Natural killer (NK) cells, the most important effectors of the innate lymphoid cells (ILCs), play a fundamental role in tumor immune-surveillance, defense against viruses and, in general, in innate immune responses. NK cell activation is mediated by several activating receptors and co-receptors able to recognize ligands on virus-infected or tumor cells. To prevent healthy cells from auto-aggression, NK cells are provided with strong inhibitory receptors (KIRs and NKG2A) which recognize HLA class I molecules on target cells and, sensing their level of expression, allow killing of targets underexpressing HLA-class I. In vivo, NK cell-mediated anti-tumor function may be suppressed by tumor or tumor-associated cells via inhibitory soluble factors/cytokines or the engagement of the so called immune-check point molecules (e.g., PD1-PDL1). The study of these immune check-points is now offering new important opportunities for the therapy of cancer. In haemopoietic stem cell transplantation, alloreactive NK cells (i.e., those that express KIRs, which do not recognize HLA class I molecules on patient cells), derived from HSC of haploidentical donors, are able to kill leukemia blasts and patient's DC, thus preventing both tumor relapses and graft-versus-host disease. A clear correlation exists between size of the alloreactive NK cell population and clinical outcome. Thus, in view of the recent major advances in cancer therapy based on immuno-mediated mechanisms, the phenotypic analysis of cells and molecules involved in these mechanisms plays an increasingly major role. © 2017 International Clinical Cytometry Society.

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer.

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1+ NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1−, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56dim than in CD56bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.

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