A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88

Abstract

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.

Cancer‐testis antigens in canine histiocytic sarcoma and other malignancies

“…Of those, one was a homologue of a scantly‐characterized open reading frame (C30H15orf39), and three (ATXN2L; CASC1; ZNF570) were considered likely to have mixed tissue expression based on human data, so these four were not evaluated. Peptides from the remaining four gene products (AKR1E2; SPECC1; TPX2; NT5C1B) were synthesized and tested for class I binding in a peptide‐MHC surface stabilization assay . All bound DLA‐88*508:01 to varying degrees (Table ; Figure S1C).…”

“…Isolated PBMCs were cultured in either R‐10 or RPMI 1640 that contained 1% autologous dog serum, P/S and l ‐glutamine. The cell clones 9‐15 (DH82 cells stably expressing DLA‐88*508:01‐FLAG) and BARC3 (RMA‐S cells stably expressing DLA‐88*508:01‐FLAG) had been previously generated and were cultured as described . In some experiments, cell lines and PBMCs were cultured in medium containing 10‐20 μM 5‐aza‐2′‐deoxycytidine (5‐aza‐dC; Sigma‐Aldrich, St. Louis, Missouri), diluted from a 200 mM stock solution prepared in DMSO and stored at −80°C.…”

“…Peptides were synthesized by Peptide 2.0 (Chantilly, Virginia), reconstituted in DMSO and stored as stock solutions at −80°C. A peptide‐MHC class I surface stabilization assay was performed as previously described . Briefly, BARC3 cells (10 5 per 100 μL) were cultured in R‐10 containing 100 μM peptide overnight at 27°C, and then incubated for an additional 5 hours at 37°C prior to staining with a primary murine anti‐MHC class I mAb (H58A; Monoclonal Antibody Center, Washington State University, Pullman, Washington), followed by washing and secondary incubation with an Alexa Flour 647‐labelled goat anti‐mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania).…”

“…The cell clones [9][10][11][12][13][14][15] (DH82 cells stably expressing DLA-88*508:01-FLAG) and BARC3 (RMA-S cells stably expressing DLA-88*508:01-FLAG) had been previously generated and were cultured as described. 15,16 In some experiments, cell lines and PBMCs were cultured in medium containing 10-20 μM 5-aza-2 0 -deoxycytidine (5-aza-dC; Sigma-Aldrich, St. Louis, Missouri), diluted from a 200 mM stock solution prepared in DMSO and stored at −80 C. The medium and 5-aza-dC were replaced every 12 hours for the 72-hour incubation period. All cells were grown under standard incubator conditions (37 C; humidified 5% CO 2 atmosphere), unless otherwise stated.…”

Characterization of the Canine MHC Class I DLA-88*50101 Peptide Binding Motif as a Prerequisite for Canine T Cell Immunotherapy

“…Their analysis was based on sequence comparison of 36 peptides and modeling analyses [55]. They proposed positions 2 and 3 as preferred hydrophobic residues, while the C-terminal residue of the peptide preferred a basic amino acid.…”