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WNT/β-catenin-activated hepatocellular carcinoma (W/B subclass HCC) is considered as molecularly homogeneous entity and has been linked to resistance to immunotherapy. However, recent studies have indicated possible heterogeneity in the immuno-vascular microenvironment in this subclass. We set out to test the hypothesis that specific immuno-vascular features might stratify W/B subclass HCCs into tumors having distinct aggressive natures.In this study, we analyzed 352 resected HCCs including 78 immunohistochemically defined W/B subclass HCCs. The density of tumor-infiltrating CD3The T-cell density of W/B subclass HCCs was regionally heterogenous within tumor tissues, and focally reduced T-cell density was observed in areas with VETC. VETC-positivity (defined as VETC area ratio greater than 1%) and T-cell infiltration were inversely associated in both W/B subclass and non-W/B subclass HCCs. Fibroblast growth factor 2 (FGF2) gene expression was higher in W/B subclass than in non-W/B subclass HCCs. VETC positivity and low T-cell density correlated with increased expression of FGF2 in W/B subclass HCCs. VETC-positive HCCs showed significantly shorter DFS in W/B subclass HCCs.In conclusion, the immune and vascular microenvironments are interrelated and are also correlated with clinicopathological heterogeneity in W/B subclass HCC. These results may inform clinical practice and translational research on the development of therapeutic stratification of HCCs. This article is protected by copyright. All rights reserved.

Figure legends for suppremental Figure 1 to 5.

Multivariate analysis of clinicopathological factors and infiltrating immune cells

Neoplastic cancer cells and cancer stroma (including infiltrating immune cells) determine the biology and prognosis of cancer. Various types of adaptive and innate immune cells are known to infiltrate the cancer stroma. However, the patterns and spatial distribution of immune cell infiltration as well as its association with tumor histology remain poorly understood. To address these issues, we comprehensively analyzed the infiltrating immune cells present in lung adenocarcinoma. The principal types of both adaptive and innate infiltrating immune cells were immunohistochemically evaluated in the predominant histologic components of 111 lung adenocarcinomas. The same analysis was also carried out on 143 samples of histologic subtypes making up more than 20% of tumors. As a result, plasma cells and B cells with interfollicular distribution were almost exclusively observed in invasive histologic subtypes, while an increased number of mast cells were observed in noninvasive histologic subtypes. Cluster analysis revealed four distinct immunosubtypes (CD8, mast cell, macrophage/dendritic cell, and plasma cell subtypes) based on the infiltrating immune cell profiles. These immunosubtypes correlated with histologic subtypes, and univariate and multivariate analyses identified the plasma cell subtype as an independent negative prognostic factor. These plasma cells may be one of the major producers of the immunosuppressive cytokine IL35 in cancer stroma. Cancer Immunol Res; 4(3); 234–47. ©2016 AACR.

Figure S1. Infiltration of immune cells and grades of histological subtypes. Figure S2. Infiltration of interfollicular plasma cells and Kaplan-Meier curves for DFS. Figure S3. Infiltration of interfollicular mast cells and Kaplan-Meier curves for DFS. Figure S4. Plasma cell/CD8 ratio and Kaplan-Meier curves for DFS. Figure S5. Immunohistochemical staining for immunoglobulins and PD-L1.

Supplementary Materials and Methods, Table and Figures/Figure legends

Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab′)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies.Significance:Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature.

