OBJECTIVE

Cancers have developed a number of strategies to escape immune responses including the differential expression of costimulatory molecules of the B7 family. B7-H3 and B7-H4 have recently been described in different tumor entities but the relevance for melanoma has not yet been studied so far.

METHODS

Using immunohistochemistry, B7-H3 and B7-H4 expression was studied on 29 melanoma lesions. Survival curves and log-rank tests were used to test the association of protein expression with survival. Cell lines were evaluated for B7-H3 and B7-H4 expression by PCR and flow cytometry. Functional T-cell-tumor coculture assays were carried out with in vitro generated tumor transfectants.

RESULTS

B7-H3 and B7-H4 expression was detected in primary tumor lesions (29 of 29 and 28 of 29) and in metastases (28 of 29 and 26 of 29). The numbers of CD68(+) macrophages were significantly lower in patients with low B7-H4 expression, whereas CD8(+) T-cell infiltrates were independent of expression levels. Furthermore, a survival benefit for patients with B7-H4 low expressing melanoma was found, whereas B7-H3 was not associated with any clinical parameter. All 23 melanoma cell lines analyzed expressed B7-H3 and B7-H4 mRNA and protein, but B7-H4 was restricted to intracellular compartments. On silencing of B7-H3 by specific shRNA tumor-associated antigen-specific T cell responses were unaltered. Overexpression of B7-H4 on melanoma cells did not alter the cytotoxicity of different CD8(+) effector cells, but drastically inhibited cytokine production.

CONCLUSIONS

Our study provides for the first time evidence of B7-H4 expression on melanoma cells as a mechanism controlling tumor immunity which is associated with patients' survival.

The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.

The B7-CD28 family of ligands and receptors play important roles in T-cell co-stimulation and co-inhibition. Phylogenetically they can be divided into three groups. The recent discovery of the new molecules (B7-H3 [CD276], B7x [B7-H4/B7S1], and HHLA2 [B7H7/B7-H5]/TMIGD2 [IGPR-1/CD28H]) of the group III has expanded therapeutic possibilities for the treatment of human diseases. In this review, we describe the discovery, structure, and function of B7-H3, B7x, HHLA2, and TMIGD2 in immune regulation. We also discuss their roles in important pathological states such as cancers, autoimmune diseases, transplantation, and infection. Various immunotherapeutical approaches are emerging including antagonistic monoclonal antibodies and agonistic fusion proteins to inhibit or potentiate these molecules and pathways in cancers and autoimmune diseases.

Degeneration of joint articular cartilage is a leading cause of disability worldwide, and is due in large part to the fact that adult articular cartilage is unable to undergo effective intrinsic repair. To overcome this barrier, we have developed a tissue engineering strategy which harnesses the superior anabolic activity of juvenile chondrocytes to produce a scaffold-independent, living neocartilage graft. Preclinical studies demonstrate that bioengineered neocartilage survives allogeneic and xenogeneic transplantation, suggesting the utility of universal donor-derived neocartilage for joint repair. However, the mechanism underlying neocartilage transplant tolerance remains poorly understood. We show here that neocartilage-derived chondrocytes are unable to stimulate allogeneic T cells in vitro, and they do not constitutively express cell surface molecules required for induction of T cell immune responses, including major histocompatibility complex (MHC) Class II antigens and costimulatory molecules B7-1 and B7-2. Additionally, chondrocytes suppress, in a contact-dependent manner, the proliferation of activated T cells, with suppression associated with chondrocyte expression of multiple negative regulators of immune responses, including B7 family members (B7-H1, B7-DC, B7-H2, B7-H3, and B7-H4), chondromodulin-I and indoleamine 2,3-dioxygenase. Thus, the survival of transplanted bioengineered neocartilage may depend on both passive and active mechanisms of immune evasion.

B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility were shown by reverse transcription and real-time PCR and immunohistochemistry analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double-blind study. B7-H3 messenger RNA expression was shown to be significantly increased with stage of melanoma (P<0.0001) and significantly associated with melanoma-specific survival in both stage III (P<0.0001) and stage IV (P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression of B7-H3 increased migration and invasion, whereas knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to phosphorylated signal transducer and activator of transcription-3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression and events of metastasis.

