B7-H1 And B7-H3 Expression In Non-small Cell Lung Cancer And Their Roles In Tumor Immune Evasion

Lung cancer is a malignant tumor with highest incidence and mortality in the World. Of all lung cancers, the non-small cell lung cancer (NSCLC), is the most common one, occupying 80～87% of the total. In recent years, certain gratifying achievements have been made in the treatments of NSCLC, with molecule-targeted treatments in particular. But unfortunately, the five-year survival rate for patients has not been raised significantly, therefore it is urgent to search for more effective ways for their treatments. With the deepening of tumor immune researches, although some tumor immune treatment methods have been introduced into clinical practices, most of these methods are still in the stage of exploration and perfection, with only limited clinical effects. Many tumor immune mechanisms still deserve further exploration, because they possess great significance in understanding the emergence and progression regularities of tumors and in searching for more effective methods for their treatments.The emergences and metastases of tumors are closely related to the internal and external environments of the tumor cells. The tumor microenvironment, which is composed of tumor cells themselves, mesenchyme cells, micro blood vessels, tissue liquids, and some infiltrated cells(such as dendritic cells, macrophages, T lymphocytes and other immune cells), is the internal one in which tumors emerge and progress. The infiltrated immune cells in tumor the microenvironment, are the effector cells of the immune defense, which recognize and attack tumor cells. Through modifying surface antigens themselves, expressing negative regulatory molecules, and secreting control determinants, tumor cells can escape the immune cell recognition and attack to occur and progress. T cells are the primary immune effector cells of anti-tumor, whose activation depends on the joint action of the dual signals, the special antigen signal and the co-stimulatory molecule signal. The B7 family of the co-stimulatory molecules plays a very important role in activating and inhibiting T cell immunity. The co-stimulatory molecules can not only provide the second positive signal, very important in staring, strengthening and preserving T cell immunity, but also produce signals to inhibit T cell immune response. The inhibition of T cell immune response is the major source for tumors to escape immunity. Being new members in B7, B7-H1 and B7-H3 are over expressive in many tumor cell lines and tissues, to be more and more attention. Most researches indicate that B7-H1 expressed in tumor is negative regulatory molecule that is beneficial to the emergence and progression of tumors, but its biological mechanisms need to be explored further. Indicated in earlier researches, B7-H3 could co-stimulate the cell proliferation of CD4+、CD8+T cells, and increase the activation of cytotoxic T lymphocyte(CTL). But further studies revealed that B7-H3 could also inhibit the proliferation of T cells in vitro, and lower the cell determinant secretion of IFN-γ、TNF-α、GM-CSF and other cytokines. The recent clinical studies show that B7-H3 expressed in tumor tissues is related to disease progression and poor prognosis, but the opposite reports have also been noticed. So the biological functions of B7-H3 are still controversial. Since there are not many studies on the expressive significance of B7-H1 and B7-H3 in lung cancer tissues, their biological functions and mechanisms in lung cancer emergence and progression need to be studied further.INF-y is the important effective and regulatory factor of T cells. Researches indicate that INF-y can down-regulate CXCR4 expression, which play a role in tumor proliferation, invasion and metastasis. TIA-1 (T-cell intracellular antigen 1) is a cytolytic granule protein, an important cytolytic effector of CTL and NK. TIA-1 is commonly regarded as the marker of CTL and NK, and researches find that TIA-1+ cells infiltrated in tumor decrease in tumor progression. Researches have already proved that B7-H1 and B7-H3 can regulate CTL and NK. Through detecting the expression levels of B7-H1、B7-H3 and CXCR4, and the infiltration levels of TIA-1+ and IFN-γ+ cell in NSCLC tissues, and together with the cell lines studies in vitro, this study tries to explore the clinical significance of B7-H1 and B7-H3 expression and their