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@#[摘 要] 以靶向PD-1/PD-L1和CTLA-4为代表的免疫检查点阻断治疗在实体瘤的治疗中取得了不俗疗效，但仅有不到30%的患者能够从中受益的现实表明，还存在其他免疫检查点分子介导的免疫抑制。B7H3属于免疫球蛋白超家族B7家族成员，与CTLA-4/PD-1主要表达在T细胞并介导后者的免疫抑制或耗竭不同，B7H3蛋白不仅诱导性表达在免疫细胞，还组成性高表达在多种肿瘤细胞和肿瘤相关脉管系统。B7H3不仅能够激活多条信号通路直接促进肿瘤细胞恶性表型，还可以通过重塑肿瘤免疫抑制微环境间接促进肿瘤的进展、转移和耐药。因此，基于B7H3的多项靶向抗肿瘤策略，包括特异性抗体、抗体偶联药物及CAR-T细胞等均已进入临床试验并展示出较好的应用前景。但总体而言，该领域的研究依然处于探索阶段，在相互作用受体的鉴定、降低毒性、打破耐药及联合用药策略优化等方面还面临着许多问题和挑战亟待突破。
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Objective:To investigate the effect of B7-H3 gene on the biological function of fibr-oblastlike synoviocytes (FLS) in osteoarthritis (OA).Methods:Synovial tissue of five cases of OA and synovial tissue of 4 normal knee were obtained, and the primary cell lines were isolated and cultured. The expression of B7-H3 in OA synovial tissue and primary OA-FLS were studied by immunohi-stochemistry, real time-poly merase chain reaction (PCR) and FACS. According to sites 996 and 1041 of B7-H3, corresponding siRNA was designed and the expression of B7-H3 in FLS was silenced and down-regulated. The inhibition of B7-H3 and its protein in target cells was determined by Western blot and FACS. The migration and invasion ability of B7-H3 in target cells were analyzed by scratch assay and Transwell assay. CCK8 assay was used to detect cell proliferation ability, and CBA assay was used to detect cytokines and chemokines in cell culture supernatant. GraphPad Prism 8.0 software was used to analyze the experimental data. The normal distribution data was expressed as mean±standard deviation ( Mean± SD). The comparison between data was performed by T test, and P<0.05 was considered statistically significant. Results:The abnormally high expression of B7-H3 in fibro-blast-like synoviocytes of OA was detected. Compared with siNC, si996 and si1041 inhibited the expression of B7-H3 in OA-FLS. In the Transwell migration experiment, the mean cells number of random view in the siNC group, the si996 group, and the si1041 group indicating decreased migration ability of OA-FLS [siNC vs si996 (100.3±3.7) /view vs (48.7±1.2) /view, t=13.24, P<0.001; siNC vs si1041 (100.3±3.7) /view vs (59.7±1.9) /view, t=9.80, P<0.001). In the Transwell invasion experiment, the mean cells number of random view in the siNC group, in the si996 group, and in the si1041 group indicating decreased invasion ability of OA-FLS [siNC vs si996 (127.3±5.6) /view vs (39.7±3.3) /view, t=13.49, P<0.001; siNC vs si1041 (127.3±5.6) /view vs (57.3±1.9) /view, t=11.85, P<0.001]. The secretion of IL-6 [siNC vs si996 (248±21) pg/ml vs (111±12) pg/ml, t=24.08, P=0.002; siNC vs si1041 (248±21) pg/ml vs (46±5) pg/ml, t=13.21, P=0.006], IL-8 [siNC vs si996 (118.1±15.6) pg/ml vs (47.1±5.4) pg/ml, t=6.68, P=0.022; siNC vs si1041 (118.1±15.6) pg/ml vs (10.0±1.3) pg/ml, t=13.08, P=0.006], CXCL8 [siNC vs si996 (178.8±6.4) ng/ml vs (83.2±2.7) ng/ml, t=13.77, P=0.005; siNC vs si1041 (178.8±6.4) ng/ml vs (93.5±2.8) ng/ml, t=12.23, P=0.007] and CCL2 [siNC vs si996 [(184.1±5.1) ng/ml vs (109.4±5.9) ng/ml, t=9.57, P=0.011; siNC vs si1041 (184.1±5.1) ng/ml vs (97.1±1.5) ng/ml, t=16.39, P=0.004] was decreased . Conclusion:B7-H3 may regulate the migration, invasion, cytokine secretion and other biological functions of OA-FLS, providing clues for further study of B7-H3's involvement in the pathogenesis of OA.
