Abstract
Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.
Highlights
Natural killer (NK) cells belong to the innate immunity and represent the first-line defense of the immune system in the control of pathogens, tumor growth, and metastasis diffusion
Scanning electron microscopy analysis (Figures 1C,D) showed an overview of NK-cell-derived microvesicle (NKMV) and NK cells release exosomes (NKExo) and confirmed a high density of both types of EVs in the 10,000 × g fraction (Figure 1C), while that one purified at 100,000 × g consisted prevalently of vesicles with dimensions smaller than 150 nm, which were accordant with an exosome population (Figure 1D)
We evaluated the activity of NKEVs on NK cells in peripheral blood mononuclear cells (PBMCs) depleted from monocytes, in the presence or absence of CD3/CD28 T-cell stimuli
Summary
Natural killer (NK) cells belong to the innate immunity and represent the first-line defense of the immune system in the control of pathogens, tumor growth, and metastasis diffusion. Human peripheral blood NK cells can be identified by their expression of CD56 in the CD3neg lymphocyte gate. They can be further divided into CD56brightCD16+ NK cells, which produce interferon gamma (IFNγ) and represent 10% of NK cell population, and into cytotoxic CD56dimCD16bright NK cells, which represent 90% of the NK cell population under physiological conditions [4]. NK cells constitutively express a lytic machinery able to kill target cells independently from any previous activation and can be induced to migrate toward inflammation sites and infected or transformed cells, by different chemoattractants [5]. Previous studies have shown that a low activity of NK cells in peripheral blood is associated with increased cancer risk [6]. Considerable efforts are being made to achieve better activation, proliferation, and viability of NK cells, to be used in cancer immunotherapy [9]
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