B7-H4 is a recently-identified member of B7 costimulation superfamily. B7-H4 serves as an inhibitory modulator of T cell response through suppressing the proliferation, activation, and cytokine production of T cells. B7-H4 is found to be abnormally expressed in multiple human tumors. Many studies have shown that B7-H4 expression is closely related to the progression and prognosis of urologic tumors. Investigation of B7-H4 function will provide new strategies for diagnosis and immunotherapy of urologic tumors. This article reviews the recent progress of B7-H4 research in urologic tumors.
Humans ; Interleukin-2 ; Urologic Neoplasms ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

OBJECTIVETo construct a eukaryotic expression vector encoding the gene of extracellular region of mouse B7-H4, to express it in yeast cell line and to determine its biological activity.

METHODSThe extracellular region of the mouse B7-H4 gene was amplified with Xho I and EcoR I by PCR from a mouse B7-H4 chimeric plasmid. Digested with Xho I and EcoR I, the mB7-H4 gene was inserted into the yeast expression plasmid Ppic9. The DNA sequence was confirmed by double digestion and the Ppic9-mB7-H4 plasmid was transfected into the yeast cells. The expression of mB7-H4 was confirmed by PCR, Western Blot and ELISA analysis, and its biological function was determined.

RESULTPpic9-mB7-H4 transfectants expressed mB7-H4 in yeast cells, and mB7-H4 effectively inhibited the proliferation of T lymphocytes with a fractional inhibition rate of 28.3 % and inhibited IL-2, IL-4, IL-10 and IFN-gamma production with fractional inhibition rates of 68.8%, 78.8%, 67.6% and 77.7%, respectively.

CONCLUSIONThe eukaryotic expression plasmid mouse B7-H4 has been successfully constructed and the expressed products of B7-H4 possess biological activity.

Animals ; B7-1 Antigen ; biosynthesis ; genetics ; Mice ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocytes ; immunology ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

OBJECTIVETo investigate the expression of B7-H4 in prostate cancer tissue and the relationship between the expression and the clinicopathological features.

METHODSImmunohistochemical staining was used to detect the expression of B7-H4 in prostate cancer tissue. And the relationship between the expressions and pathology was evaluated.

RESULTSThe B7-H4 was diffusely expressed in cytoplasm and/or membrane of the prostate cancer tissue; the expression was much higher than that in normal prostate tissue (P<0.05). The expression of B7-H4 in the prostate cancer tissue was higher in patients with higher tumor grade.

CONCLUSIONB7-H4 may be used as an new indicator for the diagnosis and prognosis of prostate cancer and a novel target for immunotherapy.

Aged ; Aged, 80 and over ; B7-1 Antigen ; metabolism ; Biomarkers, Tumor ; metabolism ; Humans ; Male ; Middle Aged ; Prostate ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

OBJECTIVETo prepare a monoclonal antibody (mAb) against extracellular domain of B7-H4 and to investigate the expression of B7-H4 in pancreatic cancer tissue with the prepared mAb.

METHODSBalb/c mice were immunized with 3T3-B7-H4 cells and the splenic cells of the immunized mice were fused with Sp2/0 myeloma cells by conventional hybridoma techniques. An indirect ELISA method using 3T3-B7-H4 lysate as antigen was established to screen antibody-producing hybridoma cell lines. Western blott, immunoprecipitation (IP), and immunohistochemistry (IHC) were applied to characterize the mAb. Immunohistochemical staining was used to detect the expression of B7-H4 in human pancreatic cancer tissue. The correlation of B3-H4 expressions and pathological features of pancreatic cancer was analyzed.

