Elevated unphosphorylated STAT1 and IRF9 in T and B cells of primary sjögren's syndrome: Novel biomarkers for disease activity and subsets.

OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.

Increased IgD and CD27 Double Negative (DN) B cell population in pediatric onset autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.

Neutrophils disrupt B-1a cell homeostasis by targeting Siglec-G to exacerbate sepsis.

B-1a cells, an innate-like cell population, are crucial for pathogen defense and the regulation of inflammation through their release of natural IgM and IL-10. In sepsis, B-1a cell numbers are decreased in the peritoneal cavity as they robustly migrate to the spleen. Within the spleen, migrating B-1a cells differentiate into plasma cells, leading to alterations in their original phenotype and functionality. We discovered a key player, sialic acid-binding immunoglobulin-like lectin-G (Siglec-G), which is expressed predominantly on B-1a cells and negatively regulates B-1a cell migration to maintain homeostasis. Siglec-G interacts with CXCR4/CXCL12 to modulate B-1a cell migration. Neutrophils aid B-1a cell migration via neutrophil elastase (NE)-mediated Siglec-G cleavage. Human studies revealed increased NE expression in septic patients. We identified an NE cleavage sequence in silico, leading to the discovery of a decoy peptide that protects Siglec-G, preserves peritoneal B-1a cells, reduces inflammation, and enhances sepsis survival. The role of Siglec-G in inhibiting B-1a cell migration to maintain their inherent phenotype and function is compromised by NE in sepsis, offering valuable insights into B-1a cell homeostasis. Employing a small decoy peptide to prevent NE-mediated Siglec-G cleavage has emerged as a promising strategy to sustain peritoneal B-1a cell homeostasis, alleviate inflammation, and ultimately improve outcomes in sepsis patients.

Unravelling B cell heterogeneity: insights into flow cytometry-gated B cells from single-cell multi-omics data.

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches. Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases. Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data (AlliGateR). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations. Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets.

B cell heterogeneity in human tuberculosis highlights compartment-specific phenotype and functional roles.

B cells are important in tuberculosis (TB) immunity, but their role in the human lung is understudied. Here, we characterize B cells from lung tissue and matched blood of patients with TB and found they are decreased in the blood and increased in the lungs, consistent with recruitment to infected tissue, where they are located in granuloma associated lymphoid tissue. Flow cytometry and transcriptomics identify multiple B cell populations in the lung, including those associated with tissue resident memory, germinal centers, antibody secretion, proinflammatory atypical B cells, and regulatory B cells, some of which are expanded in TB disease. Additionally, TB lungs contain high levels of Mtb-reactive antibodies, specifically IgM, which promotes Mtb phagocytosis. Overall, these data reveal the presence of functionally diverse B cell subsets in the lungs of patients with TB and suggest several potential localized roles that may represent a target for interventions to promote immunity or mitigate immunopathology.

Dynamic changes in B cell subpopulations in response to triple-negative breast cancer development.