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Objective:To investigate the expression of B7-H3 in diffuse large B-cell lymphoma (DLBCL) and its prognostic significance.Methods:The paraffin-embedded tumor tissues of 103 patients with newly diagnosed DLBCL in Linyi Central Hospital from May 2013 to May 2019 were detected by using immunohistochemistry. The association of the expression of B7-H3 protein with the clinicopathological features, progression-free survival (PFS) and overall survival (OS) of DLBCL patients was analyzed. Cox proportional hazards model was used to analyze the factors affecting PFS and OS.Results:The positive rate of B7-H3 protein in patients with DLBCL was 68.0% (70/103). There were no statistically significant differences in the positive rate of B7-H3 protein among patients with different gender, age, clinical staging, international prognostic index (IPI) score, treatment effect, B symptoms, pathological type and other clinicopathological characteristics (all P > 0.05). The 5-year PFS and 5-year OS rates were 24% and 32% in all patients, the 5-year PFS rates were 47% and 14% in B7-H3 negative and B7-H3 positive patients, respectively ( P < 0.01); and 5-year OS rates were 50% and 24% in B7-H3 negative and B7-H3 positive patients, respectively ( P < 0.001). Multi-factor Cox regression analysis showed that B7-H3 positive was an adverse affecting factor of PFS ( HR = 2.685, 95% CI 1.503 - 4.789, P = 0.001) and OS ( HR = 2.262, 95% CI 1.248 - 4.098, P = 0.007). Conclusions:The moderate and high expression of B7-H3 may be related to the poor prognosis of DLBCL patients.
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@#[摘 要] B7/CD28家族分子作为主要的共信号分子，在T细胞功能调控及免疫应答中发挥着至关重要的作用，关于其功能的研究及应用在世界范围内广泛开展。其中，CD28和CTLA-4都可以与B7-1/B7-2结合，但CD28能够促进T细胞增殖，维持Treg细胞稳态；而CTLA-4则可以抑制T细胞增殖，影响CD4+T细胞的分化；抗CTLA-4单抗ipilimumab与抗PD-1单抗联用能够用于治疗PD-L1+的非小细胞肺癌以及无症状的Ⅳ期黑色素瘤。PD-L1/PD-L2：PD-1途径则可以通过多种方式调节T细胞功能，参与CD8+T细胞“耗竭”状态的形成与维持。目前，针对PD-L1/PD-L2：PD-1途径的免疫治疗相关药物开发广泛且较为成熟，抗PD-1单抗主要通过诱导肿瘤浸润的部分耗竭的CD8+T细胞亚群的扩增发挥抗肿瘤作用。ICOS：ICOSL途径在T细胞分化、细胞因子分泌以及体液免疫应答中起着重要作用，抗ICOS抗体药物feladilimab与抗PD-1单抗联用能够有效治疗多发性或难治性骨髓瘤。B7-H3同时具有共刺激效应和共抑制效应，阻断B7-H3后能够有效增强TIL的抗肿瘤效应，其单抗enoblituzumab能够与抗PD-1单抗联用治疗非小细胞肺癌。B7-H4、人内源性逆转录病毒‑H长末端重复关联蛋白（human endogenous retrovirus‑H long terminal repeat‑associating protein，HHLA）以及含V结构域抑制T细胞活化的免疫球蛋白（V‑domain Ig‑cotaining suppressor of T cell activation，VISTA）均能够提供抑制信号，针对B7-H4靶点的CAR-T治疗以及VISTA的小分子抑制剂CA-170均有临床试验正在进行中。目前，对共信号分子的研究已经获得了长足进展，针对某些活化性分子或者抑制性分子开发了相关抗肿瘤药物并在临床中取得一定成效，但如何进一步提高其治疗有效率、减少不良反应等仍有待探索。
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Objective:To investigate the effects of B7-H3 molecule on clear cell renal cell carcinoma (786-O) metastasis.Methods:Lentiviral transfection method was used to construct 786-O cells stably expressing low level of B7-H3 (shB7-H3 group) and a negative control cell line (shNC group). RT-qPCR, flow cytometry and Western blot were used to assess the efficiency of lentiviral transfection. CCK-8 method was used to detect the proliferation of 786-O cells in the two groups. Flow cytometry was performed to detect the changes in cell cycle. Cell scratch test and Transwell assay were used to detect the differences in cell migration and invasion. Western blot was used to detect the expression of marker proteins in the process of epithelial-mesenchymal transition (epithelial-mesenchymal transition, EMT). Changes in the expression of chemokines and their receptors were analyzed by flow cytometry and RT-qPCR. Effects of anti-CCL4 antibody on cell migration and invasion were analyzed by Transwell assay.Results:Flow cytometry showed that 786-O cells highly expressed B7-H3 molecules and the lentiviral transfection method successfully constructed the cell line with lower expression of B7-H3 (786-O-shB7-H3) and control cell line (786-O-shNC). B7-H3 molecule had no significant effect on the proliferation of 786-O cells. No significant difference in cell cycle was found between the two groups. Compared with 786-O-shNC cells, the migration and invasion ability of 786-O-shB7-H3 cells was suppressed. Moreover, the expression of EMT-related marker proteins (fibronectin and N-cadherin) was reduced and the expression of E-cadherin was increased in 786-O-shB7-H3 cells. The expression of CCL4 and its receptor CCR5 in the shB7-H3 group was lower than that in the shNC group. After intervention with anti-CCL4 antibody, the migration and invasion ability of 786-O-shNC cells was reduced, while that of 786-O-shB7-H3 cells had no significant change.Conclusions:Knocking down the expression of B7-H3 molecule had no significant effect on the proliferation of 786-O cells, but could affect the EMT process of 786-O cells and reduce tumor migration and invasion ability, thereby inhibiting tumor progression.
