Evaluation of transcriptional changes following stimulation of naïve, TCM, TEM and TEMRA CD8+ T cell subsets with AB248 compared to IL-2

Evaluation of transcriptional changes following stimulation of naïve, TCM, TEM and TEMRA CD8+ T cell subsets confirmed that AB248 recapitulated the expected IL-2 signature with high fidelity Human PBMCs from 3 donors were sorted into the following subsets based on the expression of CD45RO and CD62L markers: CD8+-naïve T cells (CD62L+CD45RO-), CD8+ central memory T cells (CD62L+CD45RO+), CD8+ effector memory T cells (CD62L-CD45RO+), and CD8+ effector cells (CD62L-CD45RO-). Briefly, cells were stained with antibodies to sort the individual CD8+ T cell subsets, sorted via FACS, and flow sorted cells were collected in AIM V™ medium (Gibco) and then treated at concentrations corresponding to the EC99 by pSTAT5 for of rhIL-2 (R&D Systems) or AB248 for 24h. Briefly, 70,000 to 200,000 cells were added to 96 well plates in 100 μL AIM V medium (Thermo). At the end of stimulation, cells were pelleted, and RNA isolated per manufacturer’s instructions (Arcturus PicoPure RNA Isolation Kit, Applied Biosystems). RNA was sent to Genewiz for sequencing using Ultra-Low Input RNA-Seq protocol (detects ≥10 pg RNA). Sequencing data was aligned against the human reference genome (Ensembl GrCh38) using the STAR software package and quantified using the featureCounts package. Differential expression analysis was performed using the DESeq2 Bioconductor package.