Yoshinori Ikarashi

The Role of PGE2 in the Differentiation of Dendritic Cells: How Do Dendritic Cells Influence T‐Cell Polarization and Chemokine Receptor Expression?

The role of prostaglandin E2 (PGE2) in the function of dendritic cells (DCs), T‐cell polarization, and expression of chemokine receptors was evaluated in human cells. Immature DCs were generated from peripheral blood CD14+ cells using a combination of GM‐CSF and interleukin‐4 (IL‐4) with or without PGE2. On day 6, maturation of DCs was induced by the addition of tumor necrosis factor alpha with or without PGE2. DCs harvested on day 6 (immature DCs) or day 9 (mature DCs) were examined using functional assays. In the presence of PGE2, immature and mature DCs showed, phenotypically, a lower expression of CD1a and, functionally, a higher allostimulatory capacity at a high DC/T‐cell ratio than control cells cultured in the absence of PGE2. DCs cultured in the presence of PGE2 induced the differentiation of naïve T cells toward a helper T‐cell type 1 (Th1) response, which was independent of IL‐12 secretion in the basal state despite a slightly lower interferon gamma secretion compared with control cells. However, the function of cytotoxicity‐stimulating autologous T cells was not augmented by the addition of PGE2. Immature DCs expressed the inflammatory chemokine receptors, CCR1 and CXCR4, but not CCR6, regardless of the presence or absence of PGE2. Mature DCs expressed CCR7 equally, measured using a migration test and the measurement of calcium flux with macrophage inflammatory protein‐3β and reverse transcription‐polymerase chain reaction assay in all of the groups. All of these findings suggest that PGE2 affects the DC‐promoted differentiation of naïve T cells to a Th1 response in the basal state, without affecting chemokine receptor expression on DCs.

NKG2A Inhibits Invariant NKT Cell Activation in Hepatic Injury

Activation of invariant NKT (iNKT) cells in the liver is generally regarded as the critical step for Con A-induced hepatitis, and the role of NK cell receptors for iNKT cell activation is still controversial. In this study we show that blockade of the NKG2A-mediated inhibitory signal with antagonistic anti-NKG2A/C/E mAb (20d5) aggravated Con A-induced hepatitis in wild-type, Fas ligand (FasL)-mutant gld, and IL-4-deficient mice even with NK cell and CD8 T cell depletion, but not in perforin-, IFN-γ-, or IFN-γ- and perforin-deficient mice. Consistently, 20d5 pretreatment augmented serum IFN-γ levels and perforin-dependent cytotoxicity of liver mononuclear cells following Con A injection, but not their FasL/Fas-dependent cytotoxicity. However, blockade of NKG2A-mediated signals during the cytotoxicity effector phase did not augment cytotoxic activity. Activated iNKT cells promptly disappeared after Con A injection, whereas NK1− iNKT cells, which preferentially expressed CD94/NKG2A, predominantly remained in the liver. Pretreatment with 20d5 appeared to facilitate disappearance of iNKT cells, particularly NK1− iNKT cells. Moreover, Con A-induced and α-galactosylceramide-induced hepatic injury was very severe in CD94/NKG2A-deficient DBA/2J mice compared with CD94/NKG2A-intact DBA/2JJcl mice. Overall, these results indicated that a NKG2A-mediated signal negatively regulates iNKT cell activation and hepatic injury.

Immediate and Long-term Effects of Radiation on the Immune System of Specific-pathogen-free Mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.

