T Cell–specific Ablation of Fas Leads to Fas Ligand–mediated Lymphocyte Depletion and Inflammatory Pulmonary Fibrosis

To study the role of Fas–Fas ligand (FasL) interaction-mediated apoptosis in lymphocyte homeostasis, we generated a mutant fas allele allowing conditional inactivation of the fas gene through Cre-mediated recombination. Experiments in which Fas was ablated in T cells, B cells, T and B cells, or in a more generalized manner demonstrated that the development of lymphoproliferative disease as seen in Fas-deficient mice requires Fas ablation in lymphoid and nonlymphoid tissues. Selective inactivation of Fas in T cells led to a severe lymphopenia over time, accompanied by up-regulation of FasL on activated T cells and apoptosis of peripheral lymphocytes. In addition, the mutant animals developed a fatal wasting syndrome caused by massive leukocyte infiltration in the lungs together with increased inflammatory cytokine production and pulmonary fibrosis. Inhibition of Fas–FasL interaction in vivo completely prevented the loss of lymphocytes and initial lymphocyte infiltration in the lungs. Thus, FasL-mediated interaction of activated, Fas-deficient T cells with Fas-expressing cells in their environment leads to break down of lymphocyte homeostasis and development of a lung disease strikingly resembling idiopathic pulmonary fibrosis in humans, a common and severe disease for which the mutant mice may serve as a first animal model

Elimination of chronic viral infection by blocking CD27 signaling

Neutralizing antibody (nAb) responses to lymphocytic choriomeningitis virus (LCMV) in mice and immunodeficiency virus and hepatitis C virus in humans are usually weak and slow to develop. This may be the result of structural properties of the surface glycoprotein, a low frequency of B cells with neutralizing specificity, and the necessity of prolonged affinity maturation of specific nAbs. In this study, we show that during LCMV infection, CD27 signaling on CD4+ T cells enhances the secretion of interferon-γ and tumor necrosis factor-α. These inflammatory cytokines lead to the destruction of splenic architecture and immunodeficiency with reduced and delayed virus-specific nAb responses. Consequently, infection with the otherwise persistent LCMV strain Docile was eliminated after CD27 signaling was blocked. Our data provide a novel mechanism by which LCMV avoids nAb responses and suggest that blocking the CD27–CD70 interaction may be an attractive strategy to prevent chronic viral infection

TLR2-Dependent Induction of IL-10 and Foxp3+CD25+CD4+ Regulatory T Cells Prevents Effective Anti-Tumor Immunity Induced by Pam2 Lipopeptides In Vivo

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3+CD4+ regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine

Effects of acute exercise on expressions of functional receptors on CD56dim and CD56bright natural killer cells

PURPOSE: Mobilization and cytotoxicity of natural killer (NK) cells are regulated by cell surface receptors such as adhesion molecules and activating/inhibitory receptors. In this study, we examined the effects of acute exercise on the expression of these cell surface molecules and receptors. METHODS: Six healthy male college students (22.8 ± 0.8 years olds) exercised on the cycle ergometer for 30 min at intensities corresponding to the individual onset of blood lactate accumulation level (70-85%VO2max). Venous blood samples were collected at rest (PRE); just before the end of exercise (END) and 30 (POST 30), 60 (POST 60), 120 (POST 120) and 180 (POST 180) min post exercise. The densities of cell surface molecules and receptors on CD56dim and CD56bright NK cells were determined by flow cytometry. One-way ANOVA and MANOVA were used for statistical analyses. RESULTS: At PRE, expressions of CD16, CD56, CD44, CD62L, CD314, CD335, CD159a and CX3CR1 differed between CD56dim and CD56bright NK cells. Expressions of adhesion molecules CD62L and CX3CR1 changed significantly in both subsets during and after exercise. The expressions of CD62L tended to decrease at END, and then they increased significantly at POST 30. These changes were mainly due to the proportional changes in CD62Lnegative cells. The opposite patterns of changes were seen in the expressions of CX3CR1. Additionally, the expressions of CX3CR1 decreased at POST 120 and 180 only in CD56dim NK cells. The changes in the expressions of CD44 were similar to those seen in the expressions of CD62L. Although changes in the expression of adhesion molecules were statistically significant, they were relatively unclear in histogram analyses. With regard to the expressions of NK cell activating/inhibitory receptors, most changes were observed in CD56dim NK cells. The expressions of CD16 decreased at END and returned at POST 30. The expressions of CD212 dropped from END to POST 30. In contrast, the expressions of CD335 increased from END to POST 30. Exceptionally, changes in the expressions of CD226 were found in both subsets. The expressions decreased at POST180. CONCLUSION: These results suggest that acute exercise influences the expressions of cell surface molecules and receptors. Changes were mainly observed at END and POST 30 in CD56dim NK cell. However, the delayed changes were also seen in some receptors. The changes in several receptors were related to cell mobilization. In contrast, the changes in other receptors were not directly related to mobilization and cytotoxicity

