Multiplexed Identification of Cytotoxic Cell assay (PAINTKiller assay)

Cell mediated cytotoxicity plays important roles in host immune defence and cancer immunosurveillance. Current technologies that study cell cytotoxicity are target cell centric and lack the capability to identify and isolate cytotoxic effector cells. We developed the Multiplexed Identification of Cytotoxic Cell assay (PAINTKiller), a novel flow cytometry compatible method for direct identification and sorting of cytotoxic effector cells. We demonstrate that this technique can accurately identify cytotoxic effector cells which can be isolated, expanded and retained its superior cytotoxic capability. This technique can also be easily combined with other flow cytometry complementary assays, cell sorting and sequencing. The results indicate that PAINTKiller assay will be a powerful tool to uncover the determinants of functional immune response and will be of significant interest for cell therapy manufacturing workflow. Overall design: Human primary NK cells were enriched from PBMCs using EasySep™ Human NK Cell Enrichment Kit (STEMCELL Technologies, 19055) and expanded for 9-10 days using ImmunoCult™ NK Cell Expansion Kit (STEMCELL Technologies, 100-0711). For sequencing, expanded NK cells were modified with anti-CFSE capture antibody before co-cultured with or without CFSE-stained K562 at a ratio of 3:1. After 4 hours, cells were collected and pre-blocked by Fc-blocking reagent (Biolegend, 422301). Samples were stained with anti-FITC-PE (Southern Biotech, cat no: 6400-09) while concurrently barcoded with ß2M-targeting hashtag before pooled and stained with a cocktail of TotalSeq-A antibodies targeting PE, CD3, CD56, CD16, CD57, CCR7, CD45RA, CD107a, CD69, NKG2D, NKp44, and PD-1. Cells were washed with cold PBS/2% FBS and re-suspended in PBS before loading on the Chromium Controller (10x Genomics).