Three-dimensionally Patterned Scaffolds Modulate the Biointerface at the Nanoscale.

The main aim of cell instructive materials is to guide in a controlled way cellular behavior by fine-tuning cell-material crosstalk. In the last decades, several efforts have been spent in elucidating the relations between material cues and cellular fate at the nanoscale and in the development of novel strategies for gaining a superior control over cellular function modulation. In this context, a particular attention has been recently paid to the role played by cellular membrane rearrangement in triggering specific molecular pathways linked to the regulation of different cellular functions. Here, we characterize the effect of linear microtopographies upon cellular behavior in three-dimensional (3D) environments, with particular focus on the relations linking cytoskeleton structuration to membrane rearrangement and internalization tuning. The performed analysis shown that, by altering the cellular adhesion processes at the micro- and nanoscale, it is possible to alter the membrane physical state and cellular internalization capability. More specifically, our findings pointed out that an increased cytoskeletal structuration influences the formation of nanoinvagination membrane process at the cell-material interface and the expression of clathrin and caveolin, two of the main proteins involved in the endocytosis regulation. Moreover, we proved that such topographies enhance the engulfment of inert polystyrene nanoparticles attached on 3D patterned surfaces. Our results could give new guidelines for the design of innovative and more efficient 3D cell culture systems usable for diagnostic, therapeutic, and tissue engineering purposes.

Imatinib and Nilotinib Off-Target Effects on Human NK Cells, Monocytes, and M2 Macrophages.

Tyrosine kinase inhibitors (TKIs) are used in the clinical management of hematological neoplasms. Moreover, in solid tumors such as stage 4 neuroblastomas (NB), imatinib showed benefits that might depend on both on-target and immunological off-target effects. We investigated the effects of imatinib and nilotinib on human NK cells, monocytes, and macrophages. High numbers of monocytes died upon exposure to TKI concentrations similar to those achieved in patients. Conversely, NK cells were highly resistant to the TKI cytotoxic effect, were properly activated by immunostimulatory cytokines, and degranulated in the presence of NB cells. In NB, neither drug reduced the expression of ligands for activating NK receptors or upregulated that of HLA class I, B7-H3, PD-L1, and PD-L2, molecules that might limit NK cell function. Interestingly, TKIs modulated the chemokine receptor repertoire of immune cells. Acting at the transcriptional level, they increased the surface expression of CXCR4, an effect observed also in NK cells and monocytes of patients receiving imatinib for chronic myeloid leukemia. Moreover, TKIs reduced the expression of CXCR3 (in NK cells) and CCR1 (in monocytes). Monocytes also decreased the expression of M-CSFR, and low numbers of cells underwent differentiation toward macrophages. M0 and M2 macrophages were highly resistant to TKIs and maintained their phenotypic and functional characteristics. Importantly, also in the presence of TKIs, the M2 immunosuppressive polarization was reverted by TLR engagement, and M1-oriented macrophages fully activated autologous NK cells. Our results contribute to better interpreting the off-target efficacy of TKIs in tumors and to envisaging strategies aimed at facilitating antitumor immune responses.

miR-24-3p down-regulates the expression of the apoptotic factors FasL and BIM in human natural killer cells.

MicroRNAs are involved in the regulation of different functions in immune and non-immune cells. Here we show that miR-24-3p functionally interacts with FASLG mRNA and down-regulates its expression. This interaction occurs in human natural killer cells (NK), leading to the modulation of FasL surface expression. Moreover, miR-24-3p also modulates the mRNA and protein expression of BIM in NK cells. Thus, it likely contributes to the control of both the extrinsic and intrinsic apoptotic pathways. In line with this hypothesis, inhibition of miR-24-3p improves both initiator caspase-8 and effector caspase-3 and -7 activities, increases cell apoptosis, and reduces cell viability. Our data suggest that miR-24-3p can act as a survival factor in NK cells, affecting the FasL-mediated killing of Fas expressing cells and the BIM-dependent cell death. More generally, miR-24-3p may condition the level of cell apoptosis, which increases at the contraction phase of the immune response when the clearance of various expanded effector cells is needed.

NK Cell Function Regulation by TGF-ß-Induced Epigenetic Mechanisms.

