Abstract 141: TCR sequencing from tissue micro-regions and single cells utilizing RareCyte CytePicker and Archer Immunoverse technologies
Authors: Johnson Laura , Hartje Luke , U'Ren Lance , Ericson Nolan , Podyminogin Rebecca , George Tad , Daniel Steve 
Published: August 2019

Abstract
Background: T-cell receptor (TCR) based immunotherapies are becoming an important cornerstone of immuno-oncology. Complete TCR sequencing requires single-cell resolution to capture both α and β chains. There is great interest in obtaining single-cell TCR sequences from archived tumor tissue. This task requires technology that can not only retrieve single cells but also sequence degraded RNA from archived tissues samples at the single cell level. The RareCyte CyteFinder® platform provides integrated multi-parameter imaging and retrieval capabilities for identification and isolation of rare cells and microscopic regions of interest (ROI) for molecular analyses. Archer has developed the Immunoverse™ platform of targeted Next-Generation Sequencing (NGS) assays to characterize the human immune repertoire from partially degraded RNA inputs. Combining these two technologies affords the unique potential to accelerate engineered cell-based therapeutic by sequencing TCR from individual cells.
Methods: 40 um ROI containing 1 to 10 T-cells were isolated from fresh/frozen (OCT) melanoma or OCT and FFPE tonsil using the CyteFinder® system. In addition, single flu antigen-specific T-cells were retrieved from live cell preparations. OCT tonsil sections were stained with a 3-color panel to discriminate T cell and B cell zones, and OCT tumor sections were stained with a 6-color panel to identify immune infiltrate, while FFPE sections were stained with a nuclear marker. The Archer® Immunoverse™ TCR All Chains library preparation kit was used to generate libraries which were then sequenced on the Illumina NextSeq platform. The resultant library sequences were deduplicated, error corrected, aligned to reference V, (D), J and C regions of TCRs, and assembled to identify clonotypes from α and β chains using the Archer® Analysis tool.
Results: TCR transcripts were observed from RNA isolated from as few as one cell. CDR3 sequences with V and J segments from single flu-targeting T-cells were obtained and found to match previously published sequences. TCR transcripts were observed in all ROI retrieved from OCT and FFPE tonsil when processed with the Immunoverse™ TCR assay. The numbers of transcripts observed correlated with the number of isolated cells analyzed. The sample source also affected the number of observed clones with more clones observed from cells isolated from fresh/frozen tissue.
Conclusions: These results support the utility of the RareCyte platform and Archer® Immunoverse™ TCR assay for profiling RNA derived from low numbers of cells in either fresh/frozen tissue, FFPE tissue, or live cells. A workflow that combines RareCyte and Archer technologies shows promise as a method for pairing α and β chain TCR sequences from RNA isolated from a single cell.
Citation Format: Laura Johnson, Luke Hartje, Lance U'Ren, Nolan Ericson, Rebecca Podyminogin, Tad George, Steve Daniel. TCR sequencing from tissue micro-regions and single cells utilizing RareCyte CytePicker and Archer Immunoverse technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 141.