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