The canine MHC class Ia allele DLA-88*508:01 presents diverse self- and canine distemper virus-origin peptides of varying length that have a conserved binding motif

Abstract

Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV hemagglutinin, large polymerase, matrix, nucleocapsid, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.

The prevalent Boxer MHC class Ia allotype dog leukocyte antigen (DLA)‐88*034:01 preferentially binds nonamer peptides with a defined motif

“…A summary of the binding preferences of DLA‐88*034:01 , based on the three combined datasets, is shown in Table . This works brings the total number of DLA‐88 allotypes for which a binding motif has been deduced from eluted peptide sequences to three ( DLA‐88*501:01 ; * 508:01 and *034:01 ) . Of course, binding assays with synthetic peptides that have various combinations of favored and disfavored residues, as well as alanine substitutions at these positions, will be needed to verify the conclusions of this study.…”

Cancer‐testis antigens in canine histiocytic sarcoma and other malignancies

“…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.…”

“…The mass spectral data used here are from a previously reported study and additional peptide isolation experiments. Large‐scale culture and weekly harvests of the 9‐15 clone, affinity isolation of peptide‐DLA‐88*508:01‐FLAG from 9–15 lysates, acid elution of bound peptides and additional peptide clean‐up and concentration were performed as described . Eluates were analysed by LC‐MS/MS, using a nanoACQUITY UPLC system (Waters) and Thermo Q Exactive HF‐X mass spectrometer (Thermo Fisher Scientific).…”

“…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).…”

“…Peptide‐independent MHC class I staining of BARC3 cells at 27 and 37°C was performed to validate assay performance. The canine self peptide K‐11 (RFLDKDGFIDK) served as a positive binding control . Data was acquired on a modified FACScan flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey) and analysed with FlowJo software (FlowJo LLC, Ashland, Oregon).…”