Immune cells and tumor vessels constitute important elements in tumor tissue; however, their detailed relationship in human tumors, including hepatocellular carcinoma (HCC), is still largely unknown. Consequently, we expanded our previous study on the immune microenvironment of HCC and analyzed the relationship among the immune microenvironment, inflammatory/angiostatic factor expression, angiogenic factor expression, and tumor vessel findings, including vessels encapsulating tumor clusters (VETC) and macrotrabecular-massive (MTM) patterns. We classified HCC into four distinct immuno-vascular subtypes (Immune-high/Angiostatic [IH/AS], Immune-mid/Angio-mid [IM/AM], Immune-low/Angiogenic [IL/AG], and Immune-low/Angio-low [IL/AL]). IH/AS, IM/AM, and IL/AG subtypes were associated with decreasing lymphocytic infiltration and increasing angiogenic factor expression and VETC/MTM positivity, reflecting their reciprocal interaction in the tumor microenvironment of HCC. IL/AG subtype was further characterized by CTNNB1 mutation and activation of Wnt/β-catenin pathway. IL/AL subtype was not associated with increased lymphocyte infiltration or angiogenic factor expression. Prognostically, IH/AS subtype and VETC/MTM positivity were independently significant in two independent cohorts. Increased angiogenic factor expression was not necessarily associated with VETC/MTM positivity and poor prognosis, especially when inflammatory/angiostatic milieu co-existed around tumor vessels. These results may provide insights on the therapeutic effects of immunotherapy, anti-angiogenic therapies, and their combinations. The potential of evaluating immuno-vascular microenvironment in predicting the clinical effect of these therapies in non-resectable HCC needs to be analyzed in the future study. CONCLUSION: HCC can be classified into four distinct immuno-vascular subtypes (IH/AS, IM/AM, IL/AG, and IL/AL) that reflect the reciprocal interaction between the anti-tumor immune microenvironment and tumor angiogenesis. In addition to its clinicopathological significance, immuno-vascular classification may also provide pathological insights on the therapeutic effect of immunotherapy, anti-angiogenic therapy, and their combination.

Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab′)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies. Significance: Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature.

Background The thymus plays an essential role in removing autoreactive T cells. Autoantigen-expressing thymic epithelial cells (TECs) contribute to the tolerogenic process. The thymus transiently shrinks as an acute thymic involution (ATI) under various inflammatory conditions. However, whether ATI occurs during local skin inflammation remains unclear, as does its influence on thymic immune tolerance. Objective We investigated whether imiquimod-induced dermatitis causes ATI and impairs thymic immune tolerance against desmoglein 3 (Dsg3), an epidermal autoantigen of pemphigus vulgaris. Methods 5% imiquimod cream was applied daily, at 62.5 mg/day (high dose group) or 31.25 mg/day (low dose group), for 1 week on the back of wild-type mice, and to wild-type mice that had undergone bone-marrow transplantation from Dsg3-specific T-cell receptor (TCR) transgenic-Rag2−/− mice. Next, thymocytes, TECs and other immune cells were analyzed by flow cytometry. TEC-associated Dsg3 expression was also analyzed by immunofluorescence staining. Results Thymus weight and thymocyte number in all developmental stages decreased in a dose-dependent manner under imiquimod-induced dermatitis. The number of total TECs, specifically medullary, but not cortical, TECs, decreased in high and low dose groups. Accordingly, the number of Dsg3-experssing UEA-1+keratin 5+mTEC decreased in the thymus during imiquimod-induced dermatitis. Although Dsg3-sepcific transgenic thymocytes was usually deleted in the thymus under physiological condition by central tolerance, Dsg3-sepcific transgenic CD4+CD8− thymocytes significantly increased in number under imiquimod-induced dermatitis. Conclusion These findings indicate a crosstalk between skin and thymus in adult mice and suggest that skin inflammation may impair thymic tolerance to autoantigens, such as Dsg3.

Tumor tissue is composed of tumor cells and tumor stroma. Tumor stroma contains various immune cells and non-immune stromal cells, forming a complex tumor microenvironment which plays pivotal roles in regulating tumor growth. Recent successes in immunotherapies against tumors, including immune checkpoint inhibitors, have further raised interests in the immune microenvironment of liver carcinoma. The immune microenvironment of tumors is formed because of interactions among tumor cells, immune cells and non-immune stromal cells, including fibroblasts and endothelial cells. Different patterns of immune microenvironment are observed among different tumor subtypes, and their clinicopathological significance and intertumor/intratumor heterogeneity are being intensively studied. Here, we review the immune microenvironment of hepatocellular carcinoma, intrahepatic cholangiocarcinoma and liver metastasis of colorectal adenocarcinoma, focusing on its histopathological appearance, clinicopathological significance, and relationship with histological and molecular classifications. Understanding the comprehensive histopathological picture of a tumor immune microenvironment, in addition to molecular and genetic approaches, will further potentiate the effort for precision medicine in the era of tumor-targeting immunotherapy.

