krn-7000 and Lung-Neoplasms

Invariant natural killer T (iNKT) cells have adjuvant activity due to their ability to produce large amounts of IFN-gamma, which activates other cells in innate and acquired systems, and orchestrates protective immunity. Based on these adjuvant mechanisms, we developed iNKT cell-targeted adjuvant therapy and carried out a phase I/IIa trial on advanced lung cancer patients. The patient group with increased numbers of IFN-gamma-producing cells showed prolonged survival with a median survival time of 31.9 months. Sixty percent of the patients in this group survived for more than 2 years with only a primary treatment and without tumor progression and metastasis.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Cancer Vaccines; Clinical Trials as Topic; Galactosylceramides; Humans; Immunity, Innate; Immunotherapy; Interferon-gamma; Lung Neoplasms; Natural Killer T-Cells; Neoplasm Metastasis

Topics: Adjuvants, Immunologic; Animals; CD40 Antigens; CD40 Ligand; Cytokines; Dendritic Cells; Galactosylceramides; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Natural Killer T-Cells; Receptors, Antigen, T-Cell; Translational Research, Biomedical

Topics: Animals; Antigens, CD1; Antigens, CD1d; Clinical Trials as Topic; Dendritic Cells; Galactosylceramides; Humans; Immunotherapy; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes

Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to specific glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d-expressing antigen-presenting cells (APCs), thus orchestrating other immune cells to fight tumors. Because of their ability to induce strong antitumor responses activated by αGalCer, iNKT cells have been studied for their application in cancer immunotherapy. In our previous phase I/II trial in non-small cell lung cancer (NSCLC) patients who had completed the standard treatment, we showed a relatively long median survival time without severe treatment-related adverse events. Based on these results, we performed a phase II trial to evaluate clinical responses, safety profiles and immune responses as a second-line treatment for advanced NSCLC.. Patients with advanced or recurrent NSCLC refractory to first-line chemotherapy were eligible. αGalCer-pulsed APCs were intravenously administered four times. Overall survival time was evaluated as the primary endpoint. The safety profile and immune responses after APC injection were also monitored. This study was an open label, single-arm, phase II clinical trial performed at Chiba University Hospital, Japan.. Thirty-five patients were enrolled in this study, of which 32 (91.4%) completed the trial. No severe adverse events related to the treatment were observed. The estimated median survival time of the 35 cases was 21.9 months (95% CI, 14.8 to 26.0). One case (2.9%) showed a partial response, 14 cases (40.0%) remained as stable disease, and 19 cases (54.3%) were evaluated as progressive disease. The geometric mean number of iNKT cells in all cases was significantly decreased and the mean numbers of natural killer (NK) cells, interferon-γ-producing cells in response to αGalCer, and effector CD8+ T cells were significantly increased after the administration of αGalCer-pulsed APCs.. The intravenous administration of αGalCer-pulsed APCs was well-tolerated and was accompanied by prolonged overall survival. These results are encouraging and warrant further evaluation in a randomized phase III trial to demonstrate the survival benefit of this immunotherapy.. UMIN000007321.

Topics: Adult; Aged; Antigen-Presenting Cells; Carcinoma, Non-Small-Cell Lung; Female; Galactosylceramides; Humans; Lung Neoplasms; Male; Middle Aged; Young Adult

We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-β was upregulated in progressive disease cases.

Topics: Adult; Aged; Antigen-Presenting Cells; Bronchi; Carcinoma, Non-Small-Cell Lung; Female; Galactosylceramides; Humans; Immunotherapy; Interferon-gamma; Lung Neoplasms; Male; Middle Aged; Natural Killer T-Cells; Neoplasm Recurrence, Local; Tumor Microenvironment