Despite presenting a worse prognosis and being associated with highly aggressive tumors, triple-negative breast cancer (TNBC) is characterized by the higher frequency of tumor-infiltrating lymphocytes, which have been implicated in better overall survival and response to therapy. Though recent studies have reported the capacity of B lymphocytes to recognize overly-expressed normal proteins, and tumor-associated antigens, how tumor development potentially modifies B cell response is yet to be elucidated. Our findings reveal distinct effects of 4T1 and E0771 murine tumor development on B cells in secondary lymphoid organs. Notably, we observe a significant expansion of total B cells and plasma cells in the tumor-draining lymph nodes (tDLNs) as early as 7 days after tumor challenge in both murine models, whereas changes in the spleen are less pronounced. Surprisingly, within the tumor microenvironment (TME) of both models, we detect distinct B cell subpopulations, but tumor development does not appear to cause major alterations in their frequency over time. Furthermore, our investigation into B cell regulatory phenotypes highlights that the B10 Breg phenotype remains unaffected in the evaluated tissues. Most importantly, we identified an increase in CD19 + LAG-3 + cells in tDLNs of both murine models. Interestingly, although CD19 + LAG-3 + cells represent a minor subset of total B cells (< 3%) in all evaluated tissues, most of these cells exhibit elevated expression of IgD, suggesting that LAG-3 may serve as an activation marker for B cells. Corroborating with these findings, we detected distinct cell cycle and proliferation genes alongside LAG-3 analyzing scRNA-Seq data from a cohort of TNBC patients. More importantly, our study suggests that the presence of LAG-3 B cells in breast tumors could be associated with a good prognosis, as patients with higher levels of LAG-3 B cell transcripts had a longer progression-free interval (PFI). This novel insight could pave the way for targeted therapies that harness the unique properties of LAG-3 + B cells, potentially offering new avenues for improving patient outcomes in TNBC. Further research is warranted to unravel the mechanistic pathways of these cells and to validate their prognostic value in larger, diverse patient cohorts.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Intratumoral CD38+CD19+B cells associate with poor clinical outcomes and immunosuppression in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The widespread involvement of tumor-infiltrating B cells highlights their potential role in tumor behavior. However, B cell heterogeneity in PDAC remains unexplored. Studying TIL-Bs in PDAC aims to identify new treatment strategies. METHODS: We performed single-cell RNA sequencing to study the heterogeneity of B cells in PDAC. The prognostic and immunologic value of the identified CD38+ B cells was explored in FUSCC (n = 147) and TCGA (n = 176) cohorts. Flow cytometry was conducted to characterize the relationship between CD38+ B cells and other immune cells, as well as their phenotypic features. In vitro and in vivo experiments were performed to assess the putative effect of CD38+ B cells on antitumor immunity. FINDINGS: The presence of CD38+ B cells in PDAC was associated with unfavorable clinicopathological features and poorer overall survival (p < 0.001). Increased infiltration of CD38+ B cells was accompanied by reduced natural killer (NK) cells (p = 0.021) and increased regulatory T cells (p = 0.016). Molecular profiling revealed high expression of IL-10, IL-35, TGF-ß, GZMB, TIM-1, CD5 and CD21, confirming their putative regulatory B cell-like features. Co-culture experiments demonstrated suppression of NK cell cytotoxicity by CD38+ B cell-derived IL-10 (p < 0.001). Finally, in vivo experiments suggested adoptive transfer of CD38+ B cells reduced antitumor immunity and administration of a CD38 inhibitor hampered tumor growth (p < 0.001). INTERPRETATION: We discovered regulatory B cell-like CD38+ B cell infiltration as an independent prognostic factor in PDAC. The use of CD38 inhibitor may provide new possibilities for PDAC immunotherapy. FUNDING: This study was supported by the National Natural Science Foundation of China (U21A20374), Shanghai Municipal Science and Technology Major Project (21JC1401500), Scientific Innovation Project of Shanghai Education Committee (2019-01-07-00-07-E00057), Special Project for Clinical Research in the Health Industry of the Shanghai Health Commission (No. 20204Y0265) and Natural Science Foundation of Shanghai (23ZR1479300).

Novel B-cell subsets as potential biomarkers in idiopathic inflammatory myopathies: insights into disease pathogenesis and disease activity.

Idiopathic inflammatory myopathies are a heterogeneous group of rare autoimmune disorders characterized by progressive muscle weakness and the histopathologic findings of inflammatory infiltrates in muscle tissue. Although their pathogenesis remains indefinite, the association of autoantibodies with clinical manifestations and the evidence of high effectiveness of depleting therapies suggest that B cells could be implicated. Therefore, we explored the landscape of peripheral B cells in this disease by multiparametric flow cytometry, finding significant numerical decreases in memory and double-negative subsets, as well as an expansion of the naive compartment relative to healthy controls, that contribute to defining disease-associated B-cell subset signatures and correlating with different clinical features of patients. Additionally, we determined the potential value of these subsets as diagnostic biomarkers, thus positioning B cells as neglected key elements possibly participating in idiopathic inflammatory myopathy onset or development.

