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.

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.

Association of B7-H4 gene polymorphisms in urothelial bladder cancer.

BACKGROUND/AIM: We aimed to study polymorphisms of the B7-H4 gene in order to evaluate a possible association in urothelial carcinoma, as it is highly expressed in cancer tissues. MATERIALS AND METHODS: In this study B7-H4 gene rs10754339, rs10801935, and rs3738414 SNPs were studied by PCR-RFLP method in paraffin-embedded tumor specimens from 62 urothelial carcinoma patients and in a control group including 30 patients without bladder cancer. RESULTS: We detected that the rs10754339 polymorphism was more frequent in the cancer patients when compared with the control group (P < 0.05). Only the rs3738414 polymorphism showed a statistically significant difference in frequency between pathologic diagnostic groups. CONCLUSION: The rs10754339 AA genotype distribution was found to have a higher frequency whereas the rs3738414 AG genotype distribution was lower in the bladder cancer group (P < 0.05). None of the genotype distributions showed a significant difference from the control group for the rs10801935 polymorphism. We conclude that B7-H4 has the potential to be a useful prognostic marker in urothelial carcinoma.

B7-H4(B7x)-Mediated Cross-talk between Glioma-Initiating Cells and Macrophages via the IL6/JAK/STAT3 Pathway Lead to Poor Prognosis in Glioma Patients.

PURPOSE: The objective of this study was to evaluate clinical significance and immunosuppressive mechanisms of B7-H4 (B7x/B7S1), a B7 family member, in glioma. EXPERIMENTAL DESIGN: B7-H4 levels in glioma tissue/cerebral spinal fluid (CSF) were compared between different grades of glioma patients. Survival data were analyzed with Kaplan-Meier to determine the prognostic value of B7-H4. Cytokines from CD133(+) cells to stimulate the expression of B7-H4 on human macrophages (Mφs) were investigated by FACS, neutralizing antibodies, and Transwell chemotaxis assay. shRNA, reporter vector, and chromatin immunoprecipitation were used to determine the binding of STAT3 to the B7-H4 promoter. The function of B7-H4(+) Mφs in vitro was evaluated through phagocytosis, T-cell proliferation/apoptosis, and cytokine production as well as in the xenografted model for in vivo analysis. RESULTS: We found that B7-H4 expression in tumors was associated with prognosis of human glioblastoma and correlated directly with malignant grades. Mechanistically, glioma initiating CD133(+) cells and Mφs/microglia cointeraction activated expression of B7-H4 via IL6 and IL10 in both tumor cells and microenvironment supporting cells. IL6-activated STAT3 bound to the promoter of B7-H4 gene and enhanced B7-H4 expression. Furthermore, CD133(+) cells mediated immunosuppression through B7-H4 expression on Mφs/microglia by silencing of B7-H4 expression on these cells, which led to increased microenvironment T-cell function and tumor regression in the xenograft glioma mouse model. CONCLUSIONS: We have identified B7-H4 activation on Mφs/microglia in the microenvironment of gliomas as an important immunosuppressive event blocking effective T-cell immune responses. Clin Cancer Res; 22(11); 2778-90. ©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.

Activation of the arylhydrocarbon receptor causes immunosuppression primarily by modulating dendritic cells.

UVR suppresses the immune system in an antigen-specific manner via induction of regulatory T cells (Tregs). The specific immunosuppression by UVR harbors therapeutic potential but is associated with UVR-induced DNA damage, requiring the identification of other triggers inducing the same immunosuppressive effects without DNA damage. The aryl hydrocarbon receptor (AhR) was identified as a molecular target for UVR and its activation to be involved in UVR-induced immunosuppression. Accordingly, the AhR agonist 4-n-nonylphenol (NP) suppressed sensitization and induced Treg similar to UVR. Here we show that antigen-presenting cells are critically involved in AhR-induced immunosuppression. Injection of hapten-coupled dendritic cells (DCs) treated with NP into mice did not result in sensitization but induced Treg. NP induced the release of IL-2 by DC that subsequently triggered the release of IL-10. NP upregulated the negative regulatory molecule B7-H4 via the release of IL-2 that was functionally relevant as inhibition of B7-H4 prevented the induction of Treg. Together, this indicates that activation of the AhR switches antigen-presenting cells from a stimulatory into a regulatory phenotype, ultimately inducing Treg. Thus, AhR agonists may represent an alternative to suppress the immune system like UVR but without causing the adverse effects of UVR including DNA damage.

Endogenous expression of B7-H4 improves long-term murine islet allograft survival.