Critical Role for Tumor Necrosis Factor–related Apoptosis-inducing Ligand in Immune Surveillance Against Tumor Development

Natural killer (NK) cells and interferon (IFN)-γ have been implicated in immune surveillance against tumor development. Here we show that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) plays a critical role in the NK cell–mediated and IFN-γ–dependent tumor surveillance. Administration of neutralizing monoclonal antibody against TRAIL promoted tumor development in mice subcutaneously inoculated with a chemical carcinogen methylcholanthrene (MCA). This protective effect of TRAIL was at least partly mediated by NK cells and totally dependent on IFN-γ. In the absence of TRAIL, NK cells, or IFN-γ, TRAIL-sensitive sarcomas preferentially emerged in MCA-inoculated mice. Moreover, development of spontaneous tumors in p53+/− mice was also promoted by neutralization of TRAIL. These results indicated a substantial role of TRAIL as an effector molecule that eliminates developing tumors

PD-1とVEGFR2の同時阻害による相乗的抗腫瘍効果

Recent basic and clinical studies have shown that the programmed death ligand (PD-L)/PD-1 pathway has a significant role in tumour immunity, and its blockade has a therapeutic potential against several human cancers. We hypothesized that anti-angiogeneic treatment might augment the efficacy of PD-1 blockade. To this end, we evaluated combining the blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2) in a murine cancer model using Colon-26 adenocarcinoma. Interestingly, simultaneous treatment with anti-PD-1 and anti-VEGFR2 monoclonal antibodies (mAbs) inhibited tumour growth synergistically in vivo without overt toxicity. Blocking VEGFR2 inhibited tumour neovascularization significantly, as demonstrated by the reduced number of microvessels, while PD-1 blockade had no impact on tumour angiogenesis. PD-1 blockade might promote T cell infiltration into tumours and significantly enhanced local immune activation, as shown by the up-regulation of several proinflammatory cytokine expressions. Importantly, VEGFR2 blockade did not interfere with T cell infiltration and immunological activation induced by PD-1 blockade. In conclusion, simultaneous blockade of PD-1 and VEGFR2 induced a synergistic in-vivo anti-tumour effect, possibly through different mechanisms that might not be mutually exclusive. This unique therapeutic strategy may hold significant promise for future clinical application.博士（医学）・甲第600号・平成25年5月29日発行元の猶予期間終了の後、本文を登録予定（2014.06

A Critical Role for the Programmed Death Ligand 1 in Fetomaternal Tolerance

Fetal survival during gestation implies that tolerance mechanisms suppress the maternal immune response to paternally inherited alloantigens. Here we show that the inhibitory T cell costimulatory molecule, programmed death ligand 1 (PDL1), has an important role in conferring fetomaternal tolerance in an allogeneic pregnancy model. Blockade of PDL1 signaling during murine pregnancy resulted in increased rejection rates of allogeneic concepti but not syngeneic concepti. Fetal rejection was T cel

Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer

The CD1d reactive glycolipid, α-galactosylceramide (α-GalCer), potently activates T cell receptor-α type I invariant NKT cells that secondarily stimulate the proliferation and activation of other leukocytes, including NK cells. Here we report a rational approach to improving the antitumor activity of α-GalCer by using delayed interleukin (IL)-21 treatment to mature the α-GalCer–expanded pool of NK cells into highly cytotoxic effector cells. In a series of experimental and spontaneous metastases models in mice, we demonstrate far superior antitumor activity of the α-GalCer/IL-21 combination above either agent alone. Superior antitumor activity was critically dependent upon the increased perforin-mediated cytolytic activity of NK cells. Transfer of α-GalCer–pulsed dendritic cells (DCs) followed by systemic IL-21 caused an even more significant reduction in established (day 8) metastatic burden and prolonged survival. In addition, this combination prevented chemical carcinogenesis more effectively. Combinations of IL-21 with other NK cell–activating cytokines, such as IL-2 and IL-12, were much less effective in the same experimental metastases models, and these cytokines did not substitute effectively for IL-21 in combination with α-GalCer. Overall, the data suggest that NK cell antitumor function can be enhanced greatly by strategies that are designed to expand and differentiate NK cells via DC activation of NKT cells