The discovery of new functions for the original B7 family members, together with the identification of additional B7 and CD28 family members, have revealed new ways in which the B7:CD28 family regulates T cell activation and tolerance. B7-1/B7-2:CD28 interactions not only promote initial T cell activation but also regulate self-tolerance by supporting CD4+CD25+ T regulatory cell homeostasis. CTLA-4 can exert its inhibitory effects in both B7-1/B7-2 dependent and independent fashions. B7-1 and B7-2 can signal bidirectionally by engaging CD28 and CTLA-4 on T cells and by delivering signals into B7-expressing cells. The five new B7 family members, ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4 (B7x/B7-S1) are expressed on professional antigen-presenting cells as well as on cells within nonlymphoid organs, providing new means for regulating T cell activation and tolerance in peripheral tissues. The new CD28 families members, ICOS, PD-1, and BTLA, are inducibly expressed on T cells, and they have important roles in regulating previously activated T cells. PD-1 and BTLA also are expressed on B cells and may have broader immunoregulatory functions. The ICOS:ICOSL pathway appears to be particularly important for stimulating effector T cell responses and T cell-dependent B cell responses, but it also has an important role in regulating T cell tolerance. In addition, the PD-1:PD-L1/PD-L2 pathway plays a critical role in regulating T cell activation and tolerance. In this review, we revisit the roles of the B7:CD28 family members in regulating immune responses, and we discuss their therapeutic potential.

We describe here a newly identified member of the human B7 family, designated B7 homolog 3 (B7-H3), that shares 20-27% amino acid identity with other B7 family members. B7-H3 mRNA is not detectable in peripheral blood mononuclear cells, although it is found in various normal tissues and in several tumor cell lines. Expression of B7-H3 protein, however, can be induced on dendritic cells (DCs) and monocytes by inflammatory cytokines and a combination of phorbol myristate acetate (PMA) + ionomycin. Soluble B7-H3 protein binds a putative counter-receptor on activated T cells that is distinct from CD28, cytotoxic T lymphocyte antigen 4 (CTLA-4), inducible costimulator (ICOS) and PD-1. B7-H3 costimulates proliferation of both CD4+ and CD8+ T cells, enhances the induction of cytotoxic T cells and selectively stimulates interferon gamma (IFN-gamma) production in the presence of T cell receptor signaling. In contrast, inclusion of antisense B7-H3 oligonucleotides decreases the expression of B7-H3 on DCs and inhibits IFN-gamma production by DC-stimulated allogeneic T cells.Thus, we describe a newly identified costimulatory pathway that may participate in the regulation of cell-mediated immune responses.

Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism. However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as our model, here we show that enhancer of zeste homologue 2 (EZH2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) and DNA methyltransferase 1 (DNMT1)-mediated DNA methylation repress the tumour production of T helper 1 (TH1)-type chemokines CXCL9 and CXCL10, and subsequently determine effector T-cell trafficking to the tumour microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T-cell tumour infiltration, slows down tumour progression, and improves the therapeutic efficacy of programmed death-ligand 1 (PD-L1; also known as B7-H1) checkpoint blockade and adoptive T-cell transfusion in tumour-bearing mice. Moreover, tumour EZH2 and DNMT1 are negatively associated with tumour-infiltrating CD8(+) T cells and patient outcome. Thus, epigenetic silencing of TH1-type chemokines is a novel immune-evasion mechanism of tumours. Selective epigenetic reprogramming alters the T-cell landscape in cancer and may enhance the clinical efficacy of cancer therapy.

We investigated the in vivo function of the B7 family member B7-H3 (also known as B7RP-2) by gene targeting. B7-H3 inhibited T cell proliferation mediated by antibody to T cell receptor or allogeneic antigen-presenting cells. B7-H3-deficient mice developed more severe airway inflammation than did wild-type mice in conditions in which T helper cells differentiated toward type 1 (T(H)1) rather than type 2 (T(H)2). B7-H3 expression was consistently enhanced by interferon-gamma but suppressed by interleukin 4 in dendritic cells. B7-H3-deficient mice developed experimental autoimmune encephalomyelitis several days earlier than their wild-type littermates, and accumulated higher concentrations of autoantibodies to DNA. Thus, B7-H3 is a negative regulator that preferentially affects T(H)1 responses.

