CRISPR screening for cytotoxic granule degranulation pathway in CD8 T cell

Abstract

Abstract Degranulation of perforin-containing cytotoxic granules is important for NK cells and CD8 cytotoxic T lymphocytes (CTLs) to kill infected cells or tumors. Defects in this pathway cause the primary form of hemophagocytic lymphohistiocytosis (HLH), which is known as familial HLH (FHL) syndrome. Most of reported FHL causative genes encode molecules that are involved in the intracellular trafficking of cytotoxic granules and their fusion to the plasma membrane, followed by the delivery of their contents into the target cell through immunological synapses. However, other components essential for cytotoxic granule degranulation for CTLs are not well understood. To identify indispensable molecules for cytotoxic granule degranulation, we established an efficient in vitro CD8 amplification and degranulation system to perform a CRISPR-screening. To test this system, we transduced OT-I CD8 cells with a gRNA library targeting PIP 3binding proteins, which are important for cell adhesion, subcellular trafficking and T cell activation. Cells were harvested at short (Day 8) and medium-timepoints (Day 15) to evaluate the frequency of gRNAs in the degranulated (CD107+) and non-degranulated fractions. Of the top 10 hits of genes whose deletion suppressed degranulation, five were already listed in the 2022 IUIS classification of inborn error of immunity. We will use individual knockouts to evaluate how these genes affect degranulation. To expand to genome-wide screening, we have also subcloned a full library of sgRNAs into our high efficiency retroviral vector. Thus, we established an efficient assay system to perform a CRISPR-screening for CD8 T cells and can detect genes affected by primary immunodeficiencies. This work was supported by the Intramural Research Program of NIAID, NIH.