RESULTSA hybridoma cell line secreting mAb against B7-H4 was obtained. The subclass of this mAb was IgM, and the light chain was Kappa. Western blot and IP showed that the mAb specifically recognized B7-H4. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The B7-H4 was diffusely expressed in the cytoplasma and/or membrane of pancreatic cancer tissue, which was much higher than that expressed in normal pancreatic tissue (4.00 ± 1.44 compared with 1.12 ± 0.78, P ± 0.01). The expression of B7-H4 was higher in pancreatic cancer tissues with higher pathological grade or with lymph node metastasis as compared with that in pancreatic cancer tissues with lower grade or with no lymph mode metastasis (6.10 ± 0.72 compared with 3.55 ± 1.12,P<0.01: 6.14 ± 0.66 compared with 3.70 ± 1.25,P<0.01). The expression level of B7-H4 was not related to patients'age and gender.

CONCLUSIONMonoclonal antibody against B7-H4 with high activity and specificity has been prepared successfully. The expression of B7-H4 in pancreatic cancer tissue is up-regulated,which is closely related to the tumor grade and lymph node metastasis in pancreatic cancer.

3T3 Cells ; Animals ; Antibodies, Monoclonal ; biosynthesis ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Mice ; Mice, Inbred BALB C ; Neoplasm Grading ; Pancreatic Neoplasms ; metabolism ; pathology ; V-Set Domain-Containing T-Cell Activation Inhibitor 1 ; metabolism

OBJECTIVETo investigate the effects of B7H4 on human bone marrow mesenchymal stem cells (HBMSC) mediating immune suppression.

METHODSThe expression of the negative immunoregulatory factor B7H4 on HBMSC were analyzed by RT-PCR and flow cytometry (FCM), respectively. The blocking experiment was used to detect the effects of B7H4 on HBMSC mediating suppression on PHA induced T cell activation, proliferation and cell cycle. HBMSC inhibiting T cell proliferation was examined by transwell cell culture system.

RESULTSB7H4 was highly expressed on HBMSC. Blocking the B7H4 expression by B7H4mAb significantly attenuated the inhibitory effects of HBMSC on T cell proliferation. Compared with that of the unblocking group, T cell stimulator index (SI) of the B7H4 blocked group was significantly increased (53 +/- 5 vs 15 +/- 8, P < 0.01) and the inhibitory effects of HBMSC on T cell cycle were weakened significantly through down-regulating the cell number in G(0)/G(1) phase \[(85.6 +/- 9.9)% vs (95.8 +/- 9.9)%\] and up-regulating those in S phase\[(5.8 +/- 3.2)% vs (2.3 +/- 2.2)%, P < 0.05\]. The suppressive effects of HBMSC on T cell proliferation were significantly weakened after separating HBMSC from T cells by transwell cell culture system. Compared with the cell to cell contact group, T cell SI was significantly increased (27 +/- 17 vs 15 +/- 3, P < 0.01).

CONCLUSIONHBMSC highly express B7H4, which plays an important role in the suppressive effects of HBMSC on T cell proliferation.

B7-1 Antigen ; metabolism ; physiology ; Bone Marrow Cells ; immunology ; metabolism ; Cell Cycle ; immunology ; Cell Proliferation ; Cells, Cultured ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Mesenchymal Stromal Cells ; immunology ; metabolism ; Phytohemagglutinins ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; immunology ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

OBJECTIVETo construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities.

METHODSThe full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol I and EcoR V the B7-H4 gene was inserted into pshuttle-CMW(PSC). Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac I linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested.

RESULTSThe full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination, and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production.

CONCLUSIONThe bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocytes ; immunology ; Transfection ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

OBJECTIVETo investigate the influence of co-stimulatory molecules B7-H4 expression on prognosis of gastric cancer patients treated by cytokine-induced killer cells (CIK cells) adoptive immunotherapy.

METHODSClinical data of 156 cases of gastric cancer patients were retrospectively analyzed. Patients were divided into chemotherapy group(n=81) and chemotherapy combined with CIK cell therapy group(n=75). B7-H4 expression was detected in the surgical specimens of gastric cancer patients by immunohistochemistry assay. Disease-free survival was compared between the chemotherapy group and the CIK group at different expression levels of B7-H4.

