T-cell repertoire (TCR) sequencing by next-generation sequencing (NGS) is a valuable tool for building a deeper understanding of the adaptive immune system. As immunotherapies, particularly T-cell dependent therapies, show increasing potential in treating cancer, the ability to gain a detailed, unbiased view of the TCR becomes imperative for biomarker discovery, immune response to treatment, and study of tumor microenvironments. A key question the field seeks to understand is the relationship between circulating T-cells and infiltrating T-cells at the tumor site. Here, we present a novel approach for TCR sequencing using the Ion S5 ™ sequencer which leverages simplified library construction workflows and offers a more complete characterization of the entire V(D)J region of TCRB. This method can leverage mRNA as input, minimizing requirements in starting materials and focusing sequencing to productive TCRB arrangements. This approach targets the constant (C) and the FR1 regions, minimizing the potential for primer bias and greatly increasing the phylogenetic information content compared to techniques that exclusively characterize the CDR3 domain. Our results show that the observed circulating T-cell repertoire size is approximately 2 orders of magnitude higher than the infiltrating T-cell repertoire. Accordingly, while it is difficult to fully capture the complete repertoire of circulating T-cells due to its vast diversity, we show that it is possible to reliably capture the complete infiltrating T-cell repertoire with as high as 10 samples on the Ion 530 ™ chip. Replicate sequencing runs of infiltrating T-cells offers correlation of ~0.9, indicating that the results were reproducible, and the samples were sequenced to appropriate depth. In summary, we believe that this workflow will allow researchers to more routinely characterize the infiltrating T-cell repertoire and offers the field a better understanding of the impact of repertoire diversity on tumor elimination.

Citation Format: Geoffrey Lowman, Elizabeth Linch, Lauren Miller, Denise Topacio-Hall, Timothy Looney, Alex Pankov, Yongming Sun, Xinzhan Peng, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1631. doi:10.1158/1538-7445.AM2017-1631