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

BMC Immunol. 2020 May 18;21(1):27. doi: 10.1186/s12865-020-00357-w.


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 < 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.

Keywords: Autoimmune disease; Autoreactive CD4+ T lymphocytes; Citrullination; Multi-MHC class II tetramer assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Arthritis, Rheumatoid / drug therapy*
  • Arthritis, Rheumatoid / metabolism
  • CD4-Positive T-Lymphocytes / drug effects*
  • CD4-Positive T-Lymphocytes / metabolism
  • Citrulline / therapeutic use*
  • Epitopes, T-Lymphocyte / drug effects
  • Epitopes, T-Lymphocyte / metabolism
  • Extracellular Matrix Proteins / metabolism
  • Female
  • Fibrinogen / metabolism
  • Flow Cytometry / methods
  • Histocompatibility Antigens Class II / metabolism*
  • Humans
  • Male
  • Middle Aged
  • Pyrophosphatases / metabolism
  • Vimentin / therapeutic use


  • Epitopes, T-Lymphocyte
  • Extracellular Matrix Proteins
  • Histocompatibility Antigens Class II
  • Vimentin
  • Citrulline
  • Fibrinogen
  • Pyrophosphatases