The clinicopathological characteristics of steatosis in hepatocellular carcinoma (HCC) remain unclear. Here, we elucidate the features of macrovesicular steatosis (MaS) and microvesicular steatosis (MiS) in HCC and their relationships with background liver steatosis. A total of 165 HCC lesions were classified as MaS-HCC, MiS-HCC, or conventional HCC (cHCC) according to the cutoff value of 30% MaS or MiS in tumor cells. We analyzed the clinicopathological differences among these groups. MaS-HCC had less portal vein invasion, a higher proportion of HCC with intratumoral fibrosis, and a lower cumulative risk of recurrence than MiS-HCC or cHCC. Moreover, both MaS-HCC and MiS-HCC had lower incidences of hepatitis virus infection and higher levels of HbA1c than cHCC. Background liver steatosis was also higher in MaS-HCC than in cHCC. Immunohistochemical expression of perilipin (Plin1) and adipophilin (ADRP), major proteins expressed on lipid droplet membranes, revealed that almost all lipid droplets in HCC were Plin1 negative, whereas those in background liver were positive. In contrast, ADRP was expressed on lipid droplets in both HCC and background liver. We concluded that MaS-HCC and MiS-HCC were associated with metabolic abnormalities but exhibited different biologic behaviors. Furthermore, lipid droplets in HCC were pathophysiologically different from those in background liver.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor–transgenic mice to thymus-transplanted mice, Dsg3-specific CD4(+) T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3(−/−) mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

IL-27 is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4(+) T cells by IL-27, enhanced by TGF-β, and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient-deprivation cell death without releasing high-mobility group box-1. This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Interestingly, Ch25h expressing CD4(+) T cells that received IL-27 and TGF-β signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27 treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worsened outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

Background Recently, some studies reported the pulmonary artery hypertension (PAH)–associated genes. However, a majority of patients with familial or sporadic PAH lack variants in the known pathogenic genes. In this study, we investigated the new causative gene variants associated with PAH. Methods and Results Whole‐exome sequencing in 242 Japanese patients with familial or sporadic PAH identified a heterozygous substitution change involving c.226G>A (p.Gly76Ser) in tumor necrotic factor receptor superfamily 13B gene ( TNFRSF13B ) in 6 (2.5%) patients. TNFRSF13B controls the differentiation of B cell and secretion of inflammatory cytokines and may be involved in vascular inflammation. In silico structural analysis simulation demonstrated the structural instability of the N‐terminal region of the protein synthesized from TNFRSF13B p.Gly76Ser variant. These suggest that the TNFRSF13B p.Gly76Ser variant may be involved in the development of PAH via aberrant inflammation in pulmonary vessels. Conclusions TNFRSF13B p.Gly76Ser variant is a candidate of novel causative gene variant for PAH.

Increasing reports have noted an increased prevalence of lung cancer in human immunodeficiency virus (HIV)-positive patients with poor prognosis. A 51-year-old HIV-positive man was diagnosed with stage IV squamous cell lung cancer. He had high grade spike intermittent fever and persistent elevation of the white blood cell count as well as C-reactive protein (CRP) levels. Although we suspected opportunistic infections, we did not detect any infection. The autopsy showed positive immunostaining for Interleukin-6 (IL-6) in plasma cells of the stromal regions and G-CSF in tumor cells, which were considered responsible for his significant tumor fever, leukocytosis and high titers of CRP. This case report highlights the need to consider cytokine-producing tumor as a differential diagnosis of fever and high inflammatory status in HIV-positive cancer patients. Keywords: Human immunodeficiency virus (HIV), Tumor fever, Lung cancer, IL-6, G-CSF

Background Metastasis from one tumor into another is known as tumor-to-tumor metastasis. We report a case of a contiguous occurrence of meningioma and brain cancer metastasis. Patient A 91-year-old woman presented with a sudden onset of weakness in her right limbs and gait disturbance. Fourteen years previously, she had a tumor that was suspected to be a meningioma in the left frontal convexity with no change for several years. One year earlier to presentation she was diagnosed with lung carcinoma in the left lower lobe. Results Magnetic resonance images revealed enlargement of the tumor with perifocal edema. Histologic examination showed a meningioma with contiguous metastatic poorly differentiated adenocarcinoma, as well as a clearly defined border between the two components. The clinical origin of the metastasis was presumed to be from the left lower lobe of the lung. Conclusion Although our case does not strictly fulfill the definition of tumor-to-tumor metastasis, we suggest a contiguous occurrence develops by the same mechanism and may be a subtype of this process. Although previous reports suggested the loss of epithelial cadherin (E-cadherin) expression in the recipient tumor as the cause of contiguous metastasis, E-cadherin expression was positive in our case and did not seem to be involved in the localization of the metastasis.

