<em>Correcting functional gaps in iPSC-derived NK cells through CRISPR screens and transcriptional reference mapping</em> — ASN Events
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  3. Correcting functional gaps in iPSC-derived NK cells through CRISPR screens and transcriptional reference mapping

Correcting functional gaps in iPSC-derived NK cells through CRISPR screens and transcriptional reference mapping (#271)

Giovanna Perinetti Casoni 1 2 , Camille Philippon 2 , Herman Netskar 1 2 , Minoru Kanaya 2 , Aline Pfefferle 1 3 , Karl-Johan Malmberg 1 2 3
  1. The Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
  2. Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital and University of Oslo, Oslo, Norway
  3. Center for Hematology and Regenerative Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

 

Induced pluripotent stem cells (iPSCs) provide an unlimited source of customizable natural killer (NK) cells for immunotherapy. Differentiating NK cells from a few well-characterized iPSC clones ensures greater homogeneity and consistency than those derived from peripheral blood (PB). The iPSC platform also facilitates multiplexed genetic manipulation, enhancing cell specificity, persistence, and homing. However, the development of the functional template in iPSC-derived NK cells (iNK) and how this is tuned by acquisition of inhibitory receptors remains poorly defined.

In this study, we examined receptor repertoires, transcriptional states, and functions in various genetically engineered iNK lines. Transcriptional mapping positioned iNK cells near cytokine-activated "early" NKG2A+CD56dim PB-derived NK cells. Although early in differentiation, iNK cells exhibited a strong cytotoxic program with high expression of Eomes, granzyme B, DNAM-1, and NKG2D. However, gaps in transcription factors and regulons, including TBX21 and KLF3, linked to fully mature PB NK cells were observed.

Interestingly, iNK cells from reprogrammed T cell populations with fixed KIR repertoires gradually reacquired a diverse KIR repertoire. Driven by epigenetic promoter silencing, this variability resulted in iNK cells with KIR distributions mirroring the natural diversity seen in donors. Although KIR signaling and education were attenuated in iNK cells after feeder-cell based expansion, KIR expression influenced missing-self specificity against targets lacking the cognate HLA-C ligands. Thus, stochastic KIR acquisition during iNK differentiation leads to a genetically diverse KIR repertoire that may impact iPSC-based NK cell immunotherapy specificity. By combining gene editing to correct transcriptional immaturity with insights into the natural stochastic process of KIR diversification, our strategy establishes a robust framework for creating iNK cells with enhanced cytotoxicity and tumor-targeting specificity for adoptive immunotherapy.