Xist Deletion in B Cells Results in Systemic Lupus Erythematosus Phenotypes.

Systemic lupus erythematosus (SLE) is an autoimmune disease preferentially observed in females. X-linked gene expression in XX females is normalized to that of XY males by X-Chromosome Inactivation (XCI). However, B cells from female SLE patients and mouse models of SLE exhibit mislocalization of Xist RNA, a critical regulator of XCI, and aberrant expression of X-linked genes, suggesting that impairment of XCI may contribute to disease. Here, we find that a subset of female mice harboring a conditional deletion of Xis t in B cells ("Xist cKO") spontaneously develop SLE phenotypes, including expanded activated B cell subsets, disease-specific autoantibodies, and glomerulonephritis. Moreover, pristane-induced SLE-like disease is more severe in Xist cKO mice. Activated B cells from Xist cKO mice with SLE phenotypes have increased expression of proinflammatory X-linked genes implicated in SLE. Together, this work indicates that impaired XCI maintenance in B cells directly contributes to the female-bias of SLE.

ABCs begin with ZEB2.

Age-associated B cells (ABCs) are a stable subset of memory B lymphocytes that develop during microbial infections and in autoimmune diseases. Despite growing appreciation of their phenotypic and functional characteristics, the transcriptional networks involved in ABC fate commitment and maintenance have remained elusive. In their recent publication, Dai et al. tackle this problem, leveraging both mouse models and human diseases to reveal zinc finger E-box-binding homeobox 2 (ZEB2) as a key transcriptional regulator of ABC lineage specification. In aggregate, their results show that ZEB2, a member of the zinc finger E homeobox binding family, promotes ABC differentiation by repressing alternative differentiative fates and targeting genes important for ABC character and function. Moreover, their results strengthen the case for causal links between ABC fate and function in autoimmune pathologies.

T-bet B or not T-bet B, is that the question in obesity-related metabolic disease?

Obesity is linked to inflammation and downstream metabolic dysregulation. In this issue of Cell Metabolism, Hägglöf et al. show that iNKT cells enable the accumulation of T-bet+ B cells in white adipose tissue, which in turn produce chemokine and antibody mediators that exacerbate the onset and severity of metabolic disease.

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Trivalent nucleoside-modified mRNA vaccine yields durable memory B cell protection against genital herpes in preclinical models.

Nucleoside-modified mRNA vaccines have gained global attention because of COVID-19. We evaluated a similar vaccine approach for preventing a chronic, latent genital infection rather than an acute respiratory infection. We used animal models to compare an HSV-2 trivalent nucleoside-modified mRNA vaccine with the same antigens prepared as proteins, with an emphasis on antigen-specific memory B cell responses and immune correlates of protection. In guinea pigs, serum neutralizing-antibody titers were higher at 1 month and declined far less by 8 months in mRNA- compared with protein-immunized animals. Both vaccines protected against death and genital lesions when infected 1 month after immunization; however, protection was more durable in the mRNA group compared with the protein group when infected after 8 months, an interval representing greater than 15% of the animal's lifespan. Serum and vaginal neutralizing-antibody titers correlated with protection against infection, as measured by genital lesions and vaginal virus titers 2 days after infection. In mice, the mRNA vaccine generated more antigen-specific memory B cells than the protein vaccine at early times after immunization that persisted for up to 1 year. High neutralizing titers and robust B cell immune memory likely explain the more durable protection by the HSV-2 mRNA vaccine.

Memory B cells and plasma cells: The differentiative continuum of humoral immunity.

Immunological memory is a composite of lasting antibody titers maintained by plasma cells in conjunction with memory T and B cells. Memory B cells are a critical reservoir for plasma cell generation in the secondary response. Identification of memory B cells requires that they be distinguished from naïve, activated, and germinal center precursors and from plasma cells. Memory B cells are heterogeneous in isotype usage, immunoglobulin mutational content, and phenotypic marker expression. Phenotypic subsets of memory B cells are defined by PD-L2, CD80, and CD73 expression in mice, by CD27 and FCRL4 expression in humans and by T-bet in both mice and humans. These subsets display marked functional heterogeneity, including the ability to rapidly differentiate into plasma cells versus seed germinal centers in the secondary response. Memory B cells are located in the spleen, blood, other lymphoid organs, and barrier tissues, and recent evidence indicates that some memory B cells may be dedicated tissue-resident populations. Open questions about memory B cell longevity, renewal and progenitor-successor relationships with plasma cells are discussed.

The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients.