Since the discovery of triggering receptor expressed on myeloid cells (TREM)-1 in 2000, evidence documenting the profound ability of the TREM and TREM-like receptors to regulate inflammation has rapidly accumulated. Monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells, neutrophils, microglia, osteoclasts, and platelets all express at least one member of the TREM family, underscoring the importance of these proteins in the regulation of innate resistance. Recent work on the TREM family includes: characterization of a new receptor expressed on plasmacytoid dendritic cells; definition of a key role for TREM in inflammatory bowel disease and multiple sclerosis; an expanded list of diseases associated with the release of soluble forms of TREM proteins; and identification of the first well characterized TREM ligand: B7-H3, a ligand for TREM-like Transcript (TLT)-2. Moreover, analysis of TREM signaling has now identified key regulatory components and defined pathways that may be responsible for the complex functional interactions between the TREM and toll-like receptors. In addition, there is expanding evidence of a role for TREM in the regulation of integrin function via Plexin-A1. Together these new findings define the TREM and TREM-like receptors as pluripotent modifiers of disease through the integration of inflammatory signals with those associated with leukocyte adhesion.

B7-H3 and B7x are recently discovered members of the B7-CD28 family thought to dampen peripheral immune responses via negative costimulation. We evaluated their potential expression in human prostate cancer using a large cohort of patients with 7 years of follow-up. We identified 823 patients with tissue available treated with radical prostatectomy between 1985 and 2003. Immunohistochemistry was performed on tissue microarray sections using anti-B7-H3 and -B7x. The percentage and intensity of immunoreactivity by tumor cells were blindly evaluated by two urological pathologists, and outcome analyses were conducted. Both B7-H3 and B7x were highly expressed; 93% and 99% of tumors had aberrant expression, respectively. The median percentage of tumor cells staining positive was 80% for each molecule. Strong intensity for B7-H3 and B7x was noted in 212 (26%) and 120 (15%) patients, respectively. Patients with strong intensity for B7-H3 and B7x were significantly more likely to have disease spread at time of surgery (P < 0.001 and P = 0.005, respectively). Additionally, patients with strong intensity for B7-H3 and B7x were at significantly increased risk of clinical cancer recurrence (P < 0.001 and P = 0.005) and cancer-specific death (P = 0.004 and P = 0.04, respectively). To our knowledge, we present the largest investigation of B7 family molecules in a human malignancy and a previously undescribed evaluation of B7x in prostate cancer. B7-H3 and B7x are abundantly expressed in prostate cancer and associated with disease spread and poor outcome. Given the proposed immune-inhibitory mechanisms of B7-H3 and B7x, these molecules represent attractive targets for therapeutic manipulation in prostate cancer.

Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body's immunological activity against tumors. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clinical activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or molecules targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clinical trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.

Inhibitory regulatory functions of B7-H3 and B7-H4 regarding T-cell activation have been reported recently. Little is known about the significance of human B7-H3 and B7-H4 expression in non-small-cell lung cancer (NSCLC), and we conducted the present study to address this issue in cell lines and tumor tissue from 70 patients. B7-H3 is over-expressed by all six NSCLC cell lines on both mRNA and protein level. B7-H4 is only transcripted by one cell line in which an alternatively spliced form was discovered. In tumor tissues, expression of B7-H3 and B7-H4 was found both on the cell membrane and in the cytoplasm. Thirty-seven percent of the specimens expressed B7-H3 and 43% B7-H4. The number of T infiltrating lymphoid cells (TILs) in the tumor tissues that expressed B7-H3 or B7-H4 was much lower than those who did not. There was a significant positive relation between the expression of B7-H3 and B7-H4, and high B7-H3 or B7-H4 expression was significantly more common in cases with lymph node metastasis. These observations suggest the contribution of B7-H3 and B7-H4 to immune dysfunction and tumor progression in NSCLC patients. B7-H3 and B7-H4 may be an important target for diagnosis and/or therapy of NSCLC.

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

T cell activation is regulated by the innate immune system through positive and negative costimulatory molecules. B7-H3 is a novel B7-like molecule with a putative receptor on activated T cells. Human B7-H3 was first described as a positive costimulator, most potently inducing IFN-gamma production and cellular immunity. In this study we examined the expression and function of mouse B7-H3. B7-H3 is mostly expressed on professional APCs; its expression on dendritic cells appears to be up-regulated by LPS. In contrast to human B7-H3, we found that mouse B7-H3 protein inhibited T cell activation and effector cytokine production. An antagonistic mAb to B7-H3 enhanced T cell proliferation in vitro and led to exacerbated experimental autoimmune encephalomyelitis in vivo. Therefore, mouse B7-H3 serves as a negative regulator of T cell activation and function.

