Autoantigen In Patients Research Articles

6489 Type 1 Diabetes Mellitus And Multiple Sclerosis: A Case Report Showing The Concurrent Association Of Two Autoimmune Diseases

Abstract Disclosure: P.B. Singh: None. B. Sabates: None. A. Sharifzadeh: None. R.F. Rodriguez: None. Introduction: Both Type 1 Diabetes Mellitus (T1DM) and Multiple Sclerosis (MS) represent persistent autoimmune conditions. T1DM is characterized by the presence of specific autoantibodies targeting pancreatic β-cells. In MS, autoreactive cells mediate a demyelinating inflammatory response directed against the central nervous system. Despite differences in demographics and clinical characteristics, their common root in autoimmunity has led researchers to discover commonalities in genetic, environmental and immunological features. Case Presentation We present a case of a 40-year-old female with medical history of T1DM diagnosed at 7 years of age and MS diagnosed at 32 years of age. She presented to the hospital with clinical features of optic neuritis and symptoms of squeezing ribcage pressure consistent with acute MS exacerbation. After confirmatory neuroimaging, high-dose intravenous corticosteroids were initiated, and Endocrinology services were consulted for inpatient glycemic management. As the patient’s diabetes was treated via continuous subcutaneous insulin infusion (CSII), her pump settings were adjusted to maintain euglycemia during hospitalization. This case is reported to highlight and discuss the association between T1DM and MS. Discussion About 4-5% of the general population is affected by autoimmune disorders, among which T1DM and MS comprise a substantial portion. Multiple studies demonstrate the association and shared etiopathogenesis between T1DM and MS. One study revealed that the prevalence of T1DM is five times higher in patients with MS in comparison to the general population. In another prospective cohort study, individuals with T1DM had a three times higher chance of subsequently developing MS. Additionally, first-degree relatives of MS patients had a roughly 40% higher risk of developing T1DM. Women with T1DM had a 20-fold higher risk of having MS than women without the condition, per another study. As T-cell mediated autoimmune disorders, both T1DM and MS are characterized by autoantigen specific T1-helper cell responses, decreased T-cell suppressor activity, and the presence of various autoantibodies, some of which are seen in both diseases. One study showed that autoreactive T cells could target both pancreatic islet and central nervous system autoantigens in patients with MS, patients with T1DM, and relatives of patients with T1DM. Since MS- and T1DM-associated HLA haplotypes differ between populations, HLA class II alleles per se may not alone be responsible for disease susceptibility. It is suggested that variation at other non-HLA class II loci and/or unknown environmental factors might contribute significantly to the co-occurrence of these 2 diseases. In conclusion, individuals who already have one autoimmune condition are more likely to have other autoimmune conditions, with T1DM and MS being associated more often than expected. Presentation: 6/2/2024

The gut microbiota posttranslationally modifies IgA1 in autoimmune glomerulonephritis.

Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila. IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1KI-CD89tg) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.

ADIPOPHILIN PEPTIDE (ADPH 129-137) IS NOT A TARGET ANTIGEN FOR CD8+ T-CELLS IN PATIENTS WITH OBESITY.

In obesity, the infiltration of leukocytes into adipose tissue seems to play a key role in the development of inflammation and insulin resistance. Over-expression of adipophilin (ADPH) in adipose tissue, a protein which regulates lipid droplet structure and formation, has been reported in some studies. To investigate the role of ADPH 129-137 as a target for CD8+ T-cells in PBMCs of patients with obesity. PBMCs were obtained from 9 non-diabetic obese patients and 11 healthy subjects expressing the HLA-A0201 molecule. The ELISPOT assay used to monitor the presence of IFN-γ producing CD8+ T-cells against a HLA class I-restricted epitope derived from Adipophilin (ADPH 129-137) and two control peptides: Flu MP58-66 and Melan-A27-35. The outcomes showed no significant difference between patient group and healthy donors in response to ADPH 129-137. These results demonstrated that ADPH 129-137 peptide possibly does not act as an autoantigen in patients with obesity.