TGF-ß is a potent immunosuppressive cytokine that severely affects the function of NK cells. Tumor cells can take advantage of this ability, enriching their surrounding microenvironment with TGF-ß. TGF-ß can alter the expression of effector molecules and of activating and chemokine receptors, influence metabolism, induce the NK cell conversion toward the less cytolytic ILC1s. These and other changes possibly occur by the induction of complex gene expression programs, involving epigenetic mechanisms. While most of these programs are at present unexplored, the role of certain transcription factors, microRNAs and chromatin changes determined by TGF-ß in NK cells start to be elucidated in human and/or mouse NK cells. The deep understanding of these mechanisms will be useful to design therapies contributing to restore the full NK function.

Engineered Cell-Based Therapeutics: Synthetic Biology Meets Immunology.

Synthetic Biology has enabled new approaches to several medical applications including the development of immunotherapies based on bioengineered cells, and most notably the engineering of T-cells with tumor-targeting receptors, the Chimeric Antigen Receptor (CAR)-T cells. CAR-T-cells have successfully treated blood tumors such as large B-cell lymphoma and promise a new scenario of therapeutic interventions also for solid tumors. Learning the lesson from CAR-T cells, we can foster the reprogramming of T lymphocytes with enhanced survival and functional activity in depressing tumor microenvironment, or to challenge diseases such as infections, autoimmune and chronic inflammatory disorders. This review will focus on the most updated bioengineering approaches to increase control, and safety of T-cell activity and to immunomodulate the extracellular microenvironment to augment immune responses. We will also discuss on applications beyond cancer treatment with implications toward the understanding and cure of a broader range of diseases by means of mammalian cells engineering.

Novel Immunoregulatory Functions of IL-18, an Accomplice of TGF-ß1.

TGF-ß1 is a pleiotropic factor exerting a strong regulatory role in several cell types, including immune cells. In NK cells it profoundly alters the surface expression of crucial activating and chemokine receptors. To understand which soluble signals might better contrast these effects, we cultured human NK cells in the presence of TGF-ß1 and different innate and adaptive cytokines, generally referred as "immunostimulatory". These included IL-2, IL-15, IL-21, IL-27, and IL-18. Unexpectedly, IL-18 strengthened rather than contrasting important TGF-ß1-mediated functions. In particular, IL-18 further reduced the expression of CX3CR1 and NKp30, leading to the virtual abrogation of the triggering capability of this activating receptor. Moreover, IL-18 further increased the expression of CXCR4. The IL-18-mediated additive effect on NKp30 and CXCR4 expression involved transcriptional regulation and activation of MEK/ERK and/or p38MAPK. A proteomic approach quantified both surface and intracellular proteins significantly modified in cytokine-treated NK cells, thus giving global information on the biological processes involving TGF-ß1 and IL-18. Our data support the concept that IL-18 may have a different behavior depending on the type of soluble factors characterizing the microenvironment. In a TGF-ß1 rich milieu such as tumors, it may contribute to the impairment of both NK cells recruitment and killing capability.

Membrane Poration Mechanisms at the Cell-Nanostructure Interface.

3D vertical nanostructures have become one of the most significant methods for interfacing cells and the nanoscale and for accessing significant intracellular functionalities such as membrane potential. As this intracellular access can be induced by means of diverse cellular membrane poration mechanisms, it is important to investigate in detail the cell condition after membrane rupture for assessing the real effects of the poration techniques on the biological environment. Indeed, differences of the membrane dynamics and reshaping have not been observed yet when the membrane-nanostructure system is locally perturbed by, for instance, diverse membrane breakage events. In this work, new insights are provided into the membrane dynamics in case of two different poration approaches, optoacoustic- and electro-poration, both mediated by the same 3D nanostructures. The experimental results offer a detailed overview on the different poration processes in terms of electrical recordings and membrane conformation.

TGF-ß1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells.

Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-ß1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-ß1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX3CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-ß1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX3CR1. We demonstrated the functional interaction of miR-27a-5p with the 3' untranslated region (3'UTR) of CX3CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX3CR1 3'UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-ß1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX3CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX3CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-ß1-induced regulator of CX3CR1 expression.

A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice.

Myeloid-derived suppressor cells (MDSCs) exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells. Their mechanism of differentiation and identification of MDSC-specific therapeutic targets are major areas of interest. We have devised a highly efficient and rapid method to produce very large numbers of melanoma-infiltrating MDSCs ex vivo without inducing tumors in mice. These MDSCs were used to study their differentiation, immunosuppressive activities and were compared to non-neoplastic counterparts and conventional dendritic cells using unbiased systems biology approaches. Differentially activated/deactivated pathways caused by cell type differences and by the melanoma tumor environment were identified. MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase. These studies uncovered more than 60 potential novel therapeutic targets. As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients. Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.