B7 coregulatory ligands can be aberrantly expressed in human disease. In the context of cancer, these ligands may act as antigen-specific inhibitors of T-cell-mediated antitumoral immunity. We recently reported that B7-H1 expression by carcinomas of the kidney and bladder portends aggressive disease and diminished survival. The expression of these proteins in prostate cancer, however, has not been investigated. We evaluated B7-H3 and B7-H1 protein expression in the pathologic specimens of 338 men treated for clinically localized prostate cancer between 1995 and 1998 with radical retropubic prostatectomy. Expression levels of B7-H3 in prostate cancer were correlated with pathologic indicators of aggressive cancer as well as clinical outcome. We report that B7-H3 is uniformly and aberrantly expressed by adenocarcinomas of the prostate, high-grade prostatic intraepithelial neoplasia, and four prostate cancer cell lines, whereas B7-H1 is rarely expressed. B7-H3 is expressed by benign prostatic epithelia, although at a more reduced level relative to neoplastic tissue. Increasing levels of B7-H3 intensity correlate with worsening clinicopathologic features of prostate cancer. Marked B7-H3 intensity, present in 67 (19.8%) specimens, confers a >4-fold increased risk of cancer progression after surgery (risk ratio, 4.42; P < 0.001). A survey of normal tissues revealed that B7-H3 is expressed within the liver, urothelium, and fetal kidney. In summary, B7-H3 is aberrantly expressed in all prostate cancers and represents an independent predictor of cancer progression following surgery. Moreover, B7-H3 encompasses a novel diagnostic and potential therapeutic target for the clinical management of prostate cancer and, perhaps, other malignancies as well.

B7-H3, a surface immunomodulatory glycoprotein, inhibits natural killer cells and T cells. The monoclonal antibody (mAb) 8H9 is specific for 4Ig-B7-H3, the long and principal form of B7-H3. Early results from radioimmunotherapy using 8H9 have shown promise in patients with metastatic solid tumors to the central nervous system. Whereas B7-H3 transcript was ubiquitously expressed in a wide spectrum of human solid tumors as well as human normal tissues, B7-H3 protein was preferentially expressed only in tumor tissues. By quantitative reverse transcription-PCR, all three isoforms of microRNA miR-29 (a, b, and c) were highly expressed in normal tissues. However, they were down-regulated in a broad spectrum of solid tumors, including neuroblastoma, sarcomas, brain tumors, and tumor cell lines. B7-H3 protein expression was inversely correlated with miR-29 levels in both cell lines and tumor tissues tested. Using luciferase reporter assay, miR-29a was shown to directly target B7-H3 3' untranslated region, and knock-in and knockdown of miR-29a led to down-regulation and up-regulation, respectively, of B7-H3 protein expression. The ability of miR-29 to control B7-H3 protein expression has implications in immune escape by solid tumors. Differential modulation of this key immunoinhibitory molecule in tumor versus normal tissues may advance both cell-mediated immunotherapy and antibody-based targeted strategies using the B7-H3-specific mAb 8H9.

T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-gamma production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.

In this study, in an attempt to identify neuroblastoma-associated surface antigens, we generated mAbs against the ACN neuroblastoma cell line. A mAb was selected (5B14) that reacted with all neuroblastoma cell lines analyzed and allowed detection of tumor cell infiltrates in bone marrow aspirates from neuroblastoma patients. In cytofluorimetric analysis, unlike anti-disialoganglioside mAb, 5B14 mAb did not display reactivity with normal bone marrow hematopoietic cell precursors, thus representing a highly specific marker for identifying neuroblastoma cells. Molecular analysis revealed that the 5B14 mAb-reactive surface glycoprotein corresponded to the recently identified 4Ig-B7-H3 molecule. Remarkably, mAb-mediated masking of the 4Ig-B7-H3 molecule on cell transfectants or on freshly isolated neuroblastoma cells resulted in enhancement of natural killer-mediated lysis of these target cells. These data suggest that 4Ig-B7-H3 molecules expressed at the tumor cell surface can exert a protective role from natural killer-mediated lysis by interacting with a still undefined inhibitory receptor expressed on natural killer cells.