Anti-citrullinated α-enolase peptide as a highly sensitive autoantigen in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease in Taiwan. Anti-cyclic citrullinated peptide (anti-CCP) assay is widely used for RA diagnosis; however, not all anti-CCPs are detectable in RA-joint lesions. Citrullinated α-enolase peptide (CEP), which has a unique immunodominant epitope, can be detected in synovial fluid. Here, we aimed to evaluate the potential of anti-CEP as a serologic marker for the early diagnosis of RA and a prognostic predictor of joint destruction. We also determined the association of single-nucleotide polymorphisms (SNPs) in genes with the serological status and clinical characteristics of RA. Clinical records of 30 patients with RA were collected, and their serum and DNA samples were evaluated using enzyme-linked immunosorbent assay (ELISA) and SNP cross-reaction analysis. A considerable amount of anti-CEP was detected in patients with RA, a trend similar to that of anti-CCP. Moreover, anti-CEP was considerably associated with the protein-arginine deiminase type-2 SNP rs1005753.

An update on malignant tumor-related stiff person syndrome spectrum disorders: clinical mechanism, treatment, and outcomes.

Stiff person syndrome (SPS) is a rare central nervous system disorder associated with malignancies. In this review, we retrieved information from PubMed, up until August 2023, using various search terms and their combinations, including SPS, stiff person syndrome spectrum disorders (SPSSDs), paraneoplastic, cancer, and malignant tumor. Data from peer-reviewed journals printed in English were organized to explain the possible relationships between different carcinomas and SPSSD subtypes, as well as related autoantigens. From literature searching, it was revealed that breast cancer was the most prevalent carcinoma linked to SPSSDs, followed by lung cancer and lymphoma. Furthermore, classic SPS was the most common SPSSD subtype, followed by stiff limb syndrome and progressive encephalomyelitis with rigidity and myoclonus. GAD65 was the most common autoantigen in patients with cancer and SPSSDs, followed by amphiphysin and GlyR. Patients with cancer subtypes might have multiple SPSSD subtypes, and conversely, patients with SPSSD subtypes might have multiple carcinoma subtypes. The first aim of this review was to highlight the complex nature of the relationships among cancers, autoantigens, and SPSSDs as new information in this field continues to be generated globally. The adoption of an open-minded approach to updating information on new cancer subtypes, autoantigens, and SPSSDs is recommended to renew our database. The second aim of this review was to discuss SPS animal models, which will help us to understand the mechanisms underlying the pathogenesis of SPS. In future, elucidating the relationship among cancers, autoantigens, and SPSSDs is critical for the early prediction of cancer and discovery of new therapeutic modalities.

Extracellular vesicles opsonized by monomeric C-reactive protein (CRP) are accessible as autoantigens in patients with systemic lupus erythematosus and associate with autoantibodies against CRP

The pentraxin C-reactive protein (CRP) is a pentameric protein now known to be able to undergo dissociation into a monomeric, modified isoform, referred to as mCRP. In carefully assessing the bioactivities of each isoform, mCRP has strong pro-inflammatory activities while pCRP has mild anti-inflammatory activities. Systemic lupus erythematosus (SLE) is a disease characterized by a vast number of autoantibodies, including anti-CRP autoantibodies which have been associated with SLE disease activity and lupus nephritis. The origin of these autoantibodies is currently unknown. Extracellular vesicles (EVs) have been implicated in SLE pathogenesis as they can expose nuclear antigens on their outside surface, thereby being a potential adjuvant for the generation of autoantibodies. Herein, we studied exposure of both pCRP and mCRP on EVs in SLE plasma and the implications of each in disease activity, organ damage and clinical manifestations. We used flow cytometry to detect CRP isoforms on EV surfaces in 67 well-characterized SLE patients and 60 sex- and age-matched healthy controls. Autoantibodies against mCRP were measured using ELISA. We found an abundance of both pCRP and mCRP on SLE EVs compared to controls. Furthermore, mCRP+ but not pCRP+ EVs were elevated in patients with active disease and in anti-CRP positive patients. The proportions of mCRP+ EVs were lower in patients with acquired organ damage, especially in patients with lupus nephritis (LN), and displayed an inverse relationship with disease duration in LN and patients with active disease. Speculatively, these data suggest EV-bound mCRP as a relevant factor in SLE pathogenesis, which could contribute to development of anti-CRP autoantibodies by stimulating an immune response.