possible roles in tumor immune evasionObjectivesTo detect the expression levels B7-H1 and B7-H3 in NSCLC tissues, and to explore the clinical significance of the two biomarker expressions and their possible roles in tumor immune evasion Methods1、Collect 128 cases of resected NSCLC tissue samples from March 2005 to October 2007. Immunohistochemical method was used to detect the expression level of B7-H1, B7-H3 and CXCR4 and the infiltration degree of TIA-1 and IFN-γcells in NSCLC tissues. Questionnaires were designed to record the patients’ clinic-pathologic information, including age, sex, operation time, tumor location and size, pathological type, differentiation degree, lymph metastasis, and TNM stage. Also, the follow-up was conducted to investigate the patients’ survival conditions. The relationships between these biomarkers expression levels and clinic-pathologic factors were analyzed.2、pGPU6/GFP/Neo plasmid vectors was selected to construct recombinant B7-H3 shRNA vector. With liposome transfection method, H1299 cell line with high expressed B7-H3 transfected with recombinant B7-H3 shRNA vector to establish B7-H3 gene silencing cell H1299-B7-H3-KD. pIRES2-EGFP plasmid vectors was selected to construct recombinant B7-H3 expression vector. With liposome transfection method, SPC-A1 cell line with low expression of B7-H3 transfect with recombinant B7-H3 expression vector to establish B7-H3 gene transfected cell SPC-A1-B7-H3. Detect, with 3H-TdR incorporation assay, the proliferation in vitro of T cell inspired by gene silencing H1299-B7-H3-KD and gene transfected SPC-A1-B7-H3 cells, also with ELISA, the INF-γconcentration in supernatant. Analyze, with flow cytometry, the circle change of the inspired T cell. Detect, with flow cytometry and RT-PCR, the expression level of B7-H3 gene silencing and gene transfected cells, and also CXCR4 expression level of cell lines HI299 under the action of IFN-γ. Detect, with Transwell experiments, the influence of IFN-γin vitro migration of cell lines H1299.Results1、Of 128 cases of NSCLC,93cases (72.7%) were of over expressed B7-H1, and 89 cases (69.5%) were of over expressed B7-H3. Out of adjacent tissues, B7-H1 was mainly of low expression. B7-H3 was of non-or-low expression. The expression levels of B7-H1、B7-H3 in NSCLC tissues were significantly higher than those in the adjacent tissues (P<0.001,<0.001). The expression levels of B7-H1、B7-H3 in NSCLC tissues increased with lymph node metastasis and TNM pathologic stage advance (P<0.05,<0.05), showing no relevance to other clinic-pathologic factors. 2、The over expression of B7-H1 and B7-H3 in NSCLC tissues showed significant relevance (P<0.05). Of 128 cases of NSCLC tissues,68 (53.1%) showed co-expression of B7-H1 and B7-H3, and no co-expression cases of B7-H1 and B7-H3 was found in the adjacent tissues. The co-expression rate (72.9%) of B7-H1 and B7-H3 in patients with lymph node metastasis was significantly higher than that in patients without lymph node metastasis (29.31%)(P<0.001). The co-expression rate of B7-H1 and B7-H3 increased steadily (26.7%,53.5% and 82.5%)(P<0.001) with the advance of TNM pathologic stages (Ⅰ,Ⅱ,Ⅲ). The co-expression of B7-H1 and B7-H3 showed no significant relevance to age, gender, pathological type, differentiation, and tumor size.3, The rates of TIA-1+ cell and IFN-y+ cell high infiltration (47.7%,35.9%) in NSCLC tissues were significantly higher than those of adjacent tissues (P<0.01,<0.01). TIA-1+ cell and IFN-y+cell in the adjacent tissues showed low infiltration. The infiltration levels of TIA-1+and IFN-y+cells in NSCLC tissues decreased with lymph node metastasis and TNM pathologic stage advance (P<0.05, 0.05,0.05,0.05), but the rate of TIA-1+cell high infiltration in squamous cell carcinomas (57.4%) was obviously higher than that in adenocarcinomas (38.8%)(P<0.05).4、The over expression (55.5%) of CXCR4 in NSCLC tissues was significantly higher than that in the adjacent tissues (P<0.01). CXCR4 of the adjacent tissues all showed low expression. The expression level of CXCR4 increased with TNM pathologic stage advances (P<0.05).5、The univariate analysis of clinicopathological factors associated with survival found the following:The medium survival for patients with low proliferation tumor was significantly shorter than that for patients with medium and high proliferation tumor (27.1 vs 38.3 vs 37.5 months)(p <0.05); The medium survival for