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@#B7-H3 is an immune checkpoint molecule overexpressed on the surface of a variety of tumors, and is is an ideal target for tumor immunotherapy. In this study, nitrolated T cell epitope designed in the early stage of the laboratory was used to construct an epitope vaccine that can target immune checkpoint B7-H3. The vaccine can significantly inhibit tumor growth in the CT26 colon cancer model, and has a significant synergistic effect with the PD-L1 protein vaccine. B7-H3 vaccine can increase the proportion of CD4+ T cells in splenic T lymphocytes and the proportion of CD8+ T cells in tumor-infiltrating T lymphocytes, while reducing the proportion of suppressor Treg cells in tumor-infiltrating CD4+ T lymphocytes, which effectively improves tumor immunosuppressive microenvironment. Research results suggest that the B7-H3 epitope vaccine can be used as an effective tumor vaccine candidate molecule.
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Objective@#To investigate the expression of B7H3 in neuroblastoma (NB) tissues and its relationship between clinical characteristics and prognosis of patients.@*Methods@#The clinical data and pathological wax of 100 cases with neuroblastoma admitted from January 2000 to December 2015 in Sun Yet-Sen University Cancer Center were collected.The expression of B7H3 in tumor tissues was detected by immunohistochemistry (IHC) and then the expression of B7H3 and its relation to pathological parameters and survival rate of patients were analyzed.@*Results@#(1) The positive rates of B7H3 in tumor tissues were 79% (79/100 cases), of which 37 were weak positive, 31 were mode-rate positive, 11 were strong positive, 58%(58/100 cases) showed low expression and 42%(42/100 cases) showed high expression.(2)The positive rate of B7H3 was positively correlated with the risk stratification, age, stage, primary site and N-MYC gene status (r=0.621, 0.350, 0.730, 0.224, 0.335; all P<0.05). (3)The high expression rates of B7H3 were positively correlated with the risk, stage, N-MYC gene status and tumor size (r=0.177, 0.016, 0.175, 0.284; all P<0.05). (4)B7H3 negative and positive 4-year event free survival (EFS) rates of 100 patients were 89.5% and 19.9% (χ2=31.260, P<0.001), 4-year overall survival(OS) rates were 94.7% and 48.3% (χ2=20.212, P<0.001), for 33 non-high-risk patients, B7H3 negative and positive 4-year EFS rates were 100.0% and 47.3% (χ2=8.760, P=0.003), and 4-year OS rates were 100.0% and 93.8% (χ2=1.063, P=0.003), respectively.Sixty-seven high-risk patients with B7H3 negative and positive 4-year EFS were 50.0% and 15.4% (χ2=4.093, P=0.043), 4-year OS were 75.0% and 42.0%, respectively (χ2=1.872, P=0.171). (5)The 4-year EFS rates of 100 patients with B7H3 low expression and high expression were 41.8% and 27.1% (χ2=3.801, P=0.051), and 4-year OS rates were 58.6% and 63.8% (χ2=0.111, P=0.739), respectively.The 4-year EFS and OS rates for 33 non-high-risk patients with B7H3 low expression and high expression were 94.7% and 44.2% (χ2=9.122, P=0.003), 100.0% and 90.9% (χ2=2.000, P=0.157), respectively.The 4-year EFS and OS rates of 67 high-risk patients with high expression and low expression of B7H3 were 13.9% and 22.1% (χ2=0.061, P=0.805), 36.3% and 57.7%(χ2=2.060, P=0.151), respectively.(6)Multivariate analysis showed that OS and EFS in B7H3 positive patients were lower than those in B7H3 negative patients[RR 95%CI: 28.110 (2.430-325.148); P=0.008; RR 95%CI: 12.834 (2.669-61.715), P=0.001].@*Conclusions@#The positive rate of B7H3 in neuroblastoma is high, and the expression level of B7H3 is closely related to the clinical characteristics of the patients.Positive B7H3 in tumor tissues is an independent poor prognostic factor.B7H3 may be one of the new prognostic indicators for NB.
