Tumor-Infiltrating γδ T Cells Suppress T and Dendritic Cell Function via Mechanisms Controlled by a Unique Toll-like Receptor Signaling Pathway

Summaryγδ T cells are important contributors to innate immunity against cancer, but their regulatory role in controlling immune responses remains largely unknown. Here we report that a dominant γδ1 T cell population among lymphocytes infiltrating breast tumors possessed a potent ability to suppress naive and effector T cell responses and to block the maturation and function of dendritic cells. Adoptive cotransfer experiments demonstrated their in vivo suppressive activity. However, their immunosuppressive activity could be reversed by human Toll-like receptor (TLR) 8 ligands both in vitro and in vivo. siRNA-mediated knockdown experiments revealed that MyD88, TRAF6, IKKα IKKβ, and p38α molecules in γδ1 cells were required for these cells to respond to TLR8 ligands, whereas TAK1, JNK, and ERK molecules did not appear to be involved in functional regulation. These results provide new insights into the regulatory mechanisms of tumor-specific γδ T cells and identify a unique TLR8 signaling pathway linking to their functional regulation

Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第945号）・境澤 香里BACKGROUND: The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients. METHODS: High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45(+) cells, CTC were identified by staining with MART-1-and gp100-specific antibodies (HMW-MAA(+), CD45(-), MART-1/gp100(+)). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis. RESULTS: CTC (HMW-MAA(+), CD45(-), MART-1/gp100(+)) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient. CONCLUSION: Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy. British Journal of Cancer (2012) 106, 939-946. doi:10.1038/bjc.2012.12 www.bjcancer.com Published online 26 January 2012 (C) 2012 Cancer Research UKArticleBRITISH JOURNAL OF CANCER. 106(5):939-946 (2012)journal articl

Tumor-Specific Human CD4+ Regulatory T Cells and Their Ligands Implications for Immunotherapy

AbstractRegulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4+ regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4+ Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4+ Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4+ T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4+ effector cells. These findings suggest that the presence of tumor-specific CD4+ Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer

Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells

Angiogenesis is a complex process involving an ECM and vascular endothelial cells (EC), and is regulated by various angiogenic factors including VEGF. The ability to form a capillary/tube-like network is a specialized function of EC. Therefore, in vitro angiogenesis was assessed by a capillary/tube-like network formation assay. There are three angiogenic parameters: capillary length, number of capillaries, and relative capillary area per field. We evaluated capillary length per field in the assay. VEGF promoted capillary/tube-like network formation of EC in a type I collagen gel matrix in vitro. Moreover, we demonstrated the involvement of ILK in a VEGF signaling pathway mediating capillary/tube-like network formation of EC using dominant-negative, kinase deficient ILK. This is a straightforward assay to monitor responses of human vascular endothelial cells

Tumor-Infiltrating γδ T Cells Suppress T and Dendritic Cell Function via Mechanisms Controlled by a Unique Toll-like Receptor Signaling Pathway

Summaryγδ T cells are important contributors to innate immunity against cancer, but their regulatory role in controlling immune responses remains largely unknown. Here we report that a dominant γδ1 T cell population among lymphocytes infiltrating breast tumors possessed a potent ability to suppress naive and effector T cell responses and to block the maturation and function of dendritic cells. Adoptive cotransfer experiments demonstrated their in vivo suppressive activity. However, their immunosuppressive activity could be reversed by human Toll-like receptor (TLR) 8 ligands both in vitro and in vivo. siRNA-mediated knockdown experiments revealed that MyD88, TRAF6, IKKα IKKβ, and p38α molecules in γδ1 cells were required for these cells to respond to TLR8 ligands, whereas TAK1, JNK, and ERK molecules did not appear to be involved in functional regulation. These results provide new insights into the regulatory mechanisms of tumor-specific γδ T cells and identify a unique TLR8 signaling pathway linking to their functional regulation

Immunotherapy with MVA-BN®-HER2 induces HER-2-specific Th1 immunity and alters the intratumoral balance of effector and regulatory T cells

