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With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments. Immune checkpoint inhibitors are now routinely used in cancer therapy, however, the dosage and integration into conventional cancer therapy is determined via empirical experience rather than mechanistic rationale. Here authors establish an advanced single-molecule imaging method, by with which they are directly monitoring and evaluating the effect of immune checkpoint inhibitors on T cell signaling. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Immunomodulators blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) have improved the treatment of a broad spectrum of cancers. These immune checkpoint inhibitors (ICIs) reactivate the immune system against tumour cells but can also trigger autoimmune side effects, including type 1 diabetes. Mesenchymal stem cell (MSC) therapy is the most prevalent cell therapy, with tissue-regenerating, anti-fibrosis and immunomodulatory functions provided by the secretome of the cells. Here, we examined whether systemic MSC treatment could prevent the development of type 1 diabetes in a NOD mouse model. Methods: The purified PD-L1 monoclonal antibody was administered to induce diabetes in male NOD mice which normally do not develop diabetes. Human adipose-derived MSCs were administered by tail vein injections. T cells, macrophages and monocyte-derived macrophages expressing C-X-C motif chemokine ligand 9 (CXCL9) in pancreatic sections of NOD mice and a cancer patient who developed diabetes following the ICI treatments were analysed by immunofluorescence. Tissue localisation of the injected MSCs, plasma exosome levels and plasma cytokine profiles were also investigated. Results: PD-1/PD-L1 blockade induced diabetes in 16 of 25 (64%) NOD mice which received anti-PD-L1 mAb without hMSCs [MSC(−)], whereas MSC administration decreased the incidence to four of 21 (19%) NOD mice which received anti-PD-L1 mAb and hMSCs [MSC(+)]. The PD-1/PD-L1 blockade significantly increased the area of CD3-positive T cells (6.2-fold) and macrophage-2 (Mac-2) antigen (2.5-fold)- and CXCL9 (40.3-fold)-positive macrophages in the islets. MSCs significantly reduced T cell (45%) and CXCL9-positive macrophage (67%) accumulation in the islets and the occurrence of diabetes. The insulin content (1.9-fold) and islet beta cell area (2.7-fold) were also improved by MSCs. T cells and CXCL9-positive macrophages infiltrated into the intricate gaps between the beta cells in the islets by PD-1/PD-L1 blockade. Such immune cell infiltration was largely prevented by MSCs. The most striking difference was observed in the CXCL9-positive macrophages, which normally did not reside in the beta cell region in the islets but abundantly accumulated in this area after PD-1/PD-L1 blockade and were prevented by MSCs. The CXCL9-positive macrophages were also observed in the islets of a cancer patient who developed diabetes following the administration of ICIs but few CXCL9-positive macrophages were observed in a control patient. Mechanistically, the injected MSCs accumulated in the lung but not in the pancreas and strongly increased plasma exosome levels and changed plasma cytokine profiles. Conclusions/interpretation: Our results suggest that MSCs can prevent the incidence of diabetes associated with immune checkpoint cancer therapy and may be worth further consideration for new adjuvant cell therapy. [ABSTRACT FROM AUTHOR]

The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1–PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1–PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale. Takehara et al performed imaging analysis of microcluster formation between the PD-L1 and PD-L2, which are known to play a role in T cell activation in response to tumour cell signaling. Their analysis showed that the cluster formation inhibited T cell receptor signaling and could serve as a visual index for PD-L1/2-targeted cancer therapies. [ABSTRACT FROM AUTHOR]

The expression of PD-L1 on tumor cells or within the tumor microenvironment has been associated with good prognosis and sustained clinical responses in immunotherapeutic regimens based on PD-L1/PD-1/CD80 immune checkpoint blockade. To look into the current controversy in cancer immunotherapy of the relative importance of PD-L1 expression on tumor cells versus non-tumor cells of the tumor microenvironment, a hematological mouse tumor model was chosen. By combining a genetic CRISPR/Cas9 and immunotherapeutic approach and using a syngeneic hematopoietic transplantable tumor model (E.G7-cOVA tumor cells), we demonstrated that dual blockade of PD-L1 interaction with PD-1 and CD80 enhanced anti-tumor immune responses that either delayed tumor growth or led to its complete eradication. PD-L1 expression on non-tumor cells of the tumor microenvironment was required for the promotion of tumor immune escape and its blockade elicited potent anti-tumor responses to PD-L1 WT and to PD-L1-deficient tumor cells. PD-L1+ tumors implanted in PD-L1-deficient mice exhibited delayed tumor growth independently of PD-L1 blockade. These findings emphasize that PD-L1 expression on non-tumor cells plays a major role in this tumor model. These observations should turn our attention to the tumor microenvironment in hematological malignancies because of its unappreciated contribution to create a conditioned niche for the tumor to grow and evade the anti-tumor immune response. [ABSTRACT FROM AUTHOR]