Activated T cells have been implicated in chronic rhinosinusitis (CRS) and asthma and physically interact with epithelial cells in the airways. We now report that human airway epithelial cells display significant constitutive cell-surface expression of costimulatory ligands, B7-H1, B7-H2, B7-H3, and B7-DC. Expression of B7-H1 and B7-DC was selectively induced by stimulation of either BEAS2B or primary nasal epithelial cells (PNEC) with interferon (IFN)-gamma (100 ng/ml). The combination of IFN-gamma and tumor necrosis factor-alpha (100 ng/ml) selectively induced expression better than IFN-gamma alone. Fluticasone treatment (10(-7) M) reduced the baseline expression and inhibited the induction of B7-H1 and B7-DC in BEAS2B cells. In vitro exposure of PNEC to IFN-gamma also resulted in selective induction of B7-H1 and B7-DC. Monoclonal antibody blockade of B7-H1 or B7-DC enhanced IFN-gamma expression by purified T cells in co-culture experiments, suggesting that these two B7 homologs inhibit T cell responses at the mucosal surface. Immunohistochemical staining of human sinonasal surgical tissue confirmed the presence of B7-H1, B7-H2, and B7-H3 in the epithelial cell layer, especially in samples from patients diagnosed with Samter's Triad, a severe form of CRS. Real-time PCR analysis of sinonasal tissue revealed elevated levels of B7-H1 and B7-DC in CRS compared with controls. These results demonstrate that epithelial cells express functional B7 costimulatory molecules and that expression of selected B7 family members is inducible in vitro and in vivo. Epithelial B7 homologs could play a role in regulation of lymphocytic activity at mucosal surfaces.

The field of immunotherapy is a continually expanding niche in cancer biology research. In the last two decades, there has been significant progress in identifying better targets and creating more specific agents for therapy in the field. B7-H3 (CD276) is an immune checkpoint from the B7 family of molecules, many of whom interact with known checkpoint markers including CTLA4, PD-1, and CD28. This is an exciting molecule that is overexpressed in many cancers, although the receptor of B7-H3 has not been characterized. Initially, B7-H3 was thought to co-stimulate the immune response, but recent studies have shown that it has a co-inhibitory role on T-cells, contributing to tumor cell immune evasion. Therefore, its overexpression has been linked to poor prognosis in human patients and to invasive and metastatic potential of tumors in in vitro models. Moreover, recent evidence has shown that B7-H3 influences cancer progression beyond the immune regulatory roles. In this review, we aim to characterize the roles of B7-H3 in different cancers, its relationship with other immune checkpoints, and its non-immunological function in cancer progression. Targeting B7-H3 in cancer treatment can reduce cell proliferation, progression, and metastasis, which may ultimately lead to improved therapeutic options and better clinical outcomes.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells, and they promote an immunosuppressive environment in tumor-bearing hosts. To characterize MDSCs in melanoma, we examined the expression of inhibitory B7 molecules by CD11b(+)Gr1(+) cells isolated from mice with transplantable ret tumors. B7 molecules were expressed on CD11b(+)Gr1(+) cells, which also expressed CD124 and inducible nitric oxide synthase, thus verifying their relation to MDSCs. In developing melanomas, CD11b(+)Gr1(+) cells express only low levels of B7-H1. In contrast, B7-H1 is upregulated in large tumors, and functional analysis demonstrates that CD11b(+)Gr-1(+) cells suppress the proliferation of CD4(+) T cells through B7-H1. Depletion of regulatory T cells (Tregs) significantly downregulated the expression of B7-H1, B7-H3, and B7-H4 on MDSCs and reduced tumor growth, indicating a concerted immunosuppressive activity of Tregs and MDSCs. No differences in the suppressive function of MDSCs between CD25-depleted and non-depleted mice were recorded. Instead, tumor-derived MDSCs from Treg-depleted hosts produced less IL-10 and more IFN-γ as compared with Treg-harboring mice. These studies indicate that Tregs in tumors not only suppress effector T cells directly, but also modify the phenotype of tumor-infiltrating CD11b(+) cells to express inhibitory B7-H molecules and to produce IL-10.

