Brief treatment in-vitro with Dasatinib does not appreciably alter gene expression by CD8+ T cells

Pre-incubation of T cells with the protein kinase inhibitor dasatinib prevents internalisation of T cell receptors upon binding by peptide-MHC multimers and enhances multimer staining. If used with downstream sc-RNAseq of sorted T cells, there is a possibility that this treatment could influence gene expression. No significant changes in gene expression were detected between dasatinib-treated and untreated cells. To assess the effect of dasatinib, we combined mouse liver leukocytes from 3 male B10.BR individuals (H-2k) following a prime-boost against H-2Kb, and separated them into two pools. One was incubated with dasatinib (50nM) for 30 minutes at ambient temperature prior to staining with a panel of antibodies, the other was pre-incubated with staining buffer only. Treated and untreated cells were sorted into 3 populations of CD8+ T cells each (activated, virtual-central memory and naïve) using the BD Influx cytometer, followed by Sample Tag labelling. The tagged samples were pooled prior to capture on a BD Rhapsody instrument, and library preparation using the Rhapsody Whole Transcriptome Analysis kit, according to the manufacturer’s instructions. Libraries were sequenced using an Illumina NextSeq 1000 with subsequent processing via Seven Bridges.