Multifunctional T cell reactivity with native and glycosylated type II collagen in rheumatoid arthritis

Abstract

Type II collagen (CII) is a cartilage-specific protein to which a loss of immune tolerance may trigger autoimmune reactions and cause arthritis. The major T cell epitope on CII, amino acids 259-273, can be presented by several HLA-DRB1 04 alleles in its native or posttranslational glycosylated form. The present study was undertaken to functionally explore and compare CII-autoreactive T cells from blood and synovial fluid of patients with rheumatoid arthritis (RA). Peripheral blood was obtained from HLA-DRB1 04-positive RA patients (n = 10) and control subjects (n = 10) and stimulated in vitro with several variants of the CII(259-273) epitope, i.e., unmodified, glycosylated on Lys-264, glycosylated on Lys-270, or glycosylated on both Lys-264 and Lys-270. Up-regulation of CD154 was used to identify responding T cells. These cells were further characterized by intracellular staining for interleukin-17 (IL-17), interferon-γ (IFNγ), and IL-2 by flow cytometry. Synovial T cells from RA patients were investigated in parallel. Multifunctional T cell responses toward all examined variants of the CII(259-273) peptide could be detected in RA patients and, to a lesser extent, also in healthy HLA-matched controls (P < 0.001). In RA patients, a comparison between blood- and joint-derived T cell function revealed a significant increase in levels of the proinflammatory cytokine IFNγ in synovial T cells (P = 0.027). Studies of longitudinally obtained samples showed that T cell responses were sustained over the course of disease, and even included epitope spreading. The identification of inflammatory T cell responses to both glycosylated and nonglycosylated variants of the major CII epitope in RA patients suggests that CII autoreactivity in RA may be more common than previously recognized.