The protein disulfide isomerase A3 and osteopontin axis promotes influenza-induced lung remodelling.

BACKGROUND AND PURPOSE: Fibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral-fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza-induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral-mediated fibrotic remodelling. EXPERIMENTAL APPROACH: A retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)-infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony-stimulating factor (M-CSF) were used as a cell culture model. KEY RESULTS: The TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu-infected acute respiratory distress syndrome (ARDS) patients compared with non-ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza-infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1. CONCLUSION AND IMPLICATIONS: The PDIA3-SPP1 axis promotes post-influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus-induced lung fibrotic sequela.

Mitoquinone mesylate attenuates pathological features of lean and obese allergic asthma in mice.

Obesity is associated with severe, difficult-to-control asthma, and increased airway oxidative stress. Mitochondrial reactive oxygen species (mROS) are an important source of oxidative stress in asthma, leading us to hypothesize that targeting mROS in obese allergic asthma might be an effective treatment. Using a mouse model of house dust mite (HDM)-induced allergic airway disease in mice fed a low- (LFD) or high-fat diet (HFD), and the mitochondrial antioxidant MitoQuinone (MitoQ), we investigated the effects of obesity and ROS on HDM-induced airway inflammation, remodeling, and airway hyperresponsiveness (AHR). Obese allergic mice showed increased lung tissue eotaxin, airway tissue eosinophilia, and AHR compared with lean allergic mice. MitoQ reduced airway inflammation, remodeling, and hyperreactivity in both lean and obese allergic mice, and tissue eosinophilia in obese-allergic mice. Similar effects were observed with decyl triphosphonium (dTPP+), the hydrophobic cationic moiety of MitoQ lacking ubiquinone. HDM-induced oxidative sulfenylation of proteins was increased particularly in HFD mice. Although only MitoQ reduced sulfenylation of proteins involved in protein folding in the endoplasmic reticulum (ER), ER stress was attenuated by both MitoQ and dTPP+ suggesting the anti-allergic effects of MitoQ are mediated in part by effects of its hydrophobic dTPP+ moiety reducing ER stress. In summary, oxidative signaling is an important mediator of allergic airway disease. MitoQ, likely through reducing protein oxidation and affecting the UPR pathway, might be effective for the treatment of asthma and specific features of obese asthma.

Protein Disulfide Isomerase A3 Regulates Influenza Neuraminidase Activity and Influenza Burden in the Lung.

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3-/- in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis.

BACKGROUND: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated. OBJECTIVES: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis. METHODS: Role of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice. RESULTS: PDIA3 and club cell secretory protein (SCGB1A1) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3, specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice. CONCLUSIONS: Our study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis.

DRP1-Mediated Mitochondrial Fission Regulates Lung Epithelial Response to Allergen.

Mitochondria regulate a myriad of cellular functions. Dysregulation of mitochondrial control within airway epithelial cells has been implicated in the pro-inflammatory response to allergens in asthma patients. Because of their multifaceted nature, mitochondrial structure must be tightly regulated through fission and fusion. Dynamin Related Protein 1 (DRP1) is a key driver of mitochondrial fission. During allergic asthma, airway epithelial mitochondria appear smaller and structurally altered. The role of DRP1-mediated mitochondrial fission, however, has not been fully elucidated in epithelial response to allergens. We used a Human Bronchial Epithelial Cell line (HBECs), primary Mouse Tracheal Epithelial Cells (MTECs), and conditional DRP1 ablation in lung epithelial cells to investigate the impact of mitochondrial fission on the pro-inflammatory response to house dust mite (HDM) in vitro and in vivo. Our data suggest that, following HDM challenge, mitochondrial fission is rapidly upregulated in airway epithelial cells and precedes production of pro-inflammatory cytokines and chemokines. Further, deletion of Drp1 in lung epithelial cells leads to decreased fission and enhanced pro-inflammatory signaling in response to HDM in vitro, as well as enhanced airway hyper-responsiveness (AHR), inflammation, differential mucin transcription, and epithelial cell death in vivo. Mitochondrial fission, therefore, regulates the lung epithelial pro-inflammatory response to HDM.