Disease activity in chronic inflammatory demyelinating polyneuropathy: association between circulating B-cell subsets, cytokine levels, and clinical outcomes.

Chronic inflammatory demyelinating polyneuropathy (CIDP), a common and treatable autoimmune neuropathy, is frequently misdiagnosed. The aim of this study is to evaluate the relationship between immunological markers and clinical outcome measures in a mixed cohort of patients with typical CIDP and CIDP variants at different disease stages. Twenty-three typical, 16 multifocal and five distal CIDP patients were included. Twenty-five sex and age-matched healthy controls and 12 patients with Charcot-Marie-Tooth type 1A (CMT1A) disease served as controls. Peripheral B-cell populations were analyzed by flow cytometry. IL6, IL10, TNFA mRNA and mir-21, mir-146a, and mir-155-5p expression levels were evaluated by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMC) and/or skin biopsy specimens. Results were then assessed for a possible association with clinical disability scores and intraepidermal nerve fiber densities (IENFD) in the distal leg. We detected a significant reduction in naive B cells (P ≤ 0.001), plasma cells (P ≤ 0.001) and regulatory B cells (P < 0.05), and an elevation in switched memory B cells (P ≤ 0.001) in CIDP compared to healthy controls. CMT1A and CIDP patients had comparable B-cell subset distribution. CIDP cases had significantly higher TNFA and IL10 gene expression levels in PBMC compared to healthy controls (P < 0.05 and P ≤ 0.01, respectively). IENFDs in the distal leg showed a moderate negative correlation with switched memory B-cell ratios (r = -0.51, P < 0.05) and a moderate positive correlation with plasma cell ratios (r = 0.46, P < 0.05). INCAT sum scores showed a moderate positive correlation with IL6 gene expression levels in PBMC (r = 0.54, P < 0.05). Altered B-cell homeostasis and IL10 and TNFA gene expression levels imply chronic antigen exposure and overactivity in the humoral immune system, and seem to be a common pathological pathway in both typical CIDP and CIDP variants.

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells.

During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. Here we use label-free proteomics to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription, and translation initiation factors that define cellular identity and function. Cross-examination of the full-length transcriptome and proteome identifies mRNAs related to B cell activation and antibody secretion that are not accompanied by detection of the encoded proteins. In addition, proteomic data further suggests that the translational repressor PDCD4 restrains B cell responses, in particular those from marginal zone B cells, to a T-cell independent antigen. In summary, our molecular characterization of B cell maturation presents a valuable resource to further explore the mechanisms underpinning the specialized functions of B cell subsets, and suggest the presence of 'poised' mRNAs that enable expedited B cell responses.

Extrafollicular IgD-CD27-CXCR5-CD11c- DN3 B cells infiltrate inflamed tissues in autoimmune fibrosis and in severe COVID-19.

Although therapeutic B cell depletion dramatically resolves inflammation in many diseases in which antibodies appear not to play a central role, distinct extrafollicular pathogenic B cell subsets that accumulate in disease lesions have hitherto not been identified. The circulating immunoglobulin D (IgD)-CD27-CXCR5-CD11c+ DN2 B cell subset has been previously studied in some autoimmune diseases. A distinct IgD-CD27-CXCR5-CD11c- DN3 B cell subset accumulates in the blood both in IgG4-related disease, an autoimmune disease in which inflammation and fibrosis can be reversed by B cell depletion, and in severe COVID-19. These DN3 B cells prominently accumulate in the end organs of IgG4-related disease and in lung lesions in COVID-19, and double-negative B cells prominently cluster with CD4+ T cells in these lesions. Extrafollicular DN3 B cells may participate in tissue inflammation and fibrosis in autoimmune fibrotic diseases, as well as in COVID-19.

B1-cell-produced anti-phosphatidylserine antibodies contribute to lupus nephritis development via TLR-mediated Syk activation.

