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Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1–2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms. • Cost-effective protocol for canine T cell receptor beta chain analysis. • Profiling canine T cell receptor beta using next-generation sequencing. • Sample multiplexing using 5′ and 3′ barcodes. • Unique molecular identifiers for error-correction. [ABSTRACT FROM AUTHOR]

Spontaneous canine malignant melanoma provides an excellent pre-clinical model to study DNA vaccines for melanoma immunotherapy. A USDA-approved xenogeneic human tyrosinase (hu TYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of hu TYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti-hu TYR ELISA was used to test pre- and post-treatment sera. Biopsies of treated skin were obtained for detection of hu TYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. hu TYR-expressing cells were observed in biopsies of hu TYR DNA microseeding sites. Increased humoral anti-hu TYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy. [ABSTRACT FROM AUTHOR]