Development of dog leukocyte antigen (DLA) typing assay for comparative immunogenomics
Naturally occurring canine cancers hold great potential as a model in clinical treatment development for immunotherapy studies. However, the canine immunogenomic landscape has not been extensively characterized. Of particular interest are the dog leukocyte antigen (DLA) genes which encode the major histocompatibility complex (MHC) proteins. MHCs present antigens on the cell surface to T-cells. The DNA sequences encoding peptide binding regions of these complexes are highly variable, and contribute to the MHC’s ability to bind a variety of peptides. Four known DLA genes encode class I MHC’s; DLA88, DLA64, DLA79, and DLA12. In cancer cells, accumulated mutations may lead to the production of neoantigens, which are proteins not previously recognized by the immune system. By integrating which allele of MHC genes present to immune cells with the mutations present in that patient’s tumor, we can predict what neoantigens are being presented on the surface by the MHC class I molecule. Development of the canine system as an immuno-oncology model necessitates a more in depth understanding of these genes. We adapted Illumina’s HLA allele-typing method for use with the DLA genes. This method utilizes a single pair of primers in a long range PCR to amplify the variable region of the four class I DLA genes. After confirming amplification on an agarose gel, amplicon libraries were prepared for sequencing on the MiSeq using the tagmentation based Nextera system. This process yields individual sequences for each sample, providing insight into variation between individuals. We employed this method to sequence germline DNA from 15 dogs with osteosarcoma and nine dogs with sun-shielded melanoma. These data will provide preliminary information critical for expanding our understanding of the canine immunogenomic landscape. In the future, this assay will provide a reliable method for immunogenomic analyses in dogs, including neoantigen analyses.