Guangbo Zhang

Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma.

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses, and was shown to either promote or inhibit T-cell responses in various experimental systems. B7-H3 was expressed in some human cancers and correlated with poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we studied the expression of B7-H3 in the pathologic specimens of 102 patients treated for colorectal carcinoma (CRC) by immunohistochemistry. Strong B7-H3 expression was found in cancer tissues from 54.3% CRC patients, while minimal expression was found in adjacent normal colorectal tissues. Higher B7-H3 expression in tumor positively correlated with a more advanced tumor grade. In addition, consistent with a role of B7-H3 in suppressing tumor immune surveillance, the expression of B7-H3 in cancer cells negatively correlated with the intensity of tumor infiltrating T lymphocytes in both tumor nest and tumor stroma. Furthermore, we found that the level of soluble B7-H3 in sera from CRC patients was higher than healthy donors. TNF-α, an important cancer-promoting inflammatory molecule, was subsequently found to significantly increase the release of soluble B7-H3 in colon cancer cell lines. Therefore, our data suggest that both soluble and membranous B7-H3 proteins are involved in colon cancer progression and evasion of cancer immune surveillance.

Soluble CD276 (B7-H3) is released from monocytes, dendritic cells and activated T cells and is detectable in normal human serum.

Expression of membrane CD276 (mB7‐H3) has been reported on dendritic cells (DCs), monocytes, activated T cells, and various carcinoma cells. However, reports concerning its in vivo function have been inconsistent. Moreover, whether there is a soluble form of this protein is not known. In this study, using a sensitive dual monoclonal antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to detect the soluble form of B7‐H3 (sB7‐H3), we demonstrated the release of sB7‐H3 by monocytes, DCs, activated T cells, and various mB7‐H3+ but not mB7‐H3– carcinoma cells. Release from cells was blocked by addition of a matrix metalloproteinase inhibitor (MMPI), which concomitantly caused the accumulation of B7‐H3 on the cell surface. To determine the level of circulating sB7‐H3, more than 200 serum samples were included in the study. The results indicated that sB7‐H3 was present at high levels in all serum samples. Western blotting of sB7‐H3 from cell culture supernatants or sera of healthy donors indicated that the molecular size was approximately 16 kDa. Soluble B7‐H3 was able to bind to the B7‐H3 receptor (B7‐H3R) on activated T cells, which showed that sB7‐H3 is a functionally active form. These results indicate that release of sB7‐H3 from the cell surface is mediated by a matrix metalloproteinase and probably regulates B7‐H3R/B7‐H3 interactions in vivo. Cleavage of sB7‐H3 to an active soluble form would alter both proximal and distal cellular responses.

B7-H3 Augments the Inflammatory Response and Is Associated with Human Sepsis

B7-H3, a new member of the B7 superfamily, acts as both a T cell costimulator and coinhibitor, and thus plays a key role in the regulation of T cell-mediated immune responses. However, it is unclear whether B7-H3 is involved in the innate immune monocyte/macrophage-mediated inflammatory response. In this paper, we show that, although B7-H3 alone failed to stimulate proinflammatory cytokine release from murine macrophages, it strongly augmented both LPS- and bacterial lipoprotein-induced NF-κB activation and inflammatory response. This occurred in both a TLR4- and TLR2-dependent manner. Blockage of B7-H3 in vivo attenuated LPS-induced proinflammatory cytokine release and endotoxic shock-related lethality. Furthermore, we found that patients diagnosed with sepsis, in contrast to healthy individuals, exhibited significant levels of raised plasma soluble B7-H3 (sB7-H3) and that this level correlated with the clinical outcome and levels of plasma TNF-α and IL-6. In addition, a putative receptor for B7-H3 was detected on monocytes and peritoneal macrophages from septic patients but not on monocytes from healthy donors. Stimulation of human monocytes with LPS and inflammatory cytokines led to a substantial release of sB7-H3. Taken together, our data indicate that significantly elevated plasma sB7-H3 in septic patients may predict a poor outcome. Furthermore, we demonstrate that B7-H3 functions as a costimulator of innate immunity by augmenting proinflammatory cytokine release from bacterial cell wall product-stimulated monocytes/macrophages and may contribute positively to the development of sepsis.

