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.

KIR3DL1 is a natural killer (NK) cell receptor that recognizes the Bw4 epitope of human leukocyte antigen (HLA) class I molecules. Following hematopoietic stem cell transplantation for patients lacking Bw4, KIR3DL1‐expressing NK cells from Bw4‐positive donors can be alloreactive and eliminate tumor cells. However, KIR3DL1 alleles having T instead of C at nucleotide 320 (encoding leucine 86 instead of serine 86) are not expressed on the cell surface. Thus, not all individuals testing positive for KIR3DL1 are optimal donors for Bw4‐negative recipients. Therefore, we developed a method for genotyping codon 86, which was validated by its perfect correlation with NK cell phenotype for 100 donors of diverse KIR3DL1/S1 genotype. We typed 600 donors and found that ∼12.2% had the KIR3DL1 gene, but did not express cell‐surface KIR3DL1. By contrast, high‐expressing allotypes were identified when haplotypes from four families with duplicated KIR3DL1/S1 genes were characterized at high resolution. Identifying donors who have KIR3DL1 but lack cell‐surface KIR3DL1 would refine donor selection. With this technique, the number of individuals identified who may not be optimal donors for Bw4‐negative patients increases by threefold, when compared with standard methods. Taken together, we propose that allele typing of killer cell Ig‐like receptor (KIR) polymorphisms should become a standard practice when selecting donors.

Toll-like receptors (TLR)s are homo- or heterodimeric proteins, whose structure and function were widely described in the antigen presenting cells (APC), such as Dendritic cells (DC). Recently, the expression and the role of TLRs in fighting against pathogens, was described also in NK cells. Their activation and functional properties can be directly and indirectly modulated by agonists for TLRs. In particular CD56^bright NK cells subset, that is the most abundant NK cell subset in tissues and tumor microenvironment (TME), was mostly activated in terms of pro-inflammatory cytokine production, proliferation and cytotoxicity, by agonists specific for endosomal TLR8. The interplay between DC and NK, that depends on both cell-to-cell contact and soluble factors such as cytokines, promote both DC maturation and NK cell activation. Based on this concept, a TLR based immunotherapy aimed to activate NK-DC axis, may modulate TME by inducing a pro-inflammatory phenotype, thus improving DC ability to present tumor-associated antigens to T cells, and NK cell cytotoxicity against tumor cells. In this mini-review, we report data of recent literature about TLRs on human NK cells and their application as adjuvant in cancer vaccines or in combined tumor immunotherapy.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA—compared with control—siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

Toll-like receptors (TLRs) are the most well-defined pattern recognition receptors (PRR) of several cell types recognizing pathogens and triggering innate immunity. TLRs are also expressed on tumor cells and tumor microenvironment (TME) cells, including natural killer (NK) cells. Cell surface TLRs primarily recognize extracellular ligands from bacteria and fungi, while endosomal TLRs recognize microbial DNA or RNA. TLR engagement activates intracellular pathways leading to the activation of transcription factors regulating gene expression of several inflammatory molecules. Endosomal TLR agonists may be considered as new immunotherapeutic adjuvants for dendritic cell (DC) vaccines able to improve anti-tumor immunity and cancer patient outcomes. The literature suggests that endosomal TLR agonists modify TME on murine models and human cancer (clinical trials), providing evidence that locally infused endosomal TLR agonists may delay tumor growth and induce tumor regression. Recently, our group demonstrated that CD56^bright NK cell subset is selectively responsive to TLR8 engagement. Thus, TLR8 agonists (loaded or not to nanoparticles or other carriers) can be considered a novel strategy able to promote anti-tumor immunity. TLR8 agonists can be used to activate and expand in vitro circulating or intra-tumoral NK cells to be adoptively transferred into patients.

Killer immunoglobulin‐like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIR^pos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti‐KIR mAb termed HP‐DM1. Testing its binding to HEK‐293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP‐DM1 mAb exclusively reacts with KIR2DL1. Using site‐directed mutagenesis, we identified the four amino acids relevant for HP‐DM1 recognition: M44, S67, R68, and T70. HP‐DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA‐C K80 recognition by KIR2DL1. Based on the HP‐DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP‐DM1 binding, continuously updating of its reactivity will be facilitated.