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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.

HLA-DRB1 alleles have been associated with several autoimmune diseases. In anti-citrullinated protein antibody positive rheumatoid arthritis (ACPA-positive RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to investigate whether expression of different alleles of major histocompatibility complex (MHC) Class II genes influence functions of 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. Existing methods for HLA-DR allele-specific protein expression were evaluated but were not mature enough to provide appropriate complementary information at the protein level. 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.

Background HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. Results Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-β, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 Conclusions Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA ‘at risk’ subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

Background:Idiopathic inflammatory myopathies (IIM) also known as myositis, are rare chronic autoimmune disorders which are represented by muscle weakness and extra-muscular features such as skin rash, interstitial lung disease (ILD) and arthritis. One of the most common autoantibodies in myositis, with a prevalence of 25-35%, is the anti Jo-1 autoantibodies, targeting the histidyl-transfer RNA synthetase (HisRS). Although the exact mechanism of how these antibodies are developed is unknown, we have previously shown that upon stimulation of both peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid cells (BALF) with HisRS protein, CD4+ T cells were activated and produced inflammatory cytokines. Hitherto the presence of antigen specific autoreactive T cells has not been established in myositis, however previous studies by our group showed a strong indication of their presence with a reactivity to a specific HisRS peptide.Objectives:The main aim of this project is to detect and characterize HisRS specific CD4+T cells using HLA Class II tetramers. HLA Class II tetramers allow the detection of rare antigen specific CD4+ T cells and are widely used in studies of immunity, vaccine development, allergy monitoring and in autoimmunity. These cells are of specific interest to understand autoimmunity and to develop new therapies in autoimmune diseasesMethods:HLA-DRB1*03:01 monomers with selected tetanus and HisRS peptides were in-house in E.coli system. The peptides of interest were attached to the N-terminus of the HLA b-chain via a flexible peptide linker. HLA-tetramers were assembled using a commercial fluorescently labeled streptavidin. The efficacy of the peptide-HLA tetramers was validated by stimulating PBMCs from HLA-matched healthy controls with tetanus peptide. The drequency of tetanus specific CD4+ T cells were detected at different time points (6,13 and 21 days) from the cultures using tetanus peptides bound HLA-DRB1*0301 tetramers. The presence of tetanus specific T cells was confirmed by the secretion of significantly higher IFNg levels upon re-stimulation of cells with tetanus peptide. The same protocol is applied for the HisRS-peptide tetramers. Peripheral blood cells are analysed from anti-Jo1+ and HLA-DRB1*0301 positive patients with IIM.Results:Applying this method, our preliminary findings demonstrate the presence of HisRS+CD4+ T cells in peripheral blood from Jo-1+ patients (n=3) using HisRS tetramers following stimulation with the respective peptide. We are now including more patient samples to confirm our findings, and further characterize their phenotype and functionalities by flow cytometry and ELISA/fluorospot assaysConclusion:Myositis is a rare and chronic autoimmune disorder, with no currently available cure. Previous studies indicate the importance of T cells in this disease. However, the phenotype, functionality and role of these cells in the disease pathogenesis has not been fully established. Characterization of this autoreactive T-cell population will help us enhance our understanding of the disease pathogenesis and thus to develop better treatment options.Acknowledgements:This work has been supported by grants from Karolinska Instiutet Resarch Foundation, Professor Nanna Svartz Stiftelse, Hjärt-Lung Fonden and Vetenskapsrådet in Sweden.Disclosure of Interests:Begum Horuluoglu: None declared, Angeles Shunashy Galindo-Feria: None declared, Karine Chemin: None declared, Genadiy Kozhukh: None declared, Anatoly Dubnovitsky: None declared, Vivianne Malmström: None declared, Ingrid E. Lundberg Consultant of: Consulting fees from Corbus Pharmaceuticals, Ind, Grant/research support from: Research grants from Bristol Myers Squibb and Astra Zeneca.

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within alpha-enolase(326-340) in the context of HLA-DRB1*04:01 and assessed the corresponding CD4(+) T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated alpha-enolase(326-340) peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of a enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno(326-340) were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno(326-340) was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within alpha-enolase(326-340) does not appear to lead to complete negative selection of cognate CD4(+) T cells. In RA patient samples, only T cells recognizing the citrullinated version of alpha-enolase(326-340) were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance. (C) 2018 Elsevier Ltd. All rights reserved.

Background Previous studies have demonstrated that CD4+ T cells from peripheral blood of anti-histidyl-tRNA synthetase (anti-His-tRNA) also known as anti-Jo-1 positive patients proliferate in response to stimulation with full-length His-tRNA and a N-terminal fragment comprising residues 1–60. In this study we present a novel epitope that identifies patients with moderate-severe interstitial lung disease (ILD). Materials and Methods Sixteen anti-Jo-1 positive patients with antisynthetase syndrome followed at the Karolinska University Hospital were enrolled. As controls we included HLA-DRB1*03-positive healthy individual (HCs, n=8). Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density centrifugation and in vitro stimulated with: a) full length His-tRNA protein; b) a novel HLA-DR*03:01 binding peptide from native His-tRNA; c) an altered peptide ligand (APL) variant of His-tRNA designed to prevent recognition by HLA-DR3/His-tRNA-specific TCRs. T cell activation was assessed by CD40L up-regulation and expression of pro-inflammatory cytokines (IFN-g, IL-2 and IL-17A) by flow cytometry. Clinical and laboratory data were documented and analysed by Student’s T test or Mann-Withney U-test. Results At the time of blood sampling the patients had a mean age of 58 years (48–83 years), median disease duration of 50 months (11–70 months), MMT8 score 80 (79-80), HAQ 0.25 (0 13-0.75). Eighty-four percent were female, 13/16 patients had ILD and 13/16 had muscle weakness. T cell activation towards the novel His-tRNA peptide was observed in 2/16 anti-Jo-1 positive patients. When stimulating with the APL, no T cell activation was observed in one of the patients that was reactive for the peptide. For evaluation of pro-inflammatory features, the His-tRNA-specific T cells displayed significant up-regulated levels of IFN-γ (p Conclusion In this study, we demonstrate the presence of His-tRNA-reactive CD4+ T cells in peripheral blood from anti-Jo-1 positive patients and a novel His-tRNA peptide, characterised by the expression of IFN-g. This phenotype seemed to correlate to a moderate-severe clinical progression of ILD.

Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from alpha-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified alpha-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue. Correction in: Frontiers in Immunology, Volume: 8, Article Number: 1236, DOI: 10.3389/fimmu.2017.01236

Additional file 1. Supplementary information.

Additional file 1. Supplementary information.