Introduction

Precisely tracking tumor-specific immune responses in clinical trials requires 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 identify the specific target epitope. Gene expression data give insights into the activity and polarization of the T cell.

Methods

Samples from two responding dogs in a trial of an autologous deglycosylated melanoma vaccine were selected to demonstrate applicability of scTCRseq in cancer immunotherapy. A single-cell suspension of cryopreserved peripheral blood mononuclear cells (PBMC) was prepared for 10X single cell sequencing. Libraries were amplified using a custom-designed nested PCR of the alpha/beta V(D)J region. This enriched product was added to the gene ([removed]GEX) library and scTCRseq performed on a NovaSeq 6000.

Results

The 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). Forty-three TRAJ, 29 TRAV, 12 TRBJ, and 22 TRBV genes were observed representing 72.9%, 51.8%, 100%, and 62.9% of all known V and J segments respectively. GEX enriched libraries successfully defined large clusters of CD8+ and CD4+ T cells consistent with previously reported results in human PBMC samples. Both dogs exhibit a small number of highly abundant T cell clones suggesting the presence of an anti-tumor T cell population.

Conclusion

The developed reagents successfully generated scTCRseq data that 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 responses in canine cancer immunotherapy trials.

Funding Information

Funded by the Siteman Investment Program jointly by Siteman Cancer Center and Ellis Fischel Cancer Center.