CD276 is an important player in macrophage recruitment into the tumor and an upstream regulator for PAI-1.

More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7-H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.

The Role of V-Domain Ig Suppressor of T Cell Activation (VISTA) in Cancer Therapy: Lessons Learned and the Road Ahead.

Immune checkpoints (ICs) have pivotal roles in regulating immune responses. The inhibitory ICs in the tumor microenvironment (TME) have been implicated in the immune evasion of tumoral cells. Therefore, identifying and targeting these inhibitory ICs might be critical for eliminating tumoral cells. V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel inhibitory IC that is expressed on myeloid cells, lymphoid cells, and tumoral cells; therefore, VISTA can substantially regulate innate and adaptive anti-tumoral immune responses. Besides, growing evidence indicates that VISTA blockade can enhance the sensitivity of tumoral cells to conventional IC-based immunotherapy, e.g., cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors. In this regard, the current study aimed to review the current evidence about the structure and expression pattern of VISTA, its role in TME, the clinicopathological significance of VISTA, and its prognostic values in various cancers. Besides, this review intended to collect the lessons from the recent pre-clinical and clinical studies and propose a strategy to overcome tumor immune-resistance states.

The Regulatory Cross-Talk between microRNAs and Novel Members of the B7 Family in Human Diseases: A Scoping Review.

The members of the B7 family, as immune checkpoint molecules, can substantially regulate immune responses. Since microRNAs (miRs) can regulate gene expression post-transcriptionally, we conducted a scoping review to summarize and discuss the regulatory cross-talk between miRs and new B7 family immune checkpoint molecules, i.e., B7-H3, B7-H4, B7-H5, butyrophilin like 2 (BTNL2), B7-H6, B7-H7, and immunoglobulin like domain containing receptor 2 (ILDR2). The current study was performed using a six-stage methodology structure and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. PubMed, Embase, Scopus, Cochrane, ProQuest, and Google Scholar were systematically searched to obtain the relevant records to 5 November 2020. Two authors independently reviewed the obtained records and extracted the desired data. After quantitative and qualitative analyses, we used bioinformatics approaches to extend our knowledge about the regulatory cross-talk between miRs and the abovementioned B7 family members. Twenty-seven articles were identified that fulfilled the inclusion criteria. Studies with different designs reported gene-miR regulatory axes in various cancer and non-cancer diseases. The regulatory cross-talk between the aforementioned B7 family molecules and miRs might provide valuable insights into the pathogenesis of various human diseases.

Langerhans Cells Suppress CD8+ T Cells In Situ during Mucocutaneous Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGVHD) induced by allogenic hematopoietic stem cell transplantation is an immunological disorder in which donor lymphocytes attack recipient organs. It has been proven that recipient nonhematopoietic tissue cells, such as keratinocytes, are sufficient as immunological targets for allogenic donor T cells, whereas Langerhans cells (LCs) are potent professional hematopoietic antigen-presenting cells existing in the target epidermis and eliminated during the early phase of mucocutaneous aGVHD. Moreover, LCs have been reported to negatively regulate various types of immune responses. Here, we present data showing that initial depletion of recipient LCs exacerbates mucocutaneous lesions in a murine model of allogenic bone marrow transplantation-induced aGVHD. Furthermore, another murine model of mucocutaneous aGVHD induced in mice with keratinocytes genetically expressing chicken ovalbumin by transfer of ovalbumin-specific CD8+ OT-I cells also showed that LC-depleted recipient mice develop aggravated mucocutaneous disease owing to decreased apoptosis of skin-infiltrating OT-I cells. Moreover, coexisting LCs directly induce apoptosis and inhibit the proliferation of OT-I cells in vitro partially via B7 family proteins. Collectively, our results indicate that LCs negatively regulate mucocutaneous aGVHD-like lesions in situ by inhibiting the number of infiltrating CD8+ T cells.

Genetic Polymorphism in Cytokines and Costimulatory Molecules in Stem Cell and Solid Organ Transplantation.

There is growing evidence supporting the genetic variability outside of HLA system that is contributing to the variation in transplant outcomes. Determining novel predictors could help to identify patients at risk and tailor their immunosuppressive regimens. This article discusses the various single nucleotide polymorphisms in costimulatory molecules and cytokines that have been evaluated for their effect on transplantation. An overview of how gene polymorphism studies are conducted and factors to consider in the experimental design to ensure meaningful data can be concluded are discussed.

Immunoregulatory protein B7-H3 regulates cancer stem cell enrichment and drug resistance through MVP-mediated MEK activation.

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

Tumor cell-associated immune checkpoint molecules - Drivers of malignancy and stemness.

Inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor cells in different tumor types. It was thought that the main function of tumor cell-associated immune checkpoint molecules would be the modulation (down- or upregulation) of antitumor immune responses. In recent years, however, it has become clear that the expression of immune checkpoint molecules on tumor cells has important consequences on the biology of the tumor cells themselves. In particular, a causal relationship between the expression of these molecules and the acquisition of malignant traits has been demonstrated. Thus, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells, the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize. Herein, we review this evidence, with a main focus on PD-L1, the most intensively investigated tumor cell-associated immune checkpoint molecule and for which most information is available. Then, we discuss more concisely other tumor cell-associated immune checkpoint molecules that have also been shown to induce the acquisition of malignant traits, such as PD-1, B7-H3, B7-H4, Tim-3, CD70, CD28, CD137, CD40 and CD47. Open questions in this field as well as some therapeutic approaches that can be derived from this knowledge, are also addressed.

Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells.

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

Roles of coinhibitory molecules B7-H3 and B7-H4 in esophageal squamous cell carcinoma.

The coinhibitory molecules, B7-H3 and B7-H4, have shown negative regulation in T cell activation and tumor-associated macrophage (TAM) polarization in tumor-specific immunity. Here, we investigated the expression of B7-H3 and B7-H4 in human and murine esophageal squamous cell carcinoma (ESCC) tissues to define their clinical significance and mechanism in a tumor microenvironment. In the present study, B7-H3 and B7-H4 were expressed in 90.6 and 92.7 % samples, respectively. High B7-H3 and B7-H4 expression was associated with advanced TNM stage and lymph node metastasis (p < 0.05, respectively). Patients with both B7-H3 and B7-H4 high-expressed tumors had the poorest prognosis (26.7 months), whereas those with both low-expressed tumors had the best survival (56.7 months). B7-H3 and B7-H4 expression were inclined to be positively related to the infiltration intensity of Treg cells and TAMs (p < 0.05, respectively), and B7-H3 expression is negatively associated with the intensity of CD8(+) T cells (p < 0.05). In 4-nitroquinoline 1-oxide (4-NQO)-induced murine models, high B7-H3 expression could only be detected at carcinoma stage, but abnormal B7-H4 expression appeared a little earlier at dysplasia stage. In vitro studies revealed that knockdown of B7-H3 on tumor cells suppressed ESCC cell migration and invasion, while knockdown of B7-H4 could inhibit ESCC cell growth. Overall, B7-H3 and B7-H4 are involved in ESCC progression and development and their coexpression could be valuable prognostic indicators. Interference of these negative regulatory molecules might be a new strategy for treating ESCC.

VISTA is an immune checkpoint molecule for human T cells.

V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T-cell function that is expressed on hematopoietic cells. VISTA levels are heightened within the tumor microenvironment, in which its blockade can enhance antitumor immune responses in mice. In humans, blockade of the related programmed cell death 1 (PD-1) pathway has shown great potential in clinical immunotherapy trials. Here, we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells and blunted the production of T-cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T-cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer.

VISTA Regulates the Development of Protective Antitumor Immunity.

V-domain Ig suppressor of T-cell activation (VISTA) is a novel negative checkpoint ligand that is homologous to PD-L1 and suppresses T-cell activation. This study demonstrates the multiple mechanisms whereby VISTA relieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highly expressed on myeloid cells and Foxp3(+)CD4(+) regulatory cells, but not on tumor cells within the tumor microenvironment (TME). VISTA monoclonal antibody (mAb) treatment increased the number of tumor-specific T cells in the periphery and enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME by decreasing the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cells within the tumor microenvironment. In addition, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3(+)CD4(+) regulatory T cells. Consequently, VISTA mAb administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy.

Induced expression of B7-H3 on the lung cancer cells and macrophages suppresses T-cell mediating anti-tumor immune response.

Macrophages are the prominent components of solid tumors and have complex dual functions in their interaction with cancer cells. Strong evidence suggests that TAM is a part of inflammatory circuits that promote tumor progression. B7-homologue 3 (B7-H3), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert co-stimulatory and immune regulatory functions. Here, we showed that a fraction of macrophages in tumor stroma expressed surface B7-H3 molecule. Normal macrophages, which did not express B7-H3, would be induced expressing B7-H3 molecule when culturing with tumor cell. Although a lung cancer cell line constitutively expressed B7-H3 mRNA and protein in plasma, primary tumor cell isolated from the transplanted lung carcinoma model expressed B7-H3 on the surface. Interestingly, in transplanted lung carcinoma model, the expression of membrane-bound B7-H3 in tumor cells was increased as prolonging of tumor transformation. In support, IL-10 released from TAM could stimulate cancer cell expression of membrane bound B7-H3. Furthermore, Lung cancer and TAM-related B7-H3 was identified as a strong inhibitor of T-cell effect and influenced the outcome of T cell immune response. In conclusion, TAM-tumor cell interaction-induced membrane-bound B7-H3 represents a novel immune escape mechanism which links the pro-inflammatory response to immune tolerance in the tumor milieu.

CD28-B7 interaction modulates short- and long-lived plasma cell function.

