The Effect Of Head & Neck Squamous Cell Carcinoma On The Expression Of Toll-like Receptor 3 In Human Natural Killer Cell

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers in the world and over the last 40 years the 5-year survival rate has only marginally improved. Cells of head and neck cancer are known to develop several molecular strategies to escape efficient immune responses. It is supposed that various tumor secreted immunosuppressive mediators contribute to massively affected immune functions within the malignant transformation process.Natural killer (NK) cells are a component of the innate immunity and play an important role in anti-infection activity and tumor surveillance. NK cells can be triggered through various receptors depending on specific ligands presented by target cells in a given encounter. However, NK cells isolated from cancer patients exhibit strongly impaired anti-tumor functions.Toll-like receptors (TLRs) are pattern-recognition receptors that trigger innate immune responses to recognize conserved molecular patterns of microbial origin. Ten TLRs have been described in human, and for most of them specific ligands have been identified to date. Recently it has been shown that treatment with the syntheticdouble-stranded RNA (dsRNA) polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, significantly upregulates both natural and CD16 mediated cytotoxicity of highly purified human NK cells. Poly I:C is known to be recognized by Toll-like receptor 3 (TLR3), and TLR3 stimulation was shown to rapidly upregulate NK cell in vivo functions. However in HNSCC micro-environment, the protein expression of TLRs in NK cell and NK cell ability to directly respond to TLRs' Hgands are still mostly unknown.There were three sections in this work, in which the effect of HNSCC on the subcellular distribution of TLR3 in NK cell was focused on.Section I TLR protein expression of human NK cell in the presence of HNSCCTo investigate TLR protein expression of human NK cell in the presence of HNSCC.Untouched NK cells from human peripheral blood of healthy donors were isolated by magnetic bead separation. TLR protein expression of human NK cell was studied by Western blot. In addition, isolated NK cells were incubated in tumor cell line supernatants (BHY, PCI-1 and PCI-13) for 24 hours before they were used for protein analysis.High purity of isolated NK cells from human peripheral blood of healthy donors was achieved using the technique of magnetic bead separation. Among human TLR proteins we found only TLR1, TLR2, TLR3, and TLR7 to be significantly expressed in native NK cells. Our investigations revealed a constitutive expression of these TLR proteins in NK cells which was not affected in response to 24 hours of incubation in different HNSCC supernatants.Section II Subcellular localization of TLR3 in human NK cellTo investigate the subcellular localization of TLR3 and the regulation of TLR3 in response to polyI:C in NK cell.Flow cytometric analysis to subcellular distribution of TLR3 was used. The CD69 expression in NK cells responding to polyI:C stimulation was examined by FACS and activation of NF-κB was analyzed by Western blot. In addition, NK cells were pretreated with anti-TLR3 mAb 30 min at 37℃, then stimulated with 50 μg/ml poly(I:C) for 24 h.. After that, CD69 expression in NK cell was further studied by Western blot and FACS.TLR3 was identified as a predominantly surface expressed receptor in native NK cells, in which TLR3 could as well be found in low level in the cellular lumen. Our data demonstrated CD69 expression significantly increased in NK cells in response to polyI:C stimulation. The CD69 MFI in polyI:C-stimulated NK cell, in which TLR3 on the surface was blocked by anti-TLR3 mAb, was 687±102, whereas the CD69 MFI in native NK cell was 532±78. There was no significant difference between them, which was consistent with the result of CD69 protein analysis. It showed that CD69 expression was not up-regulated in NK cells with surface TLR3 blockage responding to polyI.C stimulation. In the polyI:C-stimulated NK cells with normal medium incubation, IκBα degradation was detected at the time point of 60 min and by 90 min most of the IκBα protein was degradated. This is consistent with the fact that stimulation of TLRs results in NF-κB activation.Section III The effect of HNSCC on the subcellular localization of TLR3 in human NK CellTo investigate the effect of HNSCC supernatants on the subcellular localization of TLR3 in human NK Cell.Isolated NK cells were incubated in tumor cell line supernatants (BHY, PCI-1and PCI-13) or and polyI:C for 24 hours, then flow cytometric analysis to subcellular distribution of TLR3 and CD69 expression was performed. Mouse fibroblasts NIH3T3 were used for transfection with plasmid pUNO-hTLR3, then they were incubated in tumor cell line supernatants (BHY and PCI-1) for 24 hours, and subcellular distribution of TLR3 was further examined by FACS. The cytokines in HNSCC supernatants were analyzed by ELISA.Incubation of isolated NK cells in HNSCC supernatants for 24 hours resulted in an internalization of TLR3. TLR3 expression was rapidly down-regulated on the cell surface and correspondingly, increased cytoplasmic protein levels could be detected after incubation with HNSCC supernatants. Identical findings could be achieved using supernatants of three different HNSCC cell lines. The internalization of TLR3 in response to HNSCC could as well be observed in fibroblasts expressing heterologous TLR3 protein.Our data indicated antagonistic effects of poly I:C and HNSCC supernatants. Poly I:C stimulated NK cells revealed a significantly increased surface expression of TLR3 in the presence of HNSCC , which shows that poly I:C is able to impair the HNSCC induced internalization of TLR3. Our data demonstrated that poly I:C stimulation resulted in a significant upregulation of surface CD69 on the analyzed NK cells, which was not significantly affected by HNSCC supernatants.Our data revealed that IL-6 and IL-8 were secreted in huge amounts by three HNSCC cell lines. High secretion levels of IL-6 of 3750 ± 265 pg/ml were detected in BHY supernatants and lower, but still significant, levels of IL-6 were detected in the PCI-1 (340 ± 52 pg/ml) and PCI-13 (450 ± 78 pg/ml) cell lines. Determination of IL-8 in these supernatants revealed similar cytokine levels of 820 ±111 pg/ml (BHY), 200 ± 21 pg/ml (PCI-1) and 760 ± 72 pg/ml (PCI-13). The detected other cytokines were found to be secreted only at very low levels, in a range of 0-3 pg/ml, in three cell lines. So we presume that IL-6 and IL-8 secreted by HNSCC participate in the induction of TLR3 internalization in NK cell.Summary1. The technique of magnetic bead separation is a reliable method to achieve high purity of isolated NK cells from human peripheral blood of health donors.2. Among human TLR proteins, only TLR1, TLR2, TLR3, and TLR7 are significantly detected in native NK cells, and their stable protein expressions could not be affected by HNSCC.3. TLR3 expression predominantly localizes on the surface of native NK cells, and surface TLR3 stimulation by polyI:C could induce activation of NK cell with significant CD69 up-regulation. This process is achieved depending on NF-κB activation.4. HNSCC could result in an intracellular migration of surface TLR3 in NK cell, which could be identified in fibroblasts expressing heterologous TLR3. It suggests that this rapid internalisation of TLR3 of NK cell in response to HNSCC represents a novel immune escape mechanism of head and neck cancer.5. Poly I:C could not only impair the HNSCC induced intemalization of TLR3, but also trigger NK cell activation in the presence of HNSCC.6. It is presumed that some cytokines secreted by HNSCC, such as IL-6 and IL-8, participate in the induction of TLR3 intemalization in NK cell.