NK co-culture CRISPR screen in the 721.221 B-cell lymphoblast
Therefore, to gain a deeper understanding of the interaction between NK cells and B-cell malignancies, we performed here a genome-wide CRISPR screen in the 721.221 cell line (referred to as 221), an EBV-transformed lymphoblastoid B-cell line. This cell line had been selected for the loss of classical HLA-I genes [14] and is therefore sensitive to lysis by NK cells. As expected, the loss of ligands for NK activation receptors resulted in reduced sensitivity. However, deletion of a few other genes, including Signal Peptide Peptidase-Like 3 (SPPL3), had the greatest impact on resistance to lysis by NK cells. Here, we describe how SPPL3 is a crucial controller of the interaction between NK cells and their targets. We found that the increased amount of complex N-glycans in SPPL3-deleted tumor cells limits the binding of NK cell receptors and facilitates immune evasion. We further identified glycosyl transferases that were responsible for the resistant phenotype through a secondary screen in SPPL3-deleted cells and analysis of mutations that restored sensitivity to lysis by NK cells. Overall design: GeCKO V2 library-transduced 221 cells were selecteted by co-culturing with NK cells. 221 cells expressing CRIPSR library but not co-cultured with NK cells were used as control.  For glycosylation-focused secondary CRISPR screen, the glycoGene library was used. SPPL3-deleted or sgNT 221 cells expressing lentiBlast-Cas9 were transduced with library. Cells were treated with doxycycline for 10 days to induce sgRNA expression before co-incubation with NK cells. For RNA-seq analysis, transcriptome of SPPL3-KO and control (sgNT) 221 cells were compared.