Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy.

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Key role for neutrophils in radiation-induced antitumor immune responses: Potentiation with G-CSF.

Radiation therapy (RT), a major modality for treating localized tumors, can induce tumor regression outside the radiation field through an abscopal effect that is thought to involve the immune system. Our studies were designed to understand the early immunological effects of RT in the tumor microenvironment using several syngeneic mouse tumor models. We observed that RT induced sterile inflammation with a rapid and transient infiltration of CD11b+Gr-1high+ neutrophils into the tumors. RT-recruited tumor-associated neutrophils (RT-Ns) exhibited an increased production of reactive oxygen species and induced apoptosis of tumor cells. Tumor infiltration of RT-Ns resulted in sterile inflammation and, eventually, the activation of tumor-specific cytotoxic T cells, their recruitment into the tumor site, and tumor regression. Finally, the concurrent administration of granulocyte colony-stimulating factor (G-CSF) enhanced RT-mediated antitumor activity by activating RT-Ns. Our results suggest that the combination of RT and G-CSF should be further evaluated in preclinical and clinical settings.

Rationale and evidence to combine radiation therapy and immunotherapy for cancer treatment.

Cancer immunotherapy exploits the immune system's ability to differentiate between tumor target cells and host cells. Except for limited success against a few tumor types, most immunotherapies have not achieved the desired clinical efficacy until recently. The field of cancer immunotherapy has flourished with a variety of new agents for clinical use, and remarkable progress has been made in the design of effective immunotherapeutic regimens. Furthermore, the therapeutic outcome of these novel agents is enhanced when combined with conventional cancer treatment modalities including radiotherapy (RT). An increasing number of studies have demonstrated the abscopal effect, an immunologic response occurring in cancer sites distant from irradiated areas. The present work reviews studies on the combination between RT and immunotherapy to induce synergistic and abscopal effects involved in cancer immunomodulation. Further insight into the complex interactions between the immune system and cancer cells in the tumor microenvironment, and their modulation by RT, may reveal the abscopal effect as a clinically relevant and reproducible event leading to improved cancer outcome.

Restoration of thymic T-cell development by bone marrow transplantation in mouse radiation lymphomagenesis.

Fractionated total body irradiation (TBI) with X-rays induces thymic lymphoma/leukemia (TL) in C57BL/6 mice. Radiation-induced mouse TL (RITL) can be prevented by bone marrow transplantation (BMT) of unirradiated BM cells. However, the mechanisms underlying the prevention of RITL with BMT remain unclear. Here, we show that BMT restores thymic T-cell differentiation in mice subjected to TBI. TBI (four times of 1.8 Gy X-rays weekly) was conducted with C57BL/6 mice. BMT was performed immediately after the last irradiation of TBI in mice by transplantation of BM cells isolated from enhanced green fluorescence protein (eGFP) transgenic mice. Thymic cell numbers were drastically decreased in TBI and TBI + BMT mice compared to those in non-irradiated mice. Flow cytometry showed a dramatic decrease in double negative (DN, CD4-CD8-) thymocytes, especially DN2 (CD25+CD44+) and DN3 (CD25+CD44-) subpopulations, in the TBI mice on Day 10 after the last irradiation. In contrast, the DN2 and DN3 populations were recovered in TBI + BMT mice. Interestingly, these restored DN2 and DN3 cells mainly differentiated from eGFP-negative recipient cells but not from eGFP-positive donor cells, suggesting that transplanted BM cells may interact with recipient cells to restore thymic T-cell development in the RITL model. Taken together, our findings highlight the significance of restoring thymic T-cell differentiation by BMT in RITL prevention.

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.

Th Balance-Related Host Genetic Background Affects the Therapeutic Effects of Combining Carbon-Ion Radiation Therapy With Dendritic Cell Immunotherapy.

PURPOSE: The goal of this study is to clarify the underlying mechanisms of metastasis suppression by carbon-ion radiotherapy combined with immature dendritic cell immunotherapy (CiDC), which was shown previously to suppress pulmonary metastasis in an NR-S1-bearing C3H/He mouse model. METHODS AND MATERIALS: Mouse carcinoma cell lines (LLC, LM8, Colon-26, and Colon-26MGS) were grafted into the right hind paw of syngeneic mice (C57BL/6J, C3H/He, and BALB/c). Seven days later, the tumors on the mice were locally irradiated with carbon ions (290 MeV/n, 6 cm spread-out Bragg peak, 1 or 2 Gy). At 1.5 days after irradiation, bone marrow-derived immature dendritic cells (iDCs) were administrated intravenously into a subset of the mice. The number of lung metastases was evaluated within 3 weeks after irradiation. In vitro-cultured cancer cells were irradiated with carbon ions (290 MeV/n, mono-energy, LET approximately 70-80 keV/µm), and then cocultured with iDCs for 3 days to determine the DC maturation. RESULTS: CiDC effectively repressed distant lung metastases in cancer cell (LLC and LM8)-bearing C57BL/6J and C3H/He mouse models. However, Colon-26- and Colon-26MGS-bearing BALB/c models did not show enhancement of metastasis suppression by combination treatment. This result was evaluated further by comparing LM8-bearing C3H/He and LLC-bearing C57BL/6J models with a Colon-26-bearing BALB/c model. In vitro coculture assays demonstrated that all irradiated cell lines were able to activate C3H/He- or C57BL/6J-derived iDCs into mature DCs, but not BALB/c-derived iDCs. CONCLUSIONS: The genetic background of the host could have a strong effect on the potency of combination therapy. Future animal and clinical testing should evaluate host genetic factors when evaluating treatment efficacy.

