Tsuguhide Takeshima

The critical role of type-1 innate and acquired immunity in tumor immunotherapy

The discovery of a large array of tumor antigens has demonstrated that host lymphocytes can indeed recognize and destroy tumor cells as originally proposed in the cancer immunosurveillance hypothesis. Recent reports that led to the cancer immuno‐editing concept also strongly support the immunosurveillance hypothesis, and they further indicate that the host immune system plays a critical role not only in promoting host protection against cancer but also in selecting tumors that can better escape from immune attack. Thus, it is now clear that T cells have the ability to recognize and destroy spontaneously arising tumors. However, the generation of antitumor immunity is often difficult in the tumor‐bearing host because of various negative regulatory mechanisms. Here, we review our recent work on tumor immunotherapy, which utilizes the activation of type‐1 innate and/or acquired immunity as a potent strategy to overcome immunosuppression in the tumor‐bearing host. We have established a variety of tumor therapeutic protocols that aim to activate type‐1 immunity, such as tumor‐vaccine therapy with CpG encapsulated in liposomes, cell therapy using tumor‐specific Th1 cells, and gene therapy using gene‐engineered Th1 cells. We found that CpG encapsulated in liposomes stimulated IL‐12‐producing DC and induced IFN‐γ‐producing NK cells, NKT cells, and tumor‐specific CTL. Th1 cell therapy was also shown to be beneficial for acceleration of APC/Th1 cell‐cell interaction in the DLN, which was critical for inducing tumor‐specific CTL at the tumor site. Therefore, we conclude that the activation of type‐1 innate and acquired immunity is crucial for tumor immunotherapy in order to overcome strong immunosuppression in the tumor‐bearing host.

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.

Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity.

Unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) exhibit potent immunostimulating activity by binding with Toll-like receptor 9 (TLR9) expressed on antigen-presenting cells. Here, we show that CpG-ODN encapsulated in cationic liposomes (CpG-liposomes) improves its incorporation into CD11c+ dendritic cells (DCs) and induces enhanced serum interleukin (IL)-12 levels compared with unmodified CpG-ODN. CpG-liposome potently activated natural killer (NK) cells (84.3%) and NKT cells (48.3%) to produce interferon-γ (IFN-γ), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-γ–producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-γ. In contrast with the NKT cell agonist α-galactosylceramide, which induces both IFN-γ and IL-4 production by NKT cells, CpG-liposome only induced IFN-γ production by NKT cells. Such potent adjuvant activities of CpG-liposome were absent in TLR9-deficient mice, indicating that CpG-liposome was as effective as CpG-ODN in stimulating type 1 innate immunity through TLR9. In addition to TLR9, at least two other factors, IL-12 production by DCs and direct contact between DCs and NK or NKT cells, were essential for inducing type 1 innate immunity by CpG-liposome. Furthermore, ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor cells. These results indicate that CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.

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.

Generation and Targeting of Human Tumor-Specific Tc1 and Th1 Cells Transduced with a Lentivirus Containing a Chimeric Immunoglobulin T-Cell Receptor

CD4+ Th cells, in particular IFN-γ-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3ζ. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-γ in response to CEA+ tumor cell lines but not CEA− tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-γ and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2−/− mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods 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.

Real-time 4-D radiotherapy for lung cancer.

Respiratory motion considerably influences dose distribution, and thus clinical outcomes in radiotherapy for lung cancer. Breath holding, breath coaching, respiratory gating with external surrogates, and mathematical predicting models all have inevitable uncertainty due to the unpredictable variations of internal tumor motion. The amplitude of the same tumor can vary with standard deviations >5 mm occurring in 23% of T1–2N0M0 non‐small cell lung cancers. Residual motion varied 1–6 mm (95th percentile) for the 40% duty cycle of respiratory gating with external surrogates. The 4‐D computed tomography is vulnerable to problems relating to the external surrogates. Real‐time 4‐D radiotherapy (4DRT), where the temporal changes in anatomy during the delivery of radiotherapy are explicitly considered in real time, is emerging as a new method to reduce these known sources of uncertainty. Fluoroscopic, real‐time tumor‐tracking technology using internal fiducial markers near the tumor has ±2 mm accuracy, and has achieved promising clinical results when used with X‐ray therapy. Instantaneous irradiation based on real‐time verification of internal fiducial markers is considered the minimal requisite for real‐time 4DRT of lung cancers at present. Real‐time tracking radiotherapy using gamma rays from positron emitters in tumors is in the preclinical research stage, but has been successful in experiments in small animals. Real‐time tumor tracking via spot‐scanning proton beam therapy has the capability to cure large lung cancers in motion, and is expected to be the next‐generation real‐time 4DRT. (Cancer Sci 2012; 103: 1–6)

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)

Combination tumor immunotherapy with radiotherapy and Th1 cell therapy against murine lung carcinoma

Mice bearing established Lewis lung carcinoma (LLC) expressing model tumor antigen, ovalbumin (OVA) (LLC-OVA) marginally responded to local radiotherapy, but none of the mice was cured. In contrast, treatment of the tumor-bearing mice with intratumoral injection of tumor-specific T helper type 1 (Th1) cells and tumor antigen (OVA) after radiotherapy dramatically prolonged the survival days and induced complete cure of the mice at high frequency (80%). Radiation therapy combined with Th1 cells or OVA alone showed no significant therapeutic activity against LLC-OVA. Such a strong therapeutic activity was not induced by intratumoral injection of Th1 cells plus OVA. Compared with other treatment, radiation therapy combined with Th1 cells and OVA was superior to induce the generation of OVA/H-2b tetramer+ tumor-specific cytotoxic T lymphocyte (CTL) with a strong cytotoxicity against LLC-OVA in draining lymph node (DLN). Moreover, the combined therapy is demonstrated to inhibit the growth of tumor mass, which grew at contralateral side. These results indicated that radiotherapy combined with Th1 cell/vaccine therapy induced a systemic antitumor immunity. These findings suggested that combination therapy with radiotherapy and Th1 cell/vaccine therapy may become a practical strategy for cancer treatment.

Molecular structure of triiodomethane, CHI3, in the gas phase: an electron diffraction study

Abstract The molecular structure of triiodomethane (iodoform, CHI 3 ) has been determined by gas electron diffraction. The structural parameters ( r g /A and ∠ α /°) with the estimated limits of error (3 σ ) are: r (C–H)=1.111 (assumed), r (C–I)=2.145(8), r (I…I)=3.549(2), ∠ICI=111.9(7), ∠ICH=107.0(7). Mean amplitudes, l (C–I) and l (I⋯I), have also been determined. The C–I distance of CHI 3 is equal to that of CI 4 within experimental uncertainties.