How mucosal epithelia deal with stress: Role of NKG2D/NKG2D ligands during inflammation

Mucosal epithelia encounter both physicochemical and biological stress during their life and have evolved several mechanisms to deal with them, including regulation of immune cell functions. Stressed and damaged cells need to be cleared to control local inflammation and trigger tissue healing. Engagement of the activating NKG2D receptor is one of the most direct mechanisms involved in the recognition of stressed cells by the immune system. Indeed, injured cells promptly express NKG2D ligands that in turn mediate the activation of lymphocytes of both innate and adaptive arms of the immune system. This review focuses on different conditions that are able to modulate NKG2D ligand expression on the epithelia. Special attention is given to the mechanisms of immunosurveillance mediated by natural killer cells, which are finely tuned by NKG2D. Different types of stress, including viral and bacterial infections, chronic inflammation, and cigarette smoke exposure, are discussed as paradigmatic conditions for NKG2D ligand modulation, and the implications for tissue homeostasis are discussed

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Natural Killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human Cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+Fc∝RI≥- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterised yet. Here we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells, and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansion. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting, and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner.

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib- sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas

Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity

Capsaicin triggers autophagic cell survival which drives epithelial mesenchymal transition and chemoresistance in bladder cancer cells in an Hedgehog-dependent manner

Bladder cancer (BC) is a common urologic tumor characterized by high risk of recurrence and mortality. Capsaicin (CPS), used as an intravesical drug for overactive bladder, was demonstrated to induce cell death in different cancer cells including BC cells.Here we found that treatment of high-grade BC cells with high dose of CPS triggers autophagy. Infact, the CPS treatment alters the redox homeostasis by inducing production of radicals, mitochondrial depolarization, alterations of ADP/ATP ratio and activation of AMPK pathway stimulating the autophagic process in BC cells. The inhibition of autophagy, by using the specific inhibitor bafilomycin A or Beclin 1 knock-down, enhanced the CPS-induced cell death, demonstrating that CPS-induced autophagy acts as a pro-survival process in BC cells. By using PCR arrays and FACS analysis, we found that the CPS-treated BC cells displayed typical mesenchymal features of the epithelial mesenchymal transition (EMT) as elongated shape and over-expression of vimentin, α5 and β1 integrin subunits, integrin-like kinase and the anti-apoptotic Bcl-2 proteins. Moreover, we demonstrated that CPS treatment stimulates upregulation of Dhh/Ptch2/Zeb2 members of the Hedgehog signaling pathway, increases CD24, VEGFA and TIMP1 and decreases CD44 and ALCAM mRNA expression levels. By PTCH2 knock-down we found that the Hedgehog signaling pathway is involved in the CPS-induced autophagy and EMT phenotype.Finally, we also showed that the CPS-resistant EMT-positive BC cells displayed an increased drug-resistance to the cytotoxic effects of mitomycin C, gemcitabine and doxorubicine drugs commonly used in BC therapy

Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells

Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs

Role of distinct natural killer cell subsets in anticancer response

Natural killer (NK) cells, the prototypic member of innate lymphoid cells, are important effectors of anticancer immune response. These cells can survey and control tumor initiation due to their capability to recognize and kill malignant cells and to regulate the adaptive immune response via cytokines and chemokines release. However, several studies have shown that tumor-infiltrating NK cells associated with advanced disease can have profound functional defects and display protumor activity. This evidence indicates that NK cell behavior undergoes crucial alterations during cancer progression. Moreover, a further level of complexity is due to the extensive heterogeneity and plasticity of these lymphocytes, implying that different NK cell subsets, endowed with specific phenotypic and functional features, may be involved and play distinct roles in the tumor context. Accordingly, many studies reported the enrichment of selective NK cell subsets within tumor tissue, whereas the underlying mechanisms are not fully elucidated. A malignant microenvironment can significantly impact NK cell activity, by recruiting specific subpopulations and/or influencing their developmental programming or the acquisition of a mature phenotype; in particular, neoplastic, stroma and immune cells, or tumor-derived factors take part in these processes. In this review, we will summarize and discuss the recently acquired knowledge on the possible contribution of distinct NK cell subsets in the control and/or progression of solid and hematological malignancies. Moreover, we will address emerging evidence regarding the role of different components of tumor microenvironment on shaping NK cell response

Natural killer cell response to chemotherapy-stressed cancer cells: Role in tumor immunosurveillance.

