Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

Viral escape from NK-cell-mediated immunosurveillance: A lesson for cancer immunotherapy?

Natural killer (NK) cells are innate lymphocytes that participate in immune responses against virus-infected cells and tumors. As a countermeasure, viruses and tumors employ strategies to evade NK-cell-mediated immunosurveillance. In this review, we examine immune evasion strategies employed by viruses, focusing on examples from human CMV and severe acute respiratory syndrome coronavirus 2. We explore selected viral evasion mechanisms categorized into three classes: (1) providing ligands for the inhibitory receptor NKG2A, (2) downregulating ligands for the activating receptor NKG2D, and (3) inducing the immunosuppressive cytokine transforming growth factor (TGF)-ß. For each class, we draw parallels between immune evasion by viruses and tumors, reviewing potential opportunities for overcoming evasion in cancer therapy. We suggest that in-depth investigations of host-pathogen interactions between viruses and NK cells will not only deepen our understanding of viral immune evasion but also shed light on how NK cells counter such evasion attempts. Thus, due to the parallels of immune evasion by viruses and tumors, we propose that insights gained from antiviral NK-cell responses may serve as valuable lessons that can be leveraged for designing future cancer immunotherapies.

Small Interfering RNA Delivery Into Primary Human Natural Killer Cells for Functional Gene Analyses.

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.

Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.

BACKGROUND: 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)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. METHODS: 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. RESULTS: ADAPT-NK cells were >90% 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 CD45dim blast subtypes. CONCLUSIONS: These 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.

SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.

Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.