Hiromichi Yamanishi

Involvement of CD56brightCD11c+ Cells in IL-18–Mediated Expansion of Human γδ T Cells

γδ T cells are considered to be innate lymphocytes that play an important role in host defense against tumors and infections. We recently reported that IL-18 markedly amplified γδ T cell responses to zoledronate (ZOL)/IL-2. In an extension of this finding, we analyzed the mechanism underlying the IL-18–mediated expansion of γδ T cells. After incubation of PBMCs with ZOL/IL-2/IL-18, the majority of the cells expressed γδ TCR, and the rest mostly exhibited CD56brightCD11c+ under the conditions used in this study. CD56brightCD11c+ cells were derived from a culture of CD56intCD11c+ cells and CD14+ cells in the presence of IL-2 and IL-18 without the addition of ZOL. They expressed IL-18Rs, HLA-DR, CD25, CD80, CD83, CD86, and CD11a/CD18. In addition, they produced IFN-γ, TNF-α, but not IL-12, when treated with IL-2/IL-18, and they exerted cytotoxicity against K562 cells, thus exhibiting characteristics of both NK cells and dendritic cells. Incubation of purified γδ T cells with CD56brightCD11c+ cells in the presence of ZOL/IL-2/IL-18 resulted in the formation of massive cell clusters and led to the marked expansion of γδ T cells. However, both conventional CD56−/intCD11chigh dendritic cells induced by GM-CSF/IL-4 and CD56+CD11c− NK cells failed to support the expansion of γδ T cells. These results strongly suggest that CD56brightCD11c+ cells play a key role in the IL-18–mediated proliferation of γδ T cells.

Effect of IL-18 on Expansion of γδ T Cells Stimulated by Zoledronate and IL-2

Zoledronate (Zol) has recently been shown to expand γδ T cells that play important roles in host defenses against infection and tumors. In this study, we examined effects of interleukin-18 (IL-18) on expansion of γδ T cells in human peripheral blood mononuclear cells (PBMCs) stimulated by Zol and IL-2. The expansion of γδ T cells stimulated by Zol and IL-2 was strongly promoted by exogenous IL-18, and to the contrary, inhibited by neutralizing anti-IL-18 receptor antibody. The γδ T cells that expanded in the presence of Zol, IL-2, and IL-18 exhibited the phenotype of effector memory cells characterized by CD44 (+), CD27 (−), and CD45RA (−). In addition, they expressed NKG2D, perforin, CD94, CD25, and CD122, and 15% to 40% of them were positive for CD56. Incubation of γδ T cells in the presence with IL-18 produced GM-CSF, IFN-γ, and TNF-α at much higher levels than those incubated without IL-18. They showed strong cytotoxicity against tumor cells including mesothelioma cells and inhibited growth of xenograft of mesothelioma in mice. These observations indicate that IL-18 can efficiently promote expansion of γδ T cells with potent antitumor activity.

Augmentation of Immune Checkpoint Cancer Immunotherapy with IL18

Purpose: Recent clinical trials and animal models demonstrated that immune checkpoint blockade enhanced effector cell responses and tumor rejection; however, further development and improvement of cancer immunotherapy is necessary for more favorable objective responses. In this study, we examined the effect of IL18 on the antitumor effect of immune checkpoint inhibitors. Experimental Design: We examined the effect of IL18 on the peritoneal dissemination of CT-26 cells or tail vein injection metastasis of B16/F10 cells using antiprogrammed death-1 ligand-1 (αPD-L1) and/or anti-CTL–associated antigen-4 (αCTLA-4) mAbs. Result: Massive ascites developed after intraperitoneal inoculation of CT-26, resulting in animal death within 30 days. Treatment of mice with αPD-L1 and/or αCTLA-4 significantly prolonged their survival, and a combination of the antibodies and IL18 provided a much greater therapeutic benefit. The combination modality led to the accumulation of precursor of mature natural killer (pre-mNK) cells in the peritoneal cavity together with increased CD8+ T and decreased CD4+CD25+Foxp3+ T cells. Depletion of the pre-mNK cells abrogated the therapeutic effects and increased the number of CD4+CD25+Foxp3+ T cells. The combination treatment also suppressed tail vein injection metastasis of B16/F10 cells. Conclusions: The results demonstrated that IL18 enhanced therapeutic effects of immune checkpoint blockade against peritoneal dissemination of carcinoma or tail vein injection metastasis of melanoma through accumulation of pre-mNK cells, memory-type CD8+ T cells, and suppression of CD4+CD25+Foxp3+ T cells. A combination of immune checkpoint inhibitors with IL18 may give a suggestion to the development of next-generation cancer immunotherapy. Clin Cancer Res; 22(12); 2969–80. ©2016 AACR.

