Department of Molecular Biomedical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, NC.
Bioinformatics Graduate Program, North Carolina State University, Raleigh, NC.
J Immunol. 2022 Feb 1;208(3):762-771. doi: 10.4049/jimmunol.2100824. Epub 2022 Jan 5.
Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.
最近，微流控和高通量测序技术的进步使得人们能够同时从人类和小鼠的数千个单个细胞中回收配对的 H 和 L 链的 Igs 和 TCR 的 VDJ 和 VJ 链。尽管恒河猴是研究人类适应性免疫反应的最成熟的模型生物之一，但由于这些多克隆受体的复杂性，高通量单细胞免疫受体库测序检测目前还不可用。我们使用了定制的引物，这些引物可以捕获所有已知的恒河猴 Ig 和 TCR 同种型和链，与商业的单细胞免疫受体库分析解决方案完全兼容。使用这些恒河猴特异性检测方法，我们对冷冻保存的恒河猴 PBMC、脾细胞和通过 FACS 分选的 B 和 T 细胞中的>60000 个细胞进行了 Ig 和 TCR 受体库测序。我们能够回收每种 Ig 同种型和 TCR 链，测量增殖 T 细胞中的克隆扩增，并将 Ig 和 TCR 受体库与同一单细胞的基因表达谱进行配对。我们的结果证明了在恒河猴中以单细胞水平进行高通量免疫受体分析的能力。