The present study aimed to identify and compare the microbial signatures between periodontally healthy and periodontitis subjects using 454 sequences of 16S rRNA genes. Subgingival plaque samples were collected from ten periodontally healthy subjects and ten matched chronic periodontitis patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. The microbial composition differed between healthy subjects and periodontitis patients at all phylogenetic levels. Particularly, 16 species, including Lautropia mirabilis and Neisseria subflava predominated in healthy subjects, whereas nine species, including Porphyromonas gingivalis and Filifactor alocis predominated in periodontitis. UniFrac, a principal coordinate and network analysis, confirmed distinct community profiles in healthy subjects and periodontitis patients. Using predicted function profiling, pathways involved in phenylpropanoid, GPI-anchor biosynthesis, and metabolism of alanine, arginine, aspartate, butanoate, cyanoamino acid, fatty acid, glutamate, methane, proline, and vitamin B6 were significantly over-represented in periodontitis patients. These results highlight the oral microbiota alterations in microbial composition in periodontitis and suggest the genes and metabolic pathways associated with health and periodontitis. Our findings help to further elucidate microbial composition and interactions in health and periodontitis. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to define the comprehensive bacterial flora of the healthy oral cavity by identifying and comparing bacterial species in different subgingival sites using 454 sequencing of 16S rRNA genes. Materials and methods: Subgingival plaque samples were taken from six target teeth (central incisor, first premolar, and first molar in both the maxilla and mandible) of 10 periodontally healthy patients. Bacterial DNA was extracted and next-generation sequencing of 16S rRNA genes was performed. Results: Bacterial composition in phylum level was similar for all sites within the same individual irrespective of tooth location. Unweighted UniFrac distance values of microbiome also showed that average distance was significantly larger between subjects than between tooth locations of the same subjects. Conclusions: The present results clarify the lack of effect of tooth location in the healthy subgingival microbiota. Results may suggest that any subgingival site can demonstrate similar subject-specific microbiota. Clinical relevance: This investigation offers a better understanding of the uniqueness of the oral microbiome. The present study will facilitate sampling in future subgingival microbiological studies. [ABSTRACT FROM AUTHOR]

Background: V-domain Ig suppressor of T cell activation (VISTA) is a novel inhibitory immune-checkpoint protein. VISTA expression on tumour cells and the associated regulatory mechanisms remain unclear. We investigated VISTA expression and function in tumour cells, and evaluated its mechanism and activity.Methods: VISTA in tumour cells was assessed by tissue microarray analysis, immunohistochemical staining and western blot. A series of in vitro assays were used to determine the function of tumour-expressed VISTA. In vivo efficacy was evaluated in syngeneic models.Results: VISTA was highly expressed in human ovarian and endometrial cancers. Upregulation of VISTA in endometrial cancer was related to the methylation status of the VISTA promoter. VISTA in tumour cells suppressed T cell proliferation and cytokine production in vitro, and decreased the tumour-infiltrating CD8+ T cells in vivo. Anti-VISTA antibody prolonged the survival of tumour-bearing mice.Conclusions: This is the first demonstration that VISTA is highly expressed in human ovarian and endometrial cancer cells, and that anti-VISTA antibody treatment significantly prolongs the survival of mice bearing tumours expressing high levels of VISTA. The data suggest that VISTA is a novel immunosuppressive factor within the tumour microenvironment, as well as a new target for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

HPV infected cervical cells secrete mediators that are gradually changed and have influence on infiltrating M2 phenotypic monocytes in cervical lesions. However, profiles of circulating immune cells in women with cervical lesions and M2 phenotypic monocyte activity in HPV infected cervical lesions are limited. This study aimed to investigate circulating monocyte populations correlated with M2 phenotype density and its activity in HPV infected cervical lesions. HPV DNA was investigated in cervical tissues using PCR. High risk HPV E6/E7 mRNA was detected using in situ hybridization. CD163 immunohistochemical staining was performed for M2 macrophage. CD163 and Arg1 mRNA expression were detected using real-time PCR. Circulating monocyte subpopulations were analyzed using flow cytometry. CD163 and Arg1 mRNA expression were increased according to cervical lesion severity and corresponding with density of M2 macrophage in HSIL and SCC in stroma and peri-tumoral areas. Additionally, the relationship between M2 macrophage infiltration and high risk HPV E6/E7 mRNA expression was found and corresponded with cervical lesion severity. Circulating CD14CD16 and CD14CD163 monocytes were elevated in No-SIL and cervical lesions. Interestingly, CD14CD64 monocyte was greatly elevated in HSIL and SCC, whereas intracellular IL-10 monocytes were not significantly different between cervical lesions. The correlation between increasing ratio of circulating CD64/CD163 monocyte and density of infiltrating CD163 monocytes was associated with severity of HPV infected cervical lesions. The elevated circulating CD64/CD163 monocyte ratio correlates to severity of HPV infected cervical lesions and might be a prognostic marker in cervical cancer progression. [ABSTRACT FROM AUTHOR]