MVA-BN®-HER2 is a new candidate immunotherapy designed for the treatment of HER-2-positive breast cancer. Here, we demonstrate that a single treatment with MVA-BN®-HER2 exerts potent anti-tumor efficacy in a murine model of experimental pulmonary metastasis. This anti-tumor efficacy occurred despite a strong tumor-mediated immunosuppressive environment characterized by a high frequency of regulatory T cells (Treg) in the lungs of tumor-bearing mice. Immunogenicity studies showed that treatment with MVA-BN®-HER2 induced strongly Th1-dominated HER-2-specific antibody and T-cell responses. MVA-BN®-HER2-induced anti-tumor activity was characterized by an increased infiltration of lungs with highly activated, HER-2-specific, CD8+CD11c+ T cells accompanied by a decrease in the frequency of Treg cells in the lung, resulting in a significantly increased ratio of effector T cells to Treg cells. In contrast, administration of HER2 protein formulated in Complete Freund’s Adjuvant (CFA) induced a strongly Th2-biased immune response to HER-2. However, this did not lead to significant infiltration of the tumor-bearing lungs by CD8+ T cells or the decrease in the frequency of Treg cells nor did it result in anti-tumor efficacy. In vivo depletion of CD8+ cells confirmed that CD8 T cells were required for the anti-tumor activity of MVA-BN®-HER2. Furthermore, depletion of CD4+ or CD25+ cells demonstrated that tumor-induced Treg cells promoted tumor growth and that CD4 effector cells also contribute to MVA-BN®-HER2-mediated anti-tumor efficacy. Taken together, our data demonstrate that treatment with MVA-BN®-HER2 controls tumor growth through mechanisms including the induction of Th1-biased HER-2-specific immune responses and the control of tumor-mediated immunosuppression

Osteoclast Activated FoxP3+ CD8+ T-Cells Suppress Bone Resorption in vitro

BACKGROUND: Osteoclasts are the body's sole bone resorbing cells. Cytokines produced by pro-inflammatory effector T-cells (T(EFF)) increase bone resorption by osteoclasts. Prolonged exposure to the T(EFF) produced cytokines leads to bone erosion diseases such as osteoporosis and rheumatoid arthritis. The crosstalk between T-cells and osteoclasts has been termed osteoimmunology. We have previously shown that under non-inflammatory conditions, murine osteoclasts can recruit naïve CD8 T-cells and activate these T-cells to induce CD25 and FoxP3 (Tc(REG)). The activation of CD8 T-cells by osteoclasts also induced the cytokines IL-2, IL-6, IL-10 and IFN-γ. Individually, these cytokines can activate or suppress osteoclast resorption. PRINCIPAL FINDINGS: To determine the net effect of Tc(REG) on osteoclast activity we used a number of in vitro assays. We found that Tc(REG) can potently and directly suppress bone resorption by osteoclasts. Tc(REG) could suppress osteoclast differentiation and resorption by mature osteoclasts, but did not affect their survival. Additionally, we showed that Tc(REG) suppress cytoskeletal reorganization in mature osteoclasts. Whereas induction of Tc(REG) by osteoclasts is antigen-dependent, suppression of osteoclasts by Tc(REG) does not require antigen or re-stimulation. We demonstrated that antibody blockade of IL-6, IL-10 or IFN-γ relieved suppression. The suppression did not require direct contact between the Tc(REG) and osteoclasts. SIGNIFICANCE: We have determined that osteoclast-induced Tc(REG) can suppress osteoclast activity, forming a negative feedback system. As the CD8 T-cells are activated in the absence of inflammatory signals, these observations suggest that this regulatory loop may play a role in regulating skeletal homeostasis. Our results provide the first documentation of suppression of osteoclast activity by CD8 regulatory T-cells and thus, extend the purview of osteoimmunology

Expression of Mucin 1 in Mycosis Fungoides Tumour Cells: A Case Report

ArticleACTA DERMATO-VENEREOLOGICA.97(6):747-748(2017)journal articl