Autoantibodies produced by B cells play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). However, both the cellular source of antiphospholipid antibodies and their contributions to the development of lupus nephritis (LN) remain largely unclear. Here, we report a pathogenic role of anti-phosphatidylserine (PS) autoantibodies in the development of LN. Elevated serum PS-specific IgG levels were measured in model mice and SLE patients, especially in those with LN. PS-specific IgG accumulation was found in the kidney biopsies of LN patients. Both transfer of SLE PS-specific IgG and PS immunization triggered lupus-like glomerular immune complex deposition in recipient mice. ELISPOT analysis identified B1a cells as the main cell type that secretes PS-specific IgG in both lupus model mice and patients. Adoptive transfer of PS-specific B1a cells accelerated the PS-specific autoimmune response and renal damage in recipient lupus model mice, whereas depletion of B1a cells attenuated lupus progression. In culture, PS-specific B1a cells were significantly expanded upon treatment with chromatin components, while blockade of TLR signal cascades by DNase I digestion and inhibitory ODN 2088 or R406 treatment profoundly abrogated chromatin-induced PS-specific IgG secretion by lupus B1a cells. Thus, our study has demonstrated that the anti-PS autoantibodies produced by B1 cells contribute to lupus nephritis development. Our findings that blockade of the TLR/Syk signaling cascade inhibits PS-specific B1-cell expansion provide new insights into lupus pathogenesis and may facilitate the development of novel therapeutic targets for the treatment of LN in SLE.

An Optimized Flow Cytometric Method to Demonstrate the Differentiation Stage-Dependent Ca2+ Flux Responses of Peripheral Human B Cells.

Calcium (Ca2+) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca2+ flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca2+ flux responses and that B-cell subsets show specific developmental-stage dependent Ca2+ flux response patterns. Naive B cells responded with a more substantial Ca2+ flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca2+ flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca2+ responses upon activation, indicating their loss of dependence on Ca2+ signaling. Ca2+ flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.

Nodal reactive proliferation of monocytoid B-cells may represent atypical memory B-cells.

BACKGROUND: Reactive lymphadenopathies such as toxoplasmosis and cytomegalovirus lymphadenitis are associated with monocytoid cell proliferation. Monocytoid cells are B-lymphocytes with an undetermined subset. METHODS: Using digital spatial profiling whole transcriptome analyses, this study compared monocytoid and control B-cells. The B-cell subset of monocytoid cells was assigned according to gene expression profiles. RESULTS: This study identified 466 differentially expressed genes between monocytoid and control B-cells. The cellular deconvolution algorithm identified monocytoid cells as memory B-cells instead of as naïve B-cells. A comparison of the upregulated genes revealed that atypical memory B-cells had the largest number of genes overlapping with monocytoid cells compared with other memory B-cell subsets. Atypical memory B-cell markers, namely TBX21 (T-bet), FCRL4 (IRTA1), and ITGAX (CD11c), were all upregulated in monocytoid cells. Similar to atypical memory B-cells, monocytoid cells exhibited (1) upregulated transcription factors (TBX21, TOX), (2) upregulated genes associated with B-cell inhibition (FCRL5, FCRL4) and downregulated genes associated with B-cell activation (PIK3CG, NFKB1A, CD40), (3) downregulated cell cycle-related genes (CDK6, MYC), and (4) downregulated cytokine receptors (IL4R). This study also analyzed the expression of monocytoid cell signature genes in various memory B-cell subsets. Atypical memory B-cells exhibited a gene expression pattern similar to that of monocytoid cells, but other memory B-cell subsets did not. Furthermore, monocytoid cells and marginal zone lymphomas differed in gene expression profiles. CONCLUSION: Spatial transcriptomic analyses indicated that monocytoid cells may be atypical memory B-cells.