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Objective To investigate the effects of costimulatory molecule B7-H3 on the prolifera-tion and invasion of human non-small cell lung cancer cell line A549. Methods Flow cytometry was used to detect the expression of B7-H3 at protein level on A549 cells. B7-H3-targeting siRNA was transfected into A549 by lentivirus to construct B7-H3-A549 cells, which were identified with Western blot and qPCR. Differences in proliferation between B7-H3-A549 and B7-H3+A549 cells were analyzed by CCK8 assay. Flow cytometry was performed to detect the changes in apoptosis and cell cycle after AnnexinⅤ-PE/propidi-um iodide ( PI) staining. Transwell assay was used to evaluate the migration and invasion of B7-H3-A549 and B 7-H 3+ A 549 cells . Expression of apoptosis-related proteins was detected by Western blot . Results (1) B7-H3 was highly expressed on A549 cells. A stable B7-H3-A549 cell line and its control cell line B7-H3+A549 were successfully prepared. (2) A549 cell proliferation was significantly reduced after knocking down B7-H3 expression. (3) The percentage of early apoptotic cells in B7-H3-A549 cell group was higher than that in B7-H3+A549 cell group, but no significant difference in the percentages of cells undergoing late apoptosis was found between the two groups. B7-H3-A549 cells were arrested at the G0/G1 phase of cell cy-cle. (4) Compared with B7-H3+A549 cells, B7-H3-A549 cells showed suppressed migration and invasion. (5) Enhanced expression of Bad and Caspase-3 and decreased expression of Bcl-2, P-AKT and MMP-9 were detected in B7-H3-A549 cells as compared with those in B7-H3+A549 cells, but no significant difference in the total AKT was observed. Conclusions Knocking down the expression of B7-H3 molecule in A549 cells could inhibit cell proliferation and invasion, induce cell cycle arrest at G0/G1 phase and promote cell apoptosis.
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Objective To investigate the expression of B7H3 in neuroblastoma(NB)tissues and its relation_ship between clinical characteristics and prognosis of patients. Methods The clinical data and pathological wax of 100 cases with neuroblastoma admitted from January 2000 to December 2015 in Sun Yet_Sen University Cancer Center were collected. The expression of B7H3 in tumor tissues was detected by immunohistochemistry(IHC)and then the ex_pression of B7H3 and its relation to pathological parameters and survival rate of patients were analyzed. Results (1) The positive rates of B7H3 in tumor tissues were 79%(79/100 cases),of which 37 were weak positive,31 were mode_rate positive,11 were strong positive,58%(58/100 cases)showed low expression and 42%(42/100 cases)showed high expression.(2)The positive rate of B7H3 was positively correlated with the risk stratification,age,stage,primary site and N_MYC gene status(r=0. 621,0. 350,0. 730,0. 224,0. 335;all P<0. 05).(3)The high expression rates of B7H3 were positively correlated with the risk,stage,N_MYC gene status and tumor size( r=0. 177,0. 016,0. 175, 0. 284;all P<0. 05).(4)B7H3 negative and positive 4_year event free survival( EFS)rates of 100 patients were 89. 5% and 19. 9%(χ2 =31. 260,P<0. 001),4_year overall survival( OS)rates were 94. 7% and 48. 3%( χ2 =20. 212,P<0. 001),for 33 non_high_risk patients,B7H3 negative and positive 4 _year EFS rates were 100. 0%and 47. 3%(χ2 =8. 760,P=0. 003),and 4_year OS rates were 100. 0% and 93. 8%( χ2 =1. 063,P=0. 003),re_spectively. Sixty_seven high_risk patients with B7H3 negative and positive 4_year EFS were 50. 0% and 15. 4%(χ2 =4. 093,P=0. 043),4_year OS were 75. 0% and 42. 0%,respectively( χ2 =1. 872,P=0. 171).(5)The 4_year EFS rates of 100 patients with B7H3 low expression and high expression were 41. 8% and 27. 1%( χ2 =3. 801, P=0. 051),and 4_year OS rates were 58. 6% and 63. 8%(χ2 =0. 111,P=0. 739),respectively. The 4_year EFS and OS rates for 33 non_high_risk patients with B7H3 low expression and high expression were 94. 7% and 44. 2%(χ2 =9. 122,P=0. 003),100. 0% and 90. 9%(χ2 =2. 000,P=0. 157),respectively. The 4_year EFS and OS rates of 67 high_risk patients with high expression and low expression of B7H3 were 13. 9% and 22. 1%(χ2 =0. 061,P=0. 805),36. 3% and 57. 7%(χ2 =2. 060,P=0. 151),respectively.(6)multivariate analysis showed that OS and EFS in B7H3 positive patients were lower than those in B7H3 negative patients[RR 95%CI:28. 110(2. 430_325. 148);P=0. 008;RR 95%CI:12. 834(2. 669_61. 715),P=0. 001]. Conclusions The positive rate of B7H3 in neuroblasto_ma is high,and the expression level of B7H3 is closely related to the clinical characteristics of the patients. Positive B7H3 in tumor tissues is an independent poor prognostic factor. B7H3 may be one of the new prognostic indicators for NB.