B7-H3 overexpression in pancreatic cancer promotes tumor progression

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses. In addition, B7-H3 is also expressed in several types of human cancers and is correlated with the poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we compared B7-H3 expression in normal pancreas and pancreatic cancer tissue specimens, and determined the effects of low B7-H3 expression on the human pancreatic cancer cell line Patu8988 using lentivirus-mediated RNA interference. B7-H3 expression in pancreatic specimens was determined by enzyme-linked immunosorbent assay (ELISA). A Patu8988 cell line with low B7-H3 expression was established by lentivirus-mediated RNA interference to investigate the effect of B7-H3 on cell proliferation, migration and invasion in vitro. By establishing subcutaneous transplantation tumor and orthotopic transplantation pancreatic cancer mouse models, the effect of B7-H3 on cell proliferation, migration and invasion was studied in vivo. B7-H3 in tissue samples was significantly higher in the pancreatic cancer group than in the normal pancreas group (mean ± SD, 193.6±9.352 vs. 87.74±7.433 ng/g; P<0.0001). B7-H3 knockdown by RNA interference decreased cell migration and Transwell invasion up to 50% in vitro. No apparent impact was observed on cell proliferation in vitro. In the subcutaneous transplantation tumor mouse model, the tumor growth rate was reduced by the knockdown of B7-H3. In the orthotopic transplantation pancreatic cancer mouse model, the effect of inhibiting metastasis by knocking down B7-H3 was assessed in terms of the average postmortem abdominal visceral metastatic tumor weight. This demonstrated that inhibition of B7-H3 expression reduced pancreatic cancer metastasis in vivo. In conclusion, B7-H3 is aberrantly expressed in pancreatic cancer. In addition to modulating tumor immunity, B7-H3 may have a novel role in regulating pancreatic tumor progression.

CD83-stimulated monocytes suppress T-cell immune responses through production of prostaglandin E2

CD83 is commonly known as a specific marker for mature dendritic cells. It has been shown to be important for CD4+ T-cell development in the thymus. However, its function in the peripheral immune system remains enigmatic. Here, we show that CD83 inhibits proliferation and production of IL-2 and IFN-γ by T cells, and the inhibitory effect of CD83 is mediated by monocytes. Prostaglandin E2 (PGE2), but not IL-10 or TGF-β, was up-regulated specifically by CD83 in monocytes. Consistent with high levels of PGE2, expression of COX-2 also was increased upon CD83 treatment. NF-κB activation also is required for induction of PGE2 by CD83. Finally, application of the COX-2–selective inhibitor NS-398 fully prevented CD83-triggered inhibition of T-cell responses. Our study establishes an immune-regulatory mechanism by CD83 via stimulation of PGE2 production in monocytes.

Silencing of B7-H3 increases gemcitabine sensitivity by promoting apoptosis in pancreatic carcinoma

In numerous types of cancer, the expression of a novel member of the B7 ligand family, the B7-H3 immunoregulatory protein, has been correlated with a poor prognosis. In the present study, we investigated the role of B7-H3 in chemoresistance in pancreatic carcinoma. Silencing of B7-H3, through lentivirus-mediated delivery of stable short hairpin RNA, was observed to increase the sensitivity of the human pancreatic carcinoma cell line Patu8988 to gemcitabine as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 caused the cancer cells to be more resistant to the drug. Subsequently, we investigated the underlying mechanisms of B7-H3-mediated gemcitabine resistance, and found that the levels of survivin decreased in cells in which B7-H3 had been knocked down. In vivo animal experiments demonstrated that tumors in which B7-H3 had been knocked down displayed a slower growth rate compared with the control xenografts. Notably, gemcitabine treatment led to a strong antitumor activity in mice with tumors in which B7-H3 had been knocked down; however, this effect was only marginal in the control group. Furthermore, survivin expression was weak in gemcitabine-treated tumors in which B7-H3 had been knocked down and apoptosis was increased, as revealed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining. In summary, the present study demonstrated that B7-H3 induces gemcitabine resistance in pancreatic carcinoma cells, at least partially by downregulating survivin expression. These results provide novel insights into the function of B7-H3 and encourage the design and investigation of approaches targeting this protein in treating pancreatic carcinoma.

A novel subset of B7-H3+CD14+HLA-DR−/low myeloid-derived suppressor cells are associated with progression of human NSCLC

Myeloid-derived suppressor cells (MDSC) potently inhibit antitumor immune responses, and thereby promoti tumor progression and metastasis. However, the nature of human tumor-infiltrating MDSC remains poorly characterized. Here, we find B7-H3 is exclusively expressed on a subset of intratumoral CD14+HLA-DR−/low MDSC but absent from adjacent normal lung tissues of patients with non-small cell lung carcinoma (NSCLC). Cytokine analysis revealed that B7-H3+CD14+HLA-DR−/low MDSC (B7-H3+MDSC) produced higher levels of IL-10 and TNFα but lower levels of IL-1β and IL-6 when compared with B7-H3−CD14+HLA-DR−/low myeloid-derived suppressor cells (B7-H3−MDSC). In a murine lung cancer model, B7-H3+MDSCs were found only in the tumor microenvironment and their frequencies increased during tumor progression. Clinical data analysis indicated that a higher frequency of B7-H3+MDSCs was associated with reduced recurrence-free survival in patients with NSCLC. Taken together, we identify a novel subset of MDSCs within the tumor microenvironment that fosters tumor progression.

