Spontaneous cancers in dogs serve as valuable models for human cancers, facilitating translational research. Immunotherapy is a crucial area of cancer treatment, and T-cell receptor (TCR) repertoire profiling is instrumental in understanding and monitoring immune responses. However, species-specific reagents are needed for effective canine TCR profiling, particularly at the single-cell level. This research aimed to develop and validate a canine-specific scTCRseq protocol for the 10x Genomics Chromium platform and apply it to a diverse set of dog samples, including healthy controls and dogs with cancer undergoing immunotherapy.

The use of companion animals, particularly dogs, as spontaneous cancer models has gained significant traction due to their genetic variability, shared environment with humans, and naturally occurring immune responses. Numerous clinical trials in dogs have explored various immunotherapies, highlighting the value of this model system for translating findings to human applications. The importance of TCR repertoire profiling in understanding T-cell mediated immune responses in cancer, including tracking malignancies and informing personalized therapies, has also been established in the literature. The tumor microenvironment (TME) and its immunosuppressive effects are increasingly recognized as factors influencing immunotherapy efficacy.

The study utilized PBMCs and lymph node aspirates from five healthy dogs, two dogs with T-cell lymphoma, and four dogs with melanoma. Single-cell suspensions were prepared, and scRNA-seq and dog-specific scTCRseq libraries were generated using the 10x Genomics Chromium platform. A nested PCR approach was used for TCR enrichment, employing custom-designed primers targeting canine TRA and TRB chains. Sequencing was performed on an Illumina NovaSeq 6000 platform. Data analysis was carried out using Cell Ranger, integrating scRNA-seq and scTCRseq data for comprehensive repertoire analysis and cell type identification using SingleR. Flow cytometry was also used to characterize immune cell populations. Further analyses included assessing clonotype diversity, gene segment usage, germline variation, and the phenotype of expanded vs non-expanded T cells.

The researchers successfully generated and sequenced scTCRseq libraries from canine samples, achieving results comparable to human studies. A total of 77,809 V(D)J-expressing cells were profiled, revealing an average of 3498 unique clonotypes per sample. The study identified 29/34, 40/40, 22/22, and 9/9 known functional TRAV, TRAJ, TRBV, and TRBJ gene segments, respectively, also observing expression of some pseudogenes and non-functional ORFs. Healthy dogs exhibited highly diverse repertoires, whereas T-cell lymphomas showed clonal expansion, and melanoma samples exhibited oligoclonal repertoires. Integration of scRNA-seq data with scTCRseq data revealed that dominant clonotypes in melanoma PBMCs primarily consisted of activated CD8+ T cells. Analysis also uncovered germline variations and suggested re-annotation of several gene segments as functional based on the observed productive CDR3 sequences.

This study successfully established a robust scTCRseq protocol for canine samples, addressing a significant gap in the field. The findings highlight the potential of canine models in translational cancer research, especially in immunotherapy studies. The observed diverse TCR repertoires in healthy dogs contrasted sharply with the clonal or oligoclonal patterns seen in lymphoma and melanoma, respectively. The identification of activated CD8+ T cells in melanoma samples undergoing immunotherapy further suggests a potential anti-tumor response. However, the observed use of pseudogene and non-functional ORF segments warrants further investigation into canine germline variation and the potential need for refining existing reference databases. Future work should concentrate on extending the methodology to encompass other TCR loci and BCR sequencing.

This study successfully established a robust single-cell T-cell receptor sequencing protocol for dogs, opening doors for more comprehensive investigations into canine immune responses in health and disease. The results demonstrate the diverse TCR repertoires in healthy animals and reveal contrasting clonal or oligoclonal patterns in lymphoma and melanoma. The activated CD8+ T cells in melanoma suggest active anti-tumor responses during immunotherapy. The study also highlighted the need for improved canine reference databases and future research directions, including extending the analysis to include BCR sequencing and other TCR loci.

The study's limitations include the relatively small sample size, particularly in the lymphoma and melanoma cohorts, which may limit the generalizability of certain findings. The reliance on the CanFam3.1 reference genome might also have led to the under-reporting of some gene segments or over-reporting of pseudogenes or non-functional ORFs, particularly considering the germline variation observed. The methodology was limited to 5'-based 10x protocols, excluding certain other 10x approaches. Further, more in-depth analyses of the functional relevance of the identified clonotypes would strengthen the conclusions regarding anti-tumor immune responses.