BACKGROUND: Allograft rejection is one of the main obstacles for islet transplantation. B7-H4 plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. In this study, we investigated whether the endogenous expression of B7-H4 in ß cells from B7-H4 transgenic mice enhances islet allograft survival. METHODS: B7-H4 transgenic C57BL/6 (B6) mice (RIP.B7-H4) were developed by inserting the entire B7-H4 open reading frame under the rat insulin promoter (RIP). B7-H4 protein expression was examined by flow cytometric analysis and immunohistochemical staining. Islet allograft survival was investigated in streptozotocin-induced diabetic recipient BALB/c (H-2d) mice transplanted with 400 islets from RIP.B7-H4 (H-2b) mice under the kidney capsule. The recipient control group received islets from wild-type B6 donors. RESULTS: B7-H4 protein was significantly up-regulated in isolated islets from RIP.B7-H4 compared with wild-type B6 mice (56%±23% vs. 3%±1.2%). B7-H4 was coexpressed with insulin, but not glucagon, suggesting that B7-H4 is expressed in a ß-cell-specific manner. Recipient BALB/c mice transplanted with RIP.B7-H4 islets established euglycemia for 42.3±18.4 days (mean±SD; n=9) compared with controls at 23.1±7.8 days (mean±SD; n=12; P<0.004, log-rank test). CONCLUSIONS: The endogenous expression of B7-H4 in donor ß cells from transgenic mice prolongs islet allograft survival, confirming the negative role of B7-H4 in regulating alloreactive T-cell responses.

Tissue-expressed B7x affects the immune response to and outcome of lethal pulmonary infection.

B7x (B7-H4 or B7S1), a member of the B7 family, inhibits in vitro T cell proliferation and cytokine production by binding to an unidentified receptor on activated T cells, but its in vivo function remains largely unclear. We show that B7x protein was expressed in epithelial cells of the lung, but not in lymphoid tissues. To investigate the role of B7x in the lung, we determined the susceptibility of B7x-deficient (B7x(-/-)) mice to a lethal pulmonary infection with Streptococcus pneumoniae. B7x(-/-), but not B7-H3-deficient, mice were significantly more resistant to S. pneumoniae pulmonary infection than their wild-type (Wt) counterparts. B7x(-/-) mice had significantly lower bacterial burdens and levels of inflammatory cytokines in lungs as early as 12 h postinfection. They also had milder immunopathology that was localized in alveolar spaces, whereas Wt mice had severe inflammation that was perivascular. Control of infection in B7x(-/-) mice was associated with a marked increase in activated CD4 and CD8 T cells and fewer neutrophils in lungs, whereas the susceptible Wt mice had the opposite cellular profile. In B7x(-/-)Rag1(-/-) mice that lack T cells, reduction in bacterial burden was no longer observed. Control of S. pneumoniae and the increased survival observed was specific to the lung, because systemically infected B7x(-/-) mice were not resistant to infection. These data indicate that lung-expressed B7x negatively regulates T cells, and that in its absence, in B7x(-/-) mice, an enhanced T cell response contributed to reduced lethality in a pulmonary infection model with S. pneumoniae.

Intracellular B7-H4 suppresses bile duct epithelial cell apoptosis in human primary biliary cirrhosis.

The expression and function of B7-H4, a recently identified co-inhibitory molecule of the B7 superfamily, in the pathogenesis of primary biliary cirrhosis (PBC) is still unclear. Here the expression of B7-H4 in sections from PBC patients (n = 16) was examined by immunohistochemistry and it was detected in primary bile duct epithelial cells (BECs) which were isolated from PBC patients by flow cytometry (FACs). Moreover, we also analyzed BECs-associated B7-H4 function through knock-down of its expression via RNA interference (RNAi) in vitro. Immunohistochemistry and FACs evidenced that the expression of B7-H4 was restricted in the cytoplasm of BECs from PBC patients, while it was completely absent in normal liver tissues. The cytoplasmic B7-H4 gene was cloned, and sequenced analysis showed it was encoded by the same gene to the membrane B7-H4. Interesting, silencing B7-H4 by specific RNAi resulted in enhanced FasL expression and BEC apoptosis. Conversely, interruption of Fas\FasL interaction with using FasL blocking antibodies (clone 4H9) reversed cell apoptosis. Our results suggested that the intracellular B7-H4 appears to prevent Fas/FasL-mediated BEC apoptosis during the progression of PBC, and indicates B7-H4 is a possible target for therapeutic intervention of this disease.