Citrulline‐Specific Th1 Cells Are Increased in Rheumatoid Arthritis and Their Frequency Is Influenced by Disease Duration and Therapy

Abstract

Objective Rheumatoid arthritis is thought to be a T cell mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell directed therapies and the presence of CD4 T cells in rheumatoid joints. The presence of ACPA in RA serum and the association of these antibodies with HLA-DR4 alleles implicates citrullinated specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the character and specificity of auto-reactive T cell responses in RA. Methods We developed a panel of HLA-DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01 transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of cit-specific T cells in RA and healthy subjects with DRB1*04:01 haplotypes using a magnetic enrichment procedure. Results Cit-specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of cit-specific T cells and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA subjects the frequency of cit-specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic therapies irrespective of disease duration. Conclusion These findings link the presence of ACPA in RA with Th1 cells specific to citrullinated epitopes and provide tools for disease-specific immunomonitoring of autoreactive T cells.

A Molecular Analysis of the Shared Epitope Hypothesis: Binding of Arthritogenic Peptides to DRB1*04 Alleles

“…This observation was confirmed by James et al (20), suggesting that the critical amino acids in susceptibility alleles might initiate RA by preferential binding of citrullinated peptides over their native forms. To test this hypothesis over a broad spectrum of HLA alleles, we measured the binding of native and citrullinated forms of vimentin [66][67][68][69][70][71][72][73][74][75][76][77][78] and a-enolase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] as well as the immunodominant peptide type II collagen [258][259][260][261][262][263][264][265][266][267][268][269][270][271][272] to 88 different HLA-DRB1, DPB1, and DQB1 protein alleles. We then performed site-directed mutagenesis of DRB1*04:01 residues at positions 67, 70, 71, 74, and 86 to the corresponding residues in *04:02, *04:03, *04:04, *04:05, and *08:01 to determine the potential role of each position in the binding of these arthritogenic peptides.…”

“…Using Luminex beads conjugated with individual DRA/DRB1 protein alleles (referred to simply as DRB1 alleles), we measured the binding of 2 pairs of citrullinated and native peptides (vimentin 66-78 and a-enolase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] ) and 1 unmodified peptide (collagen [258][259][260][261][262][263][264][265][266][267][268][269][270][271][272] ) to 29 DRB1 alleles, 31 DPA1/DPB1 alleles (DPB1 alleles), and 28 DQA1/DQB1 alleles (DQB1 alleles). Susceptibility and resistance alleles were delineated based on findings from .5,000 subjects (3).…”

“…Citrullinated peptides and their native counterparts were chosen based on studies that demonstrated ACPA presence and its ability to elicit T cell responses in HLA-DR shared epitope-positive patients with RA. These were citrullinated vimentin 66-78 (SAVRLRSSVPGVR) (18,21,22), citrullinated aggrecan 89-103 (ATEGRVRVNSAYQDK), citrullinated CILP 297-311 (ATIKAEFVRAETPYM), citrullinated a-enolase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] (IFDSRGNPTVEVDLF) (20), and unmodified collagen 258-272 (PGIAGFKGEQGPKGE) (23,24), where the underlined arginine indicates the native amino acid that was replaced with citrulline. As a reference control for HLA class II binding, we also tested hemagglutinin A (HA) 306-318 (PKYVKQNTLKLAT).…”

“…Q70D nearly abolished the preference for citrullinated vimentin [66][67][68][69][70][71][72][73][74][75][76][77][78] , again by increasing native peptide binding 6.2-fold (from mean 6 SEM MFI 303 6 78 to 1,887 6 441) rather than inhibiting citrullinated vimentin. However, Q70D had a much more modest effect on a-enolase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] , only slightly increasing native binding and reducing the preference for citrullinated a-enolase 11-25 to 9-fold. The DRB1*04:01 double-mutant L67I-Q70D eliminated the preference for citrullinated vimentin 66-78 (ratio 1.3) and reduced the preference for citrullinated a-enolase 11-25 to 3.9-fold.…”

“…We measured binding of native and citrullinated forms of vimentin [66][67][68][69][70][71][72][73][74][75][76][77][78] and a-enolase [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and noncitrullinated type II collagen [258][259][260][261][262][263][264][265][266][267][268][269][270][271][272] to 88 class II alleles on Luminex beads (which includes alleles of many varying degrees of susceptibility and resistance). We expressed DRB1*04:01, *04:02, and *08:01 in T2 cells and mutated DRB1*04:01 at positions 67, 70, 71, 74, and 86 to corresponding residues in DRB1*04:02, *04:03, *04:04, *04:05, and *08:01.…”