Background/Purpose: T-cell frequencies against citrullinated epitopes derived from major auto-antibody targets (vimentin, fibrinogen, α-enolase) are increased in rheumatoid arthritis (RA). Emerging serologic data suggests antibody reactivity against additional citrullinated proteins, including histones and aggrecan, in RA. Among these potential targets, previous studies found T-cell reactivity against aggrecan peptides in models of RA and to some extent in clinical samples (Boots AM et al., 1997; Law SC et al., 2012; Aggarwal A et al., 2013). Here, we undertook a systematic approach to verify the relevance of aggrecan specific CD4+ T-cell responses in RA.

Methods:  Citrullinated aggrecan epitopes were predicted for their binding to DRB1*04:01 and candidates confirmed using binding assays with recombinant protein. The immunogenicity of confirmed binders was then assessed using 14-day in vitro peptide stimulation cultures followed by tetramer staining and single-cell cloning from selected subjects. Epitopes eliciting a significant response were then used for ex vivo tetramer staining of PBMC from healthy controls and from CCP+ RA patients across a range of disease activities (based on RAPID3 scores).

Results:  Starting with 28 cit-aggrecan peptides predicted to bind DR04:01, we identified 6 epitopes that activated and expanded CD4+ T cells with unusual strength for (modified) self-antigens. Using the corresponding tetramers we isolated cit-aggrecan specific T-cell clones specific for these peptides from RA patients. These clones selectively recognized citrullinated peptide and exhibited a Th1-like functional phenotype. Ex vivo tetramer analysis of PBMC revealed that a subset of RA patients had significantly increased frequencies of cit-aggrecan specific T-cells in comparison to healthy controls. Ongoing studies will determine whether cit-aggrecan specific T-cells arise early or late in disease, whether they have a distinct functional profile compared with healthy controls, and whether they exhibit expanded TCR clonotypes in established disease. Additionally, we are investigating the degree to which the serum from the blood or synovium contains antigens that specifically activate cit-aggrecan specific T-cell clones.

Conclusion:  Citrullinated aggrecan, a protein associated with autoantibodies in a subset of RA patients, have the potential to elicit very strong CD4+ T cell responses. In a subset of patients these are apparent as strong ex vivo response and show a disease associated Th1-like phenotype and some signs of clonal expansion. So far the clinical correlations of these high frequencies remain unclear but are an intriguing target of our ongoing studies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullinated-aggrecan-peptides-are-targets-of-auto-reactive-cd4-t-cells-in-rheumatoid-arthritis/