OBJECTIVE

Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC.

METHODS

Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using chi2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models.

RESULTS

B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029).

CONCLUSIONS

Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.

Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents.

We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed.

Epithelial-mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4(+)Foxp3(+) regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC.

The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630-42. ©2016 AACRSee related commentary by Datar and Schalper, p. 3422.

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-gamma production.

B7-H3 belongs to the B7 superfamily, a group of molecules that costimulate or down-modulate T-cell responses. Although it was shown that B7-H3 could inhibit T-cell responses, several studies - most of them performed in murine systems - found B7-H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7-H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T-cell subsets. We show that B7-H3 does not costimulate human T cells. In the presence of strong activating signals, B7-H3 potently and consistently down-modulated human T-cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre-activated T cells. Furthermore, we demonstrate that B7-H3-T-cell interaction is characterised by an early suppression of IL-2 and that T-cell inhibition can be reverted by exogenous IL-2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT-2) has been recently described as costimulatory receptor of murine B7-H3 we have extensively analysed interaction of human B7-H3 with TREML2/TLT-2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7-H3.

The B7 family consists of structurally related, cell-surface protein ligands, which bind to receptors on lymphocytes that regulate immune responses. Activation of T and B lymphocytes is initiated by engagement of cell-surface, antigen-specific T-cell receptors or B-cell receptors, but additional signals delivered simultaneously by B7 ligands determine the ultimate immune response. These 'costimulatory' or 'coinhibitory' signals are delivered by B7 ligands through the CD28 family of receptors on lymphocytes. Interaction of B7-family members with costimulatory receptors augments immune responses, and interaction with coinhibitory receptors attenuates immune responses. There are currently seven known members of the family: B7.1 (CD80), B7.2 (CD86), inducible costimulator ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3, and B7-H4. Members of the family have been characterized predominantly in humans and mice, but some members are also found in birds. They share 20-40% amino-acid identity and are structurally related, with the extracellular domain containing tandem domains related to variable and constant immunoglobulin domains. B7 ligands are expressed in lymphoid and non-lymphoid tissues. The importance of the family in regulating immune responses is shown by the development of immunodeficiency and autoimmune diseases in mice with mutations in B7-family genes. Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunity, inflammatory diseases and cancer.

Activated in immune responses, T lymphocytes differentiate into effector cells with potent immune function. CD28 is the most prominent costimulatory receptor for T-cell activation. However, absence of CD28 costimulation did not completely impair effector function of CD4 or CD8 T cells. Moreover, increasing number of costimulatory molecules are recently found on antigen-presenting cells to regulate T-cell activation. To understand the molecular mechanisms that determine T-cell function or tolerance, we have collectively examined the roles of positive and negative costimulatory molecules. Antigen-specific naïve CD4 and CD8 T cells, only when activated in the absence of both CD28 and ICOS pathways, were completely impaired in effector function. These tolerant T cells not only were anergic with profound defects in TcR signal transduction but also completely lacked expression of effector-specific transcription factors. T-cell tolerance induction in this system requires the action by negative costimulatory molecules; T-cell proliferation and function was partially restored by inhibiting PD-1, B7-H3 or B7S1. This work demonstrates that T-cell function or tolerance is controlled by costimulatory signals.