Pathological consequences of the unfolded protein response and downstream protein disulphide isomerases in pulmonary viral infection and disease.

Protein folding within the endoplasmic reticulum (ER) exists in a delicate balance; perturbations of this balance can overload the folding capacity of the ER and disruptions of ER homoeostasis is implicated in numerous diseases. The unfolded protein response (UPR), a complex adaptive stress response, attempts to restore normal proteostasis, in part, through the up-regulation of various foldases and chaperone proteins including redox-active protein disulphide isomerases (PDIs). There are currently over 20 members of the PDI family each consisting of varying numbers of thioredoxin-like domains which, generally, assist in oxidative folding and disulphide bond rearrangement of peptides. While there is a large amount of redundancy in client proteins of the various PDIs, the size of the family would indicate more nuanced roles for the individual PDIs. However, the role of individual PDIs in disease pathogenesis remains uncertain. The following review briefly discusses recent findings of ER stress, the UPR and the role of individual PDIs in various respiratory disease states.

Conjugated bile acids attenuate allergen-induced airway inflammation and hyperresponsiveness by inhibiting UPR transducers.

Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite-induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPR-associated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl ß-muricholic acid (AßM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AßM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs.

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.

Self-reactive VH4-34-expressing IgG B cells recognize commensal bacteria.

The germline immunoglobulin (Ig) variable heavy chain 4-34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34-expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34-expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34-encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34-encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

Decreased somatic hypermutation induces an impaired peripheral B cell tolerance checkpoint.

Patients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation of autoreactive B cells in blood is unclear. Here, we analyzed B cell tolerance in AID-deficient patients, patients with autosomal dominant AID mutations (AD-AID), asymptomatic AICDA heterozygotes (AID+/-), and patients with uracil N-glycosylase (UNG) deficiency, which impairs CSR but not SHM. The low frequency of autoreactive mature naive B cells in UNG-deficient patients resembled that of healthy subjects, revealing that impaired CSR does not interfere with the peripheral B cell tolerance checkpoint. In contrast, we observed decreased frequencies of SHM in memory B cells from AD-AID patients and AID+/- subjects, who were unable to prevent the accumulation of autoreactive mature naive B cells. In addition, the individuals with AICDA mutations, but not UNG-deficient patients, displayed Tregs with defective suppressive capacity that correlated with increases in circulating T follicular helper cells and enhanced cytokine production. We conclude that SHM, but not CSR, regulates peripheral B cell tolerance through the production of mutated antibodies that clear antigens and prevent sustained interleukin secretions that interfere with Treg function.

Effect of a chemical chaperone, tauroursodeoxycholic acid, on HDM-induced allergic airway disease.

Endoplasmic reticulum (ER) stress-induced unfolded protein response plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM rechallenge (rechallenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM rechallenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases.

Rituximab does not reset defective early B cell tolerance checkpoints.

Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve ß cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti-B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti-B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti-B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti-B cell therapy.

Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.

TNF receptor superfamily member 13b (TNFRSF13B) hemizygosity reveals transmembrane activator and CAML interactor haploinsufficiency at later stages of B-cell development.

