头颈部鳞癌对自然杀伤细胞Toll样受体3表达的影响
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摘要
头颈部鳞状细胞癌(HNSCC)是人类最常见的恶性肿瘤之一，在近40年运用传统标准方式治疗(外科手术、放射治疗和化学治疗)，其5年的生存率并没有显著地提高。鳞癌细胞能利用其分子策略逃避宿主有效的免疫反应。这可能与正常鳞状上皮恶变过程中产生多种免疫抑制产物来影响免疫功能有关。
自然杀伤(NK)细胞是天然免疫系统中首要的效应者，在抗感染和肿瘤监视方面起了很重要的作用。在某一状态下NK细胞利用自身的受体与靶细胞上的配体结合诱导其活化。然而从癌症患者身上提取的NK细胞其抗肿瘤的的能力受到很大的影响。
Toll样受体(TLRs)家族在人类共发现10类受体，能识别病原微生物中保守分子，从而启动天然免疫反应。近年来有资料显示polyI：C(人工合成的dsRNA)能上调NK细胞的自然的和CD16介导的细胞毒作用。polyI：C是Toll样受体3(TLR3)的配体，TLR3的刺激能快速上调体内NK细胞的功能。然而在肿瘤微环境中NK细胞TLR的蛋白表达水平如何，通过相应的配体刺激能否激活NK细胞，被激活的NK细胞的生物学行为又如何体现，目前未见报道。
本课题由三部分组成，着重研究TLR3在NK细胞内的表达定位及头颈部鳞癌对其影响。
第一部分人类NK细胞TLR蛋白表达及其受HNSCC的影响
研究TLR在NK细胞中蛋白表达及其是否受HNSCC的影响。
利用磁珠分离技术从正常人的外周血中获得NK细胞：采用Western blot检测人类NK细胞的TLR的蛋白表达；NK细胞与正常培养基和HNSCC细胞株(BHY、PCI-1和PCI-13)上清液培养24小时，再通过Western blot检测其TLR的蛋白水平的表达。
结果显示：1．运用密度梯度离心法和磁珠分离技术能从健康人的外周血中获得高纯度的NK细胞。2．在TLR1-10中，人类NK细胞只有TLR1、2、3和7蛋白表达。3．在上述三种HNSCC肿瘤细胞株上清液和标准培养基中培养24小时的NK细胞中，各组均只有表达TLR1、2、3和7，并且表达强度无差异，因此提示TLR在NK细胞中的蛋白表达并不受HNSCC的影响。
第二部分人类NK细胞TLR3表达定位
研究了LR3在NK细胞中的表达定位，以及进一步了解polyI：C对其作用。
利用流式细胞仪检测TLR3在NK细胞中的表达定位：NK细胞经polyI：C刺激后，用流式细胞仪测CD69的表达，并且通过Western blot了解NF-κB的活化程度；利用anti-TLR3 mAb功能性封闭NK细胞表面的TLR3受体，然后用polyI：C进行刺激，最后通过流式细胞仪和Western blot测定NK细胞的CD69的表达。
结果显示：1．TLR3在天然的人类NK细胞中主要表达在细胞表面，有少量位于细胞内。2．Poly(I：C)的刺激可以导致NK细胞表面CD69表达的显著上调。3．经功能性封闭后再用polyI：C刺激的NK细胞CD69表达MFI为687±102，与正常的NK细胞CD69表达(MFI=532±78)无显著性差异，P＞0.05；该结果与Western blot显示的CD69的蛋白表达相一致：说明封闭NK细胞表面的TLR3后，polyI：C刺激不会导致CD69表达的明显上调。4．在正常培养基中经Poly(I：C)刺激的NK细胞，在60分钟这个时间点可以测得IκBα降解，在90分钟时发现IκBα降解得更加完全。这一结果符合TLR3受刺激后导致NF-κB的活化。
第三部分HNSCC对人类NK细胞TLR3表达定位的影响
讨论HNSCC细胞株的上清液对TLR3在人类NK细胞中表达定位的影响。
将NK细胞与HNSCC细胞株(BHY、PCI-1和PCI-13)上清液培养24小时，利用流式细胞仪检测TLR3在NK细胞中的表达定位，然后用polyI：C刺激上述NK细胞，再用流式细胞仪测TLR3的表达定位和CD69的表达；将质粒pUNO-hTLR3转染到小鼠纤维母细胞NIH3T3中，使其表达异源性的TLR3，然后将其与HNSCC细胞株(BHY和PCI-1)上清液培养24小时，利用流式细胞仪检测该细胞中TLR3的表达定位；通过ELISA分析HNSCC细胞株(BHY、PCI-1和PCI-13)上清液的细胞因子的含量。