Origination of New Immunological Functions in the Costimulatory Molecule B7-H3: The Role of Exon Duplication in Evolution of the Immune System

B7-H3, a recently identified B7 family member, has different isoforms in human and mouse. Mouse B7-H3 gene has only one isoform (2IgB7-H3) with two Ig-like domains, whereas human B7-H3 has two isoforms (2IgB7-H3 and 4IgB7-H3). In this study a systematic genomic survey across various species from teleost fishes to mammals revealed that 4IgB7-H3 isoform also appeared in pigs, guinea pigs, cows, dogs, African elephants, pandas, megabats and higher primate animals, which resulted from tandem exon duplication. Further sequence analysis indicated that this duplication generated a new conserved region in the first IgC domain, which might disable 4IgB7-H3 from releasing soluble form, while 2IgB7-H3 presented both membrane and soluble forms. Through three-dimensional (3D) structure modeling and fusion-protein binding assays, we discovered that the duplicated isoform had a different structure and might bind to another potential receptor on activated T cells. In T cell proliferation assay, human 2IgB7-H3 (h2IgB7-H3) and mouse B7-H3 (mB7-H3) both increased T cell proliferation and IL-2, IFN-γ production, whereas human 4IgB7-H3 (h4IgB7-H3) reduced cytokine production and T cell proliferation compared to control. Furthermore, both h2IgB7-H3 and mB7-H3 upregulated the function of lipopolysacharide (LPS)-activated monocyte in vitro. Taken together, our data implied that during the evolution of vertebrates, B7-H3 exon duplication contributed to the generation of a new 4IgB7-H3 isoform in many mammalian species, which have carried out distinct functions in the immune responses.

Detection and quantitation of soluble B7-H3 in expressed prostatic secretions: a novel marker in patients with chronic prostatitis.

PURPOSE We determined the soluble B7-H3 level and its clinical significance in serum and expressed prostatic secretions of patients with chronic prostatitis, including chronic bacterial prostatitis (type II) and chronic pelvic pain syndrome. MATERIALS AND METHODS Using enzyme-linked immunosorbent assay we measured soluble B7-H3 in 11 patients with chronic prostatitis (type II), and 26 with inflammatory (type IIIA) and 54 with noninflammatory (type IIIB) chronic pelvic pain syndrome, and healthy donors. We assessed differences between these groups using Students t test. As determined by the National Institutes of Health-Chronic Prostatitis Symptom Index, we correlated soluble B7-H3 with clinical pain using the Pearson test. RESULTS We found no significant difference between serum soluble B7-H3 in healthy donors and patients with chronic prostatitis (p = 0.897). However, soluble B7-H3 in expressed prostatic secretions was statistically significantly decreased in patients with chronic prostatitis vs controls (p <0.001). ROC using soluble B7-H3 greater than 38.82 ng/ml in expressed prostatic secretions distinguished patients with chronic prostatitis from healthy donors with 90.9% sensitivity and 83.5% specificity. Also, soluble B7-H3 levels in expressed prostatic secretions correlated negatively with the Chronic Prostatitis Symptom Index and the pain subscore. Compared to the pretreatment level soluble B7-H3 in expressed prostatic secretions was significantly increased in patients with a greater than 25% decrease in the Chronic Prostatitis Symptom Index total score (p = 0.016). CONCLUSIONS Data indicate that the soluble B7-H3 level in expressed prostatic secretions is a novel chronic prostatitis marker that correlates negatively with subjective symptoms.

Downregulation of CD40 expression contributes to the accumulation of myeloid‑derived suppressor cells in gastric tumors

An elevated number of myeloid-derived suppressor cells (MDSCs) in tumor-bearing hosts has been recognized as a crucial mediator of tumor progression due to the cells potent ability to suppress antitumor immunity. Cluster of differentiation (CD) 40, as a suppressive phenotype expressed in MDSCs, is essential for MDSC-mediated immune suppression and the expansion of T regulatory cells. However, whether CD40 exerts a direct effect on the accumulation of MDSCs remains unclear. In the present study, CD40 was observed to be highly expressed on the MDSCs obtained from mice bearing gastric tumors. Notably, a significant decrease in the level of CD40 expression was observed in addition to an increased number of MDSCs during tumor progression. Further analysis revealed that the MDSC levels were found to positively correlate with tumor progression and that CD40 expression levels inversely correlate with the accumulation of MDSCs. To confirm the potent correlation between CD40 expression and the accumulation of MDSCs, the apoptosis of the MDSCs was detected using agonistic anti-CD40 treatment. The results indicated that CD40 activation induces apoptosis in MDSCs and that the downregulation of CD40 expression may contribute to MDSC accumulation by facilitating MDSC resistance to apoptosis. Thus, these observations provide a novel mechanism to improve our understanding of the involvement of CD40 in MDSC accumulation during cancer development.