B7-H3 and B7x are members of the B7 family of immune regulatory ligands that are thought to attenuate peripheral immune responses through co-inhibition. Previous studies have correlated their overexpression with poor prognosis and decreased tumor-infiltrating lymphocytes in various carcinomas including uterine endometrioid carcinomas, and mounting evidence supports an immuno-inhibitory role in ovarian cancer prognosis. We sought to examine the expression of B7-H3 and B7x in 103 ovarian borderline tumors and carcinomas and study associations with clinical outcome. Using immunohistochemical tissue microarray analysis on tumor specimens, we found that 93 and 100% of these ovarian tumors express B7-H3 and B7x, respectively, with expression found predominantly on cell membranes and in cytoplasm. In contrast, only scattered B7-H3- and B7x-positive cells were detected in non-neoplastic ovarian tissues. B7-H3 was also expressed in the endothelium of tumor-associated vasculature in 44% of patients, including 78% of patients with high-stage tumors (FIGO stages III and IV), nearly all of which were high-grade serous carcinomas, and 26% of patients with low-stage tumors (FIGO stages I and II; P<0.001), including borderline tumors. Analysis of cumulative survival time and recurrence incidence revealed that carcinomas with B7-H3-positive tumor vasculature were associated with a significantly shorter survival time (P=0.02) and a higher incidence of recurrence (P=0.03). The association between B7-H3-positive tumor vasculature and poor clinical outcome remained significant even when the analysis was limited to the high-stage subgroup. These results show that ovarian borderline tumors and carcinomas aberrantly express B7-H3 and B7x, and that B7-H3-positive tumor vasculature is associated with high-grade serous histological subtype, increased recurrence and reduced survival. B7-H3 expression in tumor vasculature may be a reflection of tumor aggressiveness and has diagnostic and immunotherapeutic implications in ovarian carcinomas.

The B7 family member B7-H3 (CD276) plays important roles in immune responses. However, the function of B7-H3 remains controversial. We found that murine B7-H3 specifically bound to Triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2, TREML2). TLT-2 was expressed on CD8(+) T cells constitutively and on activated CD4(+) T cells. Stimulation with B7-H3 transfectants preferentially up-regulated the proliferation and IFN-gamma production of CD8(+) T cells. Transduction of TLT-2 into T cells resulted in enhanced IL-2 and IFN-gamma production via interactions with B7-H3. Blockade of the B7-H3:TLT-2 pathway with a mAb against B7-H3 or TLT-2 efficiently inhibited contact hypersensitivity responses. Our results demonstrate a direct interaction between B7-H3 and TLT-2 that preferentially enhances CD8(+) T cell activation.

OBJECTIVE

Aberrant expression of T-cell coregulatory molecules has been investigated as a mechanism by which certain cancers may evade host immune surveillance. We evaluated expression of the T-cell coregulators B7-H1, B7-H3, and PD-1 in urothelial cell carcinoma (UCC) of the bladder.

METHODS

Immunohistochemistry for B7-H1, B7-H3, and PD-1 was done on paraffin-embedded sections from 318 consecutive patients with UCC who underwent radical cystectomy. Expression was correlated with clinicopathologic outcomes and postoperative survival.

RESULTS

B7-H3 was widely expressed in UCC, as 222 of 314 (70.7%) tumors showed positive staining. Expression of B7-H3 in UCC was significantly increased compared with adjacent, nontumor urothelium, as a median of 70% of tumor cells expressed B7-H3, compared with 20% of cells in nontumor specimens (P < 0.001). The increase in B7-H3 expression was independent of tumor stage (P = 0.13). Expression of B7-H1 by UCC tumors (P < 0.001) and PD-1 by tumor-infiltrating lymphocytes (P = 0.012) were significantly associated with increased pathologic stage. Patients who had received intravesical bacillus Calmette-Guerin before cystectomy tended to show increased expression of B7-H3 (P = 0.023) and PD-1 (P = 0.071) but were less likely to express B7-H1 (P = 0.027). Moreover, for the subset of patients with organ-confined disease (n = 167), B7-H1 expression independently predicted all-cause mortality after cystectomy (hazard ratio, 3.18; 95% confidence interval, 1.74-5.79; P < 0.001).

CONCLUSIONS

B7-H3 is highly expressed in UCC across tumor stages, whereas B7-H1 and PD-1 expression are associated with advanced disease. B7-H1 expression predicts mortality after cystectomy for patients with organ-confined tumors. These molecules may represent novel diagnostic or prognostic markers, as well as therapeutic targets, for patients with UCC.

B7-H1, B7-DC, B7-H2, B7-H3, and B7-H4, all new additions to the B7 family, here termed "the new B7s," are emerging as important tools in directing immune function; each with unique, yet often overlapping functions. Clearly, each B7 molecule has developed its own indispensable niche in the immune system. The expression of both stimulatory and inhibitory B7 molecules seems to play an essential role in regulating the immune response to transformed cells through a variety of mechanisms. As specific niches of B7 family members continue to be dissected, their diagnostic and therapeutic potential becomes ever more apparent. In this review, we will discuss the role of the new B7s in activation and inhibition of antitumor immune responses, their prospects in diagnostics, and also potential and developing immunotherapy protocols.