Radiation-induced cancer cell repopulation: a possible mechanism implied by experiments using transplantable mouse-derived sarcoma cell line.

PURPOSE: Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. MATERIALS AND METHODS: A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. RESULTS: cDNA analysis showed that cyclin-dependent kinase inhibitors, p16 and p57 were down-regulated; 14.8- and 12.0-fold, respectively for the tolerant clone. Matrix metalloproteinase 3 and 13 were up-regulated; 22.5- and 25.8-fold, respectively. Transplantation ratio was 100% (5/5) for the tolerant clone whereas it was 40% (2/5) for the parent. Under light microscope, the mean mitotic cell number was 4.0+/-3.9 for the parent, and 12.8+/-3.4 for the tolerant clone (p<0.01, Student's t-test). CONCLUSIONS: This study implies that repopulation is not a temporary reaction to irradiation. It is caused probably by "clonal" gene-expression changes, though it remains unknown whether the changes are attributable to tolerant cell selection or to gene mutation/modification.

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant.

Redox-active substances and their combinations, such as of quinone/ascorbate and in particular menadione/ascorbate (M/A; also named Apatone®), attract attention with their unusual ability to kill cancer cells without affecting the viability of normal cells as well as with the synergistic anticancer effect of both molecules. So far, the primary mechanism of M/A-mediated anticancer effects has not been linked to the mitochondria. The aim of our study was to clarify whether this "combination drug" affects mitochondrial functionality specifically in cancer cells. Studies were conducted on cancer cells (Jurkat, Colon26, and MCF7) and normal cells (normal lymphocytes, FHC, and MCF10A), treated with different concentrations of menadione, ascorbate, and/or their combination (2/200, 3/300, 5/500, 10/1000, and 20/2000 µM/µM of M/A). M/A exhibited highly specific and synergistic suppression on cancer cell growth but without adversely affecting the viability of normal cells at pharmacologically attainable concentrations. In M/A-treated cancer cells, the cytostatic/cytotoxic effect is accompanied by (i) extremely high production of mitochondrial superoxide (up to 15-fold over the control level), (ii) a significant decrease of mitochondrial membrane potential, (iii) a decrease of the steady-state levels of ATP, succinate, NADH, and NAD+, and (iv) a decreased expression of programed cell death ligand 1 (PD-L1)-one of the major immune checkpoints. These effects were dose dependent. The inhibition of NQO1 by dicoumarol increased mitochondrial superoxide and sensitized cancer cells to M/A. In normal cells, M/A induced relatively low and dose-independent increase of mitochondrial superoxide and mild oxidative stress, which seems to be well tolerated. These data suggest that all anticancer effects of M/A result from a specific mechanism, tightly connected to the mitochondria of cancer cells. At low/tolerable doses of M/A (1/100-3/300 µM/µM) attainable in cancer by oral and parenteral administration, M/A sensitized cancer cells to conventional anticancer drugs, exhibiting synergistic or additive cytotoxicity accompanied by impressive induction of apoptosis. Combinations of M/A with 13 anticancer drugs were investigated (ABT-737, barasertib, bleomycin, BEZ-235, bortezomib, cisplatin, everolimus, lomustine, lonafarnib, MG-132, MLN-2238, palbociclib, and PI-103). Low/tolerable doses of M/A did not induce irreversible cytotoxicity in cancer cells but did cause irreversible metabolic changes, including: (i) a decrease of succinate and NADH, (ii) depolarization of the mitochondrial membrane, and (iii) overproduction of superoxide in the mitochondria of cancer cells only. In addition, M/A suppressed tumor growth in vivo after oral administration in mice with melanoma and the drug downregulated PD-L1 in melanoma cells. Experimental data suggest a great potential for beneficial anticancer effects of M/A through increasing the sensitivity of cancer cells to conventional anticancer therapy, as well as to the immune system, while sparing normal cells. We hypothesize that M/A-mediated anticancer effects are triggered by redox cycling of both substances, specifically within dysfunctional mitochondria. M/A may also have a beneficial effect on the immune system, making cancer cells "visible" and more vulnerable to the native immune response.

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.

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-2(b) 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.

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-gamma (IFN-gamma), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-gamma-producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-gamma. In contrast with the NKT cell agonist alpha-galactosylceramide, which induces both IFN-gamma and IL-4 production by NKT cells, CpG-liposome only induced IFN-gamma 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.

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-gamma-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 CD3zeta. 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-gamma in response to CEA+ tumor cell lines but not CEA- tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-gamma 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.

