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1.
HLA ; 97(5): 428-434, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33527745

RESUMEN

Canine spontaneous cancers may offer greater fidelity than rodent models in advancing clinical immunotherapies. Boxers in particular are distinguished as study subjects by their popularity, and high incidence of human-relevant cancers. Further, the MHC class I allele DLA-88*034:01, with a known motif, dominates the breed, facilitating discovery of shared CTL responses against mutation-origin neoepitopes by standard prediction methods. We experimentally confirmed the allomorph's binding motif by developing an MHC surface stabilization assay. The assay validated four DLA-88*034:01-presented peptides from canine distemper virus, ubiquitously administered in routine vaccines, for positive controls in future CTL studies. In turn, these viral peptides substantiated motif-based prediction for DLA-88*034:01. The study adds new tools for studying neoepitope-specific CTL in Boxers to foster canine comparative oncology.


Asunto(s)
Virus del Moquillo Canino , Alelos , Animales , Virus del Moquillo Canino/genética , Perros , Hemaglutininas , Leucocitos , Péptidos
2.
Vet Comp Oncol ; 17(3): 317-328, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30854786

RESUMEN

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Enfermedades de los Perros/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Antígenos HLA/metabolismo , Sarcoma Histiocítico/metabolismo , Animales , Línea Celular Tumoral , Cromatografía Liquida , Perros , Sarcoma Histiocítico/genética , Humanos , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espectrometría de Masas en Tándem , Testículo/metabolismo
3.
HLA ; 92(6): 403-407, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30239163

RESUMEN

Development of effective immunotherapy for chemoresistant malignancies can be advanced by studies in spontaneous cancer models, such as the dog. A crucial first step, T-cell epitope discovery, can be assisted by determination of binding motifs of common dog leukocyte antigen (DLA) class Ia allotypes. Boxers are popular, inbred dogs with increased risks of relevant target cancers and restricted MHC diversity. We sought to identify the motif of DLA-88*034:01, a breed-dominant allotype, to assist peptide prediction from tumor antigens. Mass spectrometry of eluted peptides showed a preference for nonamers with conserved amino acid preferences: basic at position (P)1; hydrophobic at P2; acidic at P4; histidine at P6; and phenylalanine at P9. This data should expedite finding epitopes restricted by this DLA-88 allotype.


Asunto(s)
Aminoácidos/química , Epítopos/química , Antígenos de Histocompatibilidad Clase I/química , Oligopéptidos/química , Secuencias de Aminoácidos , Aminoácidos/inmunología , Animales , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Perros , Epítopos/genética , Epítopos/inmunología , Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Oligopéptidos/genética , Oligopéptidos/inmunología , Unión Proteica , Linfocitos T/química , Linfocitos T/inmunología , Espectrometría de Masas en Tándem
4.
Vet Immunol Immunopathol ; 197: 76-86, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29475511

RESUMEN

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.


Asunto(s)
Presentación de Antígeno , Virus del Moquillo Canino/química , Antígenos de Histocompatibilidad Clase I/inmunología , Péptidos/química , Proteínas Virales/química , Alelos , Secuencias de Aminoácidos , Animales , Virus del Moquillo Canino/inmunología , Perros/genética , Epítopos/química , Epítopos/inmunología , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/genética , Péptidos/inmunología , Unión Proteica , Linfocitos T/inmunología , Proteínas Virales/inmunología
5.
Dev Comp Immunol ; 41(4): 578-86, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23892057

RESUMEN

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Presentación de Antígeno/genética , Lobos/genética , Alelos , Secuencia de Aminoácidos , Animales , Línea Celular , Perros , Variación Genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Isoformas de Proteínas
6.
Vet Immunol Immunopathol ; 150(3-4): 206-12, 2012 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-23062801

RESUMEN

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 ß2-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.


Asunto(s)
Bioensayo/veterinaria , Perros/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Animales , Bioensayo/métodos , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Membrana Celular , Epítopos , Regulación de la Expresión Génica/fisiología , Antígenos de Histocompatibilidad Clase I/química , Ratones , Unión Proteica , Subunidades de Proteína
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