Akemi Kosaka

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.

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.

Extraction and purification of human interleukin-10 from transgenic rice seeds

Recombinant protein production system using transgenic rice grain offers many advantages in higher accumulation, preservation, lower production cost, ease of scale up and low risk of contamination by toxic materials. We developed a transgenic rice strain whose seeds accumulate human interleukin (IL)-10, a cytokine that suppresses inflammation-related immune responses. We also developed a method of extracting and purifying IL-10 from rice seeds. A biochemical crosslinking method was used to detect the biologically active noncovalent dimer of IL-10. This method was useful for developing efficient methods of refolding and purification. The purified IL-10 comprised only noncovalent dimers and showed higher activity than the commercial IL-10. The purified IL-10 had very low endotoxin contamination and is expected to have broad clinical application.

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) α, β chains from H‐2b‐re‐stricted Ld‐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)‐γ and IL‐12) and anti‐IL‐4 monoclonal antibody showed neither cytokine production nor cytotoxicity in response to Ld‐expressing P815 tumor cells. However, 2C‐TCR gene‐modified CD8+ T cells exhibited both IFN‐γ production and cytotoxicity in response to P815 tumor cells. The antitumor activity of TCR gene‐modified Tc1 cells was also demonstrated in vivo by Winns assay. Thus, we have developed an efficient method to induce TCR gene‐modified antigen‐specific Tc1 cells that exhibit antitumor activity both in vitro and in vivo. (Cancer Sci 2003; 94: 389–393)

Interleukin‐12‐responding asialoGM1+CD8+ central memory‐type T cells as precursor cells for interferon‐γ‐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)‐γ in response to IL‐12 plus IL‐2. Although ASGM1+CD8+ T cells showed no significant responses to IL‐12 alone or IL‐2 alone, pulse incubation of ASGM1+CD8+ T cells with IL‐12 at an earlier time (0–12 h), and subsequently with IL‐2 at a later time (12–24 h), caused the same levels of proliferation, killer cell generation and IFN‐γ production as when they were incubated simultaneously with IL‐12 plus IL‐2 for 24 h. Thus, ASGM1+CD8+ T cells appeared to respond to IL‐12 directly to acquire IL‐2 responsiveness and differentiate into IFN‐γ‐producing killer T cells. Indeed, freshly isolated ASGM1+CD8+ T cells, but not ASGM1−CD8+ T cells, expressed higher levels of IL‐12R β2 mRNA. The fact that IL‐12 administration in vivo caused the generation of ASGM1+CD8+ killer T cells in an IFN‐γ‐dependent manner further indicated a physiological significance of ASGM1+CD8+ central memory‐type T cells in IL‐12‐induced immunoregulation for the therapy of tumors and infectious diseases. (Cancer Sci 2006; 97: 1236–1241)