Abstract 1211: Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform
Authors: Ericson Nolan , Kaldjian Eric , George Tad , U'Ren Lance 
Published: August 2019

Abstract
Background. The immune system provides antigen-specific protection against pathogens as well as malignancies, both of which evolve strategies to evade immune surveillance and containment. Effective immune response often depends on activation of rare antigen-specific immune cell sub-types. The RareCyte platform provides integrated multi-parameter imaging and retrieval capabilities that allow phenotypic identification and isolation of rare cells for sequence and transcript level analyses in order to study the complexity of host defense.
Methods. Antigen-specific T cells were identified by immunofluorescent staining of live purified T cells from normal donors with the following panel: CD3; CD8; exclusionary cocktail (CD4, CD14, CD15, CD20, and DAPI); and HLA-A2 restricted tetramer (MBL). Tetramers had specificity against influenza-M1 (GILGFVFTL) and the melanoma antigen Mart-1 (ELAGIGILTV). For differential expression experiments, cells were stimulated overnight with specific target peptide or control irrelevant peptide, stained with the multiparameter panel containing the relevant tetramer, then imaged using the CyteFinder® instrument. Automated image analysis identified candidate cells with positive signal in the tetramer and CD8 channels and negative signal in the exclusion channel. Single cells displaying membrane ring tetramer distributions were retrieved using the CytePicker® module, followed by single cell RNA sequencing using the SMART-Seq® v4 kit (Takara Bio) and the MiSeq® instrument (Illumina). Differential gene expression of stimulated versus unstimulated cells was performed using DESeq2 software (BioConductor). Single-cell RNA seq FASTQ files were analyzed with the TraCeR computational method to identify TCR alpha and beta chains in both stimulated and unstimulated cells.
Results. Single influenza and Mart-1 antigen-specific T cells in donor blood samples were detected and retrieved using the CyteFinder® instrument. Anti-influenza specificity was confirmed by RNA sequencing, which revealed a majority of alpha/beta TCR pairings with identical match to literature reports. Comparative gene expression analysis of activated and control flu tetramer-positive T cells revealed 73 significantly down-regulated and 687 significantly up-regulated genes. Pathway analysis of differential expression revealed involvement of TCR signaling and inflammatory response/cytokine signaling.
Conclusions. The RareCyte platform can be used to visually identify and retrieve rare antigen-specific T cells from a bulk population by using tetramers against influenza-specific T cell receptors. T cell receptor sequencing confirmed the flu-specific identity of the tetramer-positive cells. RNA signatures of activation were identified at the single T cell level after peptide stimulation.
Citation Format: Nolan Ericson, Eric Kaldjian, Tad George, Lance U'Ren. Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform [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 1211.