Hidemitsu Kitamura

Toll-like receptor–mediated regulation of zinc homeostasis influences dendritic cell function

Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.

Local Radiation Therapy Inhibits Tumor Growth through the Generation of Tumor-Specific CTL: Its Potentiation by Combination with Th1 Cell Therapy

Radiation therapy is one of the primary treatment modalities for cancer along with chemotherapy and surgical therapy. The main mechanism of the tumor reduction after irradiation has been considered to be damage to the tumor DNA. However, we found that tumor-specific CTL, which were induced in the draining lymph nodes (DLN) and tumor tissue of tumor-bearing mice, play a crucial role in the inhibition of tumor growth by radiation. Indeed, the therapeutic effect of irradiation was almost completely abolished in tumor-bearing mice by depleting CD8(+) T cells through anti-CD8 monoclonal antibody administration. In mice whose DLN were surgically ablated or genetically defective (Aly/Aly mice), the generation of tetramer(+) tumor-specific CTL at the tumor site was greatly reduced in parallel with the attenuation of the radiation-induced therapeutic effect against the tumor. This indicates that DLN are essential for the activation and accumulation of radiation-induced CTL, which are essential for inhibition of the tumor. A combined therapy of local radiation with Th1 cell therapy augmented the generation of tumor-specific CTL at the tumor site and induced a complete regression of the tumor, although radiation therapy alone did not exhibit such a pronounced therapeutic effect. Thus, we conclude that the combination treatment of local radiation therapy and Th1 cell therapy is a rational strategy to augment antitumor activity mediated by tumor-specific CTL.

Anti-IL-6 receptor mAb eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T-cell responses

CD11b+Gr‐1+ immature myeloid cells (ImCs), which are abnormally increased in tumor‐bearing mice, were classified into three different subsets according to their phenotypic and morphological characteristics: Gr‐1low F4/80+ macrophages (MΦ‐ImCs), Gr‐1mid stab neutrophils (Neutstab‐ImCs), and Gr‐1high segmented neutrophils (Neutseg‐ImCs). In the spleen, only MΦ‐ImCs but not Neutstab‐ImCs and Neutseg‐ImCs exhibited a significant immunosuppressive activity in MLR. In contrast, tumor‐infiltrating leukocytes (TILs) contained only two ImC subsets, MΦ‐ImCs and Neutseg‐ImC, both of which exhibited stronger inhibitory activity against T cells compared with spleen‐MΦ‐ImCs. Thus, we concluded that tumor‐infiltrating MΦ‐ImCs and Neutseg‐ImCs were fully differentiated myeloid‐derived suppressor cells (MDSCs) with stronger T‐cell inhibitory activity. Indeed, spleen MΦ‐ImCs were converted into stronger MΦ‐MDSCs by tumor‐derived factor (TDF). Moreover, both spleen Neutstab‐ImCs and Neutseg‐ImCs differentiated into Neutseg‐MDSCs with suppressive activity after culture with TDF. We first demonstrated that administration of anti‐IL‐6R mAb could downregulate the accumulation of MΦ‐MDSCs and Neutseg‐MDSCs in tumor‐bearing mice. The elimination of those MDSCs caused subsequent enhancement of antitumor T‐cell responses, including IFN‐γ‐production. The therapeutic effect of anti‐IL‐6R mAb was further enhanced by combination with gemcitabine (GEM). Thus, we propose that anti‐IL‐6R mAb could become a novel tool for the downmodulation of MDSCs to enhance antitumor T‐cell responses in tumor‐bearing hosts.

1α,25-Dihydroxyvitamin D3 and all-trans retinoic acid synergistically inhibit the differentiation and expansion of Th17 cells

1α,25-Dihydroxyvitamin D(3) (1,25D3), the active form of vitamin D(3), is an immunoregulatory hormone with beneficial effects on Th1 cell-mediated inflammatory diseases. Although IL-17-producing CD4(+) T helper (Th17) cells have been recently identified as novel effector cells, the immunomodulating effects of 1,25D3 on Th17 cells have not been well defined. We confirmed here that 1,25D3 inhibited the generation of Th17 cells in vitro. Interestingly, 1,25D3 synergistically suppressed the generation of Th17 cells by the combination with all-trans retinoic acid (ATRA). 1,25D3 and ATRA suppressed the development of allergen-induced contact hypersensitivity (CHS) in a mouse ear swelling model. In addition, we found that 1,25D3 and ATRA significantly inhibited the development of human Th17 cells from both naïve and memory human CD4(+) T cells. 1,25D3 and ATRA effectively suppressed mRNA expressions of IL-1R1, IL-21R, IL-23R, RORC, and AHR in human T cells. ATRA further suppressed IL-6R, whereas 1,25D3 did not. Finally, we found that 1,25D3 and ATRA remarkably blocked IL-22 as well as IL-17 mRNA expression in human memory CD4(+) T cells. Thus, we initially reveal that 1,25D3 and ATRA have synergistic effects on the generation of Th17 cells, suggesting that the combination with ATRA would provide a promising novel therapy for Th17 cell-related immune diseases including skin inflammation.

