The Expression Of ILTs And Its Ligant In Gastric Cancer: Experimental And Clinical Research

Background:Gastric Cancer is one of the most frequent malignant tumors which are harmful to human health.There are more than 9.3 million newly discovered cases annually in the world. The death rate of Gastric Cancer is approximately 29.34 per million (man 40.80 per million and woman 18.60 per million respectively) that is second larger in the world. According to Ministry of Health statistics in 2006, the number of Gastric Cancer patients is as many as 4 million, and 3 million people die of this disease every year in our country. Recently many scholars study the cause and diversion of Gastric Cancer and acquire some success. However, we are still in double regarding the mechanism of cancer, early diagnosis and estimate of prognostic. Thus, it is significant to research the mechanism of cancer and to discuss the state and function of immunocyte during the process of tumor. There is no doubt that it will be beneficial to understand and cure Gastric Cancer.At present, the pathogen of this cancer is not clear. Epidemiology research suggests the cause of Gastric Cancer relates to many factors such as food, infection and inheritance. Recently, it is considered that the process of Gastric Cancer is an interaction between immune system and cancer cells in host. It involves not only change of effector cell functions (abnormal quantity and function of T lymphocyte) and some cytokines (IFN-y and IL-10), but also the abnormal tumor cell self (high expression of FasL and lack of co-stimulatory molecules and decrease of MHC) and relevant oncogene and anti-oncogene modification. Among all the factors above, the lack of co-stimulatory molecules which are expressed on the cancer cell surface is one of the most important reasons that results in immune evasion. Positive stimulatory molecules influence activation and proliferation of T lymphocyte while negative ones inhibit the function of T lymphocyte through the follow two ways:they induce inhibitory factors to express on effect cells and tumor cells.Ig-like transcript (ILT) which is also called leukocyte inhibitory receptor(LIR) is a kind of newly discovered receptor family which recognize HLA-I. At present,9 members of the ILT family are known and they are expresses on a wide range of cells which include T lymphocyte, monocyte, NK and dendrite cells. Different kinds of ILTs play a great role in immunologic modification by transmitting inhibitory or simulative signals, for instance, in term of inhibiting activity of killer cells and regulating signal transmission between dendrite cells and T lymphocytes and so on. Inhibitory receptors (ILT2, ILT3, ILT4) consist 4 or 2 extracellular C-2 type Ig-SF domains and possess long cytoplasm tails that contain 3 immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the expression and function of these receptors have not been well understood on tumors.HLA-G molecule is one kind of nonclassical antigen and possesses a shore cytoplasm domain. HLA-G gene contains 8 extrons and 7 introns. Alternative splicing of the primary transcripts results in seven different mRNA, encoding four membrane-anchored (HLA-G1,G2,G3,G4) and three soluble (HLA-G5,G6,G7) isoforms, in which HLA-G 1 and HLA-G5 can combine ILT2 and ILT4 with high specificity. HLA-G plays an important part in establishing and maintaining immune tolerance by inhibiting the function of immune cells. HLA-G molecule exerts suppressive role via integrating with ILT2 and ILT4 which be expressed in NK cells, T lymphocytes and APC. Soluble HLA-G(sHLA-G) molecule also can link cells receptor with CD8+thus induces apoptosis of NK and T lymphocytes. Besides, the latest research suggests that HLA-G molecule can tempt the produce of Tregs and inhibitory NK cells, and protect the target cells from being damaged by NK and T lymphocytes to a large extent. HLA-G molecule can be primarily expressed on the surface of trophoblast cells and cancer cells. There are also a few HLA-G molecules which can be expressed on the surface of histiocytes, such as T lymphocyte. However, we are still wondering in the variety of HLA-G on T lymphocytes of patients with Gastric Cancer, which is main ligand for inhibitory receptor ILT2 and ILT4.Objectives and Methods:â… . We analyzed the mRNA and protein expression of well-differentiated Gastric Cancer cell lines (MKN1), moderate differentiation Gastric Cancer cell lines (SGC-7901,AGS), poorly differentiated cell lines(MGC803) and undifferentiation cell lines (HGC-27,BCG823) respectively by fluorescent quantization PCR-Sybr Greenâ… and FACS flow cytometer. And we discussed the variety of ILT2, ILT3, ILT4 in diverse differentiation of Gastric Cancer cell lines systemically, which provide experimental evidence for succedent research.â…¡. In order to explore the effect of the different degree expression of inhibiting ILTs to immune cells, we try to investigate that different leveles of inhibitory ILTs influence immunocytes in vitro. We cultureed well-differentiated Gastric Cancer cell lines (MKN1), which express slight ILT2/ILT3/ILT4, and undifferentiation Gastric Cancer cell lines (HGC-27), which express substantial ILT2/ILT3/ILT4, with PBMC and NK together respectively, and tested MTT and HLA-G in CD4+,CD8+T lymphocytes and NK to discuss the role that inhibitory ILT regulate effector cells.