A GFP-transfected HFWT cell line, GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay

An anchorage-dependent Wilms’ tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3^?CD56⁺ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard ⁵¹Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A

In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.     

Identification of an ADAM17 Cleavage Region in Human CD16 (FcγRIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1′ positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

Activated,but not resting,T cells can be recognized and killed by syngeneic NK cells

We demonstrate that IL-2-activated NK cells or lymphokine-activated killer cells recognize and kill syngeneic CD4(+) and CD8(+) T cells that have been activated by APCs. Induction with APC required TCR-specific Ag, and lysis was perforin mediated. Brefeldin A, which disrupts protein transport, inhibited the sensitivity induced by activation. In BALB/c, expression of NKG2D ligands correlated with lysis and could be inhibited by brefeldin A. As well, addition of anti-NKG2D mAb to a killing assay completely abrogated lysis. Transduction of mouse NKG2D into a human NK cell line, YTSeco, conferred upon it the ability to kill activated BALB/c T cells, indicating that NKG2D is necessary for recognition. Our data provide a basis for studying a role for NK cells in T cell regulation.     

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8α⁺ conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8⁺ T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3⁺ DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8⁺ T cells through XCR1.     

Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.     

Basic fibroblast growth factor and interleukins 4 and 6 stimulate the release of IFN-gamma by individual NK cells.

Both the secretory and cytotoxic activity of natural killer (NK) cells are known to be regulated by such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In the present study we have used the reverse hemolytic plaque assay to investigate either the direct effects of the protein kinase activator, phorbol myristate acetate (PMA), or exposure to recombinant human interleukins 2, 4, and 6 (IL-2, IL-4, and IL-6) tumour necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) on the release of IFN-gamma by individual, immunoidentified NK cells isolated from peripheral blood. This sensitive immunoassay was adapted and coupled with immunocytochemistry not only to immunophenotype and enumerate cells secreting IFN-gamma in a given cell population, but also to quantify the amount of this cytokine released per individual cell. These studies have confirmed mononuclear cells with the morphology of large granular lymphocytes and the immunophenotype of CD3-/CD16+ NK cells to be the predominant source of spontaneously released IFN-gamma in vitro. In contrast to this, fewer than 2% of the CD3+ T cells secreted detectable levels of this cytokine during the assay, irrespective of the stimulus applied. Whilst TNF-alpha had no significant effect on IFN-gamma release by NK cells, a 6-hr exposure to IL-2 or PMA stimulated an increase in the amount secreted per single cell. Furthermore, bFGF and interleukins 4 and 6 elicited a marked, dose-dependent stimulation of IFN-gamma secretion by this cell type. However, exposure to these cytokines did not alter the number of cells capable of releasing detectable levels of IFN-gamma during the assay. These studies demonstrate that (i) both the spontaneous and stimulated release of IFN-gamma by NK cells can be visualized and quantified at the single-cell level using this sensitive immunoassay, and (ii) bFGF and interleukins 2, 4, and 6, but not TNF-alpha, are potent stimulants of IFN-gamma secretion by CD3-/CD16+ NK cells.     

The expression,regulation and adhesion function of a novel CD molecule,CD226, on human endothelial cells

CD226 is a 67 kDa type I transmembrane glycoprotein mainly expressed on activated T cells, NK cells and platelets, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. Here we found that the expression of CD226 protein and CD226mRNA were very weak in resting HUVEC and ECV304 cells, whereas high level expression could be observed when these cells were stimulated. The binding activities between activated endothelial cells and activated Jurkat cells could be partly blocked by CD226/Ig fusion protein. Similarly, CD226/Ig could also partly block the adhesion between activated endothelial cells and some leukocytes or colo205 cells. These data provided the evidence that activated endothelial cells could express high level of CD226, and CD226 was involved in the endothelial cells' adhesion. The above findings suggested that CD226 is a novel inducible adhesion molecule on human endothelial cells.     