Chymase inhibition improves vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats

Objective: To clarify the role of chymase in hypertension, we evaluated the effect of a chymase inhibitor, TY-51469, on vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats (SHR-SP). Methods: SHR-SP were treated with TY-51469 (1 mg/kg per day) or placebo from 4 to 12 weeks old or until death. Wistar–Kyoto rats were used as a normal group. Results: SBP was significantly higher in both the placebo and TY-51469 groups than in the normal group, but there was no significant difference between the two treatment groups. Plasma renin, angiotensin-converting enzyme activity and angiotensin II levels were not different between the placebo and TY-51469 groups. In contrast, vascular chymase-like activity was significantly higher in the placebo than in the normal group, but it was reduced by TY-51469. Acetylcholine-induced vascular relaxation was significantly higher in the TY-51469 group than in the placebo group. There was significant augmentation of the number of monocytes/macrophages and matrix metalloproteinase-9 activity in aortic tissue from the placebo group compared with the normal group, and these changes were attenuated by TY-51469. There were also significant increases in mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; in the placebo group that were attenuated by TY-51469. Cumulative survival was significantly prolonged in the TY-51469 group compared with the placebo group. Conclusion: Chymase might play an important role in vascular dysfunction via augmentation both of matrix metalloproteinase-9 activity and monocyte/macrophage accumulation in SHR-SP, and its inhibition may be useful for preventing vascular remodeling and prolonging survival.

Gene expression in the adrenal glands of three spontaneously hypertensive rat substrains.

We examined gene expression profiles in rat adrenal glands using genome-wide microarray technology. Gene expression levels were determined in four rat strains, including one normotensive strain [Wistar-Kyoto (WKY)] and three substrains derived from WKY rats: spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and malignant SHRSP (M-SHRSP). This study represents the first attempt at using microarrays to compare gene expression profiles in SHR, SHRSP and M-SHRSP adrenal glands, employing WKY as controls. Expression measurements were made in these four rat strains at 6 and 9 weeks of age; 6 weeks of age covers the pre-hypertensive period in SHR and SHRSP, and 9 weeks of age is the period of rapidly rising blood pressure (BP). Since the aim of this study was to identify candidate genes involved in the genesis of hypertension in the SHR substrains, we identified genes that were consistently different in their expression, isolating 87 up-regulated genes showing a more than 4-fold increase and 128 down-regulated genes showing a less than 1/4-fold decrease in at least two different experiments. We classified all these up- or down-regulated genes by their expression profiles, and searched for candidate genes. At 6 weeks of age, several BP-regulating genes including sparc/osteonectin (Spock2), kynureninase (Kynu), regulator of G-protein signaling 2 (Rgs2) and gap junction protein α1 (Gja1) were identified as up-regulated, and urotensin 2 (Uts2), cytoplasmic epoxide hydrolase 2 (Ephx2), apelin (Apln), insulin-like growth factor 1 receptor (Igf1r) and angiotensin II receptor-associated protein (Agtrap) were identified as down-regulated. The Kynu and Ephx2 genes have previously been reported by other groups to be responsible for hypertension in SHR; however, our present approach identified at least seven new candidate genes.

Dysfunction of mitochondria and deformed gap junctions in the heart of IL-18-deficient mice

Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

Antagonism of IL-4 signaling by a phosphodiesterase-4 inhibitor, rolipram, in human T cells.

