自然杀伤细胞受体及其配体表达的转录和表观遗传调控

自然杀伤(natural killer cell,NK)细胞受体及其配体在NK细胞发挥抗病毒、抗肿瘤和免疫调节作用中起重要作用.NK细胞功能的发挥取决于NK细胞受体及其配体的表达水平和其所传递信号的综合.病毒、肿瘤和热休克等刺激可以通过激活相应的转录调节因子,提高启动子活性而上调NKG2家族受体及其配体的表达,而启动子区DNA的甲基化状态、组蛋白的乙酰化和甲基化等表观遗传调控,在NK细胞受体及其配体的表达方面亦起重要作用,并决定NK细胞受体的克隆性分布.深入探讨NK细胞受体及其配体的表达调控机制,将为提高NK.细胞抗肿瘤和抗感染疗效提供新的策略.     

HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

Ras activation induces expression of raet1 family NK receptor ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.     

Defining early human NK cell developmental stages in primary and secondary lymphoid tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56^brightCD16^+/− and CD56^dimCD16⁺ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.     

IL-2/IL-18 prevent the down-modulation of NKG2D by TGF-beta in NK cells via the c-Jun N-terminal kinase (JNK) pathway

TGF-beta is known to play a major role for the reduced NKG2D expression seen in cancer patients. However, the mechanisms for reduced TGF-beta-induced down-regulation of NKG2D are unclear. In this study, we observed that IL-2/IL-18 increased the NKG2D expression in the TGF-beta treated NK cell line in a dose-dependent manner. Incubation with the JNK inhibitor SP600125 inhibited the NKG2D expression induced by IL-2/IL-18 in the TGF-beta treated human NK cell line. Moreover, the NK cytotoxicity assay showed that the reduced NK cytotoxicity by TGF-beta was recovered by IL-2/IL-18 treatment. The results indicate that IL-2/IL-18 strongly prevented the TGF-beta-induced NKG2D down-regulation in NK cells via the JNK pathway. Taken together, the protected expression of NKG2D by IL-2/IL-18 provides insight into the mechanism of NKG2D regulation and it also supplied useful information for creating a novel therapeutic approach to treat TGF-beta-secreting cancer cells.     

Cancer immunoediting of the NK group 2D ligand H60a

Cancer immunoediting describes the process whereby highly immunogenic tumor cells are removed, or edited, from the primary tumor repertoire by the immune system. In immunodeficient mice, the editing process is hampered, and "unedited" tumor cells can be recovered and studied. In this study, we compared unedited and edited tumors for their expression of NK group 2D (NKG2D) ligands, a family of surface proteins expressed on tumor cells that can activate NK cell cytotoxic activity. We found that the expression of the NKG2D ligand H60a was more heterogeneous in groups of unedited 3'-methylcholanthrene sarcoma cell lines compared with that in edited 3'-methylcholanthrene sarcoma cell lines (i.e., some unedited cell lines expressed very high levels of H60a, whereas other unedited and edited cell lines expressed very low levels). We also found that some highly immunogenic cell lines displayed a bimodal distribution consisting of H60a-hi and H60a-lo cells. In one of these cell lines, the H60a-hi cells could be removed by passaging the cells through RAG2(-/-) mice, resulting in edited cell lines that were poor targets for NK cells and that displayed progressive tumor growth. This editing of H60a-hi cells required NK cells and NKG2D. Our studies show that the expression of H60a on tumors cells can be actively modulated by the immune system, thereby implicating this NKG2D ligand in tumor immunosurveillance.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.     

生物肽SP对NK细胞NKG2D/NKG2A受体表达的影响

选择NK92-MI细胞为研究体系,研究SP对NK细胞的杀伤活性及功能性受体NKG2D/NKG2A表达的影响,以探讨SP对NK细胞功能的调节作用机制。采用MTT法测定NK92-MI细胞对K562细胞的杀伤活性;采用Real-Time PCR和流式细胞术检测NK92-MI细胞活化性受体NKG2D和抑制性受体NKG2A的基因表达和膜表达。10-14～10-8 mol/L的SP在体外可明显增强NK92-MI细胞的杀伤活性。该浓度范围的SP均可上调NKG2D/NKG2A的mRNA水平;10-14～10-8 mol/L的SP均上调NKG2D/NKG2A的膜表达,较低浓度（10-14 mol/L）的SP仅使NKG2D表达上调,而NKG2A表达无明显变化;SP刺激NKG2D膜表达增加的程度高于NKG2A。生物肽SP调节NK细胞功能性受体NKG2D/NKG2A的表达,可能是SP增强NK细胞杀伤活性的一种原因。     

Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-beta1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-beta1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-beta1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-beta1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-beta1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-beta1. Impaired NK cytotoxicity by TGF-beta1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-beta1.     

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1-5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.