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Studying gene functions in human natural killer (NK) cells is key for advancing the understanding of NK cell biology and holds promise to pave the way for improving NK cell therapies against cancer. However, NK cells are challenging to manipulate, and investigation of gene functions in NK cells is hampered by variable delivery efficiencies and impaired viability upon electroporation, lipofection, or viral transduction. Here, we report a simple workflow for delivery of commercially available small interfering RNA molecules into primary human NK cells to enable functional gene analyses. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enrichment of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Preparation of small interfering RNA Basic Protocol 3: Delivery of small interfering RNA into natural killer cells Support Protocol 1: Isolation of human peripheral blood mononuclear cells from buffy coats Support Protocol 2: Thawing and recovery of cryopreserved peripheral blood mononuclear cells Support Protocol 3: Evaluation of natural killer cell purity following magnetic enrichment Support Protocol 4: Exemplary assessment of knockdown efficiency.

Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML.ADAPT-NK cells were90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.

The molecular networks that regulate natural killer (NK) cell functions are not completely understood. Here, we present a workflow for efficient delivery of siRNA into human NK cells without compromising viability. This methodology represents a promising approach for rapidly interrogating gene functions in primary human NK cells.