MKK7 mediates miR-493-dependent suppression of liver metastasis of colon cancer cells

The prognosis of advanced colon cancer patients is profoundly affected by the presence or absence of liver metastasis. miR‐493 functions as a potent suppressor of liver metastasis, and low‐level miR‐493 expression in human primary colon cancer is associated with an elevated incidence of liver metastasis. We previously showed that IGF1R is a target gene of miR‐493, and that the inhibition of IGF1R partly explains how miR‐493 suppresses liver metastasis. However, major functional targets that mediate the antimetastatic activity of miR‐493 remain elusive. Here, we extended our search for target genes and identified MKK7, a mitogen‐activated protein kinase kinase, as a novel target of miR‐493. miR‐493 inhibits MKK7 expression by targeting the binding site at the 3′‐UTR of the mkk7 gene. MKK7 was expressed in six out of seven colon cancer cell lines examined but not in non‐transformed colon epithelial cells, and its expression was required for the activating phosphorylation of JNK. RNA interference‐mediated inhibition of MKK7 resulted in marked suppression of liver metastasis of colon cancer cells. A significant decrease of metastasized cells by the MKK7 knockdown was observed, even at early stages of the metastatic settlement, in accordance with a time course of the miR‐493‐mediated inhibition of the metastasis. Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7. Thus, MKK7 is a major functional target of miR‐493, and its suppression thwarts liver metastasis of colon cancer cells.

Phenotypical and functional alterations during the expansion phase of invariant Vα14 natural killer T (Vα14i NKT) cells in mice primed with α-galactosylceramide

Invariant Vα14 natural killer T (Vα14i NKT) cells are a unique immunoregulatory T‐cell population that is restricted by CD1d. The glycolipid α‐galactosylceramide (α‐GalCer) is presented by CD1d and causes robust Vα14i NKT‐cell activation. Three days after injection of α‐GalCer, Vα14i NKT cells vigorously increase in number and then gradually decrease to normal levels. In the present study, we found that the re‐administration of α‐GalCer into mice primed 3 days earlier causes a marked increase in serum interleukin‐4 and interferon‐γ. Intracellular staining revealed that the only expanded Vα14i NKT cells are responsible for the enhanced cytokine production. The enhanced cytokine production was correlated with an increased number of Vα14i NKT cells after priming. Additionally, primed Vα14i NKT cells produced larger amounts of cytokine as compared with naive Vα14i NKT cells when cultured with α‐GalCer‐pulsed dendritic cells. Thus, we considered that a subset of expanded Vα14i NKT cells acquired a strong ability to produce cytokines. In contrast to mice primed 3 days earlier, cytokine production is markedly diminished in mice primed 7 days earlier. The expanded Vα14i NKT cells altered the surface phenotype (NK1.1– CD69–) and contained intracellular interferon‐γ. Additionally, we found that primed Vα14i NKT cells did not disappear or down‐regulate surface TCR expression when re‐injected with α‐GalCer as compared with naive Vα14i NKT cells. These results demonstrate that the function and surface phenotype of Vα14i NKT cells is dramatically altered after α‐GalCer priming.

Co-inhibitory roles for glucocorticoid-induced TNF receptor in CD1d-dependent natural killer T cells

Invariant natural killer T (iNKT) cells are a special subset of αβ T cells with invariant TCR, which recognize α‐galactosylceramide (α‐GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with α‐GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well‐known costimulatory molecule, glucocorticoid‐induced TNF receptor (GITR), on Ag‐induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA‐1 suppressed proliferation and cytokine production of iNKT cells upon α‐GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag‐priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR‐deficient iNKT cells showed enhanced proliferation and increased cytokine production upon α‐GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of α‐GalCer suppressed tumor metastasis more efficiently in GITR‐deficient mice than in wild‐type mice. Collectively, GITR plays a co‐inhibitory role in Ag‐induced iNKT cell activation.

