| 1. |
- Chemin, Karine, et al.
(författare)
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A Novel HLA-DRB1*10:01-Restricted T Cell Epitope From Citrullinated Type II Collagen Relevant to Rheumatoid Arthritis
- 2016
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Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205. ; 68:5, s. 1124-1135
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Tidskriftsartikel (refereegranskat)abstract
- Objective. Antibodies against citrullinated type II collagen (Cit-CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit-CII-restricted T cell epitopes that are relevant to RA. Methods. Peripheral blood mononuclear cells (PBMCs) from HLA-DRB1*10:01-positive patients with RA and healthy donors were stimulated in vitro with candidate CII peptides. CD154 up-regulation was measured as a marker of antigen-specific activation, and anti-HLA-DR-blocking experiments confirmed HLA restriction. Cytokine production was measured using a Luminex technique. Direct peptide-binding assays using HLA-DRB1*10:01 and HLA-DRB1*04:01 monomeric proteins were performed. The T cell receptor (TCR) beta-chain of CD154-enriched antigen-specific T cells was analyzed using high-throughput sequencing. Results. A novel Cit-CII peptide was identified based on its ability to activate CD4+ T cells from HLA-DRB1*10:01-positive individuals. When stimulated in vitro, Cit-CII autoreactive T cells produced proinflammatory cytokines. Cit-CII311-325 bound (with low affinity) to HLA-DRB1*10:01 but not to HLA-DRB1*04:01, while the native form was unable to bind either protein. In addition, highly expanded clones were identified in the TCR beta repertoire of Cit-CII311-325-stimulated PBMCs. Conclusion. These results illustrate the ability of the citrullination process to create T cell epitopes from CII, a cartilage-restricted protein that is relevant to RA pathogenesis. The exclusive binding of Cit-CII311-325 to HLA-DRB1*10:01 suggests that recognition of citrullinated epitopes might vary between individuals carrying different RA-associated HLA-DR molecules.
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| 2. |
- Circiumaru, Alexandra, et al.
(författare)
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Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens : Clonal Diversity and Inter-Individual Variation
- 2023
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 13:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients.Methods: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5.Results: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation.Conclusions: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability.
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| 3. |
- Houtman, Miranda, et al.
(författare)
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Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain
- 2022
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 152, s. 106-110
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Tidskriftsartikel (refereegranskat)abstract
- Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population.
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| 4. |
- Houtman, Miranda, et al.
(författare)
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Haplotype-Specific Expression Analysis of MHC Class II Genes in Healthy Individuals and Rheumatoid Arthritis Patients
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- HLA-DRB1 alleles have been associated with several autoimmune diseases. For anti-citrullinated protein antibody positive rheumatoid arthritis (RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to study the genetic regulation of major histocompatibility complex (MHC) Class II gene expression in immune cells, we investigated transcriptomic profiles of a variety of immune cells from healthy individuals carrying different HLA-DRB1 alleles. Sequencing libraries from peripheral blood mononuclear cells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes of 32 genetically pre-selected healthy female individuals were generated, sequenced and reads were aligned to the standard reference. For the MHC region, reads were mapped to available MHC reference haplotypes and AltHapAlignR was used to estimate gene expression. Using this method, HLA-DRB and HLA-DQ were found to be differentially expressed in different immune cells of healthy individuals as well as in whole blood samples of RA patients carrying HLA-DRB1 SE-positive versus SE-negative alleles. In contrast, no genes outside the MHC region were differentially expressed between individuals carrying HLA-DRB1 SE-positive and SE-negative alleles, thus HLA-DRB1 SE alleles have a strong cis effect on gene expression. Altogether, our findings suggest that immune effects associated with different allelic forms of HLA-DR and HLA-DQ may be associated not only with differences in the structure of these proteins, but also with differences in their expression levels.
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| 5. |
- Brauner, Susanna, et al.
(författare)
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H1N1 vaccination in Sjogren's syndrome triggers polyclonal B cell activation and promotes autoantibody production
- 2017
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 76:10, s. 1755-1763
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesVaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naive patients diagnosed with primary Sjogren's syndrome (pSS) to an H1N1 influenza vaccine.Methods Patients with Sjogren's syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naive B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology.ResultsSurprisingly, treatment-naive patients with Sjogren's syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naive B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naive B cells to chloroquine.ConclusionsThis comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjogren's syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients.
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| 6. |
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| 7. |
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| 8. |
- Ge, Changrong, et al.
(författare)
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Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies
- 2019
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Ingår i: Arthritis & Rheumatology. - : WILEY. - 2326-5191 .- 2326-5205. ; 71:2, s. 210-221
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Tidskriftsartikel (refereegranskat)abstract
- Objective Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. Methods Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. Results Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. Conclusion These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.
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| 9. |
- Hansson, Monika, et al.
(författare)
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Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides
- 2012
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Ingår i: ARTHRITIS RESEARCH & THERAPY. - : Springer Science and Business Media LLC. - 1478-6354 .- 1478-6362. ; 14:5, s. R201-
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA. Methods: The microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (<10 mu l). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software. Results: Strong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman rho typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides. Conclusions: The multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.
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| 10. |
- Hensvold, Aase, et al.
(författare)
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The human bone marrow plasma cell compartment in rheumatoid arthritis-Clonal relationships and anti-citrulline autoantibody producing cells
- 2023
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Ingår i: Journal of Autoimmunity. - : Elsevier BV. - 0896-8411 .- 1095-9157. ; 136
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Tidskriftsartikel (refereegranskat)abstract
- A majority of circulating IgG is produced by plasma cells residing in the bone marrow (BM). Long-lived BM plasma cells constitute our humoral immune memory and are essential for infection-specific immunity. They may also provide a reservoir of potentially pathogenic autoantibodies, including rheumatoid arthritis (RA)-associated anti-citrullinated protein autoantibodies (ACPA). Here we investigated paired human BM plasma cell and peripheral blood (PB) B-cell repertoires in seropositive RA, four ACPA+ RA patients and one ACPA- using two different single-cell approaches, flow cytometry sorting, and transcriptomics, followed by recombinant antibody generation. Immunoglobulin (Ig) analysis of >900 paired heavy-light chains from BM plasma cells identified by either surface CD138 expression or transcriptome profiles (including gene expression of MZB1, JCHAIN and XBP1) demonstrated differences in IgG/A repertoires and N-linked glycosylation between patients. For three patients, we identified clonotypes shared between BM plasma cells and PB memory B cells. Notably, four individuals displayed plasma cells with identical heavy chains but different light chains, which may indicate receptor revision or clonal convergence. ACPA-producing BM plasma cells were identified in two ACPA+ patients. Three of 44 recombinantly expressed monoclonal antibodies from ACPA+ RA BM plasma cells were CCP2+, specifically binding to citrullinated peptides. Out of these, two clones reacted with citrullinated histone-4 and activated neutrophils. In conclusion, single-cell investigation of B-cell repertoires in RA bone marrow provided new understanding of human plasma cells clonal relationships and demonstrated pathogenically relevant disease-associated autoantibody expression in long-lived plasma cells.
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