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Objective To investigate the diagnostic value of B7-H3 and carcinoembryonic antigen (CEA) on diagnosis of malignant pleural effusion (MPE).Methods We collected and analysed the expression of B7-H3 and CEA in 40 MPE cases and 22 cases of benign pleural effusion (BPE) using enzyme-linked immunosorbent assay (ELISA).Receiver operator characteristic curves (ROC) were drawn according to the expression of B7-H3 and CEA, calculated diagnosis sensitivity, specificity and the area under curve (AUC).Results The diagnosis sensitivity of B7-H3 was 62.5％, specificity 81.0％, with the AUC of 0.777;similarly, The diagnosis sensitivity of CEA was 72.5％, specificity 81.0％, with the AUC of 0.850.Higher AUC of 0.910 was gained in combination ROC, with sensitivity and specificity of 72.5％, 81.0％, respectively.Conclusion B7-H3 and CEA could be available diagnosing markers for MPE.Combined applications of B7-H3 and CEA have higher AUC.They may be widely applied in future clinical practice.
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Objective To investigate the expression of B7-H3 in muscle infiltration bladder transitional cell carcinoma and its clinical significance.Methods The clinical and pathological data of 115 patients with muscle infiltration bladder transitional cell carcinoma who underwent radical surgery were retrospectively analyzed.The expression of B7-H3 in bladder cancer tissues and corresponding paracancerous tissues was detected by immunohistochemical staining,and the relationship between B7-H3 and clinicopathological characteristics was analyzed.Results The positive expression of B7-H3 was mainly in the cytoplasm,presenting brownish yellow granules.B7-H3 had positive expression in all bladder cancer tissues but no expression in paracancerous tissues.The high positive rate of B7-H3 expression in bladder cancer was 68.7％.The abnormal expression of B7-H3 was correlated with the distant metastasis and intravascular tumor thrombi (all P＜0.05),but not with gender,age,tumor stage and grade,lymph node metastasis and recurrence (all P＞0.05).The survival analysis result showed that the survival time of the patients with high expression of B7-H3 in the bladder cancer tissues was significantly lower than that of patients with low expression of B7-H3,and the difference was statistically significant (P＜0.05).Conclusion The expression of B7-H3 in muscle infiltration bladder cancer tissues is up-regulated and is positively associated with the distant metastasis of bladder cancer and intravascular tumor thrombus.The expression of B7-H3 may indicate poor prognosis of the patients with bladder cancer.
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Objective:To investigate the expression and clinical significance of B7-H3 and IL-21 in serum of patients with HBV-related primary liver cancer.Methods: Gathering 121 cases of HBV-related patients,50 cases of primary hepatic carcinoma of them were considered as hepatic carcinoma group,71 cases of benign group including 12 cases with acute hepatitis,21 cases of chronic moderate to severe hepatitis,20 cases of compensatory phase cirrhosis,18 cases of decompensated cirrhosis and 20 cases of healthy persons in the same period as normal control.The content of serum B7-H3 and IL-21 were detected by ELISA.HBV DNA quantitative results were analyzed by Quantitative Real-time PCR.Results: The levels of B7-H3 and IL-21 in patients with primary hepatic carcinoma were (207.60±57.16)ng/ml and(2 357.28±805.01)pg/ml,respectively,which were significantly higher than those of the normal control subjects(P<0.001).Comparison with the normal control subjects,the content of B7-H3 and IL-21 in serum of patients with different clinical types in the benign group increased significantly(P<0.001).B7-H3 and IL-21 were positively associated with each other in serum of patients with HBV-related primary liver cancer.The expression of sB7-H3 was not significantly correlated with the degree of HBV DNA replication.The expression of IL-21 was correlated with HBV DNA replication in patients with HBV associated hepatocellular carcinoma,but was not significantly correlated with the degree of HBV DNA replication.Conclusion: HBV-related primary hepatic carcinoma express sB7-H3 and IL-21 with high level.The continuous high expression of sB7-H3 and IL-21 in the body may be related to the development and prognosis of the patients.
