A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.
Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species.
Immunology Program, Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607, USA.
Peter
P
Ross
Jennifer C
JC
Holmes
Gregory S
GS
Gojanovich
Paul R
PR
Hess
D000818:Animals; D001681:Biological Assay / Q000379:methods / Q000662:veterinary*; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane; D004285:Dogs / Q000378:metabolism*; D000939:Epitopes; D005786:Gene Expression Regulation / Q000502:physiology; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D021122:Protein Subunits
D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D021122:Protein Subunits