B7-H3 is one of the most recently identified members of the B7/CD28 superfamily of costimulatory molecules serving as an accessory modulator of T-cell response. Recently, B7-H3 expression has been reported in several human cancers indicating an additional function of B7-H3 as a regulator of antitumor immunity. However, its precise physiologic role is still elusive, because both stimulatory and inhibitory capacities have been demonstrated. This paper summarizes the available data on B7-H3 in the regulation of T-cell response focusing on its potential role in antitumor immunity.

Previous studies have suggested aberrant expression of membrane B7-H3 in tumor cells. This aim of the study was to determine the expression level of soluble B7-H3 (sB7-H3) in circulation and to subsequently evaluate the clinical significance of circulating B7-H3 in patients with non-small cell lung cancer (NSCLC). The level of circulating B7-H3 was determined with ELISA and its correlation with the clinical data was examined. Receiver operating characteristic (ROC) curve analysis was performed to compare the sensitivity and specificity in the diagnosis of NSCLC. Circulating B7-H3 levels in patients with NSCLC were significantly higher than those in patients with other pulmonary diseases (OPD, p<0.001), or those in healthy volunteers (p<0.001). Using a cutoff of 30ng/ml, the sensitivity and specificity of sB7-H3 in differentiating between patients with NSCLC and patients with OPD, and between patients with NSCLC and healthy volunteers was, 48.8 and 98.5%, and 48.0 and 93.7%, respectively. Additionally, higher levels of sB7-H3 were associated with higher tumor stage, tumor size, nodal metastasis, and distant metastasis, but not with sex, age or histological subtype. An area under the curve (AUC) for all stages of NSCLC resulting from sB7-H3 (0.862), which was significantly better than any other tumor markers tested including CA125 (0.621), CA153 (0.571), CA199 (0.459), and CEA (0.638). These results suggest circulating B7-H3 is a valuable biomarker for NSCLC and an elevated level of circulating B7-H3 suggests a poor clinical character for NSCLC.

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.

BACKGROUND

B7-homologue 3 (B7-H3), a recently identified immunoregulatory protein, has been shown to be overexpressed in human hepatocellular carcinoma (HCC). However, whether the dynamic expression pattern of B7-H3 contributes to early invasion of HCC is largely unknown. In addition, the biological roles of B7-H3 in HCC are still unclear. Herein, we are going to examine B7-H3 expression profile and its clinicopathological significance in primary and metastatic HCC, and further determine whether B7-H3 knockdown simulates different pathological states of HCC progression and metastasis.

METHODS

Using immunohistochemistry, B7-H3 expression was studied on 116 HCC containing primary and metastatic HCCs. Survival curves and log-rank tests were used to test the association of B7-H3 expression with survival. HCC cells with B7-H3 depletion were established by RNA interference to investigate the effect of B7-H3 on cell proliferation, apoptosis, migration and invasion in vitro.

RESULTS

Statistical analysis of clinical cases revealed that B7-H3 high expression group had inclinations towards late TNM stage, the presence of vascular invasion, lymph metastasis, and the formation of microsatellite tumors. Increased intensity of tumor B7-H3 staining was detected more significantly in metastatic HCC tumors. Consistently in experiments performed in vitro, B7-H3 was able to stimulate the wound healing, metastasis and invasion of hepatoma cells by targeting epithelial-to-mesenchymal transition (EMT) via JAK2/Stat3/Slug signaling pathway, while no obvious influence on cell growth and apoptosis.

CONCLUSIONS

B7-H3 in the regulation of the metastatic capacity of HCC cells makes itself a promising therapeutic target for anti-metastasis therapy.

BACKGROUND

Costimulatory signaling has been implicated as a potential regulator of antitumor immunity in various human cancers. In contrast to the negative prognostic value of aberrant B7-H1 expression by pancreatic cancer cells, the role of B7-H3 is still unknown. Therefore, we investigated the expression pattern and clinical significance of B7-H3 expression in human pancreatic cancer.

METHODS

B7-H3 expression was evaluated by immunohistochemistry in 68 patients with pancreatic cancer who underwent surgical tumor resection. Expression data was correlated with clinicopathologic features and with the number of tumor-infiltrating T cells.