IL-17/IFN-γ double producing CD8+ T (Tc17/IFN-γ) cells: A novel cytotoxic T-cell subset converted from Tc17 cells by IL-12

It has been reported that IFN-γ-producing CD8(+) T (Tc1) cells express cytotoxic molecules such as perforin and granzyme B to exhibit higher cytotoxicity against tumor cells compared with Tc2 cells. However, the critical role of IL-17-producing CD8(+) T (Tc17)-cell subsets in tumor immunity remains unclear. Tc17 cells differentiated from naive CD8(+) T cells did not possess cytotoxic molecules and exhibited no strong cytotoxicity. However, when Tc17 effector cells were further cultured with IL-12, they converted into IFN-γ-producing Tc17 cells, which mainly consisted of IL-17/IFN-γ double-producing cells (Tc17/IFN-γ). IL-12-converted Tc17 cells also acquired cytotoxic function in addition to IFN-γ producibility. Moreover, they showed strong anti-tumor activity both in vitro and in vivo as well as Tc1 cells. Among four distinct subsets in IL-12-converted Tc17 cell populations, the isolated Tc17/IFN-γ cells exhibited cytotoxicity as well as IFN-γ-producing Tc1-like cells. Thus, we first indicate direct evidence that Tc17/IFN-γ cells, which were plastically converted from non-cytotoxic Tc17 cells by IL-12, exhibited strong anti-tumor activity as well as Tc17 cell-derived Tc1-like cells.

Essential role of Toll-like receptors for dendritic cell and NK1.1(+) cell-dependent activation of type 1 immunity by Lactobacillus pentosus strain S-PT84.

Activation of type 1 immunity plays a critical role in host defense mechanisms against infectious disease and tumor. Lactic acid bacteria, existing in the gastrointestinal tract, are one of the powerful tools to induce a type-1-dominant immunity, which may improve Th2-dependent allergic diseases. In the present work, we found that an oral intake of Lactobacillus pentosus strain, S-PT84 into mice significantly enhanced NK activity of spleen cells in vivo. We further revealed that NK1.1 positive NK cells and NKT cells are responsible cells for producing IFN-gamma after stimulation with S-PT84 in vitro. S-PT84 induced IFN-gamma-producing cells through activation of IL-12 production by CD11c(+)DCs in Toll-like receptor (TLR) 2- and/or TLR4-dependent manner. Interestingly, direct interaction between DCs and NK1.1(+) cells was also essential for the IFN-gamma production in response to the S-PT84 stimulation. Therefore, we concluded that S-PT84 effectively promoted type 1 immunity through IL-12 and IFN-gamma which were produced by DCs and NK1.1(+) cells, respectively. Thus, S-PT84 would be a nice immune modulator for improving immunobalance, which plays a pivotal role for controlling allergy, infectious diseases and tumor.

The Key Role of IL-6–Arginase Cascade for Inducing Dendritic Cell–Dependent CD4+ T Cell Dysfunction in Tumor-Bearing Mice

Evaluation of immune dysfunction during the tumor-bearing state is a critical issue in combating cancer. In this study, we initially found that IL-6, one of the cachectic factors, suppressed CD4+ T cell–mediated immunity through downregulation of MHC class II by enhanced arginase activity of dendritic cells (DC) in tumor-bearing mice. We demonstrated that administration of Ab against IL-6R (anti–IL-6R mAb) greatly enhanced T cell responses and inhibited the growth of tumor in vivo. We also found that IL-6 upregulated the expression of arginase-1 and arginase activity of DC in vitro. Tumor-infiltrating CD11c+ DC exhibited upregulated mRNA expression of arginase-1 but reduced expression of MHC class II in parallel with the increase in serum IL-6 levels at the late stage in tumor-bearing hosts. However, the administration of anti–IL-6R mAb into tumor-bearing mice inhibited both the downmodulation of MHC class II and the upregulation of arginase-1 mRNA levels in DC. Furthermore, we noted that Nω-hydroxy-L-arginine or L-arginine, an arginase-1 inhibitor, blocked the reduction in MHC class II levels on CD11c+ DC during the tumor-bearing state. Finally, we demonstrated that the administration of Nω-hydroxy-L-arginine at the peritumor site significantly enhanced CD4+ T cell responses and inhibited tumor growth. Thus, IL-6–mediated arginase activation and the subsequent reduction in MHC class II expression on DC appeared to be critical mechanisms for inducing dysfunction of the immune system in the tumor-bearing state. Blockade of the IL-6–arginase cascade is a promising tool to overcome the dysfunction of antitumor immunity in tumor-bearing hosts.