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis

PurposeIn up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity. MethodsUnlabelled protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) was performed with sera of 106 patients with SSc from two tertiary referral centres that had a nuclear pattern on HEp-2 indirect immunofluorescence without previously identified autoantibody. Telomere- or telomerase-associated proteins or protein complexes precipitated by individual sera were identified. Candidate autoantigens were confirmed through immunoprecipitation-western blot (IP-WB). A custom Luminex xMAP assay for 5 proteins was evaluated with sera from persons with SSc (n = 467), other systemic autoimmune rheumatic diseases (n = 923), non-rheumatic disease controls (n = 187) and healthy controls (n = 199). ResultsEight telomere- and telomerase-associated autoantigens were identified in a total of 11 index patients, including the THO complex (n = 3, all with interstitial lung disease and two with cardiac involvement), telomeric repeat-binding factor 2 (TERF2, n = 1), homeobox-containing protein 1 (HMBOX1, n = 2), regulator of chromosome condensation 1 (RCC1, n = 1), nucleolar and coiled-body phosphoprotein 1 (NOLC1, n = 1), dyskerin (DKC1, n = 1), probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2, n = 1) and nuclear valosin-containing protein-like (NVL, n = 2). A Luminex xMAP assay for THO complex subunit 1 (THOC1), TERF2, NOLC1, NOP2 and NVL revealed high reactivity in all index patients, but also in other patients with SSc and disease controls. However, the reactivity by xMAP assay in these other patients was not confirmed by IP-WB. ConclusionIP-MS revealed key telomere- and telomerase-associated proteins and protein complexes as autoantigens in patients with SSc.

Associations of autoimmune bullous diseases and autoantibodies against epidermal autoantigens in patients with inflammatory myopathy.

Associations of autoimmune bullous diseases and autoantibodies against epidermal autoantigens in patients with inflammatory myopathy.

Strategies Towards Antigen-Specific Treatments for Membranous Nephropathy.

Membranous nephropathy (MN) is a rare but potentially severe autoimmune disease and a major cause of nephrotic syndrome in adults. Traditional treatments for patients with MN include steroids with alkylating agents such as cyclophosphamide or calcineurin inhibitors such as cyclosporine, which have an undesirable side effect profile. Newer therapies like rituximab, although superior to cyclosporine in maintaining disease remission, do not only affect pathogenic B or plasma cells, but also inhibit the production of protective antibodies and therefore the ability to fend off foreign organisms and to respond to vaccination. These are undesired effects of general B or plasma cell-targeted treatments. The discovery of several autoantigens in patients with MN offers the great opportunity for more specific treatment approaches. Indeed, such treatments were recently developed for other autoimmune diseases and tested in different preclinical models, and some are about to jump to clinical practice. As such treatments have enormous potential to enhance specificity, efficacy and compatibility also for MN, we will discuss two promising strategies in this perspective: The elimination of pathogenic antibodies through endogenous degradation systems and the depletion of pathogenic B cells through chimeric autoantibody receptor T cells.

The Bacterial Ro60 Protein and Its Noncoding Y RNA Regulators.

Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. In D. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.

Screening for autoantibody targets in post-vaccination narcolepsy using proteome arrays.

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n=39), their first-degree relatives (FDR) (n=66), population controls (n=188), and disease controls representing multiple sclerosis (n=100) and FDR to type 1 diabetes patients (n=41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1.

High Prevalence and Disease Correlation of Autoantibodies Against p40 Encoded by Long Interspersed Nuclear Elements in Systemic Lupus Erythematosus.

Long interspersed nuclear element 1 (LINE-1) encodes 2 proteins, the RNA binding protein p40 and endonuclease and reverse transcriptase (open-reading frame 2p [ORF2p]), which are both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with LINE-1 RNA and additional RNA binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether SLE patients also produce autoantibodies against LINE-1 p40. Highly purified p40 protein was used to quantitate IgG autoantibodies in serum from 172 SLE patients and from disease controls and age-matched healthy subjects by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. Antibodies reactive with p40 were detected in the majority of patients and many healthy controls. Their levels were higher in patients with SLE, but not those with systemic sclerosis, compared to healthy subjects (P = 0.01). Anti-p40 reactivity was higher in patients during a flare than in patients with disease in remission (P = 0.03); correlated with the SLE Disease Activity Index score (P = 0.0002), type I interferon score (P = 0.006), decrease in complement C3 level (P = 0.0001), the presence of anti-DNA antibodies (P < 0.0001) and anti-C1q antibodies (P = 0.004), and current or past history of nephritis (P = 0.02 and P = 0.003, respectively); and correlated inversely with age (r = -0.49, P < 0.0001). SLE patient sera also reacted with p40-associated proteins. Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step toward SLE pathogenesis.