Systemic lupus erythematous (SLE) is a female-predominant disease characterized by autoimmune B cells and pathogenic autoantibody production. Individuals with two or more X chromosomes are at increased risk for SLE, suggesting that X-linked genes contribute to the observed sex bias of this disease. To normalize X-linked gene expression between sexes, one X in female cells is randomly selected for transcriptional silencing through X-chromosome inactivation (XCI), resulting in allele-specific enrichment of epigenetic modifications, including histone methylation and the long noncoding RNA XIST/Xist on the inactive X (Xi). As we have previously shown that epigenetic regulation of the Xi in female lymphocytes from mice is unexpectedly dynamic, we used RNA fluorescence in situ hybridization and immunofluorescence to profile epigenetic features of the Xi at the single-cell level in human B cell subsets from pediatric and adult SLE patients and healthy controls. Our data reveal that abnormal XCI maintenance in B cells is a feature of SLE. Using single-cell and bulk-cell RNA sequencing datasets, we found that X-linked immunity genes escape XCI in specific healthy human B cell subsets and that human SLE B cells exhibit aberrant expression of X-linked genes and XIST RNA interactome genes. Our data reveal that mislocalized XIST RNA, coupled with a dramatic reduction in heterochromatic modifications at the Xi in SLE, predispose for aberrant X-linked gene expression from the Xi, thus defining a genetic and epigenetic pathway that affects X-linked gene expression in human SLE B cells and likely contributes to the female bias in SLE.

The Transcription Factor T-bet Resolves Memory B Cell Subsets with Distinct Tissue Distributions and Antibody Specificities in Mice and Humans.

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.

BCR-Induced Ca2+ Signals Dynamically Tune Survival, Metabolic Reprogramming, and Proliferation of Naive B Cells.

B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses.

Age-Associated B Cells.

The age-associated B cell subset has been the focus of increasing interest over the last decade. These cells have a unique cell surface phenotype and transcriptional signature, and they rely on TLR7 or TLR9 signals in the context of Th1 cytokines for their formation and activation. Most are antigen-experienced memory B cells that arise during responses to microbial infections and are key to pathogen clearance and control. Their increasing prevalence with age contributes to several well-established features of immunosenescence, including reduced B cell genesis and damped immune responses. In addition, they are elevated in autoimmune and autoinflammatory diseases, and in these settings they are enriched for characteristic autoantibody specificities. Together, these features identify age-associated B cells as a subset with pivotal roles in immunological health, disease, and aging. Accordingly, a detailed understanding of their origins, functions, and physiology should make them tractable translational targets in each of these settings.

Ancient BCMA-like Genes Herald B Cell Regulation in Lampreys.

The TNF superfamily ligands BAFF and APRIL interact with three receptors, BAFFR, BCMA, and TACI, to play discrete and crucial roles in regulating B cell selection and homeostasis in mammals. The interactions between these ligands and receptors are both specific and redundant: BAFFR binds BAFF, whereas BCMA and TACI bind to either BAFF or APRIL. In a previous phylogenetic inquiry, we identified and characterized a BAFF-like gene in lampreys, which, with hagfish, are the only extant jawless vertebrates, both of which have B-like and T-like lymphocytes. To gain insight into lymphocyte regulation in jawless vertebrates, in this study we identified two BCMA-like genes in lampreys, BCMAL1 and BCMAL2, which were found to be preferentially expressed by B-like lymphocytes. In vitro analyses indicated that the lamprey BAFF-like protein can bind to a BCMA-like receptor Ig fusion protein and to both BCMAL1- and BCMAL2-transfected cells. Discriminating regulatory roles for the two BCMA-like molecules are suggested by their differential expression before and after activation of the B-like lymphocytes in lampreys. Our composite results imply that BAFF-based mechanisms for B cell regulation evolved before the divergence of jawed and jawless vertebrates.

Nucleoside-modified mRNA encoding HSV-2 glycoproteins C, D, and E prevents clinical and subclinical genital herpes.

The goals of a genital herpes vaccine are to prevent painful genital lesions and reduce or eliminate subclinical infection that risks transmission to partners and newborns. We evaluated a trivalent glycoprotein vaccine containing herpes simplex virus type 2 (HSV-2) entry molecule glycoprotein D (gD2) and two immune evasion molecules: glycoprotein C (gC2), which binds complement C3b, and glycoprotein E (gE2), which blocks immunoglobulin G (IgG) Fc activities. The trivalent vaccine was administered as baculovirus proteins with CpG and alum, or the identical amino acids were expressed using nucleoside-modified mRNA in lipid nanoparticles (LNPs). Both formulations completely prevented genital lesions in mice and guinea pigs. Differences emerged when evaluating subclinical infection. The trivalent protein vaccine prevented dorsal root ganglia infection, and day 2 and 4 vaginal cultures were negative in 23 of 30 (73%) mice compared with 63 of 64 (98%) in the mRNA group (P = 0.0012). In guinea pigs, 5 of 10 (50%) animals in the trivalent subunit protein group had vaginal shedding of HSV-2 DNA on 19 of 210 (9%) days compared with 2 of 10 (20%) animals in the mRNA group that shed HSV-2 DNA on 5 of 210 (2%) days (P = 0.0052). The trivalent mRNA vaccine was superior to trivalent proteins in stimulating ELISA IgG antibodies, neutralizing antibodies, antibodies that bind to crucial gD2 epitopes involved in entry and cell-to-cell spread, CD4+ T cell responses, and T follicular helper and germinal center B cell responses. The trivalent nucleoside-modified mRNA-LNP vaccine is a promising candidate for human trials.