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OBJECTIVES: To investigate the change in the distribution of memory B cell subsets in children with frequently relapsing nephrotic syndrome (FRNS) during the course of the disease. METHODS: A total of 35 children with primary nephrotic syndrome (PNS) who attended the Department of Pediatrics of the Affiliated Hospital of Xuzhou Medical University from October 2020 to October 2021 were enrolled as subjects in this prospective study. According to the response to glucocorticoid (GC) therapy and frequency of recurrence, the children were divided into two groups: FRNS (n=20) and non-FRNS (NFRNS; n=15). Fifteen children who underwent physical examination were enrolled as the control group. The change in memory B cells after GC therapy was compared between groups, and its correlation with clinical indicators was analyzed. RESULTS: Before treatment, the FRNS and NFRNS groups had significantly increased percentages of total B cells, total memory B cells, IgD+ memory B cells, and IgE+ memory B cells compared with the control group, and the FRNS group had significantly greater increases than the NFRNS group (P<0.05); the FRNS group had a significantly lower percentage of class-switched memory B cells than the NFRNS and control groups (P<0.05). After treatment, the FRNS and NFRNS groups had significant reductions in the percentages of total B cells, total memory B cells, IgM+IgD+ memory B cells, IgM+ memory B cells, IgE+ memory B cells, IgD+ memory B cells, and IgG+ memory B cells (P<0.05) and a significant increase in the percentage of class-switched memory B cells (P<0.05). The FRNS group had a significantly higher urinary protein quantification than the NFRNS and control groups (P<0.05) and a significantly lower level of albumin than the control group (P<0.05). In the FRNS group, urinary protein quantification was negatively correlated with the percentage of class-switched memory B cells and was positively correlated with the percentage of IgE+ memory B cells (P<0.05). CONCLUSIONS: Abnormal distribution of memory B cell subsets may be observed in children with FRNS, and the percentages of IgE+ memory B cells and class-switched memory B cells can be used as positive and negative correlation factors for predicting recurrence after GC therapy in these children.

Dual Role of B Cells in Multiple Sclerosis.

B cells have emerged as an important immune cell type that can be targeted for therapy in multiple sclerosis (MS). Depleting B cells with anti-CD20 antibodies is effective in treating MS. Yet, atacicept treatment, which blocks B-cell Activating Factor (BAFF) and A Proliferation-Inducing Ligand (APRIL), two cytokines important for B cell development and function, paradoxically increases disease activity in MS patients. The reason behind the failure of atacicept is not well understood. The stark differences in clinical outcomes with these therapies demonstrate that B cells have both inflammatory and anti-inflammatory functions in MS. In this review, we summarize the importance of B cells in MS and discuss the different B cell subsets that perform inflammatory and anti-inflammatory functions and how therapies modulate B cell functions in MS patients. Additionally, we discuss the potential anti-inflammatory functions of BAFF and APRIL on MS disease.

Inactive disease in patients with lupus is linked to autoantibodies to type I interferons that normalize blood IFNα and B cell subsets.

Systemic lupus erythematosus (SLE) is characterized by increased expression of type I interferon (IFN)-regulated genes in 50%-75% of patients. We report that out of 501 patients with SLE analyzed, 73 (14%) present autoantibodies against IFNα (anti-IFN-Abs). The presence of neutralizing-anti-IFN-Abs in 4.2% of patients inversely correlates with low circulating IFNα protein levels, inhibition of IFN-I downstream gene signatures, and inactive global disease score. Hallmarks of SLE pathogenesis, including increased immature, double-negative plasmablast B cell populations and reduction in regulatory B cell (Breg) frequencies, were normalized in patients with neutralizing anti-IFN-Abs compared with other patient groups. Immunoglobulin G (IgG) purified from sera of patients with SLE with neutralizing anti-IFN-Abs impedes CpGC-driven IFNα-dependent differentiation of B cells into immature B cells and plasmablasts, thus recapitulating the neutralizing effect of anti-IFN-Abs on B cell differentiation in vitro. Our findings highlight a role for neutralizing anti-IFN-Abs in controlling SLE pathogenesis and support the use of IFN-targeting therapies in patients with SLE lacking neutralizing-anti-IFN-Abs.

Amelioration of Autoimmunity in a Lupus Mouse Model by Modulation of T-Bet-Promoted Energy Metabolism in Pathogenic Age/Autoimmune-Associated B Cells.