A novel mouse model of phospholipase A2 receptor 1-associated membranous nephropathy mimics podocyte injury in patients

The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments invivo.

Antigenic Function of 14-3-3ζ is a Novel Tool to Treat Rheumatoid Arthritis

Abstract Autoantigens are an integral part of autoimmunity, but their role in health and disease are mostly unknown. We identified 14-3-3ζ as an autoantigen in patients with thoracic aortic aneurysm caused by large vessel vasculitis. Like others, neither the cause nor the consequence of newly-identified antigenic function of 14-3-3ζ protein is known. To address this, we investigated the antigenicity of 14-3-3ζ by studying ex vivo effects on human peripheral blood mononuclear cells (PBMC) polarization, and in vivo using a rat autoimmune disease model. Exogenous 14-3-3ζ promoted PBMC proliferation and T cell polarization towards increasing Th1 and Th17 populations. A specific increase in Th1 cells and IFN-γ production provided evidence for MHC class II presentation of 14-3-3ζ antigen. These 14-3-3ζ-induced responses of T cell polarization and cytokine secretion were inhibited by immunosuppressive agents. Our in-depth studies further revealed that 14-3-3ζ-induced phosphorylation of MAPK led to NF-kB activation in macrophages and PBMC. In vivo administration of recombinant 14-3-3ζ in rats triggered strong innate and adaptive immune responses with significant increases in IL-17, IFN-γ, and anti-14-3-3ζ antibody. Importantly, the immunization of Lewis rats with 14-3-3ζ protected them from rheumatoid arthritis in Pristane-Induced-Arthritis (PIA) model. These collectively demonstrate that the antigenic 14-3-3ζ by acting as a DAMP promotes IFN-γ and IL-17 levels, and confers an anti-arthritic effect. To the best of our knowledge, this is the first report describing the antigenic function of 14-3-3ζ and its physiologically relevant anti-arthritic role.

Novel minor HLA DR associated antigens in type 1 diabetes

Type 1 diabetes is an autoimmune disease leading to insulin deficiency. Autoantibodies to beta cell proteins are already present in the asymptomatic phase of type 1 diabetes. Recent findings have suggested a number of additional minor autoantigens in patients with type 1 diabetes. We have established luciferase immunoprecipitation systems (LIPS) for anti-MTIF3, anti-PPIL2, anti-NUP50 and anti-MLH1 and analyzed samples from 500 patients with type 1 diabetes at onset of clinical disease and 200 healthy individuals who had a family history of type 1 diabetes but no evidence of beta cell autoantibodies. We show significantly higher frequencies of anti-MTIF3, anti-PPIL2 and anti-MLH1 in recent onset type 1 diabetes patients in comparison to controls. In addition, antibodies to NUP50 were associated with HLA-DRB1*03 and antibodies to MLH1 were associated with HLA-DRB1*04 genotypes.

Identification of Moesin as a Novel Autoantigen in Patients with Sjögren's Syndrome.

SjOgren's Syndrome (SS) is a systemic and chronic autoimmune disorder that affects the exocrine glands with massive autoantibody production. Although the pathogenesis of the disorder is incompletely understood, but some studies have reported that anti-moesin antibodies have been detected in autoimmune diseases with which SS is closely associated. Here, we have investigated moesin's potential involvement in SS. This study aims to verify whether moesin is a specific autoantigen involved in Chinese Hans SS patients. First, recombinant human moesin was expressed and purified. Next, the protein was verified as antigen by Western blotting and immunoprecipitation. The positive protein band in the immunoprecipitation was identified by (MALDI-TOF/TOF). Finally, an optimized ELISA (Enzyme- Linked Immunosorbent Assay) kit was developed to measure the titer concentration of anti-moesin antibody-positive patients in a large cohort of clinical subjects. Univariate analysis revealed that the proportion of individuals positive for serum IgG against recombinant human moesin was 42 % in a group of Chinese Hans SS patients (21 of 50), 22 % in systemic lupus erythematosus patients and (11 of 50), compared to only 4 % in healthy controls (2 of 50). An association between anti-moesin antibodies and SS manifestation have been found which may be considered a suspected serum biomarker for the development of SS.

Identification of hnRNP C1/C2 as an Autoantigen in Patients with Behcet's Disease.