Limiting dilution analysis of T-cell progenitors in the bone marrow of thymic lymphoma-susceptible B10 and-resistant C3H mice after fractionated whole-body X-irradiation

Earlier studies from this laboratory using Thy 1 congenic B10 strain mice suggested that a depletion of T cell progenitors (pre T cells) in the bone marrow in addition to the destruction of the thymus after fractionated whole body X-irradiation (IR) are the two main critical factors that cause differentiation arrest of initially repopulating intrathymic radio-resistant T cell progenitors, which then lead to the appearance of preneoplastic, prelymphoma cells, and eventually to highly neoplastic thymic lymphomas under the influence of the thymic environment. In order to explore the significance of the depletion (or reduction) of T cell progenitors in the bone marrow during pathogenesis of radiation-induced thymic lymphomas, we compared the pool size of pre T cells in the bone marrow and the spleens as well as the profiles of the regenerating thymocyte populations between thymic lymphoma induction-susceptible B10 and -resistant C3H strain mice following irradiation. The results indicated that irradiation severely depleted the pre T cells in the bone marrow and the spleens of both lymphoma induction-susceptible and -resistant mice. They also showed that in C3H mice the differentiation and maturation of intrathymic T cell progenitors which initially repopulated the depleted thymus seemed to proceed normally in spite of the poor cellularity, while this process was greatly suppressed in B10 mice. These data indicate that a depletion of pre T cells in the bone marrow combined with atrophy of the thymus in the irradiated mice is necessary, but not sufficient for development of thymic lymphoma. Implication of these findings on the possible mechanism of radiation-induced thymic lymphomagenesis is discussed.

Administration route-dependent induction of antitumor immunity by interferon-alpha gene transfer.

Type I interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis, and immunomodulation. We have demonstrated that intratumoral injection of an IFN‐α‐expressing adenovirus effectively induces cell death of cancer cells and elicits a systemic tumor‐specific immunity in several animal models. On the other hand, reports demonstrated that an elevation of IFN in the serum following an intramuscular delivery of a vector is able to activate antitumor immunity. In this study, we compared the intratumoral and systemic routes of IFN gene transfer with regard to the effect and safety of the treatment. Intratumoral injection of an IFN‐α adenovirus effectively activated tumor‐responsive lymphocytes and caused tumor suppression not only in the gene‐transduced tumors but also in distant tumors, which was more effective than the intravenous administration of the same vector. The expression of co‐stimulatory molecules on CD11c+ cells isolated from regional lymph nodes was enhanced by IFN gene transfer into the tumors. Systemic toxicity such as an elevation of hepatic enzymes was much lower in mice treated by intratumoral gene transfer than in those treated by systemic gene transfer. Our data suggest that the intratumoral route of the IFN vector is superior to intravenous administration, due to the effective induction of antitumor immunity and the lower toxicity. (Cancer Sci 2010)

Expansion of α-galactosylceramide-stimulated Vα24+ NKT cells cultured in the absence of animal materials

Vα24+ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Vα24+ natural killer (NK) T cells, which are innate lymphocytes with potential antitumor activity, require their in vitro expansion. To avoid the potential dangers posed to patients by fetal bovine serum (FBS), the authors evaluated non-FBS culture conditions for the selective and efficient expansion of human Vα24+ NKT cells. Mononuclear cells (MNCs) and plasma from the peripheral blood of normal healthy donors were used before and after G-CSF mobilization. MNCs and plasma separated from apheresis products were also used. MNCs were cultured for 12 days in AIM-V medium containing α-galactosylceramide (α-GalCer) (100 ng/mL) and IL-2 (100 U/mL) supplemented with FBS, autologous plasma, or autologous serum. The cultured cells were collected and their surface markers, intracellular cytokines, and cytotoxicity were evaluated. The highest expansion ratio for Vα24+ NKT cells was obtained from G-CSF-mobilized MNCs cultured in medium containing 5% autologous plasma. Cultures containing MNCs and autologous plasma obtained before and after G-CSF mobilization had approximately 350-fold and 2,000-fold expansion ratios, respectively. These results suggest that G-CSF mobilization conferred a proliferative advantage to Vα24+ NKT cells by modifying the biology of cells and plasma factors. Expanded Vα24+ NKT cells retained their surface antigen expression and production of IFN-γ and exhibited CD1d-independent cytotoxicity against tumor cells. Vα24+ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with α-GalCer and IL-2.

Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression.