Single Cell T Cell Receptor Repertoire Profiling for Dogs

Background Spontaneous cancers in companion dogs are increasingly recognized as robust models of human disease. This recognition has led to translational clinical trials in companion dogs with osteosarcoma, lymphoma, melanoma, squamous cell carcinoma, and soft tissue sarcoma. The ability to precisely track tumor-specific immune responses in such clinical trials would benefit from reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). This technology defines clones of T cells reacting to immune interventions and can help identify the specific epitope of response. Single cell gene expression data give insights into the activity and polarization of the T cell. To date, scTCRseq has not been demonstrated for canine samples. Methods Samples from two responding dogs in a trial of an autologous deglycosylated melanoma vaccine were selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. A single-cell suspension of cryopreserved peripheral blood mononuclear cells (PBMC) was prepared for 10X single cell sequencing. Full length 10X cDNA was amplified using a custom-designed nested PCR of the alpha/beta V(D)J region. A library made from this enriched product (scTCRseq) and a 10X gene expression (GEX) library (scRNAseq) were sequenced on the NovaSeq 6000. Results 1,850-2,172 estimated V(D)J-expressing cells yielded 87-103.7 million reads with 73.8%-75.8% mapped to a V(D)J gene (beta/alpha chains ratio 1.5:1). 43 TRAJ, 29 TRAV, 12 TRBJ, and 22 TRBV gene segments were observed representing 72.9%, 51.8%, 100%, and 62.9% of all known V and J gene segments respectively. A large diversity of clonotypes was captured with 966-1,253 TRA/TRB clonotypes identified. Both dogs also exhibited a small number of highly abundant T cell clonotypes suggesting the presence of an anti-tumor T cell population. GEX enriched libraries successfully defined large clusters of CD8+ and CD4+ T cells that overlapped with V(D)J-expressing cells. Discussion The developed reagents successfully generated scTCRseq data, for the first time, which allowed the T cell repertoire to be surveyed in dogs responding to anti-tumor immunotherapy. These reagents will allow longitudinal tracking of anti-tumor T cell dynamics in canine cancer immunotherapy trials. ### Competing Interest Statement The authors have declared no competing interest.