Ribonucleoproteins particles that form the spliceosomes are among the most frequently targeted molecules of the autoimmune response. In the last few years, autoantibodies against all A/B hnRNP proteins have been found in the sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and serve as diagnostic markers for several rheumatic diseases. However, the functional role of hnRNP C1/C2 in autoimmune diseases is still not clearly understood. To identify hnRNP C1/C2 as an autoantigen in patients with Behcet's Disease (BD). First, HaCaT and EA.hy926 cells were cultured and RNA was extracted. Second, amplification of the corresponding gene by RT-PCR, cloning, and purification techniques was applied to acquire the recombinant protein hnRNP C1/C2. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by (MALDI-TOF/). Finally, Western blotting and ELISA were performed to verify the immunoreactivity of BD serum with recombinant hnRNPC1/C2. Results demonstrated that the reactivity of BD serum against recombinant hnRNP C1/C2 protein was significantly higher as compared to healthy control (P<0.001). hnRNP C1/C2 can be considered as a self antigen which might be involved in BD pathology in Hans Chinese population.

Identification of galectin-3 as an autoantigen in patients with IgG4-related disease

The antigenic trigger that drives expansion of circulating plasmablasts and CD4+ cytotoxic T cells in patients with IgG4-related disease (IgG4-RD) is presently unknown. We sought to sequence immunoglobulin genes from single-cell clones of dominantly expanded plasmablasts and generate recombinant human mAbs to identify relevant antigens in patients with IgG4-RD by using mass spectrometry. Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as mAbs and used to purify antigens by using immunoaffinity chromatography. Affinity-purified antigens were identified by using mass spectrometry and validated by means of ELISA. Plasma levels of the antigen of interest were also determined by using ELISA. mAbs expressed from the 2 dominant plasmablast clones of a patient with multiorgan IgG4-RD stained human pancreatic tissue sections. Galectin-3 was identified as the antigen specifically recognized by both mAbs. Anti-galectin-3 autoantibody responses were predominantly of the IgG4 isotype (28% of the IgG4-RD cohort, P=.0001) and IgE isotype (11% of the IgG4-RD cohort, P=.009). No significant responses were seen from the IgG1, IgG2, or IgG3 isotypes. IgG4 anti-galectin-3 autoantibodies correlated with increased plasma galectin-3 levels (P=.001), lymphadenopathy (P=.04), total IgG level increase (P=.05), and IgG4 level increase (P=.03). Affinity chromatography using patient-derived mAbs identifies relevant autoantigens in patients with IgG4-RD. IgG4 galectin-3 autoantibodies are present in a subset of patients with IgG4-RD and correlate with galectin-3 plasma levels. The marked increases in levels of circulating IgG4 and IgE observed clinically are, at least in part, caused by the development of IgG4- and IgE-specific autoantibody responses.

T cell responses to peptides of Goodpasture autoantigen in patients with anti-glomerular basement membrane disease.

Cell-mediated autoimmunity, especially autoreactive T cells, is crucial in the initiation of anti-glomerular membrane (GBM) disease. Epitopes for T cells on Goodpasture autoantigen are not fully defined. This study investigated T cell epitopes in anti-GBM patients, aiming to identify the epitopes and their clinical significance. Peripheral blood mononuclear cells (PBMC) were collected from 13 patients with anti-GBM disease. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. PBMC response to each peptide was detected by proliferation assay. Their associations with clinical features were further analyzed. Peripheral blood mononuclear cells proliferative responses to linear peptides on α3(IV)NC1 could be detected in all patients. Five major epitopes were identified as stimulatory in over half of the patients: α3(IV)NC1127-148 (P14) (69.2%), α3(IV)NC1159-178 (77.8%), α3(IV)NC1179-198 (55.6%), α3(IV)NC1189-208 (P19) (75.0%) and α3(IV)NC1141-154 (57.1%). P14 and P19 were highly recognized in patients comparing with healthy controls (69.2% vs. 0.0%, P=0.011; 75.0% vs. 0.0%, P=0.021, respectively). T cell proliferation to linear epitopes was detected in human anti-GBM disease. α3127-148 was a mutual T and B cell epitope, implying its initial role in epitope spreading process.

Antibodies against linear epitopes on Goodpasture autoantigen in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.

In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr≤133μmol/L), 70% of patients with moderate renal dysfunction (133μmol/L<Scr≤600μmol/L), and 94% of patients with renal failure (Scr>600μmol/L) (P=0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of EA (P4) and EB (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r=0.764, P=0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P=0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.