Analog peptides of type II collagen can suppress arthritis in HLA-DR4 (DRB1*0401) transgenic mice

Abstract

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 (CII(263-270)) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII(263-270) 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: CII(256-276) (F263N, E266D) and CII(256-270) (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, CII(256-276) (F263N, E266D) or CII(256-270) (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-gamma. 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.

Figure 1

Proliferation of DR4-(DRB1*0401)-restricted T cells in response to type II collagen. A panel of analog peptides containing amino acid substitutions was tested for the ability to stimulate the proliferation of type II collagen (CII)-primed T cells. DR4 transgenic mice were immunized with CII; 10 days later, draining lymph node cells were harvested and cultured with the analog peptides. Proliferation was measured by [³H]thymidine incorporation and results are shown as means ± standard deviation (x axis). Data shown are from a representative experiment. Lymph node cells from six immunized mice were pooled before culture, and the data were subsequently confirmed in three separate experiments. Control cells cultured with no antigen had a proliferation value of 0.38 × 10^-3 d.p.m.

Figure 2

Binding of analog peptides to DRB1*0401. IC₅₀ (50% inhibitory concentration) values are the average of two determinations per peptide. Peptides binding to DRB1*0401 with an IC₅₀ of about 1,000 are represented by striped bars (264A, 266D). Peptides binding to DRB1*0401 with an IC₅₀ of 100 or less (comparable to wild-type peptide binding) are represented by white bars.

Figure 3

Co-immunization of DR4 transgenic mice with type II collagen and analog peptides. Groups of DR4 transgenic mice were co-immunized with 50 μg of type II collagen (CII) alone (n = 18; circles) or with 50 μg of CII plus 1.2 mg of the analog peptide CI_256–276, (F263N, E266D) (n = 16; upward triangles), with 50 μg of CII plus 1.2 mg of the analog peptide CII_256–276 (F263N, E266A) (n = 10; downward triangles), or with 50 μg of CII plus 1.2 mg of the analog peptide CII_256–276 (E266D) (n = 10; diamonds) and observed for arthritis. Results are incidences (upper panel) and mean severity scores (lower panel) of arthritis observed at various time points after immunization.