Background: Phosphodiesterase (PDE4) inhibitors prevent breakdown of cAMP and affect the increase in cellular levels of cAMP, which is known to regulate immune cell functions. Because IL-4 plays a causal role in the pathogenesis of allergic disorders, we were interested to study the modulatory mechanisms of a PDE4 inhibitor, rolipram, in IL-4-mediated signaling in T cells. Methods: Human peripheral T cells were stimulated with IL-4 in combination with rolipram, and RT-PCR was performed using primers specific for IL-5. To monitor activation of transcription factors, immunostaining was employed. Results: Rolipram or a cAMP-analogue, 8-Br-cAMP, significantly downregulated IL-4-induced expression of IL-5 mRNA. The rolipram-induced inhibition of IL-5 mRNA was mediated by activation of protein kinase A (PKA), because rolipram-downregulated mRNA expression of IL-5 was restored by PKA inhibitors. Immunostaining revealed that rolipram interfered with IL-4-induced nuclear translocation of activator protein (AP)-1 components. Conclusions: This is the first demonstration of suppression of IL-4 signaling by PDE4 inhibitors via prevention of nuclear translocation of AP-1.

Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56brightCD11c+ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56brightCD11c+ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56brightCD11c+ cells. CD14+ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56intCD11c+ cells to promote their differentiation to CD56brightCD11c+ cells with helper function. The development of CD56brightCD11c+ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14+ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56brightCD11c+ cells, which in turn modulate the expansion of γδ T cells. CD56brightCD11c+ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.

Genetic analysis of genes causing hypertension and stroke in spontaneously hypertensive rats: Gene expression profiles in the kidneys

Spontaneously hypertensive rats (SHRs) and stroke-prone SHRs (SHRSP) are frequently used as models not only of essential hypertension and stroke, but also of attention-deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto (WKY) rats are normally used as controls in these studies. In the present study, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD using these rats. We previously analyzed gene expression profiles in the adrenal glands and brain. Since the kidneys can directly influence the functions of the cardiovascular, endocrine and sympathetic nervous systems, gene expression profiles in the kidneys of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks old, a period in which rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between the SHRs and WKY rats and also between the SHRSP and SHRs. A total of 232 unique genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated as SHR- and SHRSP-specific genes. Candidate genes were then selected using two different web tools: the 1st tool was the Database for Annotation, Visualization and Integrated Discovery (DAVID), which was used to search for significantly enriched genes and categorized them using Gene Ontology (GO) terms, and the 2nd was Ingenuity Pathway Analysis (IPA), which was used to search for interactions among SHR- and also SHRSP-specific genes. The analyses of SHR-specific genes using IPA revealed that B-cell CLL/lymphoma 6 (Bcl6) and SRY (sex determining region Y)-box 2 (Sox2) were possible candidate genes responsible for causing hypertension in SHRs. Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), angiotensin II receptor-associated protein (Agtrap) and apolipoprotein H (Apoh) were possible candidate genes responsible for triggering strokes. Since our results revealed that SHRSP-specific genes isolated from the kidneys of rats at 6 weeks of age, included 6 genes related to Huntingtons disease, we discussed the genetic association between ADHD and Huntingtons disease.

Frontline Science: IL‐18 primes murine NK cells for proliferation by promoting protein synthesis, survival, and autophagy

Combined stimulation by IL‐2 and IL‐18 effectively promotes proliferation of NK cells, whereas singular stimulation does not. In this study, synergistic effects of these cytokines on NK cells proliferation was analyzed, focusing on the roles of IL‐18. In splenic resting NK cells from IL‐18KO mice, IL‐18 rapidly activated NF‐κB independently of IL‐2, and activated or up‐regulated various molecules downstream of PI3K/AKT and mTOR, including S6, Bcl‐XL, ATG5, and LC3II, accompanying increases in cell growth and survival. Thus, IL‐18 alone was revealed to augment various cellular processes (gene transcription, protein synthesis, survival) in the absence or presence of IL‐2. Notably, combined IL‐18 and IL‐2 promoted autophagosome formation. In addition, priming NK cells with IL‐18 augmented IL‐2R, especially CD25, and enabled cells to respond to IL‐2, resulting in activation of STAT3 and STAT5, followed by increase of cyclin B1 leading to proliferation. However, IL‐2 alone failed to activate STAT3 or STAT5 in resting IL18KO NK cells. These results clarify the distinct roles of IL‐2 and IL‐18 in NK cell proliferation, and the intrinsic roles of IL‐18 in various cellular processes, suggesting a range of functions of IL‐18 expressed in an array of nonhematopoietic cells.