Adhesion characteristics of human interleukin 2-activated natural killer cells

Culture of human monocyte-depleted peripheral blood mononuclear cells with recombinant IL2 (rIL2) induced adherence to plastic by 24 hr and subsequent proliferation in a subpopulation of lymphocytes with phenotypic and functional characteristics of activated natural killer (NK) cells. Purified human NK cells activated in the presence of IL2 for 24 hr upregulated the expression of the CD11c (p150.95) and CD11a antigen but not other cellular adhesion molecules (CAM). After further incubation with IL2, NK cells displayed upregulation of all of the antigens in the CD11/CD18 family of CAM. The process of adhesion was strictly dependent on culture in the presence of IL2, divalent cations, and active cellular metabolism. Adhesion also was dependent on expression of CAM on the cell surface, since monoclonal antibodies to CAM inhibited adhesion of activated NK cells to varying degrees (from 50 to 80%). An antibody (LeuM5) to the CD11c antigen (p150.95) gave the highest level of inhibition, and anti-CD11a (LFA-1) also was inhibitory, while anti-CD56 (NKH1) or anti-CD11b did not interfere with adhesion to plastic. Anti-CD11c was also the most effective in initiating the detachment of adherent-phase NK cells. Antibodies to CD18 or CD2 antigen also inhibited binding of NK cells to plastic. The blocking effects of anti-CD2 and anti-CD11a were additive in this system. On the surface of plastic-adherent and motile NK cells, all CAM except the CD56 antigen had a polar or bipolar distribution, as determined by staining with anti-CAM antibodies. Surface antigens CD11b, CD11c, CD2, and CD18 on nonadherent NK cells were clustered at the cellular poles by both immunofluorescence and immunogold electron microscopy, whereas CD11a (LFA-1) and CD56 antigens were distributed diffusely. CAM, especially CD11c, were also detected in cytoplasmic granules by immunostaining in IL2-activated NK cells. Thus, CAM may be stored in granules, allowing for their rapid transfer to the cell membrane in response to activation. Our results indicate that CAM are upregulated in IL2-activated NK cells and that some of these molecules (e.g., CD11c) play an important role in the development of plastic adherence by a subpopulation of these cells.     

Modulation of ongoing human immunoglobulin synthesis by natural killer cells

Freshly separated human NK cells (NKH-1+) inhibited IgE synthesis from IgE myeloma U266/AF-10 as much as 70% whereas they enhanced IgG and IgA synthesis 200 and 500% from the lymphoblastoid cell lines GM-1500 and GM-1056, respectively. The inhibition of IgE synthesis by NK cells was due to a direct cytolytic effect on AF-10. This could be reversed using K562 cells in a cold target competition assay. NK cells also inhibited spontaneous IgE as well as IgG and IgA synthesis from B cells of highly atopic donors. On the other hand the enhancement of Ig secretion by NKH-1+ cells was shown to be mediated by soluble factors released from NK cells. Furthermore when NK cells were preincubated with immune complexes (IgE-IC) constructed of human IgE and mouse IgG1 monoclonal anti-human IgE, inhibition of IgE synthesis was reversed, and in some cases actual enhancement of IgE synthesis was observed, while enhancement of IgG and IgA synthesis was not affected. In contrast to NK cells, T cells depleted of NK cells (T-NK), when activated by IgE-IC, suppressed IgE synthesis in an isotype specific fashion. Thus, NK and T-cell modulation of ongoing Ig synthesis involve distinct mechanisms.     

Identification of resting and type I IFN-activated human NK cell miRNomes reveals microRNA-378 and microRNA-30e as negative regulators of NK cell cytotoxicity

NK cells are important innate immune cells with potent cytotoxicity that can be activated by type I IFN from the host once infected. How NK cell cytotoxicity is activated by type I IFN and then tightly regulated remain to be fully elucidated. MicroRNAs (miRNAs, or miRs) are important regulators of innate immune response, but the full scale of miRNome in human NK cells remains to be determined. In this study, we reported an in-depth analysis of miRNomes in resting and IFN-α-activated human NK cells, found two abundant miRNAs, miR-378 and miR-30e, markedly decreased in activated NK cells by IFN-α, and further proved that miR-378 and miR-30e directly targeted granzyme B and perforin, respectively. Thus, IFN-α activation suppresses miR-378 and miR-30e expression to release cytolytic molecule mRNAs for their protein translation and then augments NK cell cytotoxicity. Importantly, the phenomena are also confirmed in human NK cells activated by other cytokines and even in the sorted CD16(+)CD56(dim)CD69(+) human NK cell subset. Finally, miR-378 and miR-30e were proved to be suppressors of human NK cell cytotoxicity. Taken together, our results reveal that downregulated miR-378 and miR-30e during NK cell activation are negative regulators of human NK cell cytotoxicity, providing a mechanistic explanation for regulation of NK cell function by miRNAs.     