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Objective To investigate the influence of sorafenib on the expressions of B7-H3 and B7-H4 proteins in different human hepatocellular carcinoma cell lines,including HepG2,Hep3B,BEL-7402,BEL-7404,BEL-7405,QGY-7701,QGY-7703,SMMC-7721,MHCC97H,MHCC97L,HCCLM3 and HCCLM6.Methods Western blotting and MTT assay were used to check the influence of sorafenib on the expressions of B7-H3 and B7-H4 proteins in different human hepatocellular carcinoma cell lines and to test the inhibitory effect of sorafenib on different human hepatocellular carcinoma cell lines.Results Compared with normal human liver cells (HL-7702),the expressions of B7-H3 and B-H4 proteins in different human hepatocellular carcinoma cell lines were significantly up-regulated (P＜0.01).The cytotoxic activity IC50 values of sorafenib to Hep3B,BEL-7404,MHCC97H,HCCLM3 and HCCLM6 were 14.56,9.14,9.46,17.21 and 9.29 μmol/L respectively.After treating Hep3B,BEL-7404,MHCC97H,HCCLM3 and HCCLM6 with sorafenib at the doses of 5,10 and 20 μmol/L separately,the expressions of B7-H3 and B7-H4 proteins were strikingly down-regulated when compared with the control group (P＜0.01).Conclusion The overexpressions of B7-H3 and B7-H4 proteins in different human hepatocellular carcinoma cell lines are a common finding,which can influence tumor immune escape.It may be a new target for prevention and treatment of liver cancer in future.
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Objective To explore the role of B7-H3 in the Tie2 expressing monocytes (TEMs) mediated angiogenesis of clear cell renal cell carcinoma (ccRCC).Methods Level of B7-H3 expression on TEMs surface was detected by flow cytometry in ccRCC tissues and normal renal tissues,which were obtained from April 2016 to August 2016 from 20 patients.Microvessel density (MVD) labeled by CD34 in high B7-H3 + TEMs group and low B7-H3 + TEMs group was detected by immunohistochemical examination in ccRCC specimens.B7-H3 + TEMs and B7-H3-TEMs were co-cultured with the 786-O cell lines,and B7-H3 + TEMs and B7-H3-TEMs culture supernatants were collected as conditioned medium,then the effect of B7-H3 + TEMs on angiogenesis was tested by tubule formation assay and mouse aortic ring assay.Results Flow cytometry showed that the frequency of B7-H3 expression on TEMs in ccRCC was (45.10 ± 17.78)％,and the frequency of B7-H3 expression in normal kidney tissues was (10.28 ± 4.28) ％.The frequency of B7-H3 expression on TEMs was significantly higher than that in normal renal tissues (P ＜ 0.001).The MVD of high B7-H3 + TEMs group (103.81 ± 29.28) was higher than that of low B7-H3 + TEMs group (76.55 ± 20.80) (P =0.027).The results of tube formation assay showed that the number of tubule formation of HUVEC in B7-H3 +TEMs group(55.25 ± 11.48) was significantly greater than that of B7-H3-TEMs group (31.34 ± 8.45) and blank control group (25.00 ± 6.74) (P ＜ 0.001).The results of mouse aortic ring assay showed that the number of neovascularization in B7-H3 + TEMs group(77.35 ± 18.47) was significantly greater than that of B7-H3-TEMs group (39.42 ± 8.29) and blank control group (28.79 ± 7.63) (P ＜0.001).Conclusions B7-H3 + TEMs can promote angiogenesis in ccRCC,and might act as an effective target in anti-angiogenic therapy for ccRCC.