RESULTS

B7-H3 expression was significantly upregulated in pancreatic cancer compared to normal pancreas (p < 0.05). In 60 of 68 examined tumors B7-H3 protein was detectable in pancreatic cancer cells. Patients with high tumor B7-H3 levels had a significantly better postoperative prognosis than patients with low tumor B7-H3 levels (p = 0.0067). Furthermore, tumor B7-H3 expression significantly correlated with the number of tumor-infiltrating CD8+ T cells (p = 0.018).

CONCLUSIONS

We demonstrate for the first time that B7-H3 is abundantly expressed in pancreatic cancer and that tumor-associated B7-H3 expression significantly correlates with prolonged postoperative survival. Our findings suggest that B7-H3 might play an important role as a potential stimulator of antitumor immune response in pancreatic cancer.

Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.

BACKGROUND

Respiratory syncytial virus (RSV) is associated with wheezing illness, and infections can occur repeatedly throughout life. We hypothesized that RSV infection of respiratory tract epithelial cells up-regulates B7 molecules that regulate memory immune responses and that type 1 and 2 cytokines differentially modulate this induction.

METHODS

We used flow-cytometric analysis to investigate programmed death-1 ligand (PD-L) 1, PD-L2, B7-H3, and inducible costimulatory ligand (ICOS-L) expression on tracheal (NCI-H292), bronchial (BEAS-2B), and alveolar (A549) epithelial cells; regulation of this expression by RSV, interferon (IFN)- gamma , and interleukin (IL)-4; and the effects of IFN-gamma and IL-4 on RSV-induced expression of these molecules.

RESULTS

B7-H3 was strongly expressed, PD-L1 and ICOS-L were moderately expressed, and PD-L2 was weakly expressed on unstimulated tracheal, bronchial, and alveolar epithelial cells. RSV infection up-regulated PD-L1, PD-L2, and B7-H3 expression on all cells and ICOS-L expression on bronchial and alveolar epithelial cells. IL-4 treatment alone had no effect, whereas IFN-gamma treatment alone increased PD-L1 and PD-L2 expression on all cells and decreased B7-H3 expression on bronchial and alveolar epithelial cells. On RSV-infected alevolar epithelial cells, IFN-gamma treatment increased PD-L1 and PD-L2 expression and decreased B7-H3 and ICOS-L expression, and IL-4 treatment increased PD-L2 and B7-H3 expression and decreased ICOS-L expression.

CONCLUSIONS

Respiratory tract epithelial cells express a wide range of B7 molecules. RSV infection increases their expression, and this expression is differentially regulated by IFN-gamma and IL-4. These processes may be involved in decreasing T cell antiviral immune responses to RSV and in RSV-associated wheezing.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

OBJECTIVE

B7-H3, a member of the B7 family of immune regulatory ligands regulates T cell-mediated peripheral immune response. The purpose of this study was to correlate the expression of B7-H3 and number of lymphocytes in patients with endometrial cancer.

METHODS

A total of 107 patients with primary endometrial carcinoma (type I/endometrioid, n=81; type II, n=18) and endometrial hyperplasia (n=8) were investigated. Expression of B7-H3 in endometrial hyperplasia, endometrial carcinoma, and the endothelium of tumor-associated vasculature was assessed using immunohistochemistry from paraffin-embedded tissue blocks. Detection of CD8-positive tumor-infiltrating lymphocytes (TIL) and CD8-positive tumor-associated lymphocytes (TAL) was correlated with the expression of B7-H3.

RESULTS

Patients with high grade tumors and patients with type II carcinomas expressed significantly more B7-H3 than low grade and endometrioid tumors (p=<0.0001 and p=0.0001, respectively). The expression of B7-H3 in the endothelium of identified vasculature in the tumor specimens showed similar results with strong relation to high grade tumors (p=0.001) and type II carcinomas (p=0.004). We found a significant correlation between B7-H3 expression on cancer cells and tumor T-cell infiltration (TIL) (p=0.017). In a univariate survival analysis, overexpression of B7-H3 in tumor cells was associated with shortened overall survival (p=0.005).