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. We demonstrate that Th1 cells transduced with the TCR-alpha and -beta genes from the I-A(d)-restricted ovalbumin (OVA)(323-339)-specific T-cell clone DO11.10 produce IFN-gamma but not interleukin-4 in response to stimulation with OVA(323-339) 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.

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. Here, we reevaluated a critical role of NKT and NK cells at early time after alpha-GalCer administration. Intracellular staining experiments demonstrated that NKT cells were the earliest source of both IL-4 and IFN-gamma production after alpha-GalCer administration in vivo. However, these alpha-GalCer-activated NKT cells exhibited no significant natural killing activity. In contrast, isolated NK1.1+CD3- classical NK cells exhibited greatly enhanced natural killing activity 6 h after alpha-GalCer administration. NKT cells, however, exhibited a strong cytotoxicity when they were activated and expanded with alpha-GalCer plus IL-2 in vitro. These results indicated that NKT cells act as regulatory cells via production of cytokines for activation of NK cell-mediated cytotoxicity in vivo at early phase after alpha-GalCer administration. Thus, NK cells rather than NKT cells may be a crucial early activated killer induced by alpha-GalCer in vivo.

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.

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 immunoediting 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 immunosup-pression 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 encapsulat-ed 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-gamma-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.

Successful elimination of memory-type CD8+ T cell subsets by the administration of anti-Gr-1 monoclonal antibody in vivo.

During investigating the expression of Gr-1 antigen on various subsets of mouse spleen cells, we found that Gr-1 was expressed on memory-type CD8(+)CD44(high)CD62L(high) T cells in addition to granulocytes. Intraperitoneal administration of anti-Gr-1 mAb caused almost complete elimination of Ly-6C(+) memory-type CD8(+) T cells as well as Ly-6G(+) granulocytes. Anti-Gr-1 mAb-treated mouse spleen cells exhibited greatly reduced IFN-gamma production in response to anti-CD3 mAb both in vitro and in vivo. This reduced cytokine production appeared to be derived from elimination of IFN-gamma-producing Gr-1(+)CD8(+) T cells. Indeed, CD8(+) T cells with IFN-gamma-producing activity and cytotoxicity were generated from isolated Gr-1(+)CD8(+) cells but not from Gr-1(-)CD8(+) T cells. We also demonstrated that therapeutic effect of MBL-2 tumor-immunized spleen cells was greatly reduced by anti-Gr-1 mAb-treatment. Thus, we initially demonstrated that anti-Gr-1 mAb might become a good tool to investigate a precise role for memory-type CD8(+) T cells in vivo.

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. Intracellular staining analysis demonstrated that a high frequency of IFN-gamma-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 chemoimmunotherapy 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.

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. To elucidate this issue, we designed an acute GVHD model using (B6 x D2) F1 recipient mice transferred with spleen cells derived from either wild-type or TNF-alpha(-/-) C57BL/6 mice. Surprisingly, we found that spleen cells from TNF-alpha(-/-) mice induce more severe graft versus host reaction (GVHR) than wild-type spleen cells upon transfer into B6D2F1 mice. Transplantation of TNF-alpha(-/-) mouse spleen cells was associated with enhanced anti-host CTL generation and augmented deletion of host cells. Moreover, mice receiving TNF-alpha(-/-) cells showed significantly higher levels of serum IFN-gamma, which was mainly produced by donor CD8+ T cells. We also demonstrated that TNF-alpha deficiency in donor spleen cells caused a marked elevation of TNF-alpha producing capacity by LPS-stimulated host macrophages. Such enhanced GVHR was completely prevented by using TNF-alpha(-/-)IFN-gamma(-/-) splenic cells. Our findings demonstrate, for the first time, that donor-derived TNF-alpha suppress GVHR by inhibiting IFN-gamma-dependent donor type-1 immunity which is essential for host TNF-alpha elevation.

STAT6-mediated signaling in Th2-dependent allergic asthma: critical role for the development of eosinophilia, airway hyper-responsiveness and mucus hypersecretion, distinct from its role in Th2 differentiation.

When wild-type BALB/c mice were transferred with OVA-specific Th2 cells followed by OVA inhalation, a severe eosinophilia, mucus hypersecretion and airway hyper-responsiveness (AHR) was induced in parallel with a marked elevation of IL-4, IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF). However, neither eosinophilia, AHR nor mucus hypersecretion was induced in Th2 cell-transferred STAT6-/- mice. The failure of eosinophilia was not due to the defect of Th2 cytokine production in BALF of STAT6-/- mice transferred with Th2 cells, but because of the defect of STAT6-dependent eotaxin production. Indeed, intranasal administration of eotaxin reconstituted pulmonary eosinophilia but not AHR and mucus hypersecretion in OVA-inhalated STAT6-/- mice. These results initially provided direct evidence that STAT6-dependent eotaxin production is essential for pulmonary eosinophilia. We also dissociated the role of STAT6 for eosinophilia from that for AHR and mucus hypersecretion. Thus, STAT6 also plays a critical role at late phase of Th2-dependent allergy induction.