Molecular pattern recognition in peripheral B cell tolerance: lessons from age-associated B cells.

Although central tolerance mechanisms purge self-reactive B cells during development based on BCR signal strength, mechanisms that block the differentiation of autoreactive effector and memory B cells from mature pools remain poorly understood. Prior observations implicate nucleic acid sensing TLRs in autoimmunity, and more recent findings show that TLR9 is also involved in maintaining peripheral tolerance. Studies of the immunological changes that occur during aging revealed a subset of B cells denoted Age-associated B cells which expands in settings of aging and in autoimmunity. Further studies demonstrated that TLR9 signals poise activated B cells to adopt an Age-associated B cell phenotype, but BCR-delivered TLR9 signals cause programmed cell death that, if circumvented by costimulation, allows continued differentiation to the ABC fate. Together, these observations suggest molecular pattern recognition, rather than BCR epitope specificity per se, is a fundamental mediator of tolerogenic outcomes in the peripheral B cell activation.

The E3 ubiquitin ligase Itch restricts antigen-driven B cell responses.

The E3 ubiquitin ligase Itch regulates antibody levels and prevents autoimmune disease in humans and mice, yet how Itch regulates B cell fate or function is unknown. We now show that Itch directly limits B cell activity. While Itch-deficient mice displayed normal numbers of preimmune B cell populations, they showed elevated numbers of antigen-experienced B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit naive and, to a greater extent, germinal center (GC) B cell numbers. B cells lacking Itch exhibited increased proliferation, glycolytic capacity, and mTORC1 activation. Moreover, stimulation of these cells in vivo by WT T cells resulted in elevated numbers of GC B cells, PCs, and serum IgG. These results support a novel role for Itch in limiting B cell metabolism and proliferation to suppress antigen-driven B cell responses.

IgM Plasma Cells Reside in Healthy Skin and Accumulate with Chronic Inflammation.

Antibodies are key to cutaneous host defense and inflammation. Despite their importance, the mechanisms by which skin antibodies are sustained are poorly described. Here, we identified that, in addition to antibody production in lymphoid tissues, plasma cells reside in healthy mouse and human skin. In naïve mice, IgM was the predominant isotype produced in skin. Skin plasma cells developed independently of T cells and microbiota. Importantly, chronic skin inflammation promoted the massive accumulation of IgM-secreting cells, and cutaneous immunization directed both T cell-dependent and -independent antigen-specific IgM-secreting cells into skin. Unlike their counterparts in lymphoid tissues, cutaneous IgM-secreting cells were completely dependent on survival factors such as a proliferation-inducing ligand or B cell-activating factor, which were constitutively expressed and upregulated during inflammation in skin. Our data support a model in which skin plasma cells supply natural and adaptive IgM to the cutaneous environment, thereby supporting homeostatic skin barrier functions and providing defense against pathogen intrusion. Our results are also of potential relevance for manipulation of cutaneous plasma cells in inflammatory skin diseases or cutaneous plasma cell malignancies.

T-bet+ memory B cells: Generation, function, and fate.

B cells expressing the transcription factor T-bet have emerged as participants in a number of protective and pathogenic immune responses. T-bet+ B cells characteristically differentiate in response to combined Toll-like receptor and cytokine signaling, contribute to protective immunity against intracellular pathogens via IgG2a/c production and antibody-independent mechanisms, and are prone to produce autoantibodies. Despite recent advances, a number of questions remain regarding the basic biology of T-bet+ B cells and their functional niche within the immune system. Herein, we review the discovery and defining characteristics of the T-bet+ B cell subset in both mice and humans. We further discuss their origins, the basis for their persistence, and their potential fate in vivo. Evidence indicates that T-bet+ B cells represent a distinct, germinal center-derived memory population that may serve as an important therapeutic target for the improvement of humoral immunity and prevention of autoimmunity.