The interaction of CD28, which is constitutively expressed on T cells, with B7.1/B7.2 expressed on APCs is critical for T cell activation. CD28 is also expressed on murine and human plasma cells but its function on these cells remains unclear. There are two types of plasma cells: short-lived ones that appear in the secondary lymphoid tissue shortly after Ag exposure, and long-lived plasma cells that mainly reside in the bone marrow. We demonstrate that CD28-deficient murine short- and long-lived plasma cells produce significantly higher levels of Abs than do their wild-type counterparts. This was owing to both increased frequencies of plasma cells as well as increased Ab production per plasma cell. Plasma cells also express the ligand for CD28, B7.1, and B7.2. Surprisingly, deficiency of B7.1 and B7.2 in B cells also led to higher Ab levels, analogous to Cd28(-/-) plasma cells. Collectively, our results suggest that the CD28-B7 interaction operates as a key modulator of plasma cell function.

B7-CD28 costimulatory signals control the survival and proliferation of murine and human γδ T cells via IL-2 production.

γδ T cells play key nonredundant roles in immunity to infections and tumors. Thus, it is critical to understand the molecular mechanisms responsible for γδ T cell activation and expansion in vivo. In striking contrast to their αß counterparts, the costimulation requirements of γδ T cells remain poorly understood. Having previously described a role for the TNFR superfamily member CD27, we since screened for other nonredundant costimulatory receptors in γδ T cell activation. We report in this article that the Ig superfamily receptor CD28 (but not its related protein ICOS) is expressed on freshly isolated lymphoid γδ T cells and synergizes with the TCR to induce autocrine IL-2 production that promotes γδ cell survival and proliferation in both mice and humans. Specific gain-of-function and loss-of-function experiments demonstrated a nonredundant function for CD28 interactions with its B7 ligands, B7.1 (CD80) and B7.2 (CD86), both in vitro and in vivo. Thus, γδ cell proliferation was significantly enhanced by CD28 receptor agonists but abrogated by B7 Ab-mediated blockade. Furthermore, γδ cell expansion following Plasmodium infection was severely impaired in mice genetically deficient for CD28. This resulted in the failure to mount both IFN-γ-mediated and IL-17-mediated γδ cell responses, which contrasted with the selective effect of CD27 on IFN-γ-producing γδ cells. Our data collectively show that CD28 signals are required for IL-2-mediated survival and proliferation of both CD27(+) and CD27(-) γδ T cell subsets, thus providing new mechanistic insight for their modulation in disease models.

B7-H3 contributes to the metastatic capacity of melanoma cells by modulation of known metastasis-associated genes.

B7-H3, an immunoregulatory protein, is known to play a role in tumor progression. In many cancer types, observed correlations between high B7-H3 expression and poor prognosis have been attributed to involvement in antitumor immunity. However, here we demonstrate a nonimmunological alternative function of B7-H3 in cancer metastasis. Since advanced malignant melanoma is a disease with a poor survival rate and a broad pattern of metastasis, we used this disease as a model in our studies. We found that shRNA silencing of B7-H3 reduced the in vitro migratory potential and matrigel invasiveness of MDA-MB-435 and FEMX-I melanoma cells. In an experimental metastasis model in vivo, B7-H3 silencing of MDA-MB-435 cells resulted in reduced metastatic capacity and significantly increased the median symptom-free survival of nude mice (147 vs. 65 days, p < 0.001) and rats (53 vs. 42 days, p = 0.025) injected with MDA-MB-435 cells. Furthermore, a smaller fraction of mice had microscopically detectable metastases compared to control animals, and the pattern of metastases was slightly different between the two groups but with the brain as the predominant organ. Immunohistochemistry on samples from two melanoma patients showed strong B7-H3 staining in both a primary tumor and metastases. Notably, the metastasis-associated proteins, matrix metalloproteinase (MMP)-2, signal transducer and activator of transcription 3 (Stat3), and the level of secreted interleukin-8 (IL-8) were reduced in the B7-H3 knock-down cell variants, whereas tissue inhibitor of metalloproteinase (TIMP)-1 and-2 levels were increased. Taken together, our findings indicate a novel role for B7-H3 in the regulation of the metastatic capacity of melanoma cells and it might be a potential therapeutic target for anti-metastasis therapy.

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses.

The immunoglobulin (Ig) superfamily consists of many critical immune regulators, including the B7 family ligands and receptors. In this study, we identify a novel and structurally distinct Ig superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated V-domain Ig suppressor of T cell activation (VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTA expression on APCs inhibits T cell proliferation and cytokine production in vitro. A VISTA-specific monoclonal antibody interferes with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. Furthermore, anti-VISTA treatment exacerbates the development of the T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis in mice. Finally, VISTA overexpression on tumor cells interferes with protective antitumor immunity in vivo in mice. These findings show that VISTA, a novel immunoregulatory molecule, has functional activities that are nonredundant with other Ig superfamily members and may play a role in the development of autoimmunity and immune surveillance in cancer.