Programmed death 1 (PD-1) is an inhibitory molecule expressed on activated T cells; however, the biological context in which PD-1 controls T cell tolerance remains unclear. Using two-photon laser-scanning microscopy, we show here that unlike naive or activated islet antigen–specific T cells, tolerized islet antigen–specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DCs) in pancreatic lymph nodes. Inhibition of T cell antigen receptor (TCR)-driven stop signals depended on continued interactions between PD-1 and its ligand, PD-L1, as antibody blockade of PD-1 or PD-L1 resulted in lower T cell motility, enhanced T cell–DC contacts and caused autoimmune diabetes. Blockade of the immunomodulatory receptor CTLA-4 did not alter T cell motility or abrogate tolerance. Thus, PD-1–PD-L1 interactions maintain peripheral tolerance by mechanisms fundamentally distinct from those of CTLA-4. [ABSTRACT FROM AUTHOR]

The programmed death-1 (PD-1)/B7-H1 (also called PD-L1) pathway negatively regulates T cell activation and has been suggested to play an important role in regulating antitumor host immunity. To investigate the clinical significance of B7-H1 expression to the tumor grade and postoperative prognosis of patients with urothelial cancer, we analyzed the relationship between B7-H1 expression and various clinicopathological features and postoperative prognosis. Sixty-five urothelial cancer cases were examined. B7-H1 expression in tumors and the numbers and phenotypes of tumor-infiltrating lymphocytes were evaluated by immunohistochemistry and flow cytometry. A substantial expression of B7-H1 was observed in all urothelial cancers investigated. Tumor specimens from patients with higher WHO grade or primary tumor classifications showed significantly higher percentages of tumor-associated B7-H1. Tumor-associated B7-H1 expression was significantly associated with a high frequency of postoperative recurrence and poor survival rate. Furthermore, multivariate analysis indicated that tumor-associated B7-H1 was more significant prognostic factor than WHO grade. Our results demonstrate that the aberrant expression of B7-H1 in urothelial cancer is associated with aggressive tumors, suggesting a regulatory role of tumor-associated B7-H1 in antitumor immunity. Therefore, the manipulation of tumor-associated B7-H1 may become a beneficial target for immunotherapy in human urothelial cancer. [ABSTRACT FROM AUTHOR]

Studies the monoclonal antibody IT2 that reacts with a 70K glycoprotein (B70). Cloning of B70 complementary DNA and expression; Flow cytometric analysis; Inhibition by IT2 of the binding of a CTLA4-immunoglobulin fusion protein.

B7 molecules provide an important costimulatory signal for T cell receptor/CD3-mediated T cell activation via binding to their cognate receptors, CD28 and CTLA-4. We have introduced B7-1 (CD80) into M1 cells, spontaneously-occurred mouse myelocytic leukemic cells and assessed its potential in the induction immunity to leukemia cells. Syngeneic, immunocompetent SL mice receiving pelyclonal B7-1-transduced M1 cells showed prolonged survival than control mice. Two independent B7-1-transduced monoclonal sublines, M1-B7-1+ (F20) and M1-B7-1+ (F7), were rejected in 100 % an 50% of SL mice, respectively. In vivo depletion of T cell subsets showed that both CD4+ and CD8+ T cells were indispensable for the B7-1-dependent anti-leukemic immunity. Although a single exposure to irradiated monoclonal M1-B7-1+ cells were not fully effective, multiple exposures induced protective immunity against subsequent challenge with M1 cells. Furthermore, hyperimmunization with irradiated monoclonal M1-B7-1+ (F7) cells could partly cure mice previously injected with a lethal number of M1 cells. Although other groups have demonstrated that live, proliferating B7-1-transduced leukemic cells can improve antitumor immunity, this is the first report which shows that irradiated B7-1-transduced myeloid leukemic cells can induce protective and therapeutic immunity against leukemia. [ABSTRACT FROM AUTHOR]

Bacillus Calmette-Guérin (BCG) is the only licensed vaccine for tuberculosis (TB), and is also used as an immunotherapy for bladder cancer and other malignancies due to its immunostimulatory properties. Mycobacteria spp., however, are well known for their numerous immune evasion mechanisms that limit the true potential of their therapeutic use. One such major mechanism is the induction of programmed death ligand-1 (PD-L1), which mitigates adaptive immune responses. Here, we sought to unravel the molecular pathways behind PD-L1 up-regulation on antigen-presenting cells (APCs) by BCG. We found that infection of APCs with BCG induced PD-L1 up-regulation, but that this did not depend on direct infection, suggesting a soluble mediator for this effect. BCG induced potent quantities of IL-6 and IL-10, and the downstream transcription factor STAT3 was hyper-phosphorylated. Intracellular analyses revealed that levels of PD-L1 molecules were associated with the STAT3 phosphorylation state, suggesting a causal link. Neutralisation of the IL-6 or IL-10 cytokine receptors dampened STAT3 phosphorylation and BCG-mediated up-regulation of PD-L1 on APCs. Pharmacological inhibition of STAT3 achieved the same effect, confirming an autocrine-paracrine cytokine loop as a mechanism for BCG-mediated up-regulation of PD-L1. Finally, an in vivo immunisation model showed that BCG vaccination under PD-L1 blockade could enhance antigen-specific memory CD4 T-cell responses. These novel findings could lead to refinement of BCG as both a vaccine for infectious disease and as a cancer immunotherapy. [ABSTRACT FROM AUTHOR]