing of μm is required for clathrin-mediated endocytosis. In summary, we found that Ag stimulation physically separates Ig-αβ from μm, facilitating concomitant signal transduction and Ag delivery to the endocytic compartment

Increased Hematopoietic Cells in the mertk-/- Mouse Peritoneal Cavity: A Result of Augmented Migration

The peritoneal cavity is recognized as an important site for autoreactive B cells prior to their transit to other immune tissues; however, little is known of the genes that may regulate this process. Mice lacking the receptor tyrosine kinase Mertk display a lupus-like autoimmune phenotype with splenomegaly and high autoantibodies titers. Here, we investigate whether Mertk regulates the composition of peritoneal cells that favor an autoimmune phenotype. We found an increase in the number of macrophages, DC, plasmacytoid DC, T cells and B cells in the peritoneal cavity of mertk−/− mice when compared to wild-type mice. This disparity in cell numbers was not due to changes in cell proliferation or cell death. In adoptive transfer experiments, we showed an increase in migration of labeled donor cells into the mertk−/− peritoneal cavity. In addition, bone marrow chimeric mice showed hematopoietic-derived factors were also critical for T cell migration. Consistent with this migration and the increase in the number of cells, we identified elevated expression of CXCL9, its receptor CXCR3, and IL-7 receptor on peritoneal cells from mertk−/− mice. To corroborate the migratory function of CXCR3 on cells, the depletion of CXCR3 donor cells significantly reduced the number of adoptively transferred cells that entered into the peritoneum of mertk−/− mice. This control of peritoneal cells numbers correlated with autoantibody production and was exclusively attributed to Mertk since mice lacking other family members, Axl or Tyro 3, did not display dysregulation in peritoneal cell numbers or the autoimmune phenotype

Staphylococcus aureus Protein A Disrupts Immunity Mediated by Long-Lived Plasma Cells

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for antibodies in protecting against recurring infections, but S. aureus modulates the B cell response through expression of Staphylococcal Protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of co-stimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs (LLPCs). The absence of LLPCs was associated with a rapid decline in antigen-specific, class-switched antibody. In contrast, when previously inoculated mice were challenged with isogenic Δspa S. aureus, cells proliferated in the BM survival niches and sustained long-term antibody titers. The effects of SpA on PC fate were limited to the secondary response, as antibody levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with either WT or Δspa S. aureus. Thus, failure to establish long-term protective antibody titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool

IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF

Memory B cell responses are vital for protection against infections, but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent antigen leads to IC-FcγR interactions that induce DCs to secrete BAFF which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary antibody responses. BAFF also contributed to the maintenance and/or expansion of the Tfh population, although it was dispensable for their formation. Thus, early antibody responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses

Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion

Autoimmunity results from a breakdown in tolerance mechanisms that regulate autoreactive lymphocytes. We recently showed that during innate immune responses, secretion of IL-6 by dendritic cells (DCs) maintained autoreactive B cells in an unresponsive state. In this study, we describe that TLR4-activated DCs from lupus-prone mice are defective in repressing autoantibody secretion, coincident with diminished IL-6 secretion. Reduced secretion of IL-6 by MRL/lpr DCs reflected diminished synthesis and failure to sustain IL-6 mRNA production. This occurred coincident with lack of NF-κB and AP-1 DNA binding and failure to sustain IκBα phosphorylation. Analysis of individual mice showed that some animals partially repressed Ig secretion despite reduced levels of IL-6. This suggests that in addition to IL-6, DCs secrete other soluble factor(s) that regulate autoreactive B cells. Collectively, the data show that MRL/lpr mice are defective in DC/IL-6-mediated tolerance, but that some individuals maintain the ability to repress autoantibody secretion by an alternative mechanism. The Journal of Immunology, 2007, 178: 4803–4810

Early Preplasma Cells Define a Tolerance Checkpoint for Autoreactive B Cells

Ab-secreting plasma cells (PCs) are the effectors of humoral immunity. In this study, we describe regulation of autoreactive B cells specific for the ribonucleoprotein Smith (Sm) at an early pre-PC stage. These cells are defined by the expression of the PC marker CD138 and normal levels of CD19 and B220. They are present at a high frequency in normal mouse spleen and bone marrow, are Ag dependent, and are located predominantly along the T cell-B cell border and near bridging channels. Anti-Sm pre-PCs also occur at a high frequency in nonautoimmune mice and show additional phenotypic characteristics of PC differentiation. However, while some of these pre-PCs are Ab-secreting cells, those specific for Sm are not, indicating regulation. Consistent with this, anti-Sm pre-PCs have a higher turnover rate and higher frequency of cell death than those that do not bind Sm. Regulation of anti-Sm pre-PCs occurs upstream of the transcriptional repressor, B lymphocyte-induced maturation protein-1, expression. Regulation at this stage is overcome in autoimmune MRL/lpr mice and is accompanied by an altered B lymphocyte stimulator receptor profile. These data reveal a new B cell tolerance checkpoint that is overcome in autoimmunity