Mammalian Pol III Promoter H1 can Transcribe shRNA Inducing RNAi in Chicken Cells

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. RNAi based on DNA vector is not sufficiently established in chicken species. The present study was performed to evaluate RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 in the chicken cells by using a dual fluorescence reporter assay, a plasmid encoding GFP and a plasmid encoding RFP. The evaluation of RNAi efficiency was performed in two kinds of chicken cell type: primary CEF cells and chicken DT-40 cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescent microscopy, and their mRNAs content were analyzed by quantitative RT-PCR. The intensity of the green fluorescence generated by GFP was greatly suppressed by human H1 promoter transcribed GFP-shRNA. Quantitative RT-PCR analysis showed that normalized GFP mRNA expression was reduced to 37 and 32 in primary CEF and DT-40 cells, respectively. In contrast to GFP, the intensity of the red fluorescence generated by RFP protein and the RFP mRNA levels remained unchanged. Consequently, it was concluded that the RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 is applicable to suppress the gene expression specifically and efficiently in chicken cells. Jing Yuan and Xiaobo Wang - These authors contributed equally to this work.     

Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure.

We have previously shown that human cultured trophoblast cells are resistant to lysis by natural killer (NK) cells from both peripheral blood and decidua although cells are present in decidua which do exhibit NK activity against K562(1). Using a cold-target inhibition assay and a single-cell conjugate assay we have now examined whether these trophoblast cells have NK target structures on their surfaces. Our findings indicate that first-trimester human trophoblast cells do not express surface structures recognized by decidual Leu19+ (CD56+) large granular lymphocytes (LGLs) isolated from human decidua. Immunostaining of the conjugates formed between decidual NK effectors and K562 cells confirmed that these effector cells are CD56+ LGLs.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Bone marrow cell modulation and inhibition of myelopoiesis by large granular lymphocytes and natural killer cells

Non-adherent Percoll-separated large granular lymphocytes (LGLs) fractionated by fluorescence-activated cell sorter into CD16+ CD4- natural killer (NK) cells and CD16- CD4+ T cells, were co-cultured with bone marrow (BM) cells previously depleted of adherent T and/or NK cells by immunoadsorption (panning) and plated in a clonogenic assay to assess myeloid colony formation (CFU-gm growth). LGLs, NK cells and LGL T cells [low buoyant density (LBD) T cells] each significantly reduced colony-stimulating factor (CSF)-dependent CFU-gm growth to 70% of control values (p less than 0.05). Non-LGL T cells [high buoyant density (HBD) T cells] did not affect this growth. Incubation of the effector cells with human recombinant interleukin 2 prior to co-culturing did not alter these findings. The supernatants obtained from LGLs, NK cells and LBD T cells co-cultured with BM cells also inhibited CFU-gm growth to 70% of the control, whereas supernatants from effector cells which were not co-cultured with BM had no such effect. These supernatants from the LGL:BM co-cultured cells possessed NK cytotoxic factor (NKCF), but lacked alpha and gamma interferons, tissue necrosis factor-alpha, and prostaglandin E2. These results suggest that BM cells stimulate LGLs to produce NKCF, and that LGLs, CD16+ NK cells, and CD4+ CD16- LBD T cells activated by contact with BM cells inhibit CFU-gm growth.     

The IL-12 signature: NK cell terminal CD56+high stage and effector functions

We report that human peripheral NK cells expressing high CD56 levels (CD56(+high)) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56(+low) NK cells. CD56(+high) NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56(+)) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56(+high) than on CD56(+low) NK cells. Consistent with their activated phenotype, many CD56(+high) NK cells are cycling and mediate heightened effector functions (proliferation, IFN-gamma and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56(+low) NK cells all markers constitutively expressed on the CD56(+high) NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56(-/+low) NK cells acquire a CD56(+high) NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.