Background/Purpose: Seropositive rheumatoid arthritis (RA) is a T-cell mediated disease that is characterized by the presence of antibodies to proteins that have undergone the post-translational conversion of arginine to citrulline. Certain HLA-DR major histocompatibility complex class II (MHCII) molecules, for example HLA-DRB1*0401 are closely associated with the development of seropositive RA. These molecules prefer to bind peptides with citrulline not arginine in the P4 position. Thus, citrullination of arginine can create peptides that bind to HLA-DRB1*0401 molecules better than the un-citrullinated versions.  These citrullinated peptide:HLA-DRB1*0401 complexes may form neo-antigens that activate T-cells with specific T-cell receptors. Indeed, such cells have been identified in patients with RA.
In this study, we investigated the correlation between the reported binding affinity of citrullinated peptides to HLA-DRB1*0401 molecules (Ting et al.) and the number of T-cells that recognize these complexes after immunization with citrullinated peptide in transgenic mice expressing HLA-DRB1*0401 molecules.

Methods: Mice expressing HLA-DRB*0401 were immunized with 100 mg of one of eleven citrullinated peptides. The citrullinated peptides tested all had a citrulline at the P4 position and were from human proteins known to have citrullinated antibodies raised against them in RA. These proteins were: collagen intermediate layer protein (2 peptides), fibrinogen (2 peptides), vimentin (3 peptides), LL37 (1 peptide), a-enolase (1 peptide), and aggrecan (2 peptides). Ten days after immunization, citrullinated peptide:HLA-DRB1*0401 tetramers were used to identify specific T-cells in secondary lymphoid organs by flow cytometry.

Results: There was a wide range in the number of citrullinated peptide:HLA-DRB1*0401 tetramer-binding cells isolated from immunized mice (43-5557). The peptide that generated the lowest number of tetramer-binding cells was from human aggrecan 89-103 (Cit95) and the highest number was from human fibrinogen 69-81 (Cit74). When analyzing all peptides, there was a significant correlation between the affinity of the peptide for HLA-DRB1*0401 and the log10 number of citrullinated peptide:MHC tetramer positive cells identified after immunization (R squared 0.454, p< 0.03)

Conclusion: Immunization with citrullinated peptides with higher binding affinity to an RA-associated MHCII molecule stimulated a greater number of specific T-cells. These results provide further evidence that citrullination enhances T cell immunogenicity by improving the capacity of peptides to bind MHCII molecules.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-citrullinated-peptide-specific-t-cells-in-humanized-mice-immunized-with-citrullinated-peptides/