patients with lymph node metastasis was significantly shorter than that for patients without lymph node metastasis (22.9 vs 59.2 months)(p<0.01); As TNM stage developed, the patients’medium survival gradually shortened (69.7 vs 33.5 vs 13.5 months)(p <0.01); The male patients’medium survival was shorter than that of the female ones (33.5 vs 53.8months)(p=0.077); Age, tumor size and pathological type had no significant influence on the patients’medium survival.6、The univariate analysis of biomarker expressions associated with survival found the following: The medium survival of patients with B7-H1 over expression was significantly shorter than that of patients with B7-H1 low expression (28.7 vs 60.6 months) (p<0.01); The medium survival of patients with B7-H3 over expression was significantly shorter than that of patients with B7-H3 low expression (25.0 vs 60.6 months)(p<0.01); The medium survival of patients with high TIA-1+cells infiltration was significantly longer than that of patients with low TIA-l+cells infiltration (59.2 vs 20.5 months)(p<0.01); The medium survival of patients with high IFN-y+cells infiltration was significantly longer than that of patients with low IFN-γ+ cells infiltration (44.7 vs 28.7 months)(p <0.05); The medium survival of patients with CXCR4 over expression was shorter than that of patients with CXCR4 low expression(28.7 vs 40.7 months)(p=0.08); When B7-H1 and B7-H3 were both over expressive, the medium survival was the shortest (22.7 months), and when B7-H1 and B7-H3 were both low expressive, the medium survival was the longest (60.6 months). When B7-H1 was over expressive while B7-H3 was low expressive, the medium survival was 49.5 months. When B7-H1 was low expressive while B7-H3 was over expressive, the medium survival was 37.1 months, with significant differences existing between groups (p<0.01).7、The multivariate analysis of survival indicated that sex, TNM stage, B7-H1 expression level, B7-H3 expression level, TIA-1+cell infiltration level were were independent predictors of survival (P<0.05,0.05,0.05,0.05,0.01). The survival was shorter for male patients or patients with advanced TNM stage, B7-H1 over expression, B7-H3 over expression and low TIA-1+cell infiltration.8、In NSCLC tissues, the over expression of B7-H1 and B7-H3 was significantly positive in relevance to that of CXCR4 (P<0.05,<0.001); The high infiltration of TIA-1+cells was significantly negative in relevance to the over expression of B7-H3 and CXCR4 (P<0.05) and B7-H1, but positively relevant to the high infiltration of IFN-γ+cells (P<0.05); The high infiltration of IFN-y+ cells was negatively relevant to the over expression of B7-H1 and B7-H3, but with no statistic significance.9-Lung cancer cell lines H1299, after B7-H3 gene silencing, could significantly promote the proliferation in vitro of T cells stimulated by CD3mAb and CD28mAb(P<0.05) and stimulate these cells to secrete IFN-y. Lung cancer cell lines SCP-A1 transfected by B7-H3 gene could significantly inhibit the proliferation in vitro of T cell stimulated by CD3mAb and CD28mAb (P<0.05) and depress these cells to secrete IFN-γ(P<0.05).10, IFN-y could reduce CXCR4 expression of lung cancer cell line H1299. Like AMD3100 (CXCR4 antagonist), INF-y could significantly inhibit the migration capacity (P<0.001) of H1299 stimulated by CXCL12. H1299, after B7-H3 gene silencing, showed obvious decrease in migration capacity.ConclusionsB7-H1 and B7-H3 might play negative regulatory roles in tumor immunity, and the over expression of the two molecules may promote the emergence and progression of NSCLC. Through inhibiting the infiltration, proliferation and secretion of T lymph or NK cells that express TIA-1 and INF-y in NSCLC tissues, B7-H1 and B7-H3 could restraint the anti-tumor immune responses of patients with NSCLC. The secretion decrease of INF-y would upgrade CXCR4 expression of cancer cells. The restraint of anti-tumor immune responses enabled tumor cells to escape from immunity to proliferate and progress. The expression upgrade of CXCR4 might further accelerate the proliferation, attack and metastasis of cancer cells. B7-H1 expression level, B7-H3 expression level, TIA-1+cell infiltration level were were independent predictors of survival in patients with NSCLC. The interference in the signal pathways of the two negative regulatory molecules of B7-H1 and B7-H3 might possibly become the new treatments of NSCLC.