Expression Of Chemokine Receptor CCR4 In Non-small Cell Lung Cancer And Its Mechanism

Non small cell lung cancer,accountted for about 85% of lung cancer,is the leading cause of cancer-related death worldwide.Understanding the relationship between the immune system and cancer progression has been a hot topic in the field of tumor immunology.Compelling evidence indicates that regulatory T cell(Treg)is one of the key immunosuppressive cells that promote tumor progression.However,open questions still remain regarding the functions and the underlying mechanisms of the immunosuppressive cells in tumor immune escape and metastasis.The molecular basis of the complex biochemical processes involved in breast cancer is becoming increasingly understood.Many evidences had shown that chemokine and chemokine receptors are involved in tumor cell proliferation,survival,migration,and angiogenesis.Chemokines are chemotactic cytokines that mediate cellular trafficking,leukocyte maturation,homing of lymphocytes and the development of lymphoid tissues.C-C chemokine receptor type 4(CCR4)protein is a G-protein coupled receptor that acts as the receptor for CCL17 and CCL22.Regulatory T cells(Treg)are a subset of immunosuppressive T cells that also have high CCR4 expression.Tregs play a key role in maintaining self-tolerance and modulating adaptive immune responses.They can inhibit an excessive immune response during inflammation by suppressing effector T cells.Increased numbers of Tregs are correlated with poor prognosis in patients with tumors,suggesting that Tregs may weaken antitumor immune responses and foster immune privilege.The main experiments are as follows:1.A total of 78 primary NSCLC patients,who had undergone surgical resection with curative intent between 2009 and 2011,were obtained from the First Affiliated Hospital of Chongqing Medical University,China.To determine CCR4 expression in NSCLC patients,we examined the expression of CCR4 levels in the 78 NSCLC patients by IHC and used the scoring system to consolidate the results for negative and positive staining percentage.Then,we examined the expression of CCR4 mRNA levels in the tumor tissues and tumor adjacent tissues of NSCLC patients by realtime-PCR.To assess whether CCR4 expression influenced clinical outcome after Tumor resection,as shown in the Kaplan-Meier survival curves.To reveal the correlations among CD4+CD25+FoxP3+ Treg and CCR4+ Treg in NSCLC patients,we performed to examine the fraction of those in TIL and PBMC by flow cytometry.Next,we compared the cell surface expression of CCR4 on tumor infiltrating and circulating Treg cells in NSCLC patients and healthy controls.To investigate the activation of Treg-type cytokines expressed,we measured the IL-10 and TGF-β1 concentration in tissues.2.We performed to measure the concentration of CCL17 and CCL22 in the supernatants of tissue homogenates by ELISA.To determine whether CCL17 or CCL22 was responsible for the chemotaxis of the regulatory T cells,transwell migration assay were be used.Further studies to evaluate suppressor activity mediated by CD4+CD25+Tregs in NSCLC patients compared to healthy control,we investigate their ability to suppress autologous CD4+CD25-T cells upon stimulation with CD3/CD28.Treg cell suppressive assays were performed as described in the Materials and Methods.3.We determine the frequency of CD3-CD56+NK cells from tumor tissues in NSCLC patients.We evaluate the activating receptor and inhibitory receptor in NK cells by flow cytometric.We evaluate whether Treg cells impair IL-2 mediated activation of NK cells.Human NK cells have potent cytotoxic activity against tumor cells.To assess NK cells’ cytotoxic activity,fresh isolation of NK cells from tumor tissues and matched adjacent tissues were tested for their ability to lyse the NK sensitive cell line K562 cells.Results and conclusions were as fellows:1.After investigating for possible associations between CCR4 expression and clinicopathologic features of the patients,we found that CCR4 expression was evidently correlated to clinical stage of NSCLC patients(P<0.01).However,it was not significantly associated with gender,age,histological type,tumor size or age of patients in the NSCLC(all P?>?0.05,Table 1).The result of realtime-PCR showed that the expression of CCR4 mRNA is statistically higher than that of the matched adjacent samples(P<0.001).Taken together,we concluded that CCR4 had higher expression in tumor samples in NSCLC patients.NSCLC patients who presented with positive CCR4 expression had poor overall survival rates compared with patients who with CCR4 negative expression(P?<?0.001).Treg cells accumulated in TIL.The mean percentage of CD4+CD25+FoxP3+ Treg cells in TIL and PBMC were statistically higher than that of the matched adjacent tissues and healthy controls(P = 0.0046).The expression level of CCR4 was higher in tumor infiltrating Treg cells than in adjacent tissue cells.By analysis the correlation of CCR4+Treg and Treg in TIL,Our results showed that there was a significant correlation between the percentages of CCR4+Treg and CD4+CD25+FoxP3+Treg cells among NSCLC patients(P<0.01).ELISA results indicated that there was a significantly higher level of serum TGF-β1 in the supernatant of tissue homogenate than matched adjacent tissues(P<0.001).However,the concentration of IL-10 was no significant statistical difference.2.Higher expression of CCL17 and CCL22 were observed in the tumor sites of NSCLC patients(P<0.01).However,they were no difference in serum from NSCLC patients.Transwell migration assay result demonstrated that CD4+CD25+ T cell from TIL had a significantly greater chemoattractant response compared to CD4+CD25-T cell(P<0.001).Blocking experiments further demonstrated that antiCCL17,antiCCL22,and antiCCR4 could significantly suppressed the chemotaxis of Treg cells.Taken together,these data reminded us that Treg might via CCR17 and CCR22 recruiting Treg cells to tumor sites.Tumor-infiltrating Treg cells demonstrated increased suppressive ability on proliferation of effective T cells,compared to Treg cells obtained from matched adjacent tissues or peripheral blood.Adding neutralizing anti-TGF-β antibody reduced the suppressive ability of Treg(P<0.001).3.Flow cytometry results indicated that the frequency of CD3-CD56+NK cells from tumor tissues in NSCLC patients were significantly decreased compared with matched adjacent tissues(P<0.001),however,they were the same level in peripheral blood.flow cytometric results shown that the activating receptor of NKG2 D expression levels in NK cells from tumor tissues was significantly lower than that of matched adjacent tissues(P<0.01).However,no differences in NKp30 NKp46 and NKG2 A expression levels were observed between NK cells collected from tumor tissues and matched adjacent tissues.our result showed that the cytokine IFN-γ of IL-2-activated NK cells from tumor tissues was significantly suppressed by Treg cells,but reversed by anti-TGF-? antibody(P<0.001).CD69 was an early activation marker for NK cells,also present a similar result(P<0.001).As shown in Figure 6C,the lower lytic activity was observed in cells from tumor tissues with respect to matched adjacent tissues(P<0.001),but reversed by anti-TGF-?antibody.