Introduction

Profiling the adaptive immune repertoire using high-throughput sequencing (HTS) methods has become common in human medicine, showing promise in characterizing clonal expansion of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IgH locus only. The objectives of this study were to: (1) describe a novel HTS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma.

Methods

We isolated RNA from PBMCs of healthy dogs (n=25) and dogs newly diagnosed with intermediate-to-large B-cell lymphoma (n=18) with intent to pursue chemotherapy. 5’ rapid amplification of cDNA ends (5’RACE) and next generation sequencing were employed to generate cDNA libraries and evaluate BCR repertoires. We developed an analysis pipeline to process, identify, and quantify these repertoires.

Results

In healthy controls, we observed BCR sequence properties similar to what has been previously observed, supporting the accuracy of our methods. Additionally, we observed a bimodal pattern in IGHV usage, where repertoires were dominant in either IGHV3-38 or IGHV4-1. Among dogs with lymphoma, we demonstrated an ability to quantify clonal expansion pre-treatment and contraction post-treatment.

Conclusion

The tools we developed represent novel resources to better understand canine hematopoietic malignancies. Future studies employing these tools may improve disease tracking, provide earlier recognition of relapses, and ultimately offer better-informed and targeted therapeutics.

Funding Information

• University of Minnesota Academic Health Center Faculty Seed Grant Program
• Biotechnology and Biological Sciences Research Council Doctoral Training Partnership
• Wellcome Trust