RESULTSThe difference was not statistically significant in all clinical and pathological data between the chemotherapy group and the CIK treatment group (P>0.05). The postoperative median tumor-free survival in two groups was 18.0 and 45.0 months, respectively, and the difference was statistically significant (chi(2)=11.631, P=0.001). The postoperative median survival time was 27.0 and 49.0 months, respectively, and the difference was statistically significant (chi(2)=10.907, P=0.001). In 86 patients with low B7-H4 expression, the median tumor-free survival time was 32.0 and 62.0 months, respectively, and the difference was statistically significant (chi(2)=4.663,P=0.03). In 70 patients with high B7-H4 expression, the median tumor-free survival time was 11.0 and 18.0 months, respectively, and the difference was statistically significant (chi(2)=11.971, P=0.001).

CONCLUSIONThe median tumor-free survival time of patients with gastric cancer may be further improved by chemotherapy combined with CIK cell therapy, regardless of the level of B7-H4 expression.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; B7-1 Antigen ; metabolism ; Cytokine-Induced Killer Cells ; Disease-Free Survival ; Female ; Humans ; Immunotherapy, Adoptive ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Stomach Neoplasms ; diagnosis ; metabolism ; therapy ; V-Set Domain-Containing T-Cell Activation Inhibitor 1

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium (supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor) maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum-containing medium-cultured U251 cells (24%-35% vs. 8%-11%, P < 0.001). Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium (P < 0.01). Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.
AC133 Antigen ; Antigens, CD ; metabolism ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Culture Media, Conditioned ; Culture Media, Serum-Free ; Gene Expression Regulation, Neoplastic ; Glial Fibrillary Acidic Protein ; metabolism ; Glioma ; metabolism ; pathology ; Glycoproteins ; metabolism ; Humans ; Monocytes ; cytology ; metabolism ; Neoplastic Stem Cells ; metabolism ; pathology ; Nestin ; metabolism ; Peptides ; metabolism ; SOXB1 Transcription Factors ; metabolism ; V-Set Domain-Containing T-Cell Activation Inhibitor 1 ; metabolism

BACKGROUND AND OBJECTIVEEpithelial ovarian cancer involves a number of factors. Recent studies have shown that osteopontin (OPN) is related to the occurrence and development of a variety of tumors, but few studies are on ovarian cancer. B7-H4 is a newly identified tumor marker in ovarian cancer. This study explored the expression of OPN and B7-H4 and their clinical significance in epithelial ovarian tumors.

METHODSThe expression of OPN and B7-H4 in 15 cases of normal ovarian tissue, 20 of benign ovarian tumor tissue, 20 of borderline ovarian tumor tissue, and 40 of ovarian cancer tissue were detected by immunohistochemistry, and the relationship of OPN and B7-H4 expression to clinical and pathologic features of ovarian cancer was analyzed.

RESULTSThe expression of OPN and B7-H4 were significantly higher in ovarian cancer than in borderline and benign tumors (P<0.05). The positive rates of OPN and B7-H4 were significantly higher in poorly differentiated ovarian cancer than in medium and highly differentiated ovarian cancer (P<0.05), and the levels of expression were significantly lower in tissue at stages I and III of ovarian cancer than in stages III and IV (P<0.05). The positive rate of OPN associated with a higher rate of lymph node metastasis (P<0.05), but did not relate to age and histologic type. The positive rate of B7-H4 were significantly higher in ovarian serous carcinoma than in the mucinous carcinoma (P<0.05), but did not relate to age and lymph node metastasis.

CONCLUSIONThe expression of OPN and B7-H4 increased in epithelial ovarian cancer, which could be referenced in the diagnosis of ovarian malignant tumors.

Adenocarcinoma, Mucinous ; diagnosis ; metabolism ; pathology ; Adolescent ; Adult ; Aged ; Cystadenocarcinoma, Serous ; diagnosis ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Neoplasms, Glandular and Epithelial ; diagnosis ; metabolism ; pathology ; Osteopontin ; metabolism ; Ovarian Neoplasms ; diagnosis ; metabolism ; pathology ; Ovary ; metabolism ; V-Set Domain-Containing T-Cell Activation Inhibitor 1 ; metabolism ; Young Adult