Immune cells constitute an important element of tumor tissue. Accumulating evidence indicates their clinicopathological significance in predicting prognosis and therapeutic efficacy. Nonetheless, the combinations of immune cells forming the immune microenvironment and their association with histological findings remain largely unknown. Moreover, it is unclear which immune cells or immune microenvironments are the most prognostically significant. Here, we comprehensively analyzed the immune microenvironment and its intratumor heterogeneity in 919 regions of 158 hepatocellular carcinomas (HCCs), and the results were compared with the corresponding histological and prognostic data. Consequently, we classified the immune microenvironment of HCC into three distinct immunosubtypes: Immune-high, Immune-mid, and Immune-low. The Immune-high subtype was characterized by increased B-/plasma-cell and T cell infiltration, and the Immune-high subtype and B-cell infiltration were identified as independent positive prognostic factors. Varying degrees of intratumor heterogeneity of the immune microenvironment were observed, some of which reflected the multistep nature of HCC carcinogenesis. However, the predominant pattern of immunosubtype and immune cell infiltration of each tumor was prognostically important. Of note, the Immune-high subtype was associated with poorly differentiated HCC, cytokeratin 19 (CK19)+ , and/or Sal-like protein 4 (SALL4)+ high-grade HCC, and Hoshida's S1/Boyault's G2 subclasses. Furthermore, patients with high-grade HCC of the predominant Immune-high subtype had significantly better prognosis. These results provide a rationale for evaluating the immune microenvironment in addition to the usual histological/molecular classification of HCC. Conclusion The immune microenvironment of HCC can be classified into three immunosubtypes (Immune-high, Immune-mid, and Immune-low) with additional prognostic impact on histological and molecular classification of HCC. (Hepatology 2018).

Epithelial-mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal-epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding "focal differentiation" in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E-cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co-cultured with Schwann cells to investigate cell morphology, motility, or EMT-related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E-cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co-cultured with Schwann cells demonstrated a sheet-like appearance, increased E-cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET-like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.

Background & Aims The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. Methods Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9−/− mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. Results Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9−/− mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9−/− mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. Conclusions These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. Lay summary Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.

Outcomes for patients with hepatocellular carcinoma (HCC) remain poor because the condition is often unresponsive to the available treatments. Consequently, the early and precise diagnosis of HCC is crucial to achieve improvements in prognosis. For patients with chronic liver disease, the assessment of liver fibrosis is also important to ascertain both the staging of fibrosis and the risk of HCC occurrence. Early HCC was first described in 1991 in Japan and was defined internationally in 2009. As the concept of early HCC spread, the multistage hepatocarcinogenesis process became accepted. Consequently, improvements in imaging technology made the early diagnosis of HCC possible. At present, the most appropriate therapeutic strategy for HCC is determined using an integrated staging system that assesses the tumor burden, the degree of liver dysfunction and the patient performance status; however, pathological and molecular features are not taken into account. The recent introduction of several new therapeutic agents will change the treatment strategy for HCC. Against this background, HCC subclassification based on tumor cellular and microenvironmental characteristics will become increasingly important. In this review, we give an overview of how pathological analysis contributes to understanding the development and progression of HCC and establishing a precision diagnosis of HCC.

