PKCδ Protects against Lupus Autoimmunity.

Protein kinase C delta (PKCδ) has emerged as a key protective molecule against systemic lupus erythematosus (SLE or lupus), an autoimmune disease characterized by anti-double stranded (ds) DNA IgGs. Although PKCδ-deficient mice and lupus patients with mutated PRKCD genes clearly demonstrate the requirement for PKCδ in preventing lupus autoimmunity, this critical tolerance mechanism remains poorly understood. We recently reported that PKCδ acts as a key regulator of B cell tolerance by selectively deleting anti-dsDNA B cells in the germinal center (GC). PKCδ's tolerance function is activated by sphingomyelin synthase 2 (SMS2), a lipid enzyme whose expression is generally reduced in B cells from lupus patients. Moreover, pharmacologic strengthening of the SMS2/PKCδ tolerance pathway alleviated lupus pathogenesis in mice. Here, we review relevant publications in order to provide mechanistic insights into PKCδ's tolerance activity and discuss the potential significance of therapeutically targeting PKCδ's tolerance activity in the GC for selectively inhibiting lupus autoimmunity.

Common variable immunodeficiency, cross currents, and prevailing winds.

Common variable immunodeficiency (CVID) is a heterogenous disease category created to distinguish late-onset antibody deficiencies from early-onset diseases like agammaglobulinemia or more expansively dysfunctional combined immunodeficiencies. Opinions vary on which affected patients should receive a CVID diagnosis which confuses clinicians and erects reproducibility barriers for researchers. Most experts agree that CVID's most indeliable feature is defective germinal center (GC) production of isotype-switched, affinity-maturated antibodies. Here, we review the biological factors contributing to CVID-associated GC dysfunction including genetic, epigenetic, tolerogenic, microbiome, and regulatory abnormalities. We also discuss the consequences of these biological phenomena to the development of non-infectious disease complications. Finally, we opine on topics and lines of investigation we think hold promise for expanding our mechanistic understanding of this protean condition and for improving the lives of affected patients.

B Cell Tolerance and Targeted Therapies in SLE.

Systemic Lupus Erythematosus (SLE) is a chronic systemic autoimmune disease of high clinical and molecular heterogeneity, and a relapsing-remitting pattern. The disease is currently without cure and more prevalent in women. B cell tolerance and production of autoantibodies are critical mechanisms that drive SLE pathophysiology. However, how the balance of the immune system is broken and how the innate and adaptive immune systems are interacting during lupus-specific autoimmune responses are still largely unknown. Here, we review the latest knowledge on B cell development, maturation, and central versus peripheral tolerance in connection to SLE and treatment options. We also discuss the regulation of B cells by conventional T cells, granulocytes, and unconventional T cells, and how effector B cells exert their functions in SLE. We also discuss mechanisms of action of B cell-targeted therapies, as well as possible future directions based on current knowledge of B cell biology.

B cell abnormalities and autoantibody production in patients with partial RAG deficiency.

Mutations in the recombination activating gene 1 (RAG1) and RAG2 in humans are associated with a broad spectrum of clinical phenotypes, from severe combined immunodeficiency to immune dysregulation. Partial (hypomorphic) RAG deficiency (pRD) in particular, frequently leads to hyperinflammation and autoimmunity, with several underlying intrinsic and extrinsic mechanisms causing a break in tolerance centrally and peripherally during T and B cell development. However, the relative contributions of these processes to immune dysregulation remain unclear. In this review, we specifically focus on the recently described tolerance break and B cell abnormalities, as well as consequent molecular and cellular mechanisms of autoantibody production in patients with pRD.

Covid-19 a triggering factor of autoimmune and multi-inflammatory diseases.

SARS-CoV-2 virus has attracted a lot of attention globally due to the autoimmune and inflammatory processes that were observed during the development of Covid-19 disease. Excessive activation of immune response and triggering of autoantibodies synthesis as well as an excessive synthesis of inflammatory cytokines and the onset of cytokine storm has a vital role in the disease outcome and the occurring autoimmune complications. This scenario is reminiscent of infiltration of lymphocytes and monocytes in specific organs and the increased production of autoantibodies and chemoattractants noted in other inflammatory and autoimmune diseases. The main goal of this study is to investigate the complex inflammatory processes that occur in Covid-19 disease and to find similarities with other inflammatory diseases such as multiple sclerosis (MS), acute respiratory distress syndrome (ARDS), rheumatoid arthritis (RA) and Kawasaki syndrome to advance existing diagnostic and therapeutic protocols. The therapy with Interferon-gamma (IFN-γ) and the use of S1P receptor modulators showed promising results. However, there are many unknowns about these mechanisms and possible novel therapies. Therefore, the inflammation and autoimmunity triggered by Covid-19 should be further investigated to improve existing diagnostic procedures and therapeutic protocols for Covid-19.

Activation induced cytidine deaminase: An old friend with new faces.