结果显示：1．NK细胞表面的TLR3在正常培养基中明显高于在肿瘤上清液中，经t检验，P＜0.01；而在NK细胞内的TLR3在正常培养基中则明显低于在肿瘤上清液中，经t检验，P＜0.01。在各HNSCC细胞株上清液之间，NK细胞的TLR3表达无显著性差异，经t检验，P＞0.05。提示肿瘤上清液能使表达在细胞表面的TLR3出现快速地下调，相对应地在其细胞内的TLR3蛋白水平上升。这种TLR3“内化”现象也同样存在于表达异源性TLR3的小鼠纤维母细胞中。2．HNSCC细胞株上清液和Poly(I：C)两者具有拮抗效应；在肿瘤微环境中PolyI：C的刺激能使NK细胞表面TLR3的表达显著地增加，故提示Poly(I：C)能削弱HNSCC诱导TLR3在NK细胞中的“内化”；并且Poly(I：C)的刺激可以导致NK细胞表面CD69表达的显著上调，这种NK细胞的活化不受HNSCC上清液的影响。3．在所测定的细胞因子中IL-6和IL-8在上述三种细胞株中均大量分泌。在BHY上清液中IL-6为3750±265pg／ml，在PCI-1和PCI-13中分别为340±52pg／ml和450±78pg／ml。IL-8在BHY、PCI-1和PCI-13的上清液中的含量分别为820±111pg／ml、200±21pg／ml和760±72pg／ml。而其他的细胞因子在上清液中含量极其低，处于0～3pg／ml水平。因此，我们推测HNSCC细胞株分泌的IL-6和IL-8参与了诱导NK细胞TLR3的“内化”。
总结
1．采用免疫磁珠法的MACS技术可以从外周血中分离提取高纯度的NK细胞，这一方法值得信赖。
2．在人类天然NK细胞中，只有TLR1、TLR2、TLR3和TLR7蛋白表达，并且表达是稳定的，不受HNSCC的影响。
3．TLR3主要表达定位于人类天然NK细胞的细胞表面；polyI：C刺激细胞表面的TLR3激活NK细胞，使其CD69表达显著上调，上述过程依赖于NF-κB的活化。
4．HNSCC造成NK细胞表面的TLR3迁移至细胞内；这一现象在表达异源性TLR3的纤维母细胞上得以证实。这种TLR3在NK细胞中的“内化”，暗示了NHSCC免疫逃逸的新机制。
5．polyI：C不仅能削弱HNSCC诱导TLR3在NK细胞中的“内化”，而且能激活受HNSCC影响的NK细胞。
6．HNSCC可能分泌某些细胞因子，如IL-6和IL-8，参与诱导NK细胞TLR3的“内化”。
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 synthetic
double-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 HNSCC
To 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 cell
To 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 Cell
To 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-1
and 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.
Summary
1. 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.
自然杀伤(NK)细胞是天然免疫系统中首要的效应者，在抗感染和肿瘤监视方面起了很重要的作用。在某一状态下NK细胞利用自身的受体与靶细胞上的配体结合诱导其活化。然而从癌症患者身上提取的NK细胞其抗肿瘤的的能力受到很大的影响。
Toll样受体(TLRs)家族在人类共发现10类受体，能识别病原微生物中保守分子，从而启动天然免疫反应。近年来有资料显示polyI：C(人工合成的dsRNA)能上调NK细胞的自然的和CD16介导的细胞毒作用。polyI：C是Toll样受体3(TLR3)的配体，TLR3的刺激能快速上调体内NK细胞的功能。然而在肿瘤微环境中NK细胞TLR的蛋白表达水平如何，通过相应的配体刺激能否激活NK细胞，被激活的NK细胞的生物学行为又如何体现，目前未见报道。
本课题由三部分组成，着重研究TLR3在NK细胞内的表达定位及头颈部鳞癌对其影响。
第一部分人类NK细胞TLR蛋白表达及其受HNSCC的影响
研究TLR在NK细胞中蛋白表达及其是否受HNSCC的影响。
利用磁珠分离技术从正常人的外周血中获得NK细胞：采用Western blot检测人类NK细胞的TLR的蛋白表达；NK细胞与正常培养基和HNSCC细胞株(BHY、PCI-1和PCI-13)上清液培养24小时，再通过Western blot检测其TLR的蛋白水平的表达。
结果显示：1．运用密度梯度离心法和磁珠分离技术能从健康人的外周血中获得高纯度的NK细胞。2．在TLR1-10中，人类NK细胞只有TLR1、2、3和7蛋白表达。3．在上述三种HNSCC肿瘤细胞株上清液和标准培养基中培养24小时的NK细胞中，各组均只有表达TLR1、2、3和7，并且表达强度无差异，因此提示TLR在NK细胞中的蛋白表达并不受HNSCC的影响。
第二部分人类NK细胞TLR3表达定位
研究了LR3在NK细胞中的表达定位，以及进一步了解polyI：C对其作用。
利用流式细胞仪检测TLR3在NK细胞中的表达定位：NK细胞经polyI：C刺激后，用流式细胞仪测CD69的表达，并且通过Western blot了解NF-κB的活化程度；利用anti-TLR3 mAb功能性封闭NK细胞表面的TLR3受体，然后用polyI：C进行刺激，最后通过流式细胞仪和Western blot测定NK细胞的CD69的表达。
