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Natural killer (NK) cells are physiologically involved in the immune response against viruses, intracellular bacteria, and parasites as well as against malignant diseases. In addition to the ...cytotoxic activity, NK lymphocytes mediate a variety of homeostatic effects by producing cytokines. This study focused on the differential role of CD40 and CD80 costimulatory molecules and major histocompatibility complex class I (MHC‐I) antigens in the regulation of cytotoxicity and of interferon (IFN)‐γ secretion of resting and interleukin (IL)‐2‐activated human NK cells. CD40 and CD80 molecules were observed to play a specific role in the induction of cytotoxic function but not in IFN‐γ production of IL‐2‐activated NK effectors. In addition, a critical role of CD94‐dependent MHC‐I recognition for the regulation of IFN‐γ production and target lysis was demonstrated. These data provide a possible mechanism underlying functional interactions between NK lymphocytes and CD40/CD80‐expressing cell targets, as represented by dendritic cells.
This study aims to adoptively reduce the major histocompatibility complex class I (MHC‐I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. ...A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC‐I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC‐I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC‐I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC‐I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC‐I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC‐I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC‐I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence‐activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip‐treated tumor cells.
The bottom surface of a microfluidic system is biofunctionalized with antibodies specific for major histocompatibility complex class I (MHC‐I) molecules. Injected cells roll on this surface and are subjected to shear stress. A reduction of MHC‐I expression on tumor cell membranes is observed, as well as an increase in the recognition of tumor target cells by natural killer cells.
The yearly Think Tank Meeting of the Italian Network for Tumor Biotherapy (NIBIT) Foundation, brings together in Siena, Tuscany (Italy), experts in immuno-oncology to review the learnings from ...current immunotherapy treatments, and to propose new pre-clinical and clinical investigations in selected research areas. MAIN: While immunotherapies in non-small cell lung cancer and melanoma led to practice changing therapies, the same therapies had only modest benefit for patients with other malignancies, such as mesothelioma and glioblastoma. One way to improve on current immunotherapies is to alter the sequence of each combination agent. Matching the immunotherapy to the host's immune response may thus improve the activity of the current treatments. A second approach is to combine current immunotherapies with novel agents targeting complementary mechanisms. Identifying the appropriate novel agents may require different approaches than the traditional laboratory-based discovery work. For example, artificial intelligence-based research may help focusing the search for innovative and most promising combination partners.
Novel immunotherapies are needed in cancer patients with resistance to or relapse after current immunotherapeutic drugs. Such new treatments may include targeted agents or monoclonal antibodies to overcome the immune-suppressive tumor microenvironment. The mode of combining the novel treatments, including vaccines, needs to be matched to the patient's immune status for achieving the maximum benefit. In this scenario, specific attention should be also paid nowadays to the immune intersection between COVID-19 and cancer.
We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the ...embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells.Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.
In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require ...expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.
Abstract
40-50% of patients with melanoma are identified with BRAF mutations. About 80% of the BRAF mutations carry the V600E mutation. These patients are treated with BRAF inhibitors alone or in ...combination with other cell signaling pathway inhibitors. However, a relapse of the disease is observed within 6-8 months after the commencement of the therapy. Some studies hint that NK cell based therapy can be effective in controlling melanoma. In this study multidisciplinary approaches were tested to understand the mechanism of Vemurafenib resistance by a BRAFV600E melanoma. Vemurafenib is a BRAFV600E inhibitor. Vemurafenib resistant melanoma cell lines were raised from the sensitive cell lines by drug treatment till 80-90% of cells showed resistance. Both the resistant and sensitive cell lines were tested in a NK cell cytotoxicity assay in vitro by standard 51Cr-release assay. The Vemurafenib resistant cell lines were found to have a reduced sensitivity to NK cell killing. Though there was no significant difference in expression of NK cell activating ligands on the surface between the resistant and sensitive cell lines, there was a tendency of higher expression of MHC class I on Vemurafenib resistant cell lines. BRAF exon15 sequencing shows no difference between Vemurafenib resistant and sensitive cell types. Experiments are in progress to understand the mechanism(s) of the reduced sensitivity of the Vemurafenib resistant cell lines to NK cell killing.
Natural killer (NK) cells are most efficient if their targets do not express self MHC class I, because NK cells carry inhibitory receptors that interfere with activating their cytotoxic pathway. ...Clinicians have taken advantage of this by adoptively transferring haploidentical NK cells into patients to mediate an effective graft-versus-leukemia response. With a similar rationale, antibody blockade of MHC class I-specific inhibitory NK cell receptors is currently being tested in clinical trials. Both approaches are challenged by the emerging concept that NK cells may constantly adapt or "tune" their responsiveness according to the amount of self MHC class I that they sense on surrounding cells. Hence, these therapeutic attempts would initially result in increased killing of tumor cells, but a parallel adaptation process might ultimately lead to impaired antitumor efficacy. We have investigated this question in two mouse models: inhibitory receptor blockade in vivo and adoptive transfer to MHC class I-disparate hosts. We show that changed self-perception via inhibitory receptors in mature NK cells reprograms the reactivity such that tolerance to healthy cells is always preserved. However, reactivity against cancer cells lacking critical MHC class I molecules (missing self-reactivity) still remains or may even be increased. This dissociation between activity against healthy cells and tumor cells may provide an answer as to why NK cells mediate graft-versus-leukemia effects without causing graft-versus-host disease and may also be utilized to improve immunotherapy.
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