Activation induced cytidine deaminase (AID) protein is a member of APOBEC family. AID converts cytidine to uracil, which is a key step for somatic hypermutation (SHM) and class switch recombination (CSR). AID also plays critical roles in B cell precursor stages, removing polyreactive B cells from immune repertoire. Since the main function of AID is inducing point mutations, dysregulation can lead to increased mutation load, translocations, disturbed genomic integrity, and lymphomagenesis. As such, expression of AID as well as its function is controlled strictly at various molecular steps. Other members of the APOBEC family also play crucial roles during carcinogenesis. Considering all these functions, AID represents a bridge, linking chronic inflammation to carcinogenesis and immune deficiencies to autoimmune manifestations.

Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

Interleukins 4 (IL-4) and 21 (IL-21) belong to the common gamma chain cytokine family which are highly involved in the progression of autoimmune diseases. While IL-4 is well known to be involved in the suppression of apoptosis of autoreactive B cells, the role played by IL-21 remains unclear. In the current study, we activated the human Burkitt's lymphoma Ramos B cells with anti-IgM to mimic B cell hyperactivation observed in patients of autoimmune diseases. Consistent with other reported findings, anti-IgM led to the downregulation of proteins involved in B cell survival and proliferation, as well as the activation of caspase 3 activity and DNA damage, resulting in apoptotic cell death after 48-hour treatment. Although both IL-4 and IL-21 reversed anti-IgM-induced apoptosis and cell cycle arrest, they did so via different mechanisms: while IL-4 could directly suppress anti-IgM-induced caspase 3 activation and marker indicative of DNA damage, IL-21 could induce B cell proliferation in the presence of anti-IgM. Importantly, IL-21 also suppressed activation induced cell death in human primary B cells. Pre-treatment with clinically validated JAK inhibitors completely reversed the effects of IL-4 and IL-21 to rescue anti-IgM induced cell death and DNA damage. The results indicate the underlying mechanisms of how IL-4 and IL-21 differentially promote survival of hyperactivated B cells and provide hints to treat autoimmune diseases.

B cells in the balance: Offsetting self-reactivity avoidance with protection against foreign.

Antibodies are theoretically limitless in their diversity and specificity to foreign antigens; however they are constrained by the need to avoid binding to self. Germinal centers (GC) allow diversification and maturation of the antibody response towards the foreign antigen. While self-tolerance mechanisms controlling self-reactivity during B cell maturation are well recognized, the mechanisms by which GCs balance self-tolerance and foreign binding especially in the face of cross-reactivity between self and foreign, remain much less well defined. In this review we explore the extent to which GC self-tolerance restricts affinity maturation. We present studies suggesting that the outcome is situationally dependent, affected by affinity and avidity to self-antigen, and the extent to which self-binding and foreign-binding are interdependent. While auto-reactive GC B cells can mutate away from self while maturing towards the foreign antigen, if no mutational trajectories allow for self-reactive redemption, self-tolerance prevails and GC responses to the foreign pathogen are restricted, except when self-tolerance checkpoints are relaxed. Finally, we consider whether polyreactivity is subject to the same level of restriction in GC responses, especially if polyreactivity is linked to an increase in foreign protection, as occurs in certain broadly neutralizing antibodies. Overall, the outcomes for GC B cells that bind self-antigen can range from redemption, transient relaxation in self-tolerance or restriction of the antibody response to the foreign pathogen.

LAPTM5 mediates immature B cell apoptosis and B cell tolerance by regulating the WWP2-PTEN-AKT pathway.

Elimination of autoreactive developing B cells is an important mechanism to prevent autoantibody production. However, how B cell receptor (BCR) signaling triggers apoptosis of immature B cells remains poorly understood. We show that BCR stimulation up-regulates the expression of the lysosomal-associated transmembrane protein 5 (LAPTM5), which in turn triggers apoptosis of immature B cells through two pathways. LAPTM5 causes BCR internalization, resulting in decreased phosphorylation of SYK and ERK. In addition, LAPTM5 targets the E3 ubiquitin ligase WWP2 for lysosomal degradation, resulting in the accumulation of its substrate PTEN. Elevated PTEN levels suppress AKT phosphorylation, leading to increased FOXO1 expression and up-regulation of the cell cycle inhibitor p27Kip1 and the proapoptotic molecule BIM. In vivo, LAPTM5 is involved in the elimination of autoreactive B cells and its deficiency exacerbates autoantibody production. Our results reveal a previously unidentified mechanism that contributes to immature B cell apoptosis and B cell tolerance.

Alphavirus Replicon Particle Vaccine Breaks B Cell Tolerance and Rapidly Induces IgG to Murine Hematolymphoid Tumor Associated Antigens.

