Cells of the innate immunity play an important role in tumor immunotherapy. Thus, NK cells can control tumor growth and metastatic spread. Thanks to their strong cytolytic activity against tumors, different approaches have been developed for exploiting/harnessing their function in patients with leukemia or solid tumors. Pioneering trials were based on the adoptive transfer of autologous NK cell-enriched cell populations that were expanded in vitro and co-infused with IL-2. Although relevant results were obtained in patients with advanced melanoma, the effect was mostly limited to certain metastatic localizations, particularly to the lung. In addition, the severe IL-2-related toxicity and the preferential IL-2-induced expansion of Treg limited this type of approach. This limitation may be overcome by the use of IL-15, particularly of modified IL-15 molecules to improve its half-life and optimize the biological effects. Other approaches to harness NK cell function include stimulation via TLR, the use of bi- and tri-specific NK cell engagers (BiKE and TriKE) linking activating NK receptors (e.g. CD16) to tumor-associated antigens and even incorporating an IL-15 moiety (TriKE). As recently shown, in tumor patients, NK cells may also express inhibitory checkpoints, primarily PD-1. Accordingly, the therapeutic use of checkpoint inhibitors may unleash NK cells against PD-L1⁺ tumors. This effect may be predominant and crucial in tumors that have lost HLA cl-I expression, thus resulting "invisible" to T lymphocytes. Additional approaches in which NK cells may represent an important tool for cancer therapy, are to exploit the unique properties of the "adaptive" NK cells. These CD57⁺ NKG2C⁺ cells, despite their mature stage and a potent cytolytic activity, maintain a strong proliferating capacity. This property revealed to be crucial in hematopoietic stem cell transplantation (HSCT), particularly in the haplo-HSCT setting, to cure high-risk leukemias. T depleted haplo-HSCT (e.g. from one of the parents) allowed to save the life of thousands of patients lacking a HLA-compatible donor. In this setting, NK cells have been shown to play an essential role against leukemia cells and infections. Another major advance is represented by chimeric antigen receptor (CAR)-engineered NK cells. CAR-NK, different from CAR-T cells, may be obtained from allogeneic donors since they do not cause GvHD. Accordingly, they may represent "off-the-shelf" products to promptly treat tumor patients, with affordable costs. Different from NK cells, helper ILC (ILC1, ILC2 and ILC3), the innate counterpart of T helper cell subsets, remain rather ambiguous with respect to their anti-tumor activity. A possible exception is represented by a subset of ILC3: their frequency in peri-tumoral tissues in patients with NSCLC directly correlates with a better prognosis, possibly reflecting their ability to contribute to the organization of tertiary lymphoid structures, an important site of T cell-mediated anti-tumor responses. It is conceivable that innate immunity may significantly contribute to the major advances that immunotherapy has ensured and will continue to ensure to the cure of cancer.

In the last years, immunotherapy with antibodies against programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has shown remarkable efficacy in the treatment of different types of tumours, representing a true revolution in oncology. While its efficacy has initially been attributed only to unleashing T cell responses, responsivity to PD-1/PD-L1 blockade was observed in some tumours with low Human Leukocyte Antigen (HLA) I expression and increasing evidence has revealed PD-1 surface expression and inhibitory function also in natural killer (NK) cells. Thus, the contribution of anti-PD-1/PD-L1 therapy to the recovery of NK cell anti-tumour response has recently been appreciated. Here, we summarize the studies investigating PD-1 expression and function in NK cells, together with the limitations and perspectives of immunotherapies. A better understanding of checkpoint biology is needed to design next-generation therapeutic strategies and to improve the clinical protocols of current therapies.

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.

Immune evasion is a key strategy adopted by tumor cells to escape the immune system while promoting their survival and metastatic spreading. Indeed, several mechanisms have been developed by tumors to inhibit immune responses. PD-1 is a cell surface inhibitory receptor, which plays a major physiological role in the maintenance of peripheral tolerance. In pathological conditions, activation of the PD-1/PD-Ls signaling pathway may block immune cell activation, a mechanism exploited by tumor cells to evade the antitumor immune control. Targeting the PD-1/PD-L1 axis has represented a major breakthrough in cancer treatment. Indeed, the success of PD-1 blockade immunotherapies represents an unprecedented success in the treatment of different cancer types. To improve the therapeutic efficacy, a deeper understanding of the mechanisms regulating PD-1 expression and signaling in the tumor context is required. We provide an overview of the current knowledge of PD-1 expression on both tumor-infiltrating T and NK cells, summarizing the recent evidence on the stimuli regulating its expression. We also highlight perspectives and limitations of the role of PD-L1 expression as a predictive marker, discuss well-established and novel potential approaches to improve patient selection and clinical outcome and summarize current indications for anti-PD1/PD-L1 immunotherapy.

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.

The highly destructive mechanisms by which the immune system faces microbial infections is under the control of a series of inhibitory receptors. While most of these receptors prevent unwanted/excessive responses of individual effector cells, others play a more general role in immunity, acting as true inhibitory checkpoints controlling both innate and adaptive immunity. Regarding human NK cells, their function is finely regulated by HLA-class I-specific inhibitory receptors which allow discrimination between HLA-I+, healthy cells and tumor or virus-infected cells displaying loss or substantial alterations of HLA-I molecules, including allelic losses that are sensed by KIRs. A number of non-HLA-specific receptors have been identified which recognize cell surface or extracellular matrix ligands and may contribute to the physiologic control of immune responses and tolerance. Among these receptors, Siglec 7 (p75/AIRM-1), LAIR-1 and IRp60, recognize ligands including sialic acids, extracellular matrix/collagen or aminophospholipids, respectively. These ligands may be expressed at the surface of tumor cells, thus inhibiting NK cell function. Expression of the PD-1 checkpoint by NK cells requires particular cytokines (IL-15, IL-12, IL-18) together with cortisol, a combination that may occur in the microenvironment of different tumors. Blocking of single or combinations of inhibitory receptors unleashes NK cells and restore their anti-tumor activity, with obvious implications for tumor immunotherapy.