This study was conducted to identify epitopes derived from joint-related autoantigens that induce T-cell reactivity in patients with JIA. We recorded significant T-cell recognition of self-epitopes derived from aggrecan, fibrillin, and MMP-3 in patients with JIA irrespective of MHC genotype. Each of these self-epitopes shows a distinct profile of T-cell recognition in JIA and in health.
The proteoglycan aggrecan is one of the major constituents of the extracellular matrix (ECM). Degradation products of aggrecan can be detected in body fluids, including synovial fluid, where they reflect aggrecan turnover [38
]. Previous studies show that the proteoglycan aggrecan is a target for autoreactive T cells in RA and ankylosing spondylitis [40
]. Here, we demonstrate that aggrecan activates autoreactive T cells in JIA as well. T-cell proliferative responses to the aggrecan peptide identified were independent of JIA subtype and significantly different from those induced in healthy controls. It is well known that autoimmune disease progression is accompanied by an accumulation of neo-autoreactivity directed against target determinants not involved in disease initiation, a process known as epitope-spreading, while spontaneous regression of primary autoreactivity can occur [43
]. We noted an inverse relation between responders to the aggrecan peptide and disease duration, which suggests that the aggrecan peptide in particular could be a target of primary autoreactivity. The induction of the pro-inflammatory cytokines IFN-γ/IL-17 and inhibition of the anti-inflammatory cytokine IL-10 indeed suggest the induction of autoaggressive T cells.
Fibrillins, also part of the ECM, are believed to guide elastogenesis and are involved in tissue homeostasis and morphogenesis. Fibrillin can be detected in the synovial lining of joints in health and disease [45
]. Remarkably, T-cell proliferative responses to fibrillin are found primarily in polyarticular JIA independent of disease duration. A possible explanation for this observation may be a limited availability of fibrillin epitopes presented in oligoarticular JIA and healthy controls due to minimal or no joint destruction. To our knowledge, this is the first time that an autoantigen is described that primarily drives autoreactive T-cell responses in the more severe subtype of JIA, namely polyarticular JIA, and this autoreactivity is persistent throughout the disease course.
The MMPs are thought to be key enzymes involved in remodeling of the ECM in physiological and pathological situations. New views on the function of MMPs, however, indicate that this family of enzymes regulates various inflammatory and repair processes; matrix degradation is only one among the many functions that MMPs have [46
]. MMP-3 is expressed in both JIA and normal synovial tissue, and its expression correlates with the degree of inflammation [47
]. Recent studies identified MMP-3 as a target for T-cell recognition in both experimental arthritis and RA but also in age-matched healthy adult controls [27
]. In line with these findings, we now demonstrate that the epitope derived from MMP-3 induces T-cell proliferative responses in both patients with JIA and age-matched healthy children as well. The universal recognition of the MMP-3 epitope in JIA, RA, and healthy controls (adults and children) underlines the role of MMP-3 as a target for the immune response in health and chronic arthritis. Analysis of cytokine induction via MMP-3-specific short-term T-cell lines showed primarily production of IFN-γ. This may suggest a disease-promoting role of MMP-3-specific T cells in JIA. Further studies will be necessary to determine the relevance of this finding for the pathogenesis of JIA.
Although the method of peptide selection using 'predicted' sequences in AA as a mold is slightly unusual and evidently may not yield even a semi-complete list of potential peptide sequences, the positive results support this concept. The molecular mimicry may be a critical component of T-cell responses to peptide sequences of joint-related antigens. An extensive BLAST (Basic Local Alignment Search Tool) search, however, looking for foreign peptides with an obvious sequence homology to the aggrecan, fibrillin, or MMP-3 peptide, did not yield positive results. As such, this does not support the 'molecular mimicry' hypothesis. On the other hand, it is now known that a single TCR can recognize multiple peptides that may share only one contact residue [48
]. Clearly, when using this definition of molecular mimicry, our BLAST search must have been incomplete and potential mimicry epitopes must have been missed.
We analyzed T-cell responses to self-peptides derived from locally expressed non-immunodominant antigens in the joint. As such, it was not surprising that we were unable to detect significant cytokine production after direct incubation (without using any pre- or co-stimulation) of PBMC with the peptides derived from aggrecan, fibrillin, and MMP-3. The expected precursor frequency of these T cells will be low and involve low-avidity self-specific T cells [49
]. This problem was overcome via generation of short-term peptide-specific T-cell lines in selected patients. Using this method, we were able to confirm the reality of the recorded T-cell proliferative responses and show that T cells indeed are activated and do secrete cytokines.
With the identification of self-epitopes that are recognized in patients with JIA, we may now use techniques like T-cell capture or tetramer staining [50
] to sort the antigen-specific T cells directly and explore their qualities. Given that we expect the responding T cells to be low-affinity T cells, the use of tetramers will present difficulties. The T-cell capture technique, however, exploits so-called 'artificial APCs' (antigen-presenting cells) that contain high numbers of MHC-peptide complexes (signal 1) and include the presence of co-stimulatory molecules at the constructed MHC molecules (signal 2) [52
]. This reflects the in vivo
situation and indeed may allow binding of high-affinity as well as low-affinity T cells [52