Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health

Abstract

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index > or =2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-gamma (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.

Figure 1

Percentage of patients with juvenile idiopathic arthritis (JIA) and healthy controls showing positive T-cell proliferative responses. Transparent bar: 2 ≤ stimulation index (SI) < 3; black bar: 3 ≤ SI < 4; gray bar: SI ≥ 4. The highest frequencies were found for the peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3.

Figure 2

T-cell proliferative responses in patients with juvenile idiopathic arthritis (JIA) and healthy controls. Proliferative responses are presented as interquartile range for each group of data, with horizontal lines showing the median. The aggrecan and fibrillin peptides induced significant differences in responses of peripheral blood mononuclear cells (PBMC) from patients with JIA and healthy controls and are indicated as follows: **p = 0.005, *p = 0.015. The matrix metalloproteinase (MMP)-3 peptide induced proliferative responses in PBMC of patients with JIA as well as in PBMC of controls. Transparent boxes: JIA; gray boxes: healthy controls.

Figure 3

Relation of disease subtype and T-cell proliferative responses induced by aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3. Proliferative responses to the aggrecan peptide of peripheral blood mononuclear cells (PBMC) from both oligoarticular and polyarticular juvenile idiopathic arthritis (JIA) patients differed significantly with those from healthy controls (p = 0.034 and p = 0.036, respectively). Only PBMC from patients with polyarticular JIA showed significant T-cell proliferative responses to the fibrillin peptide (p < 0.0001 versus healthy controls, p = 0.003 versus oligoarticular JIA). Positive proliferative responses to the MMP-3 peptide were seen in both JIA subgroups and healthy controls. Black bars: oligoarticular JIA; gray bars: polyarticular JIA; transparent bars: healthy controls. Asterisks indicate significant difference.

Figure 4

Relation of disease duration and T-cell proliferative responses induced by aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3. Proliferative activity of peripheral blood mononuclear cells from patients with juvenile idiopathic arthritis was dichotomized as follows: stimulation index (SI)-non-responsive < 2 ≤ SI-responsive. Responders to the aggrecan peptide had the lowest disease duration (p = 0.016). Responders to the MMP-3 peptide showed a similar trend, which was close to reaching statistical significance (p = 0.056). Black bars: responders; transparent bars: non-responders. Asterisk indicates significant difference.

Figure 5

Cytokine production in short-term peptide-specific T-cell lines from patients with juvenile idiopathic arthritis (JIA). Short-term peptide-specific T-cell lines were generated from peripheral blood mononuclear cells of five patients with polyarticular JIA. After 14 days of culture, supernatants were taken for multiplex cytokine analysis and cells were stained for fluorescence-activated cell sorting (FACS) analysis. The aggrecan peptide induced significant production of interferon-γ (IFN-γ)/interleukin (IL)-17 and inhibition of IL-10 production (p < 0.05). The matrix metalloproteinase (MMP)-3 peptide induced significant production of IFN-γ. FACS analysis confirmed the presence of activated T cells. Asterisks indicate significant difference.