Identification Of The Novel Immune Molecules In Regulating Natural Killer Cell Cytotoxicity Against Tumor Cells And Their Mechanisms

As an important component of the innate immune system, NK cells play avital role in anti-tumor immunity via two main mechanisms. Firstly, NK cellsdeliver the lethal hit to target cells through the granules containing perforin andgranzyme by exocytosis pathway; besides, NK cells secrete cytokines tomodulate the functions of the other immunocytes. Thereby, how to regulate thecytoxic activity of NK cells to tumor cells and reinforce the sensitivity of tumorcells to NK cell cytolysis, are the key subjects and need to be investigated inanti-tumor immunotherapy.The cytoxic activity of NK cells is modulated by the NK cell receptors andcytokines. When the NK cell receptors or the cytokine receptors are activated,they could transfer signals to control the transcription and expression of NK cellfunction related genes and regulate the development, maturation and activation ofNK cells by transcriptional factors such as Ets-1(E26transformation-specific1),MEF (myocyte enhancer factor), AP-1(activator protein1) and homeoboxcontaining1(HMBOX1).Recently, miRNAs have been focused on for their important modulatoryfunctions in the development and function of immune system. It has been reported that miRNAs are involved in the development, differentiation andactivation of T cells, B cells and macrophages. However there are few reports onhow miRNAs affect NK cell cytotoxicity. To investigate the effect of miRNA onNK cell cytotoxicity, we detected the miRNA expression profile in NKL cellsafter NKL cells were incubated with the pre-coated anti-CD226mAb LeoA1andfound that six miRNA molecules (including let-7c, miR-21, miR-30c,miR-30c-1*, miR-181d and miR-200a*) showed apparent variation bymicroRNA array and RT-PCR. Among them, we explored that miR-30c-1*couldincrease the expression level of mTNF-α on NK cells via targeting the inhibitorytranscriptional factor HMBOX1and thereby promote the cytolysis of NKL cellsto human hepatoma cells in vitro and in vivo.During the development of solid tumor, tumor cells’ rapid proliferation coupledwith poor blood supply usually leads to hypoxia, decreased glucose and nutritionsupply and induces the ER stress in tumor tissues. To overcome the ER stress andto survive, the tumor cells prime the unfolded protein response (UPR). As acell-intrinsic mechanism which is crucial to tumorigenesis, UPR keeps thesurvival and development of tumor cells, induces the resistance of tumor cells tochemotherapy and radiotherapy, and affects the anti-tumor immune responses.However, the effects of UPR on the regulation of the sensitivity of tumor cells toNK cells’ cytolysis have not been established yet. In this study, firstly we showedthat the expression levels of MICA/B, CD112and CD155on hepatoma cellswere significantly down-regulated during UPR, which consequently led to moreresistance of hepatoma cells to NK cell cytotoxicity. Secondly, we found thatamong the three UPR sensors, ATF6and IRE1α were the key molecules inregulating the expressions of MICA/B, CD112and CD155. Thirdly, we exploredthat the ERAD molecule HRD1which was induced by IRE1α pathway, was involved in the degradation of MICA/B, CD112and CD155. Lastly, wedemonstrated that the expression levels of GRP78and HRD1were significantlyhigher in hepatoma cells at the advanced stages (stage T3and T4) compared withthat in hepatoma cells at the early stages (stage T1and T2). Contrast with theexpression profiles of GRP78and HRD1, the expression levels of MICA/B,CD112and CD155were significantly higher in hepatoma cells at the early stages(stage T1and T2) compared with that in hepatoma cells at the advanced tumorstages (stage T3and T4). Correlation analysis indicated that the expression ofHRD1was positively correlated with the stages of hepatoma and GRP78expression. Whereas, the expression of HRD1was negatively correlated withMICA/B, CD112and CD155expression levels.In summary, on one hand, we demonstrated that miRNA molecules are indeedinvolved in the regulation of NK cell activation and cytotoxicity. For the firsttime, we found that miR-30c-1*could promote the NK cell cytolysis andexplored its modulating mechanisms. On the other hand, we found that tumorcells could also regulate the activation and cytotoxicity of NK cells. We provedthat with the progression of hepatoma, the UPR in hepatoma cells coulddown-regulate the expression levels of NK cell activating receptors’ ligands andimpaire the sensitivity of hepatoma cells to NK cells’ killing. This may beinvolved in the immune escape of hepatoma cells. More importantly, we foundthat the decreased expression levels of MICA/B, CD112and CD155were due tothe ATF6and IRE1α pathways and the IRE1α pathway contributed to theincreased expression level of HRD1and facilitated the ERAD of MICA/B,CD112and CD155. The results we got would not only lead to new ideas tounderstand the pathogenesis process and immune escape mechanisms ofhepatoma, but also provide the theoretical and experimental evidences for the immunotherapy of hepatoma patients.