Research progress of negative costimulator B7-H4 in urologic tumors / 浙江大学学报·医学版

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.

Preparation of anti-B7-H4 monoclonal antibody to investigate B7-H4 expression in pancreatic cancer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Balb/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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>Monoclonal 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.</p>

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium / 癌症

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.

Expressions of osteopontin and B7-H4 in epithelial ovarian neoplasm and their significance / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Epithelial 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The expression of OPN and B7-H4 increased in epithelial ovarian cancer, which could be referenced in the diagnosis of ovarian malignant tumors.</p>

Expression of B7-H4 in prostate cancer and its clinical significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of B7-H4 in prostate cancer tissue and the relationship between the expression and the clinicopathological features.</p><p><b>METHODS</b>Immunohistochemical staining was used to detect the expression of B7-H4 in prostate cancer tissue. And the relationship between the expressions and pathology was evaluated.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>B7-H4 may be used as an new indicator for the diagnosis and prognosis of prostate cancer and a novel target for immunotherapy.</p>

Influence of co-stimulatory molecules B7-H4 expression on the prognosis of patients with gastric cancer treated with cytokine-induced killer cells adoptive immunotherapy / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Clinical 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

Expression and identification of recombinant mouse gene B7-H4 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULT</b>Ppic9-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.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid mouse B7-H4 has been successfully constructed and the expressed products of B7-H4 possess biological activity.</p>

The effects of B7H4 on human bone marrow mesenchymal stem cell inhibiting proliferation of PHA activated T cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of B7H4 on human bone marrow mesenchymal stem cells (HBMSC) mediating immune suppression.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>B7H4 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).</p><p><b>CONCLUSION</b>HBMSC highly express B7H4, which plays an important role in the suppressive effects of HBMSC on T cell proliferation.</p>

Construction and identification of recombinant adenoviral vector encoding B7-H4 gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.</p>