Aim Emerging evidence suggests a promising role for tumor stromal factors in characterizing patients with various types of malignancies, including hepatocellular carcinoma (HCC). We quantified the amount of collagen and elastin fibers in HCC samples with the aim of clarifying the clinico-patho-radiological significance of fiber deposition in HCC. Methods We computed the amount of collagen and elastin fibers using digital image analysis of whole-slide images of Elastica van Gieson-stained tissues from 156 surgically resected HCCs. Furthermore, we assessed the correlations between the fiber content of HCC samples and clinical, pathological, and radiological features, including immunohistochemistry-based molecular subtypes and immunosubtypes. Results The intratumoral area ratio of collagen in HCC tissues (median 3.4%, range 0.1-22.2%) was more than threefold that of elastin (median 0.9%, range 0.1-9.0%); there was a strong positive correlation between the amounts of collagen and elastin. Higher levels of combined collagen and elastin were significantly associated with the confluent multinodular macroscopic tumor type, the absence of a fibrous capsule, intratumoral steatosis, scirrhous tumor stroma, dense inflammatory-cell infiltrates, and the biliary/stem cell markers-positive HCC subtype. The associations of higher collagen levels with radiological findings, including heterogeneous enhancement and persistent enhancement on dynamic computed tomography, were significant. In contrast, the associations of radiological findings with elastin fibers were not significant. Intratumoral fibrous stroma in HCC comprised septum-like and perisinusoidal fibrosis; these two forms represented distinct distribution patterns of fibers and fibroblasts. Conclusion Quantitative analysis suggested that stromal fiber-rich HCCs likely represent a distinct clinico-patho-radiological entity.

Transforming growth factor-β (TGF-β) is a pivotal cytokine in the differentiation of regulatory T cells, and Foxo transcription factors positively regulate this process. On the other hand, the function of Foxo transcription factors is negatively regulated by PI3K/Akt signaling, which is activated by TGF-β in many types of cells; yet the role of TGF-β in Akt activity and its downstream substrates in CD4+ T cells, including Foxo transcription factors, remains to be determined. Herein, we demonstrate that TGF-β selectively induces Akt phosphorylation at Ser473 but not at Thr308 in a class IA PI3K-dependent manner in CD4+ T cells, resulting in the phosphorylation and inhibition of Foxo transcription factors and negatively regulating the differentiation of induced regulatory T cells (iTregs). These observations reveal a novel negative regulatory mechanism involving Akt and Foxo transcription factors induced by TGF-β in the iTreg differentiation process.

Neoplastic cancer cells and cancer stroma (including infiltrating immune cells) determine the biology and prognosis of cancer. Various types of adaptive and innate immune cells are known to infiltrate the cancer stroma. However, the patterns and spatial distribution of immune cell infiltration as well as its association with tumor histology remain poorly understood. To address these issues, we comprehensively analyzed the infiltrating immune cells present in lung adenocarcinoma. The principal types of both adaptive and innate infiltrating immune cells were immunohistochemically evaluated in the predominant histologic components of 111 lung adenocarcinomas. The same analysis was also carried out on 143 samples of histologic subtypes making up more than 20% of tumors. As a result, plasma cells and B cells with interfollicular distribution were almost exclusively observed in invasive histologic subtypes, while an increased number of mast cells were observed in noninvasive histologic subtypes. Cluster analysis revealed four distinct immunosubtypes (CD8, mast cell, macrophage/dendritic cell, and plasma cell subtypes) based on the infiltrating immune cell profiles. These immunosubtypes correlated with histologic subtypes, and univariate and multivariate analyses identified the plasma cell subtype as an independent negative prognostic factor. These plasma cells may be one of the major producers of the immunosuppressive cytokine IL35 in cancer stroma. Cancer Immunol Res; 4(3); 234–47. ©2016 AACR.

