Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy

Structures of the HC gp-39(263–275) sequence (compound 1) and of a series of modified peptides at anchor position 1 (P1, Phe265; compounds 24 to 35) and associated bioactivities

<p><b>Copyright information:</b></p><p>Taken from "Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation"</p><p>http://arthritis-research.com/content/9/4/R71</p><p>Arthritis Research & Therapy 2007;9(4):R71-R71.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC2206373.</p><p></p> Binding of MHC anchor variant peptides to HLA-DR4(B1*0401) was determined in a competition binding assay. IC= 50% inhibitory concentration. All peptides were found to bind HLA-DR4 with high relative affinity (ICranged between 0.001 and 0.38 μM). The hybridoma response (IL-2 production) to wild-type (WT) peptide and MHC anchor variant peptides presented by HLA-DRB1*0401 expressing B lymphoblastoid cells as source of antigen-presenting cells is expressed as stimulation index (SI). The SI values are based on mean fluorescence counts derived from duplicate or triplicate measurements and calculated as the ratio of mean fluorescence counts of antigen stimulated cultures and control cultures. Background (no peptide added) values for hybridoma 5G11, 8B12 and 14G11 were 29,203, 16,288 and 7,152 mean fluorescence units/counts, respectively. The standard deviation of measurements did not exceed 15%. Values greater or less than 30% (2 × the standard deviation) of the positive control (response to WT peptide) are defined as super agonists (+30%) and partial agonists (-30%) respectively and are indicated in bold

Treatment parameters for both non-arthritic and arthritic mice treated with placebo, prednisolone (10 mg/kg) or ORG 37663 (12 mg/kg) for 21 days.

<p>Both non-arthritic and arthritic mice are treated with placebo, prednisolone (10 mg/kg/day) or ORG 37663 (12 mg/kg/day) for 21 days. Each experimental group consists of 12 mice; in total 36 non-arthritic and 36 arthritic mice were included. Values represent means ± SD during the blood glucose kinetics test except BW, which is represented as means ± SD before the test.</p><p>* Significant difference (p≤0.05) when compared to the placebo-treated group of that same parameter.</p>#<p>Significant difference (p≤0.05) when compared to the placebo-treated non-arthritic mice.</p><p>Treatment parameters for both non-arthritic and arthritic mice treated with placebo, prednisolone (10 mg/kg) or ORG 37663 (12 mg/kg) for 21 days.</p

The formulas used to calculate the concentration <i>vs</i>. time curves and the kinetic parameters in a first order absorption process in an one-compartment model.

<p>C_t, D-[6,6-²H]-glucose concentration at time point t; M_t, fractional contribution of D-[6,6-²H]-glucose at time point t; [glc]_t, blood glucose concentration at time point t. C(0)^ab, initial concentration of D-[6,6-²H]-glucose determined by extrapolation of the absorption period; C(0)^el, initial concentration of D-[6,6-²H]-glucose determined by extrapolation of the elimination period; k^ab, absorption rate constant; k^el, absorption rate constant. D, dose D-[6,6-²H]-glucose administrated; BG, average blood glucose concentration during the test; Ra = EGP.</p><p>The formulas used to calculate the concentration <i>vs</i>. time curves and the kinetic parameters in a first order absorption process in an one-compartment model.</p

Effects of ORG 37663 and prednisolone on glucose kinetics after 21 days of treatment.

<p>(<b>A</b>) Blood glucose concentrations (<b>B</b>) MCR (<b>C</b>) EGP levels (<b>D</b>) C-peptide concentrations (<b>E</b>) C-peptide HOMA-IR. Results represent means ± SD and are outlined for each experimental group (n = 12). * Significant difference p≤0.05, ** significant difference p≤0.01, *** significant difference p≤0.001, ^#significant difference (p≤0.05) when compared to the placebo-treated non-arthritic mice, NS  =  not significant.</p

Effects of prednisolone on glucose kinetics after 21 days of treatment.

<p>(<b>A</b>) Blood glucose concentrations (<b>B</b>) MCR (<b>C</b>) EGP levels MCR (<b>D</b>) C-peptide concentrations (<b>E</b>) C-peptide HOMA-IR. Results represent means ± SD and are outlined for each experimental group (n = 12). * Significant difference p≤0.05, ** significant difference p≤0.01, *** significant difference p≤0.001, ^#significant difference (p≤0.05) when compared to the placebo-treated non-arthritic mice, NS  =  not significant.</p

Treatment parameters for both non-arthritic and arthritic mice treated with prednisolone for 21 days.

<p>Both non-arthritic and arthritic mice are treated with prednisolone (0, 1.5, 10 and 30 mg/kg/day) for 21 days. Each experimental group consists of 12 mice; in total 48 non-arthritic and 48 arthritic mice were included. Values represent means ± SD during the blood glucose kinetics test except BW, which is represented as means ± SD before the test.</p><p>* Significant difference (p≤0.05) when compared to the placebo-treated group of that same parameter.</p>#<p>Significant difference (p≤0.05) when compared to the placebo-treated non-arthritic mice.</p><p>Treatment parameters for both non-arthritic and arthritic mice treated with prednisolone for 21 days.</p

Arthritis development.

<p>Arthritis development in mice subjected to blood glucose kinetics three times and in mice in which no experiments were performed. (<b>A</b>) AUC of the overall arthritis score, corrected for base line, after 21 days. (<b>B</b>) X-ray analysis to assess bone destruction. Results are presented as mean ± SEM (n = 20). No significant differences between the two groups was observed. NS  =  not significant.</p

Effects of prednisolone on bodyweight, thymus weight and arthritis development after 21 days of treatment.

<p>(<b>A</b>) Bodyweight (<b>B</b>) thymus weight (<b>C</b>) AUC of the overall arthritis score (<b>D</b>) X-ray analysis to assess bone destruction. Results are represented as means ± SD for bodyweight and thymus weight and as mean ±SEM for AUC arthritis score and X-ray score and are outlined for each experimental group (n = 12). * Significant difference p≤0.05, ** significant difference p≤0.01, *** significant difference p≤0.001, NS  =  not significant.</p

Effects of ORG 37663 and prednisolone on bodyweight, thymus weight and arthritis development after 21 days of treatment.

<p>(<b>A</b>) Bodyweight (<b>B</b>) thymus weight (<b>C</b>) AUC of the overall arthritis score (<b>D</b>) X-ray analysis to assess bone destruction. Results are represented as means ± SD for bodyweight and thymus weight and as mean ±SEM for AUC arthritis score and X-ray score and are outlined for each experimental group (n = 12). * Significant difference p≤0.05, ** significant difference p≤0.01, *** significant difference p≤0.001, ^#significant difference (p≤0.05) when compared to the placebo-treated non-arthritic mice, NS  =  not significant.</p