Lauren Miller

Haplotype Analysis of the TRB Locus by TCRB Repertoire Sequencing

Polymorphism within the T cell receptor beta variable gene (TRBV) has been implicated in autoimmune disease and immuneCrelated adverse events (IRAEs) during immunotherapy. Previous efforts to evaluate TRBV polymorphism by whole genome sequencing (WGS) have been hampered by the repetitive nature of the TCRB locus. We present a novel longCamplicon TCRB repertoire sequencing approach to evaluate TRBV polymorphism from peripheral blood, which we use to identify TRBV allele haplotypes in 81 Caucasians.

Abstract 4668: Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma

Introduction T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we evaluate evidence for T cell convergence within tumor biopsy research samples from a set of 85 subjects with melanoma. Methods Total RNA from 85 tumor biopsy research samples (non-FFPE) was extracted for use in long-amplicon TCRB chain sequencing (mean amplicon length of 330bp covering CDR1, 2 and 3) via the Ion AmpliSeq Immune Repertoire Assay Plus, TCRB. To evaluate T cell convergence within each biopsy, we searched for instances where TCRB chains were identical in amino acid space (shared variable gene identity and CDR3 amino acid sequence) but had distinct nucleotide sequences owing to N-addition and exonucleotide chewback within the V-D and D-J junctions of the CDR3. To provide context, we evaluated evidence for T cell convergence with T cell repertoires derived from healthy donor peripheral blood leukocytes (PBL). Results Sequencing of melanoma biopsies yielded an average of 6029 clones per sample. 11 of 85 samples yielded fewer than 100 clones and were eliminated from downstream analysis. Convergent T cell receptors were identified in 68/74 (92%) of tumor infiltrating T cell repertoires having greater than 100 detected clones. The frequency of convergent rearrangements was approximately 50-fold greater in the melanoma-infiltrating T cell repertoire than healthy PBL samples (p Conclusions These data suggest that T cell convergence may be a common feature of the melanoma infiltrating T cell repertoire. Convergence was more frequently observed within the TME than T cell repertoires derived from healthy PBL, consistent with elevated antigen-driven T cell selection within the TME. The extent to which convergence is a feature of the TME in other cancers is not yet known. T cell receptor convergence may be driven by T cell responses to tumor neoantigen within the TME. In such case, in silico identification of convergent T cell receptors by long-amplicon sequencing may serve as an approach for rapid identification of antigen-specific T cell receptors for future therapeutic use. For research use only. Citation Format: Timothy J. Looney, Sean Glenn, Sarabjot Pabla, Jeff Conroy, Carl Morrison, Alice Zheng, Lauren Miller, Elizabeth Linch, Denise Topacio, Geoff Lowman, Fiona Hyland, Mark Anderson. Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4668.

Abstract 1631: Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM

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

Abstract 3567: Sequencing the human TCRβ repertoire on the Ion S5™ System

Next-generation Sequencing (NGS) is proving an important tool in increasing understanding of the human immune system, and thereby cancer immunology. αβ-T cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβ-T cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with Leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’RACE protocol than the commonly used BIOMED2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Denise S. Topacio-Hall, Tim Looney, Yongming Sun, Lauren Miller, Elizabeth Linch, Geoffrey Lowman, Lifeng Lin, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the human TCRβ repertoire on the Ion S5™ System [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 3567. doi:10.1158/1538-7445.AM2017-3567