Regulatory T cells (Tregs) play pivotal roles in inhibiting anti-tumor immunity at different phases and locations (priming phase in lymph node and effector phase in tumor tissue) through their ability to suppress the function of T, NK, and antigen-presenting cells. However, our knowledge about the roles of Tregs in tumor immunity has largely derived from analyses after systemic depletion of Tregs or systemic manipulation of Treg functions in murine tumor models. The specific roles that intra- and extra-tumor Tregs play remain unclear due to technical difficulties to deplete or manipulate Tregs in a spatially specific manner. In order to achieve spatially specific depletion of Tregs in a clinically applicable manner, we have previously developed an anti-CD25 F(ab')2 near-infrared photoimmunotherapy (NIR-PIT), in which an intravenous injection of IR700-conjugated anti-CD25 F(ab')2 followed by irradiation of near-infrared light on tumors achieves selective depletion of intratumor Tregs and suppresses tumor growth. The suppression of tumor growth by the anti-CD25 F(ab')2 NIR-PIT depends on CD8 T and NK cells and their production of IFN-γ; however, the exact target of IFN-γ and the mechanisms of tumor regression remain to be elucidated. In this study, we aimed to dissect the mechanisms of the anti-tumor effect of intratumor Treg depletion by anti-CD25 F(ab')2 NIR-PIT using a series of Ifngr1 knockout mice models including bone-marrow chimera and conditional knockout models. Unexpectedly, we found that the target of IFN-γ in anti-CD25 F(ab')2 NIR-PIT was endothelial cells of tumor vessels. In both MC38 and EO771 tumor models (murine colon and breast cancer, respectively), CD8 T cells, NK cells, and Tregs were mainly located along the tumor vessels, and depletion of intratumor Tregs by anti-CD25 F(ab')2 NIR-PIT induced IFN-γ expression from these perivascular CD8 T and NK cells for up to 6 hours. This transient but synchronized induction of IFN-γ/STAT1 signaling along tumor vessels caused rapid vessel regression, intratumor ischemia, and apoptotic death of tumor cells. Combination with IL-15 treatment further eradicated residual tumor cells to achieve complete remission of MC38 and EO771 tumors. Citation Format: Yutaka Kurebayashi, Peter L. Choyke, Hisataka Kobayashi, Noriko Sato. Intratumor regulatory T cells prevent IFN-gamma-dependent tumor vessel regression and can be selectively targeted by anti-CD25 near-infrared photoimmunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3444.

Epithelial-mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal-epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding "focal differentiation" in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E-cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co-cultured with Schwann cells to investigate cell morphology, motility, or EMT-related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E-cadherin membrane expression (P 0.001) and decreased nuclear accumulation of SMAD3 (P 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co-cultured with Schwann cells demonstrated a sheet-like appearance, increased E-cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET-like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-β and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.

We have been continuously observing the daily Be-7 concentrations at Bangkok in Thailand ($13.765^\circ$ N, $100.526^\circ$ E) since June 2014 to investigate the relationship between solar activities and the variation of cosmogenic nuclides in low latitude. The Be-7 concentrations show a clear seasonal variation with the high and low concentrations during January to May and June to December, respectively, for each year. In order to understand the seasonal variations, we calculated back trajectories of air mass by HYSPLIT. The calculation indicates that the back trajectories are distinguished three patterns in a year, which are "Indian Ocean trajectories", "Continental trajectories", and "Pacific Ocean trajectories". Moreover, we estimated monthly amount of Be-7 coming to Bangkok for each trajectories by EXPACS. From these findings, the amount of Be-7 is high during Continental and Pacific Ocean trajectories. On the other hand, one is low during Indian Ocean trajectories. This phase of calculated Be-7 can explain the seasonal variation. Comparing this phase and the phase of observed Be-7 concentrations, there is a time lag of one or two months.

Interleukin (IL)-17-producing helper T (Th17) cells serve as a Th subset involved in epithelial cell- and neutrophil-mediated immune responses against extracellular microbes and in the development of various autoimmune diseases. The differentiation of Th17 cells is controlled by a number of intracellular signaling cascades and a complex network of transcription factors. Recently, it has been shown that PI3K, Akt, and mammalian target of rapamycin (mTOR) complexes, such as mTORC1 and mTORC2, also positively regulate Th17 differentiation both in vivo and in vitro via multiple mechanisms; here, we review the current knowledge regarding the mechanisms through which these molecules enhance Th17 differentiation.

SummaryThe PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4+ T cells upon stimulation through TCR and CD28. Here, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.

