Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): preparation and immunological analysis of vaccine efficacy.

To elucidate the immunologic mechanisms of artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which indicated a great vaccine efficacy in human cancers, we prepared ovalbumin (OVA)-H/K-HELP by conjugating killer and helper epitopes of OVA-model tumor antigen via a glycine-linker. Vaccination of C57BL/6 mice with OVA-H/K-HELP (30 amino acids) but not with short peptides mixture of class I-binding peptide (8 amino-acids) and class II-binding peptide (17 amino-acids) combined with adjuvant CpG-ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotides), induced higher numbers of OVA-tetramer-positive CTL with concomitant activation of IFN-γ-producing CD4(+) Th1 cells. However, replacement of glycine-linker of OVA-H/K-HELP with other peptide-linker caused a significant decrease of vaccine efficacy of OVA-H/K-HELP.

Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine.

We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes.

Identification of a meiosis-specific protein, MEIOB, as a novel cancer/testis antigen and its augmented expression in demethylated cancer cells.

Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen.

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.

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.

IFN-gamma-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).

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.

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.

Natural killer T cells from interleukin-4-deficient mice are defective in early interferon-gamma production in response to alpha-galactosylceramide.

Discovery of the natural killer (NK) T cell-specific ligand, alpha-galactosylceramide (alpha-GalCer) has enabled us to investigate the functional regulation of NKT cells. However, the detailed mechanism of cytokine production by NKT cells remains to be elucidated. Here we evaluated the role of interleukin (IL)-4 in the production of interferon (IFN)-gamma from NKT cells using IL-4-deficient C57BL/6 mice (IL-4(-/-) mice).

AsialoGM1+CD8+ central memory-type T cells in unimmunized mice as novel immunomodulator of IFN-gamma-dependent type 1 immunity.

In unimmunized specific pathogen-free mice, there are unique memory-type CD8(+) T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (T(CMT)) judging from their expression profile of CD44, IL-2Rbeta, CD62L and CCR7 cell-surface molecules. Among CD44(high)CD8(+) so-called memory CD8(+) T cell population, ASGM1(+)CD44(high)CD8(+) T(CMT), but not ASGM1(-)CD44(high)CD8(+) memory T cells, produced IFN-gamma by stimulation with anti-CD3 mAb.

Interleukin-12-responding asialoGM1+CD8+ central memory-type T cells as precursor cells for interferon-gamma-producing killer T cells.

While investigating CD8(+) memory T cells in unimmunized C57BL/6 mice, we found that there were unique memory-type CD8(+) T cells expressing asialoGM1 (ASGM1), CD62L and CCR7 cell surface molecules, which occupied approximately 10% of CD8(+) T cells and 35% of CD44(+) memory CD8(+) T cells. Culture of freshly isolated ASGM1(+)CD8(+) T cells with interleukin (IL)-12 plus IL-2 caused the proliferation and generation of killer T cells. Moreover, ASGM1(+)CD8(+) T cells, but not ASGM1(-)CD8(+) T cells, produced high levels of interferon (IFN)-gamma in response to IL-12 plus IL-2.

Development of a functional cDNA array for evaluation of the Th1/Th2 balance.

The immune balance controlled by CD4(+) helper T cell subsets (T helper 1 (Th1) and T helper 2 (Th2)) is crucial for immunoregulation and its imbalance causes various immune diseases including infections, allergic disorders and autoimmune diseases. Therefore, it is of great importance to develop a system of diagnosing Th1/Th2 imbalances for curing immune diseases. Here we developed a functional cDNA array filter useful for assessing the Th1/Th2 balance in mice.

[Development of DNA array filter useful for the analysis of Th1/Th2 balance].

DNA arrays are useful for determining the expression levels of a number of genes at once. We utilized this technique to evaluate the Th1/Th2 balance in vivo. Immune responses are controlled by two types of helper T cells, Th1 and Th2.

NKT cells act as regulatory cells rather than killer cells during activation of NK cell-mediated cytotoxicity by alpha-galactosylceramide in vivo.

Administration of NKT cell ligands, alpha-galactosylceramide (alpha-GalCer) resulted in the activation of both cytokine production and natural killing. These responses were abolished in both CD1d-deficient mice and Valpha14NKT-deficient mice. Therefore, NKT cells have been considered to be responsible cells for both cytokine production and natural killing.

Generation of leukemia-specific T-helper type 1 cells applicable to human leukemia cell-therapy.

Leukemic dendritic cells (DC) were induced from the peripheral blood (PB) or bone marrow (BM) of leukemia patients by culture with (i) GM-CSF + IL-3 (neutral condition); (ii) GM-CSF + IL-3 + IL-12 + IFN-gamma (type 1-condition); or (iii) GM-CSF + IL-3 + IL-4 (type 2-condition). Although leukemic cells rapidly differentiated into adhesive leukemic DC in all culture conditions, type1-conditions were the most suitable for inducing leukemic DC expressing high levels of HLA and costimulatory molecules. Addition of IL-2 after 2 days of culture induced a preferential growth of minor T cell populations interacting with leukemic DC.

Unexpected role of TNF-alpha in graft versus host reaction (GVHR): donor-derived TNF-alpha suppresses GVHR via inhibition of IFN-gamma-dependent donor type-1 immunity.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation, leading to significant morbidity and mortality. Host-derived TNF-alpha play a role in the induction of allo-reactive donor T cell activation and the pathogenesis of GVHD. On the other hand, the precise role of donor-derived TNF-alpha in GVHD remains unclear.

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)-alpha and TCR-beta genes.

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 re-stimulated with the model tumor antigen (OVA) for 4 days. Spleen cells obtained from mice cured by Th1-cell therapy produced high levels of IFN-gamma, while spleen cells from mice cured by Th2-cell therapy produced high levels of IL-4.

An efficient method to prepare T cell receptor gene-transduced cytotoxic T lymphocytes type 1 applicable to tumor gene cell-therapy.

Genes encoding 2C T cell receptor (TCR) alpha, beta chains from H-2(b)-restricted L(d)-specific CD8(+) cells were successfully transduced into polyclonally activated CD8(+) cells by retroviral modification to generate antigen-specific cytotoxic T lymphocytes (CTL). Antigen-nonspecific CD8(+) T cells polyclonally expanded in the presence of interleukin (IL)-2, Th1 cytokines (interferon (IFN)-gamma and IL-12) and anti-IL-4 monoclonal antibody showed neither cytokine production nor cytotoxicity in response to L(d)-expressing P815 tumor cells. However, 2C-TCR gene-modified CD8(+) T cells exhibited both IFN-gamma production and cytotoxicity in response to P815 tumor cells.

Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets.

We found that T(h)1 cells derived from ovalbumin (OVA)-specific TCR transgenic (DO11.10) mice showed significantly higher levels of VLA-2 (CD49b/CD29) expression than T(h)2 cells. In the early days (until 6 days) during induction of T(h)1 or T(h)2 cells, the expression of VLA-2 was gradually increased on both T(h) subsets.

[Genetically engineered mice and its application to bioindustry].

More than 2 decades have past since the first transgenic mouse was created. In this review we will focus on recent technologies in the field of mouse genetic engineering and on its application to bioindustry. The most important milestone in this field was the invention of the gene targeting technique.