Deep profiling of single T cell receptor repertoire and phenotype with targeted RNA-seq (TECH2P.927)

Abstract

Abstract Analysis of TCR repertoires is useful for monitoring T cell responses. Integrating TCR sequencing with expression of targeted genes at the single cell level allows comprehensive analysis of T cell function and specificity. RT-PCR reactions of single T cells were performed using 76 primers from VJC regions and 136 primers for targeted T cell genes. A series of nested PCR reactions incorporated with barcodes and adapters for pooled amplicons sequencing. A bioinformatics pipeline was used data analysis. 2880 single T cells were collected and tested. TCR sequences were obtained in up to 90% of the wells. Based on the CDR3 region, multiple dominant TCR clones were identified. 52 of 68 target genes were validated with sorted target-specific T cells. FOXP3, IL10, PRF1, IL13, and RUNX3 were highly expressed in memory activated cells. The specific CD8+ T cells had high frequency of CCR9, LAG3, CD8, CD62L, and IFNG expression. TGFB, TNFA, IL12, FOXP3, PD1, TBET, CTLA4, MKI67, RUNX1, and GATA3 were major biomarkers in the specific CD4 cells. The gene expression patterns were often associated with TCR repertoire CDR3 variations and/or the TCRβ and TCRα chain usage although the same TCRα/β sequences may have different target genes expressed. We expanded a method enabling sequencing of TCR repertoire and multiple functional genes in single, sorted, T cells through targeted RNA-seq technology. This approach could reveal important insights into T cell functions and clonal development.