Naive CD4(+) T cells are activated by antigen-presenting cells (APCs) and differentiate into distinct types of helper T (T(h)) cells in the lymph node or spleen. Oxygen (O(2)) tension is generally low in these secondary lymphoid tissues compared with the bloodstream or atmosphere. However, the effect of changes in O(2) concentration on the differentiation of T(h) cells remains unclear. Here, we established a novel model of T(h)-cell differentiation, which mimics physiological O(2) conditions. We primed naive CD4(+) T cells under 5% O(2), which has been observed in the lymph node or spleen and reoxygenated under normoxia that mimicked the O(2) concentration in blood. In this model, the differentiation of T(h)17 cells, but not T(h)1 or iTreg cells, was enhanced. Under the condition of 5% O(2), mammalian target of rapamycin complex 1 (mTORC1) was activated and led to the stabilization of hypoxia-inducible factor 1α (HIF-1α) in T(h)17 cells. The activation of mTORC1 and the acceleration of T(h)17-cell differentiation, which occurred when cells were primed under 5% O(2), were not observed in the absence of HIF-1α but were accelerated in the absence of von Hippel-Lindau tumor suppressor protein (vHL), a factor critical for HIF-1α degradation. Thus, a positive feedback loop between HIF-1α and mTORC1 induced by hypoxia followed by reoxygenation accelerates T(h)17-cell differentiation.

Spring weight-saving will be achieved by increased design stress. However, in case of the conventional spring steel, high strength steel reduces corrosion fatigue life and toughness, and it gets down the reliability of springs in actual environment. In this paper, we propose newly developed spring material with emphasis on improvement of shot peening to satisfy higher strength and corrosion fatigue life simultaneously.The chemical elements of developed material were designed under the following philosophies, where Si stays at 1.8% for sag-resistance, C is reduced to 0.4% for the corrosion fatigue life, Ni is added by 0.5% for the controled forming of corrosive pit, and a trace of B and Nb is added for the strength of austenitic grain boundary which be way of corrosion fatigue's crack propagation and fine grain size respectively.Experimental results by straight test piece and coil spring under high stress shows that the developed spring material is effective for improving corrosive fatigue life and delayed fracture strength. Developed spring material can achieve 20% weight-saving under 1200MPa design stress comparing with the conventional spring steel.

As for automotive suspension coil spring, its weight saving has been achieved by increasing the design stress, since the increasing of design stress requires higher fatigue strength and sag resistance. However, in the case of the conventional spring steel, the high tensile strength over 1900MPa can dramatically reduce the corrosion fatigue strength and delayed fracture strength. In this paper, a newly developed spring steel material, satisfying higher strength and corrosion fatigue life of spring, is proposed, and its application to automotive suspension coil spring under the appropriate spring manufacturing process is introduced. Carbon content of developed steel is less than conventional steels to increase of corrosion fatigue life and fracture toughness. Nickel is added to restrain corrosive pit forming. Boron and niobium are added to improve delayed fracture strength. Experimental results by both the test piece and spring under high strength design, shows that the developed spring material is effective for improving the corrosive fatigue life and delayed fracture strength, and can achieve the 20% weight saving under 1200MPa design stress, comparing with conventional spring steel.

The effect of induction-hardened depth on torsional fatigue of hole notched specimens was investigated. The results are as follows. (1) Induction-hardened specimens show the best torsional fatigue strength when the effective case depth / radius ratio is about 0.5. (2) In the case of shallow case depth, the cracks initiate at the hardened / non-hardened boundary. On the other hand, the fracture of deeply hardened specimen originates at the surface layer. (3) The cracks initiate at the hole surface in the normal direction of principal stress, because the principal shear stress is smaller than principal stress at hole surface.

The purpose of our research was to develop a new steel which keeps good cold formability without spheroidization annealing. Based on fundamental research about the influence of hot working condition and chemical composition on rolled bar hardness, Jominy hardenability, case hardenability, cold formability and mechanical properties, a new case hardening steel has been developed which can be forged without spheloidization annealing. The developed steel contains boron, and the silicon and manganese contents are less than conventional case hardening steel. The developed steel shows fatigue strength equivalent to the conventional case hardening steel and better toughness and machinability.

The effect of induction-hardened depth on torsional fatigue strength of notched specimens were investigated. The results are as follows. (1) Induction-hardened specimens shows the best torsional fatigue strength when the effective case depth / radius ratio is about 0.5. (2) In the case ofshallow case depth, the cracks initiate at the hardened / unhardened bound-ary. On the other hand, the fracture ofdeeply hardened specimen originates from the surface