Yuji Togashi

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.

Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells

Adoptive immunotherapy using antigen-specific T-helper type 1 (Th1) cells has been considered as a potential strategy for tumor immunotherapy. However, its application to tumor immunotherapy has been hampered by difficulties in expanding tumor-specific Th1 cells from tumor-bearing hosts. Here, we have developed an efficient protocol for preparing mouse antigen-specific Th1 cells from nonspecifically activated Th cells after retroviral transfer of T-cell receptor (TCR)-α and TCR-β genes. We demonstrate that Th1 cells transduced with the TCR-α and -β genes from the I-Ad-restricted ovalbumin (OVA)323–339-specific T-cell clone DO11.10 produce IFN-γ but not interleukin-4 in response to stimulation with OVA323–339 peptides or A20 B lymphoma (A20-OVA) cells expressing OVA as a model tumor antigen. TCR-transduced Th1 cells also exhibited cytotoxicity against tumor cells in an antigen-specific manner. Moreover, adoptive transfer of TCR-transduced Th1 cells, but not mock-transduced Th1 cells, exhibited potent antitumor activity in vivo and, when combined with cyclophosphamide treatment, completely eradicated established tumor masses. Thus, TCR-transduced Th1 cells are a promising alternative for the development of effective adoptive immunotherapies.

A Critical Role for Mouse CXC Chemokine(s) in Pulmonary Neutrophilia During Th Type 1-Dependent Airway Inflammation

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

Critical role of the Th1/Tc1 circuit for the generation of tumor-specific CTL during tumor eradication in vivo by Th1-cell therapy

Th1 and Th2 cells obtained from OVA‐specific T cell receptor transgenic mice completely eradicated the tumor mass when transferred into mice bearing A20‐OVA tumor cells expressing OVA as a model tumor antigen. To elucidate the role of Tc1 or Tc2 cells during tumor eradication by Th1‐ or Th2‐cell therapy, spleen cells obtained from mice cured of tumor by the therapy were restimulated with the model tumor antigen (OVA) for 4 days. Spleen cells obtained from mice cured by Th1‐cell therapy produced high levels of IFN‐γ, while spleen cells from mice cured by Th2‐cell therapy produced high levels of IL‐4. Intracellular staining analysis demonstrated that a high frequency of IFN‐γ‐producing Tc1 cells was induced in mice given Th1‐cell therapy. In contrast, IL‐4‐producing Tc2 cells were mainly induced in mice after Th2‐cell therapy. Moreover, Tc1, but not Tc2, exhibited a tumor‐specific cytotoxicity against A20‐OVA but not against CMS‐7 fibrosarcoma. Thus, immunological memory essential for CTL generation was induced by the Th1/Tc1 circuit, but not by the Th2/Tc2 circuit. We also demonstrated that Th1‐cell therapy is greatly augmented by combination therapy with cyclophosphamide treatment. This finding indicated that adoptive chemoimmuno‐therapy using Th1 cells should be applicable as a novel tool to enhance the Th1/Tc1 circuit, which is beneficial for inducing tumor eradication in vivo.

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.

First clinical trial of cancer vaccine therapy with artificially synthesized helper/ killer-hybrid epitope long peptide of MAGE-A4 cancer antigen

A patient with pulmonary metastasis of colon cancer was treated with artificially synthesized helper/killer‐hybrid epitope long peptide (H/K‐HELP) of MAGE‐A4 cancer antigen. The patient was vaccinated with MAGE‐A4‐H/K‐HELP combined with OK432 and Montanide ISA‐51. There were no severe side‐effects except for a skin reaction at the injection site. MAGE‐A4‐H/K‐HELP induced MAGE‐A4‐specific Th1 and Tc1 immune responses and the production of MAGE‐A4‐specific complement‐fixing IgG antibodies. Tumor growth and carcinoembryonic antigen tumor marker were significantly decreased in the final diagnosis. This is the first report that artificially synthesized MAGE‐A4‐H/K‐HELP induces Th1‐dependent cellular and humoral immune responses in a human cancer patient. (Cancer Sci 2012; 103: 150–153)

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)

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timms method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timms method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timms method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.

Abnormal Copper Accumulation in the Liver of LEC Rats: A Rat Form of Wilson’s Disease

Since the hepatitis in LEC rats is controlled by a single autosomal recessive gene [1], a genetic metabolic disorder is likely to be the cause of the hepatitis. Although interesting biochemical changes associated with the development of the hepatitis have been reported [2–4], they have been unable to provide any direct evidence about the pathogenesis of the rats.