OBJECTIVE: Emerging evidence indicates that a distinct CD11c+T-bet+ B cell subset, termed age/autoimmune-associated B cells (ABCs), is the major pathogenic autoantibody producer in lupus. Human lupus is associated with significant metabolic alterations, but how ABCs orchestrate their typical transcription factors and metabolic programs to meet specific functional requirements is unclear. We undertook this study to characterize the metabolism of ABCs and to identify the regulators of their metabolic pathways in an effort to develop new therapies for ABC-mediated autoimmunity. METHODS: We developed a T-bet-tdTomato reporter mouse strain to trace live T-bet+ B cells and adoptively transferred CD4+ T cells from bm12 mice to induce lupus. We next sorted CD11c+tdTomato+ B cells and conducted RNA sequencing and an extracellular flux assay. A metabolic restriction to constrain ABC formation was tested in human and mouse B cells. We used a bm12-induced lupus mouse model to conduct the metabolic intervention. RESULTS: ABCs exhibited a hypermetabolic state with enhanced glycolytic capacity. The increased glycolytic rate in ABCs was promoted by interferon-γ (IFNγ) signaling. T-bet, a downstream transcription factor of IFNγ, regulated the gene program of the glycolysis pathway in ABCs by repressing the expression of Bcl6. Functionally, glycolysis restriction could impair ABC formation. The engagement of glycolysis promoted survival and terminal differentiation of antibody-secreting cells. Administration of a glycolysis inhibitor ameliorated ABC accumulation and autoantibody production in the lupus-induced bm12 mouse model. CONCLUSION: T-bet can couple immune signals and metabolic programming to establish pathogenic ABC formation and functional capacities. Modulation of ABCs favored a metabolic program that could be a novel therapeutic approach for lupus.

Pla2g2a promotes innate Th2-type immunity lymphocytes to increase B1a cells.

Newborns require early generation of effective innate immunity as a primary physiological mechanism for survival. The neonatal Lin28+Let7- developmental pathway allows increased generation of Th2-type cells and B1a (B-1 B) cells compared to adult cells and long-term maintenance of these initially generated innate cells. For initial B1a cell growth from the neonatal to adult stage, Th2-type IL-5 production from ILC2s and NKT2 cells is important to increase B1a cells. The Th17 increase is dependent on extracellular bacteria, and increased bacteria leads to lower Th2-type generation. Secreted group IIA-phospholipase A2 (sPLA2-IIA) from the Pla2g2a gene can bind to gram-positive bacteria and degrade bacterial membranes, controlling microbiota in the intestine. BALB/c mice are Pla2g2a+, and express high numbers of Th2-type cells and B1a cells. C57BL/6 mice are Pla2g2a-deficient and distinct from the SLAM family, and exhibit fewer NKT2 cells and fewer B1a cells from the neonatal to adult stage. We found that loss of Pla2g2a in the BALB/c background decreased IL-5 from Th2-type ILC2s and NKT2s but increased bacterial-reactive NKT17 cells and MAIT cells, and decreased the number of early-generated B1a cells and MZ B cells and the CD4/CD8 T cell ratio. Low IL-5 by decreased Th2-type cells in Pla2g2a loss led to low early-generated B1a cell growth from the neonatal to adult stage. In anti-thymocyte/Thy-1 autoreactive µκ transgenic (ATAµκ Tg) Pla2g2a+ BALB/c background C.B17 mice generated NKT2 cells that continuously control CD1d+ B1 B cells through old aging and lost CD1d in B1 B cells generating strong B1 ATA B cell leukemia/lymphoma. Pla2g2a-deficient ATAµκTg C57BL/6 mice suppressed the initial B1a cell increase, with low/negative spontaneous leukemia/lymphoma generation. These data confirmed that the presence of Pla2g2a to control bacteria is important to allow the neonatal to adult stage. Pla2g2a promotes innate Th2-type immunity lymphocytes to increase early generated B1a cells.