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Objective To study the impacts of B7-H3 molecule on the proliferation of T lymphocytes in different activation conditions and on the secretion of relevant cytokines.Methods Peripheral blood samples were collected from healthy subjects to separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation.T lymphocytes were isolated from some of the PBMCs and purified with T Cell Enrichment Kit.PBMC and purified T lymphocytes were activated by anti-CD3/CD28 monoclonal antibodies (McAb) in vitro.Flow cytometry analysis was used to detect the expression of CD28 and CTLA-4 on T lymphocytes at different time points for further analyzing the activation states of T lymphocytes.On this basis, human B7-H3-Fc fusion protein was added into the mixed co-cultivation system on days 0, 1, 2, 3 and 4, and Hu-Fc fusion protein was used as isotype control.CCK-8 method was performed to detect the proliferation of T lymphocytes in each group.ELISA method was used to detect the secretion of cytokines (IL-2, IL-10 and IFN-γ) and to analyze the immune responses induced by stimulating T lymphocytes at different states of activation with B7-H3.Results B7-H3 molecule significantly inhibited the quiescent T lymphocytes from secreting IL-2 and IL-10, but had no significant impact on IFN-γ secretion.Moreover, it significantly promoted the activated T lymphocytes to secret IL-2 and IFN-γ, but had no obvious impact on IL-10 secretion.Results of the cell proliferation assay showed that B7-H3 molecule inhibited the in vitro proliferation of T lymphocytes in the PBMC, but had no obvious impact on purified T lymphocytes.ConclusionThe regulatory effects of B7-H3 molecule on the immune functions of T lymphocytes vary with the activation states of T lymphocytes.
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Objective:To investigate the effect and mechanism of targeted B7-H3 gene silencing on the tumorigenesis and metastasis of human hematological malignancy xenograft tumor in nude mice.Methods: Real-time fluorogentic quantitative PCR (qPCR) and flow cytometry (FCM) were used to detect the expression of B7-H3 in 13 strains of malignant hematologic cells.Then,U937,Maver and Z138 cells which expressed high level of B7-H3 were screened out.Targeted B7-H3 knockdown in U937,Ma-ver and Z138 was performed by lentivirus transduction and the effect of B7-H3 silencing in stable cell lines was tested by qPCR and FCM.Injecting the nine groups subcutaneously into the nude mice to establish xenograft models after dividing the U937,Maver and Z138 into non-infected control group (CON),B7-H3 knockdown group (KD) and negative non-targeted control infected group (NC),respectively,for detecting the tumorigenicity and metastasis in vivo.Furthermore,the expression of Ki-67 in xenograft tumors was detected by immunohistochemistry (IHC).The expression of metalloproteinase 2 (MMP-2) was detected by western blot.Results: The stable B7-H3 silencing cell lines of U937,Maver and Z138 were successfully established.Compared with the NC group,the KD groups of U937,Maver and Z138 had an obviously slower tumor growth.The average tumor inhibition rates at the end of observation period were 61.83％ (F=43.78,P0.05).The liver distant metastasis of all the xenograft tumor models in nude mice was the most common and the rates of distant metastasis in KD groups were significantly lower than that of the corresponding NC groups.The Ki-67 indexes of the KD groups were significantly lower than those of the relative NC groups in three cell lines (U937: 40.3％±5.2％ vs.79.1％±6.3％,q=30.31,P0.05).The expressions of MMP-2 were also significantly lower in the KD groups than in the NC groups (U937: q=14.59,P0.05).Conclusion: Targeted B7-H3 gene silencing could inhibit the tumorigenesis and metastasis of human hematological malignancy xenograft tumor in nude mice.The mechanism may be related to the down-regulation of Ki-67 and MMP-2.
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B7-H3,a newly discovered co-stimulatory regulatory protein member of the B7 family.Its mRNA is ubiquitously expressed in a wide spectrum of tissues.As a co-stimulatory or co-inhibitory signal molecule which can regulate immune response,B7-H3 plays an important role in the immune system.Besides,B7-H3 can be also involved in cancer progression via non-immunological.Recently,the aberrant expression of B7-H3 has been described in various malignancies,and significantly correlated with poor prognosis and cancer progression.Therefore,B7-H3 is considered as an early diagnostic and prognostic marker and therapeutic target for tumors,but the specific molecular mechanisms of B7-H3 regulation are poorly understood.The immune and gene therapy of tumor by target B7-H3 has made some progress.