BACKGROUND: Heterozygous C104R or A181E TNF receptor superfamily member 13b (TNFRSF13B) mutations impair removal of autoreactive B cells, weaken B-cell activation, and convey to patients with common variable immune deficiency (CVID) an increased risk for autoimmunity. How mutant transmembrane activator and CAML interactor (TACI) influences wild-type TACI function is unclear; different models suggest either a dominant negative effect or haploinsufficiency. OBJECTIVE: We investigated potential TACI haploinsufficiency by analyzing patients with antibody-deficient Smith-Magenis syndrome (SMS) who possess only 1 TNFRSF13B allele and antibody-deficient patients carrying one c.204insA TNFRSF13B null mutation. METHODS: We tested the reactivity of antibodies isolated from single B cells from patients with SMS and patients with a c.204insA TNFRSF13B mutation and compared them with counterparts from patients with CVID with heterozygous C104R or A181E TNFRSF13B missense mutations. We also assessed whether loss of a TNFRSF13B allele induced haploinsufficiency in naive and memory B cells and recapitulated abnormal immunologic features typical of patients with CVID with heterozygous TNFRSF13B missense mutations. RESULTS: We found that loss of a TNFRSF13B allele does not affect TACI expression, activation responses, or establishment of central B-cell tolerance in naive B cells. Additionally, patients with SMS and those with a c.204insA TNFRSF13B mutation display normal regulatory T-cell function and peripheral B-cell tolerance. The lack of a TNFRSF13B allele did result in decreased TACI expression on memory B cells, resulting in impaired activation and antibody secretion. CONCLUSION: TNFRSF13B hemizygosity does not recapitulate autoimmune features of CVID-associated C104R and A181E TNFRSF13B mutations, which likely encode dominant negative products, but instead reveals selective TACI haploinsufficiency at later stages of B-cell development.

Wiskott-Aldrich Syndrome protein deficiency perturbs the homeostasis of B-cell compartment in humans.

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.

Signaling lymphocytic activation molecule (SLAM)/SLAM-associated protein pathway regulates human B-cell tolerance.

BACKGROUND: Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) can mediate the function of SLAM molecules, which have been proposed to be involved in the development of autoimmunity in mice. OBJECTIVE: We sought to determine whether the SLAM/SAP pathway regulates the establishment of human B-cell tolerance and what mechanisms of B-cell tolerance could be affected by SAP deficiency. METHODS: We tested the reactivity of antibodies isolated from single B cells from SAP-deficient patients with X-linked lymphoproliferative disease (XLP). The expressions of SAP and SLAM family members were assessed in human bone marrow-developing B cells. We also analyzed regulatory T (Treg) cell function in patients with XLP and healthy control subjects. RESULTS: We found that new emigrant/transitional B cells from patients with XLP were enriched in autoreactive clones, revealing a defective central B-cell tolerance checkpoint in the absence of functional SAP. In agreement with a B cell-intrinsic regulation of central tolerance, we identified SAP expression in a discrete subset of bone marrow immature B cells. SAP colocalized with SLAMF6 only in association with clustered B-cell receptors likely recognizing self-antigens, suggesting that SLAM/SAP regulate B-cell receptor-mediated central tolerance. In addition, patients with XLP displayed defective peripheral B-cell tolerance, which is normally controlled by Treg cells. Treg cells in patients with XLP seem functional, but SAP-deficient T cells were resistant to Treg cell-mediated suppression. Indeed, SAP-deficient T cells were hyperresponsive to T-cell receptor stimulation, which resulted in increased secretion of IL-2, IFN-γ, and TNF-α. CONCLUSIONS: SAP expression is required for the counterselection of developing autoreactive B cells and prevents their T cell-dependent accumulation in the periphery.

CVID-associated TACI mutations affect autoreactive B cell selection and activation.

Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6-expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications.

Specific peripheral B cell tolerance defects in patients with multiple sclerosis.

Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. B cells have recently emerged as major contributors to disease pathogenesis, but the mechanisms responsible for the loss of B cell tolerance in patients with MS are largely unknown. In healthy individuals, developing autoreactive B cells are removed from the repertoire at 2 tolerance checkpoints during early B cell development. Both of these central and peripheral B cell tolerance checkpoints are defective in patients with rheumatoid arthritis (RA) and type 1 diabetes (T1D). Here, we found that only the peripheral, but not the central, B cell tolerance checkpoint is defective in patients with MS. We show that this specific defect is accompanied by increased activation and homeostatic proliferation of mature naive B cells. Interestingly, all of these MS features parallel defects observed in FOXP3-deficient IPEX patients, who harbor nonfunctional Tregs. We demonstrate that in contrast to patients with RA or T1D, bone marrow central B cell selection in MS appears normal in most patients. In contrast, patients with MS suffer from a specific peripheral B cell tolerance defect that is potentially attributable to impaired Treg function and that leads to the accumulation of autoreactive B cell clones in their blood.

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells.

Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.