CONCLUSIONS

B7-H3 is overexpressed on cancer cells and in the endothelium of tumor-associated vasculature in high grade tumors (G3) and type II carcinomas. B7-H3 expression on cancer cells is correlated with the number of T cells infiltrating the tumor. Endometrium tumor development and progression may be associated with downregulation of T-cell-mediated antitumor immunity through B7-H3.

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.

T cells are the main effector cells in immune response against tumors. The activation of T cells is regulated by the innate immune system through positive and negative costimulatory molecules. Targeting immune checkpoint regulators such as programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) and CTL antigen 4 (CTLA-4) has achieved notable benefit in a variety of cancers, which leads to multiple clinical trials with antibodies targeting the other related B7/CD28 family members. Recently, five new B7 family ligands, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7, were identified. Here we review recent understanding of new B7 family checkpoint molecules as they have come to the front of cancer research with the concept that tumor cells exploit them to escape immune surveillance. The aim of this article is to address the structure and expression of the new B7 family molecules as well as their roles in controlling and suppressing immune responses of T cells as well as NK cells. We also discuss clinical significance and contribution of these checkpoint expressions in human cancers. Mol Cancer Ther; 16(7); 1203-11. ©2017 AACR.

BACKGROUND

B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer.

METHODS

MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients).

RESULTS

We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts.

CONCLUSIONS

We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.

Co-signaling molecules in the B7-CD28 family have been intensively studied over the past decade and have brought much excitement to the field of immune regulation. The discovery of new functions for the classical pathways CD80/CD86/CD28/CTLA-4 and the identification of novel pathways of the family, including B7-H1/B7-DC/PD-1, B7-H2/ICOS, B7-H3, B7-H4 and BTLA, are greatly broadening our understanding of the control of T cell-mediated immune responses and tolerance.

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3-mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners.

The B7 homolog B7-H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7-H3 in alloresponses. Immunohistological and molecular analyses showed B7-H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7-H3 in rejecting allografts, we generated B7-H3-/- mice and showed that targeting of B7-H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12-14 days of survival in wild-type controls but led to permanent cardiac and islet allograft survival in B7-H3-/- mice. Cardiac allografts in treated B7-H3-/- mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild-type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7-H3-/- as compared to wild-type recipients. Lastly, in addition to the expected antigen-presenting cell expression of B7-H3, CD4 and CD8 T cells showed B7-H3 induction upon cell activation, and both dendritic cell- and T cell-expressed B7-H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7-H3 promotes T cell-mediated immune responses and the development of acute and chronic allograft rejection.

B7-H3, a novel B7 family member, positively or negatively regulates T-cell responses. We investigated the clinical relevance and prognostic significance of B7-H3 in hepatocellular carcinoma (HCC). Western blotting showed B7-H3 upregulation in 17 of 24 (70.8 %) HCC tissues compared with nontumor liver tissues (p = 0.028). B7-H3 immunostaining on tissue microarrays containing 240 HCC patient samples indicated that 225 (93.8 %) tumors had aberrant B7-H3 expression, with strong intensity in 79 (32.9 %) cases, whereas B7-H3 expression in peritumor liver cells was weak in most cases (226; 94.2 %). Notably, patients with high/moderate tumor cell B7-H3 expression showed significantly poorer survival (p = 0.009) and increased recurrence (p = 0.002). After multivariable adjustment, high/moderate B7-H3 expression remained significant for an increased risk of recurrence (hazard ratio = 1.79; 95 % confidence interval = 1.19-2.70; p = 0.005). B7-H3 expression correlated with invasive phenotypes like vascular invasion and advanced tumor stage, and the metastatic potential of HCC cell lines. Flow cytometry showed that B7-H3 expression is inversely correlated with proliferation and interferon-γ production by infiltrating T cells. Interferon-γ stimulation significantly upregulated B7-H3 expression in HCC cells in vitro, implicating B7-H3 expression as a feedback mechanism to evade anti-tumor immunity. Importantly, the prognostic value of B7-H3 expression was validated in an independent cohort of 206 HCC patients. Collectively, our data suggest that B7-H3 was abundantly expressed in HCC and was associated with adverse clinicopathologic features and poor outcome. Thus, B7-H3 represents an attractive target for diagnostic and therapeutic manipulation in human HCC.