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Objective To investigate the therapeutic effects of anti-B7-H3 blocking monoclonal antibody(McAb) in a murine model of neutrophilic asthma. Methods Twenty-four female BALB/c mice were randomly divided into four groups: PBS control group (group A), neutrophilic asthma group (group B),anti-B7-H3 McAb group (group C) and IgG isotype control group (group D). Those in group A were sensitized by injection of PBS and challenged with PBS through inhalation,while the other mice were sensi-tized by injection of ovalbumin (OVA) plus airway instillation of lipopolysaccharide(LPS),and then chal-lenged with OVA. Moreover,mice in group C and group D were respectively injected with anti-B7-H3 McAb and IgG isotype control in the induction period. Each mouse′s performance was observed. Samples of bron-choalveolar lavage fluid (BALF) and lung tissues were collected. Total and differential cell counts in BALF were determined by using microscope. Levels of cytokines including IFN-γ,IL-4,IL-6,IL-17,tumor nec-rosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) in BALF were measured by ELISA. Lung sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to identify tissue inflammation and mucus production,respectively. Immunohistochemistry was used to measure the expression of B7-H3 in frozen mouse lung sections. Results Mice in group B and group D showed asth-matic symptoms such as breathlessness,dysphoria and incontinence after nebulization,while these symptoms in group C were alleviated due to anti-B7-H3 McAb treatment. No abnormalities were observed in group A. Compared with group A,the other three groups showed increased total cell count in BALF and higher per-centages of neutrophil and eosinophil(P<0.05). These three indicators in group C were lower than those in group B and group D (P<0.05). With regard to lung infiltration by Th1,Th2 and Th17 cells,the levels of IFN-γ,IL-4,IL-6,IL-17,TNF-α and G-CSF in BALF were increased in group B,group C and group D as compared with those in group A. Compared with group C,group B and group D showed higher levels of IL-6, TNF-α,IL-17 and G-CSF(P<0.05),but lower level of IL-4(P<0.05). No statistical difference in the level of IFN-γ was observed among group B, group C and group D. Histological staining of lung sections showed that no obvious inflammatory cells and mucus secretion was observed in group A. Massive infiltration of inflammatory cells and neutrophils and mucus hypersecretion were detected in group B and group D. Treatment with anti-B7-H3 McAb inhibited the accumulation of neutrophils and mucus hypersecretion in lung tissues of group C. Compared with group A, levels of B7-H3-positive cells were significantly increased in group B and group D. Anti-B7-H3-treated mice showed reduced levels of B7-H3-positive cells in lung tissues as compared with those in group B and group D(P<0.05). Conclusion Treatment with anti-B7-H3 bloc-king McAb in an early stage can relieve the asthmatic syndrome, reduce airway inflammatory cells, inhibit mucus production and down-regulate Th17 cell-related cytokine secretion,which helps to alleviate airway and systematic inflammation in mice with NA,and partially inhibit the development of NA.
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Background and purpose:B7-H3 is a newly identiifed member of the B7-family of co-stimulatory molecule, and however, its exact role in human osteosarcoma is still unclear. The purpose of this study was to examine the expression of B7-H3 in osteosarcoma tissues and to investigate its correlations with clinicopathological factors and overall survival in patients with osteosarcoma.Methods:The expression of B7-H3 and the intensity of tumor-inifltrating T lymphocytes (TILs) in pathologic specimens of osteosarcoma, osteochondroma and bone ifbrous dysplasia tissues were evaluated by immunohistochemical assay.Results:The expression rate of B7-H3 was 91.8% (56/61) in osteosarcoma lesions, while B7-H3 was barely expressed in adjacent normal tissues and bone ifbrous dysplasia tissues. The intensity of B7-H3 expression in osteochondroma was 56.8%, which was signiifcantly decreased compared with osteosarcoma tissues. Tumor B7-H3 expression was associated with Ennecking stage and pulmonary metastasis, while inversely correlated with the number of tumor-inifltrating CD8+ T cells (P<0.05). Moreover, patients with high tumor B7-H3 levels had a signiifcantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (P<0.05).Conclusion:B7-H3 is overexpressed in human osteosarcoma tissues, and B7-H3 expression is highly correlated with tumor development and overall patients’ prognosis. Moreover, overexpression of B7-H3 in tissues can relfect CD8+T cell inifltration and may help tumor cells avoid immune surveillance.
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Objective To investigated the significance of serum B7-H3 in differential diagnosis of patients with benign and malignant obstructive jaundice.Methods The B7-H3 levels in 60 patients with obstructive jaundice who were divided into benign (32 patients) and malignant (28 patients) obstructive jaundice group were tested by enzyme-linked immunosorbent assay before and after operation.Results The serum B7-H3 levels of malignant obstructive jaundice group were significantly higher than that of benign group (9334.57 ± 1009.57) pg/mL vs (4450.81 ± 406.92) pg/mL,(P ＜ 0.01).The B7-H3 levels of benign and malignant obstructive jaundice group after operation were significantly lower than those before operation (2531.03 ± 415.64) pg/mL vs (4450.81 ±406.92) pg/mL,(5833.00 ± 2190.87) pg/mL vs (9334.57±1009.57) pg/mL,(P＜0.01).Conclusion B7-H3 participate in the inflammatory immune and tumor immune response,The serum B7-H3 can be used as differential diagnosis index of benign and malignant obstructive jaundice.