â…¢. Immunohistochemistry was used to detect the expression of ILT2/ILT3/ILT4 in pathologic tissue with diverse differentiation from patients who surfer Gastric Cancer to make further efforts to explore the value of ILT2/ILT3/ILT4 in clinical application.â…£. We also introduced TaqMan probe FQ-PCR and FACS flow cytometer to inspect the expression of HLA-G that is ligand for ILTs in PBMC and CD4+,CD8+T lymphocyte, and to research the changes of HLA-G and analyse the relation between Gastric Cancer phase and the expression of HLA-G molecule preliminarily. In a word, from this part of research we discussed systemically the variety of ILT2, 3,4 in diverse differentiation of Gastric Cancer cell lines and their effect on immunocytes. It is expected to provide experimental evidence to illustrate the mechanism for the growth and metastasis of tumor and theoretical basis simultaneously for the diagnosis and treatment of Gastric Cancer and the exploiture of new drugs.Results and Conclusions:I. Expression of inhibitory ILTs mRNA and protein of human gastric cancer:an experiment study in vitro1. Using fluorescent quantization PCR-Sybr Green I, we detected the expression of ILT2,3,4 mRNA from 6 Gastric Cancer cell lines with diverse differentiation. From the results, we can see that all the differentiation groups express various extents ILT mRNA, and ILT2 mRNA is expressed the highest especially. In the mean time, well-differentiated Gastric Cancer cell lines (MKN1) express the lowest amount of ILTs among them and, on the contrary, undifferentiation Gastric Cancer cell lines (HGC-27 and BCG823) express the highest amount of ILTs. There is significant difference in 6 cell lines with diverse differentiation.2. We found that ILT2,3,4 are expressed with different extent in nearly all differentiation groups except undifferentiation Gastric Cancer cell lines (BCG823), which express little ILT3 and ILT4, and the different levels of ILTs relates to the differentiation of tumor cells by FACS flow cytometer.Experimental results above demonstrate that malignant Gastric Cancer cells can express ILT2/ILT3/ILT4 through some common pathway, which influence the function of effector cells and their killer activity to tumor cells.II Inhibitory ILTs regulation to effector cells:an experiment study in vitrol.By coculture ILT2highILT3highILT4high HGC-27 cell and ILT2lowILT3lowILT4low MKN1 cell with PBMC,NK respectively, we found that the killer activity of effector cells was reduced distinctly, and the former was more prominence than latter. Thus, we considered that varying degree of ILT2/ILT3/ILT4 that were expressed by human stomach cancer cell influences the proliferation and killer activity of PBMC and NK cells.2. Coculture were obtained as described above for 24 hours, we conclued that HLA-G at the surface of T. and NK cells varies markedly and ILT2highILT3highILT4hig HGC-27 had a more significant effect on HLA-G than those by ILT2lowILT3lowILT4low MKN1. Inaddition, HLA-G that is expressed at the surface of T lymphocytes mainly located CD4+T lymphocytes, and there was little even no HLA-G at the surface of CD8+T lymphocytes. Besides, HLA-G expressed on CD56+NK cells is lower than that in T lymphocytes.Experimental results indicated that ILT2/ILT3/ILT4 have an impact on the killer activity of effector cells, and may regulate the expression of HLA-G..III. Expression of inhibitory ILTs on gastric cancers:a clinical studyThe expression of ILT2/ILT3/ILT4 from 50 Gastric Cancer pathological tissues were tested. From the results, we can see that it is dissimilar with diverse differentiation and type of tumor, and ILT2/ILT3/ILT4 cound be expressed either in cytoplasm or membrane of Gastric Cancer cells and even both. The positive rate of ILT2,ILT3 and ILT4 expression among 50 Gastric Cancer pathological tissues also varies a lot, and the positive rate of ILT2 is the highest-62.0%(31/50), while the positive rate of ILT3 and ILT4 is 18.0%(9/50) and 34%(17/50) respectively. There was no ILT2/ILT3/ILT4 on normal tissues, which indicated that inhibitory ILTs, especially ILT2, may be a valuable index for diagnosing Gastric Cancer and provide a new target to treat gastric cancer.IV. Expression of non-classical HLA-G on PBLs of gastric cancers:a clinical study1. By Fluorescent quantitation RT-PCR, we considered that HLA-G mRNA in both lymphocytes from Gastric Cancer and healthy control was weak, and there was no significant difference betweenâ… +â…¡Gastric Cancer and healthy control (P>0.05) However, HLA-G mRNA inâ…¢+â…£Gastric Cancer was lower than that inâ… +â…¡(P< 0.05). Also, there is no distinct change between well-differentiated and poorly differentiated Gastric Cancer in term of clinical differentiation.2. By FACS Flow Cytometry, we found that the expression of HLA-G on lymphocytes both from Gastric Cancer and healthy control was low, and there was nearly no HLA-G expression on CD8+T lymphocytes of patients of Gastric Cancer. On the contrary, HLA-G molecules were mainly expressed on CD4+T lymphocytes, and the positive rate was also different in diverse patients, which was associated with tumor microenvironment and other influence factor.