Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform
Transcript-level study of rare antigen-specific T cells needs the intact cell and not just its image; this AACR 2019 poster reports single influenza- and Mart-1-tetramer-positive T cells identified and retrieved from normal donor blood on the RareCyte platform, with the influenza-tetramer-positive cells RNA sequenced to confirm their flu-specific identity.
Presented by RareCyte.
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- Antigen-specific T cells were identified by membrane ring tetramer staining on a multiparameter imaging panel. Live purified T cells from normal donors were stained with CD3, CD8, an HLA-A2 restricted tetramer against influenza-M1 (GILGFVFTL) or Mart-1 (ELAGIGILTV), and an exclusion cocktail of CD4, CD14, CD15, CD20 and DAPI; automated image analysis then kept only cells positive in the tetramer and CD8 channels and negative in the exclusion channel.
- Every flu tetramer-positive cell retrieved yielded a productive T cell receptor pair. 10 of the 10 single cells picked with the CytePicker module gave productive TCR alpha/beta pairs after whole transcriptome amplification and MiSeq sequencing, and 7 of 10 carried exact or near-exact matches to published influenza-specific TCR sequences under TraCeR analysis.
- Peptide stimulation left a readable activation signature in the retrieved single cells. Comparing stimulated with control flu tetramer-positive T cells returned 760 differentially expressed genes, 73 down-regulated and 687 up-regulated, and STRING analysis of the top 50 mapped onto TCR signaling and inflammatory response/cytokine signaling pathways.
