Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain

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

Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis

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 α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-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

Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

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 alpha-enolase, fibrinogen-beta, 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 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. 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

Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted alpha-Enolase T Cell Epitope in Rheumatoid Arthritis

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</p