De novo immune responses to myeloid and other blood-borne tumors are notably limited and ineffective, making our ability to promote immune responses with vaccines a major challenge. While focus has been largely on cytotoxic cell-mediated tumor eradication, B-cells and the antibodies they produce also have roles in anti-tumor responses. Indeed, therapeutic antibody-mediated tumor cell killing is routinely employed in patients with hematolymphoid cancers, but whether endogenous antibody responses can be incited to blood-born tumors remains poorly studied. A major limitation of immunoglobulin therapies is that cell surface expression of tumor-associated antigen (TAA) targets is dynamic and varied, making promotion of polyclonal, endogenous B cell responses appealing. Since many TAAs are self-antigens, developing tumor vaccines that enable production of antibodies to non-polymorphic antigen targets remains a challenge. As B cell responses to RNA vaccines are known to occur, we employed the Viral Replicon Particles (VRP) which was constructed to encode mouse FLT3. The VRP-FLT3 vaccine provoked a rapid IgG B-cell response to this self-antigen in leukemia and lymphoma mouse models. In addition, IgGs to other TAAs were also produced. Our data suggest that vaccination with RNA viral particle vectors incites a loss of B-cell tolerance that enables production of anti-tumor antibodies. This proof of principle work provides impetus to employ such strategies that lead to a break in B-cell tolerance and enable production of broadly reactive anti-TAA antibodies as potential future therapeutic agents for patients with hematolymphoid cancers.

Analysis of a miR-148a Targetome in B Cell Central Tolerance.

A microRNA (miRNA) often regulates the expression of hundreds of target genes. A fundamental question in the field of miRNA research is whether a miRNA exerts its biological function through regulating a small number of key targets or through small changes in the expression of hundreds of target genes. We addressed this issue by performing functional analysis of target genes regulated by miR-148a. We previously identified miR-148a as a critical regulator of B cell central tolerance and found 119 target genes that may mediate its function. We selected 4 of them for validation and demonstrated a regulatory role for Bim, Pten, and Gadd45a in this process. In this study, we performed functional analysis of the other miR-148a target genes in in vitro and in vivo models of B cell central tolerance. Our results show that those additional target genes play a minimal role, if any, in miR-148a-mediated control of B cell central tolerance, suggesting that the function of miRNAs is mediated by a few key target genes. These findings have advanced our understanding of molecular mechanisms underlying miRNA regulation of gene expression and B cell central tolerance.

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance.

Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.

B-cell intrinsic and extrinsic signals that regulate central tolerance of mouse and human B cells.

The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM+ immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here, we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.

Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice.

Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH-DH-JH and Vκ-Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.

Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

SMS2 deficiency impairs PKCδ-regulated B cell tolerance in the germinal center.

B cell tolerance prevents autoimmunity by deleting or deactivating autoreactive B cells that otherwise may cause autoantibody-driven disorders, including systemic lupus erythematosus (lupus). Lupus is characterized by immunoglobulin Gs carrying a double-stranded (ds)-DNA autospecificity derived mainly from somatic hypermutation in the germinal center (GC), pointing to a checkpoint breach of GC B cell tolerance that leads to lupus. However, tolerance mechanisms in the GC remain poorly understood. Here, we show that upregulated sphingomyelin synthase 2 (SMS2) in anti-dsDNA GC B cells induces apoptosis by directly activating protein kinase C δ (PKCδ)'s pro-apoptotic activity. This tolerance mechanism prevents lupus autoimmunity in C57/BL6 mice and can be stimulated pharmacologically to inhibit lupus pathogenesis in lupus-prone NZBWF1 mice. Patients with lupus consistently have substantially reduced SMS2 expression in B cells and to an even greater extent in autoimmune-prone, age-associated B cells, suggesting that patients with lupus have insufficient SMS2-regulated B cell tolerance.

Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice.

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ+ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

Developmental changes in the rules for B cell selection.

The autoimmune checkpoint during B cell maturation eliminates self-antigen reactive specificities from the mature B cell repertoire. However, an exception to this rule is illustrated by B-1 cells, an innate-like self-reactive B cell subset that is positively selected into the mature B cell pool in a self-antigen-driven fashion. The mechanisms by which B-1 cells escape central tolerance have puzzled the field for decades. A key clue comes from their restricted developmental window during fetal and neonatal life. Here we use B-1 cells as a prototypic early life derived B cell subset to explore developmental changes in the constraints of B cell selection. We discuss recent advancements in the understanding of the molecular program, centered around the RNA binding protein Lin28b, that licenses self-reactive B-1 cell output during ontogeny. Finally, we speculate on the possible link between the unique rules of early life B cell tolerance and the establishment of B cell - microbial mutualism to propose an integrated model for how developmental and environmental cues come together to create a protective layer of B cell memory involved in neonatal immune imprinting.

Single-cell approaches to investigate B cells and antibodies in autoimmune neurological disorders.

Autoimmune neurological disorders, including neuromyelitis optica spectrum disorder, anti-N-methyl-D-aspartate receptor encephalitis, anti-MOG antibody-associated disorders, and myasthenia gravis, are clearly defined by the presence of autoantibodies against neurological antigens. Although these autoantibodies have been heavily studied for their biological activities, given the heterogeneity of polyclonal patient samples, the characteristics of a single antibody cannot be definitively assigned. This review details the findings of polyclonal serum and CSF studies and then explores the advances made by single-cell technologies to the field of antibody-mediated neurological disorders. High-resolution single-cell methods have revealed abnormalities in the tolerance mechanisms of several disorders and provided further insight into the B cells responsible for autoantibody production. Ultimately, several factors, including epitope specificity and binding affinity, finely regulate the pathogenic potential of an autoantibody, and a deeper appreciation of these factors may progress the development of targeted immunotherapies for patients.