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Updated: Jun 28, 2025
T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing
Published on: January 12, 2021
Single-cell T-cell receptor repertoire profiling in dogs
My H Hoang1,2, Zachary L Skidmore1,2, Hans Rindt3
1Division of Oncology, Department of Medicine, Washington University School of Medicine, St Louis, MO, USA.
Abstract:
Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.
Insights
This study demonstrates single-cell T cell receptor sequencing (scTCRseq) in companion dogs with cancer, enabling tracking of tumor-specific immune responses. Findings reveal distinct T cell receptor repertoires in healthy, lymphoma, and melanoma dogs, crucial for cancer immunotherapy research.
Area of Science:
- Comparative oncology
- Immunogenomics
- Canine cancer research
Background:
- Spontaneous canine cancers serve as valuable models for human diseases.
- Tracking tumor-specific immune responses is essential for advancing cancer immunotherapy.
- Species-specific reagents for single-cell T cell receptor sequencing (scTCRseq) are needed for canine cancer models.
Purpose of the Study:
- To demonstrate the applicability of scTCRseq in companion dogs with cancer.
- To integrate scTCRseq with scRNA data in a canine cancer immunotherapy context.
- To characterize T cell receptor repertoires in healthy dogs and dogs with T cell lymphoma and melanoma.
Main Methods:
- Single-cell suspensions from PBMCs and lymph node aspirates were analyzed.
- Profiling involved dog-specific scTCRseq primers and single-cell RNA (scRNA) sequencing.
- V(D)J gene segments and T cell receptor clonotypes were identified and quantified.
Main Results:
- Over 77,000 V(D)J-expressing cells were detected, with thousands of unique clonotypes per sample.
- A high percentage of known functional TRAV, TRAJ, TRBV, and TRBJ gene segments were observed.
- Healthy dogs showed diverse repertoires, lymphomas had clonal repertoires, and melanoma dogs exhibited dominant clonotypes, primarily CD8+ T cells with activated phenotypes.
Conclusions:
- scTCRseq is applicable and valuable for canine cancer immunotherapy research.
- Distinct T cell receptor repertoire profiles correlate with cancer type and treatment.
- Identified T cell populations in melanoma suggest potential anti-tumor activity, warranting further investigation.
