Tineke Cantaert

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.

Anti-TNF treatment blocks the induction of T cell-dependent humoral responses

Objective Experimental and human data suggest that tumour necrosis factor (TNF) blockade may affect B cell responses, in particular the induction of T cell-dependent (TD) humoral immunity. This study aimed to assess this hypothesis directly in patients with arthritis by analysing longitudinally the effect of TNF blockade on B cell activation and the maturation of humoral responses against TD and T cell-independent vaccines. Materials and methods Peripheral blood samples were obtained from 56 spondyloarthritis patients before and after treatment with either non-steroidal anti-inflammatory drug (NSAID) alone or TNF blockers and analysed for B cell activation, plasma cell differentiation, germinal centre versus extra-follicular B cell maturation, and somatic hypermutation. Vaccine responses to hepatitis B and Streptococcus pneumoniae were measured by ELISA. Results TNF blockade augmented B cell activation as reflected by the expression of early activation markers, CD40, and costimulatory molecules, without affecting differentiation towards plasmablasts. This was associated with a specific increase of the unswitched fraction of circulating memory B cells and a decreased level of somatic hypermutation in anti-TNF treated patients, indicating an impairment of the germinal centre-dependent B cell maturation. In agreement with these findings, TNF blockade profoundly suppressed the response to the TD vaccination against hepatitis B, whereas the T cell-independent response against pneumococcal polysaccharides was only modestly affected. Conclusions These data indicate that TNF blockade severely impedes the induction of primary TD humoral responses, probably by interfering with the germinal centre reaction.

Disease-specific and inflammation-independent stromal alterations in spondylarthritis synovitis

OBJECTIVEnThe molecular processes driving the distinct patterns of synovial inflammation and tissue remodeling in spondylarthritis (SpA) as compared to rheumatoid arthritis (RA) remain largely unknown. Therefore, we aimed to identify novel and unsuspected disease-specific pathways in SpA by a systematic and unbiased synovial gene expression analysis.nnnMETHODSnDifferentially expressed genes were identified by pan-genomic microarray and confirmed by quantitative polymerase chain reaction and immunohistochemical analyses of synovial tissue biopsy samples from patients with SpA (n=63), RA (n=28), and gout (n=9). The effect of inflammation on gene expression was assessed by stimulating fibroblast-like synoviocytes (FLS) with synovial fluid and by analysis of synovial tissue samples at weeks 0 and 12 of etanercept treatment.nnnRESULTSnUsing very stringent statistical thresholds, microarray analysis identified 64 up-regulated transcripts in patients with SpA synovitis as compared to those with RA synovitis. Pathway analysis revealed a robust myogene signature in this gene set. The myogene signature was technically and biologically reproducible, was specific for SpA, and was independent of disease duration, treatment, and SpA subtype (nonpsoriatic versus psoriatic). Synovial tissue staining identified the myogene expressing cells as vimentin-positive, prolyl 4-hydroxylase β-positive, CD90+, and CD146+ mesenchymal cells that were significantly overrepresented in the intimal lining layer and synovial sublining of inflamed SpA synovium. Neither in vitro exposure to synovial fluid from inflamed SpA joints nor in vivo blockade of tumor necrosis factor modulated the SpA-specific myogene signature.nnnCONCLUSIONnThese data identify a novel and disease-specific myogene signature in SpA synovitis. The fact that this stromal alteration appeared not to be downstream of local inflammation warrants further analysis of its functional role in the pathogenesis of the disease.

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 β 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.

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

BACKGROUNDnSignaling 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.nnnOBJECTIVEnWe 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.nnnMETHODSnWe 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnSAP expression is required for the counterselection of developing autoreactive B cells and preventsxa0their T cell-dependent accumulation in the periphery.

Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance

Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

Potential roles of activation-induced cytidine deaminase in promotion or prevention of autoimmunity in humans

Autoimmune manifestations are paradoxical and frequent complications of primary immunodeficiencies, including T and/or B cell defects. Among pure B cell defects, the Activation-induced cytidine Deaminase (AID)-deficiency, characterized by a complete lack of immunoglobulin class switch recombination and somatic hypermutation, is especially complicated by autoimmune disorders. We summarized in this review the different autoimmune and inflammatory manifestations present in 13 patients out of a cohort of 45 patients. Moreover, we also review the impact of AID mutations on B-cell tolerance and discuss hypotheses that may explain why central and peripheral B-cell tolerance was abnormal in the absence of functional AID. Hence, AID plays an essential role in controlling autoreactive B cells in humans and prevents the development of autoimmune syndromes.

Presence and Role of Anti–Citrullinated Protein Antibodies in Experimental Arthritis Models

OBJECTIVEnAnti-citrullinated protein antibodies (ACPAs) are the serologic hallmark of rheumatoid arthritis. Functional studies on the role of ACPAs in experimental arthritis have yielded conflicting results, and therefore the present study was undertaken to assess systematically whether citrullinated proteins can really induce ACPAs and modulate arthritis in mice.nnnMETHODSnBalb/c, SJL, and DBA/1 mice were immunized with either native or citrullinated fibrinogen, myelin basic protein (MBP), and type II collagen (CII). ACPAs were detected with a peptide-based enzyme-linked immunosorbent assay (ELISA) and with Western blotting using fibrinogen as substrate. Arthritis was induced in mice by immunization with CII in Freunds complete adjuvant or by injection of anticollagen antibodies.nnnRESULTSnAnalysis of the sera of mice immunized with citrullinated proteins revealed false-positive results with the citrulline peptide-based ELISA. In contrast, Western blot analysis using either citrullinated or native fibrinogen as substrate reliably detected ACPAs in Balb/c mice immunized with citrullinated fibrinogen, MBP, and CII. However, these ACPAs failed to induce or aggravate disease in Balb/c mice in the anticollagen antibody-induced arthritis model. Immunization with citrullinated fibrinogen induced ACPAs but did not lead to arthritis development in SJL and DBA/1 mice. In contrast, immunization with citrullinated CII failed to induce ACPAs or enhance disease in these strains in the collagen-induced arthritis model.nnnCONCLUSIONnMice can develop genuine ACPAs, but detection of ACPAs is highly dependent on strain, immunogen, immunization protocol, and detection assay. Murine ACPAs are not overtly pathogenic, since neither preexisting ACPAs nor the use of citrullinated collagen as immunogen modulates the clinical course of arthritis.

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.