Natural killer (NK) cells are innate cytotoxic lymphoid cells that actively prevent neoplastic development, growth, and metastatic dissemination in a process called cancer immunosurveillance. An equilibrium between immune control and tumor growth is maintained as long as cancer cells evade immunosurveillance. Therapies designed to kill cancer cells and to simultaneously sustain host antitumor immunity are an appealing strategy to control tumor growth. Several chemotherapeutic agents, depending on which drugs and doses are used, give rise to DNA damage and cancer cell death by means of apoptosis, immunogenic cell death, or other forms of non-apoptotic death (i.e., mitotic catastrophe, senescence, and autophagy). However, it is becoming increasingly clear that they can trigger additional stress responses. Indeed, relevant immunostimulating effects of different therapeutic programs include also the activation of pathways able to promote their recognition by immune effector cells. Among stress-inducible immunostimulating proteins, changes in the expression levels of NK cell-activating and inhibitory ligands, as well as of death receptors on tumor cells, play a critical role in their detection and elimination by innate immune effectors, including NK cells. Here, we will review recent advances in chemotherapy-mediated cellular stress pathways able to stimulate NK cell effector functions. In particular, we will address how these cytotoxic lymphocytes sense and respond to different types of drug-induced stresses contributing to anticancer activity

Memory NK cell features exploitable in anticancer immunotherapy

Besides their innate ability to rapidly produce effector cytokines and kill virus-infected or transformed cells, natural killer (NK) cells display a strong capability to adapt to environmental modifications and to differentiate into long-lived, hyperfunctional populations, dubbed memory or memory-like NK cells. Despite significant progress in the field of NK cell-based immunotherapies, some factors including their short life span and the occurrence of a tumor-dependent functional exhaustion have limited their clinical efficacy so that strategies aimed at overcoming these limitations represent one of the main current challenges in the field. In this scenario, the exploitation of NK cell memory may have a considerable potential. This article summarizes recent evidence in the literature on the peculiar features that render memory NK cells an attractive tool for antitumor immunotherapy, including their long-term survival and in vivo persistence, the resistance to tumor-dependent immunosuppressive microenvironment, the amplified functional responses to IgG-opsonized tumor cells, and in vitro expansion capability. Along with highlighting these issues, we speculate that memory NK cell-based adoptive immunotherapy settings would greatly take advantage from the combination with tumor-targeting therapeutic antibodies (mAbs), as a strategy to fully unleash their clinical efficacy

Polyfunctional Melan-A-specific tumor-reactive CD8+ T cells elicited by dacarbazine treatment before peptide-vaccination depends on AKT activation sustained by ICOS

The identification of activation pathways linked to anti-tumor T-cell polyfunctionality in long surviving patients is of great relevance in the new era of immunotherapy. We have recently reported that dacarbazine (DTIC) injected one day before peptide-vaccination plus IFN-α improves the anti-tumor lytic activity and enlarges the repertoire of Melan-A-specific T-cell clones, as compared with vaccination alone, impacting the overall survival of melanoma patients. To identify the mechanisms responsible for this improvement of the immune response, we have analyzed the endogenous and treatment-induced antigen-specific response in a panel of Melan-A-specific CD8+ T-cell clones in terms of differentiation phenotype, inhibitory receptor profile, polyfunctionality and AKT activation. Here we show that Melan-A specific CD8+ T cells isolated from patients treated with chemoimmunotherapy possess a late differentiated phenotype as defined by the absence of CD28 and CD27 co-stimulatory molecules and high levels of LAG-3, TIM-3 and PD-1 inhibitory receptors. Nevertheless they show higher proliferative potential and an improved anti-tumor polyfunctional effector profile in terms of co-production of TNF-α, IFN-γ and Granzyme-B compared with cells derived from patients treated with vaccination alone. Polyfunctionality is dependent on an active AKT signalling related to the engagement of the co-stimulatory molecule ICOS. We suggest that this phenotypic and functional signature is dictated by a fine-tuned balance between TCR triggering, AKT activation, co-stimulatory and inhibitory signals induced by chemoimmunotherapy and may be associated with anti-tumor T cells able to protect patients from tumor recurrence