Introduction

We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).

Methods

DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.

Results

Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.

Conclusions

In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in diarthrodial joints. We have previously established that mice transgenic for the human DR genes associated with RA are susceptible to collagen-induced arthritis (CIA) and we have identified a determinant of type II collagen (CII263–270) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII263–270 would suppress disease in DR1 transgenic mice. Because the immunodominant determinant is the same for both DR1 transgenic and DR4 transgenic mice, we attempted to determine whether the analog peptide that was suppressive in DR1 transgenic mice would also be effective in suppressing CIA in DR4 transgenic mice. We treated DR4 transgenic mice with two analog peptides of CII that contained substitutions in the core of the immunodominant determinant: CII256–276 (F263N, E266D) and CII256–270 (F263N, E266A). Mice were observed for CIA, and T-cell proliferative responses were determined. Either peptide administered at the time of immunization with CII significantly downregulated arthritis. Binding studies demonstrated that replacement of the phenylalanine residue in position 263 of the CII peptide with asparagine significantly decreased the affinity of the peptide for the DR4 molecule. In contrast, replacement of the glutamic acid residue in position 266 with aspartic acid or with alanine had differing results. Aspartic acid reduced the affinity (35-fold) whereas alanine did not. Both peptides were capable of suppressing CIA. With the use of either peptide, CII256–276 (F263N, E266D) or CII256–270 (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-γ. We have identified two analog peptides that are potent suppressors of CIA in DR4 transgenic mice. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of HLA-DR4 that can suppress autoimmune arthritis.