The development of IL-17/IFN-γ-double producing CTLs from Tc17 cells is driven by epigenetic suppression of Socs3 gene promoter

The plasticity of T lymphocytes induced by epigenetic modifications of gene promoters may play a pivotal role in controlling their effector functions, which are sometimes causally associated with immune disorders. IL ‐17‐producing T cells, which induce type 17 immune responses, are newly identified pathogenic effector cells. The type 1 signature cytokine IFN‐γ strongly inhibits their differentiation, indicating a mutually exclusive relationship between type 17‐ and type 1‐immune responses. However, many reports indicate the presence of a unique IL‐17/IFN‐γ‐double producing T‐cell subset in various inflammatory settings, although the mechanisms responsible for their development and their precise functions remain unclear. Here, we demonstrate that IL‐12 permits the conversion of mouse IL‐17‐producing CD8+ T (Tc17) cells to IL‐17/IFN‐γ‐double producing CD8+ T (Tc17/IFN‐γ) cells, and that this conversion is due to repressive epigenetic modifications of Socs3 gene promoters. Moreover, we show that SOCS3 strongly regulates the capability of Tc17 cells to produce IL‐17, in addition to regulating the expression of the type 17‐master regulator RORγt. These findings elucidate the mechanisms underlying the conversion of Tc17 cells into Tc17/IFN‐γ cells. As these cells are known to have potent antitumor activities, manipulation of these conversion mechanisms for therapeutic tumor immunity may be possible.

IFN-γ-dependent type 1 immunity is crucial for immunosurveillance against squamous cell carcinoma in a novel mouse carcinogenesis model

3-Methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCCs). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intra-dermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of interferon (IFN)-gamma-dependent type 1 immunity in immunosurveillance against SCC from the following results: (i) The incidence of SCC was accelerated in IFN-gamma-deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-oligodeoxynucleotides (CpG-ODN) caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity and (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN-gamma-deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer.

Combination immunotherapy with radiation and CpG-based tumor vaccination for the eradication of radio- and immuno-resistant lung carcinoma cells

Unmethylated cytosine‐phosphorothioate‐guanine containing oligodeoxynucleotides (CpG‐ODN) is known as a ligand of toll‐like receptor 9 (TLR9), which selectively activates type‐1 immunity. We have already reported that the vaccination of tumor‐bearing mice with liposome‐CpG coencapsulated with model‐tumor antigen, ovalbumin (OVA) (CpG + OVA‐liposome) caused complete cure of the mice bearing OVA‐expressing EG‐7 lymphoma cells. However, the same therapy was not effective to eradicate Lewis lung carcinoma (LLC)‐OVA‐carcinoma. To overcome the refractoriness of LLC‐OVA, we tried the combination therapy of radiation with CpG‐based tumor vaccination. When LLC‐OVA‐carcinoma intradermally (i.d.) injected into C57BL/6 became palpable (7–8 mm), the mice were irradiated twice with a dose of 14 Gy at intervals of 24 h. After the second radiation, CpG + OVA‐liposome was i.d. administered near the draining lymph node (DLN) of the tumor mass. The tumor growth of mice treated with radiation plus CpG + OVA‐liposome was greatly inhibited and approximately 60% of mice treated were completely cured. Moreover, the combined therapy with radiation and CpG + OVA‐liposome allowed the augmented induction of OVA‐tetramer+ LLC‐OVA‐specific cytotoxic T lymphocyte (CTL) in DLN of tumor‐bearing mice. These results indicate that the combined therapy of radiation with CpG‐based tumor vaccine is a useful strategy to eradicate intractable carcinoma. (Cancer Sci 2009; 100: 934–939)