结果显示：1．TLR3在天然的人类NK细胞中主要表达在细胞表面，有少量位于细胞内。2．Poly(I：C)的刺激可以导致NK细胞表面CD69表达的显著上调。3．经功能性封闭后再用polyI：C刺激的NK细胞CD69表达MFI为687±102，与正常的NK细胞CD69表达(MFI=532±78)无显著性差异，P＞0.05；该结果与Western blot显示的CD69的蛋白表达相一致：说明封闭NK细胞表面的TLR3后，polyI：C刺激不会导致CD69表达的明显上调。4．在正常培养基中经Poly(I：C)刺激的NK细胞，在60分钟这个时间点可以测得IκBα降解，在90分钟时发现IκBα降解得更加完全。这一结果符合TLR3受刺激后导致NF-κB的活化。
第三部分HNSCC对人类NK细胞TLR3表达定位的影响
讨论HNSCC细胞株的上清液对TLR3在人类NK细胞中表达定位的影响。
将NK细胞与HNSCC细胞株(BHY、PCI-1和PCI-13)上清液培养24小时，利用流式细胞仪检测TLR3在NK细胞中的表达定位，然后用polyI：C刺激上述NK细胞，再用流式细胞仪测TLR3的表达定位和CD69的表达；将质粒pUNO-hTLR3转染到小鼠纤维母细胞NIH3T3中，使其表达异源性的TLR3，然后将其与HNSCC细胞株(BHY和PCI-1)上清液培养24小时，利用流式细胞仪检测该细胞中TLR3的表达定位；通过ELISA分析HNSCC细胞株(BHY、PCI-1和PCI-13)上清液的细胞因子的含量。
结果显示：1．NK细胞表面的TLR3在正常培养基中明显高于在肿瘤上清液中，经t检验，P＜0.01；而在NK细胞内的TLR3在正常培养基中则明显低于在肿瘤上清液中，经t检验，P＜0.01。在各HNSCC细胞株上清液之间，NK细胞的TLR3表达无显著性差异，经t检验，P＞0.05。提示肿瘤上清液能使表达在细胞表面的TLR3出现快速地下调，相对应地在其细胞内的TLR3蛋白水平上升。这种TLR3“内化”现象也同样存在于表达异源性TLR3的小鼠纤维母细胞中。2．HNSCC细胞株上清液和Poly(I：C)两者具有拮抗效应；在肿瘤微环境中PolyI：C的刺激能使NK细胞表面TLR3的表达显著地增加，故提示Poly(I：C)能削弱HNSCC诱导TLR3在NK细胞中的“内化”；并且Poly(I：C)的刺激可以导致NK细胞表面CD69表达的显著上调，这种NK细胞的活化不受HNSCC上清液的影响。3．在所测定的细胞因子中IL-6和IL-8在上述三种细胞株中均大量分泌。在BHY上清液中IL-6为3750±265pg／ml，在PCI-1和PCI-13中分别为340±52pg／ml和450±78pg／ml。IL-8在BHY、PCI-1和PCI-13的上清液中的含量分别为820±111pg／ml、200±21pg／ml和760±72pg／ml。而其他的细胞因子在上清液中含量极其低，处于0～3pg／ml水平。因此，我们推测HNSCC细胞株分泌的IL-6和IL-8参与了诱导NK细胞TLR3的“内化”。
总结
1．采用免疫磁珠法的MACS技术可以从外周血中分离提取高纯度的NK细胞，这一方法值得信赖。
2．在人类天然NK细胞中，只有TLR1、TLR2、TLR3和TLR7蛋白表达，并且表达是稳定的，不受HNSCC的影响。
3．TLR3主要表达定位于人类天然NK细胞的细胞表面；polyI：C刺激细胞表面的TLR3激活NK细胞，使其CD69表达显著上调，上述过程依赖于NF-κB的活化。
4．HNSCC造成NK细胞表面的TLR3迁移至细胞内；这一现象在表达异源性TLR3的纤维母细胞上得以证实。这种TLR3在NK细胞中的“内化”，暗示了NHSCC免疫逃逸的新机制。
5．polyI：C不仅能削弱HNSCC诱导TLR3在NK细胞中的“内化”，而且能激活受HNSCC影响的NK细胞。
6．HNSCC可能分泌某些细胞因子，如IL-6和IL-8，参与诱导NK细胞TLR3的“内化”。
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 synthetic
double-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 HNSCC
To 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 cell
To 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 Cell
To 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-1
and 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.
Summary
1. 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.
引文
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