The study of complex immunologic diseases and tumor microenvironment has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B cell antibodies (Igs) and T-cell receptors (TCRs). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from tissue and PBMCs were used to generate immune sequencing libraries. Using a unique molecule index (UMI) to discretely barcode each mRNA molecule, PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. The B cell genes were enriched during library preparation by IGH, IGK and IGL primes, including isotype-specific primers (IgA, IgD, IgE, IgG and IgM). The T-cell genes were enriched by TCRα and TCRβ specific primers. To investigate the applicability of minimal residual disease assessment, we obtained Jurkat RNA, a homogenous population of leukemic Jurkat T-cells and spiked it into a PBMC RNA sample at varying proportions of Jurkat RNA (10%, 1%, 0.1%, 0.01% and 0.001%). The RNA mixtures were made into TCR libraries, sequenced on both Illumina MiSeq and Oxford Nanopore MinION, and analyzed to assess the TCR repertoire. Utilization of UMIs enabled absolute quantification of the B cell antibody/TCR clones and accurate ranking of their abundance. For B cell repertoire sequencing, the use of isotype-specific primers enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody analysis enabled measurement of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. For TCR repertoire sequencing, distinct and shared clonal sequences were quantitatively detected in PBMC samples. The method also accurately and sensitively detected the control TCR clone spiked into both Illumina and Oxford Nanopore libraries at levels appropriate for minimal residual disease assessment.This immune repertoire sequencing approach allows accurate clonal determination for both Igs and TCRs. This technique is applicable for a variety of applications including design of antibody chains for in vitro synthesis, investigation of T-cell infiltration of tumor microenvironments, and monitoring of minimal residual disease.

Citation Format: Chen Song, Luo Sun, Pingfang Liu, Bradley Langhorst, Andrew Barry, Theodore B. Davis, Eileen T. Dimalanta. Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B046.