As the toxicity associated with the α-GalCer-pulsed dendritic cell (DC) therapy could be considered to be negligible, its addition to postoperative adjuvant chemotherapy would be expected to greatly improve the therapeutic effect, and could result in prolonged survival. The aim of the present study is to compare the therapeutic efficacy of alpha-galactosylceramide-pulsed DC therapy in patients who have undergone a complete resection of stage II-IIIA non-small-cell lung cancer (NSCLC) followed by postoperative adjuvant therapy with cisplatin plus vinorelbine, to that in patients who did not receive additional treatment (surgical resection plus postoperative adjuvant chemotherapy only).. Subsequent to the complete resection of NSCLC, followed by the administration of cisplatin plus vinorelbine dual-agent combination adjuvant chemotherapy, patients who satisfy the inclusion criteria will be randomly allocated to either the α-GalCer-pulsed DC immune therapy group, or the standard treatment group. In total, 56 patients will be included in the study. The primary endpoint is recurrence-free survival, and the secondary endpoints are natural killer T-cell-specific immune response, the frequency of toxic effects and safety, and overall survival.. In order to determine the efficacy of α-GalCer-pulsed DC therapy, the present study compares patients with stage II-III NSCLC who underwent complete surgical resection followed by postoperative adjuvant therapy with cisplatin plus vinorelbine, to those who did not receive additional treatment (surgical resection plus postoperative adjuvant chemotherapy only).. UMIN000010386 ( R000012145 ). Registered on 1 April 2013. UMIN-CTR is officially recognized as a registration site which satisfies ICMJE criteria.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Cisplatin; Clinical Protocols; Dendritic Cells; Disease-Free Survival; Female; Galactosylceramides; Humans; Immunotherapy, Adoptive; Japan; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Natural Killer T-Cells; Neoplasm Recurrence, Local; Neoplasm Staging; Pneumonectomy; Research Design; Time Factors; Treatment Outcome; Vinblastine; Vinorelbine; Young Adult

The intravenous administration of α-Galactosylceramide (α-GalCer)-pulsed antigen presenting cells (APCs) is well tolerated and the increased IFN-γ producing cells in the peripheral blood after the treatment appeared to be associated with prolonged survival. An exploratory study protocol was designed with the preoperative administration of α-GalCer-pulsed APCs to clarify the mechanisms of these findings, while especially focusing on the precise tumor site.. Patients with operable advanced lung cancer received an intravenous injection of α-GalCer-pulsed APCs before surgery. The resected lung and tumor infiltrating lymphocytes (TILs) as well as peripheral blood mononuclear cells were collected and the invariant NKT (iNKT) cell-specific immune responses were analyzed.. Four patients completed the study protocol. We observed a significant increase in iNKT cell numbers in the TILs and augmented IFN-γ production by the α-GalCer-stimulated TILs.. The administration of α-GalCer-pulsed APCs successfully induced the dramatic infiltration and activation of iNKT cells in the tumor microenvironment.

Topics: Adenocarcinoma; Aged; Antigen-Presenting Cells; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Galactosylceramides; Humans; Immunotherapy; Lung Neoplasms; Lymph Nodes; Male; Natural Killer T-Cells; Receptors, Antigen, T-Cell; Tumor Microenvironment

To evaluate the safety, immune responses, and antitumor responses after the administration of alpha-galactosylceramide (alphaGalCer) KRN7000-pulsed PBMC cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs), a phase I-II study in patients with non-small cell lung cancer was conducted. Patients with advanced non-small cell lung cancer or recurrent lung cancer refractory to the standard therapy were eligible. alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs (1 x 10(9)/m(2)) were i.v. administered four times. Immune responses were monitored weekly. Twenty-three patients were enrolled in this study and 17 cases (73.9%) completed. No severe adverse event related to the treatment was observed. After the injection of alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs, an increased number of IFN-gamma-producing cells in the peripheral blood were detected in 10 patients (58.8%). Five cases remained as stable disease, and the remaining 12 cases were evaluated as progressive disease. The estimated median survival time (MST) of the 17 cases was 18.6 mo (range, 3.8 to 36.3 mo). Ten patients who displayed increased IFN-gamma-producing cells (> or =2-fold) showed prolonged MST (31.9 mo; range, 14.5 to 36.3 mo) as compared with poor-responder patients (n = 7) MST (9.7 mo; range, 3.8 to 25.0 mo) (log-rank test, p = 0.0015). The administration of alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs was well tolerated and was accompanied by the successful induction of NKT cell-dependent immune responses. The increased IFN-gamma-producing cells that result from alphaGalCer stimulation in PBMCs were significantly associated with prolonged MST. These results are encouraging and warrant further evaluation for survival benefit of this immunotherapy.

Topics: Adult; Aged; Cancer Vaccines; Carcinoma, Non-Small-Cell Lung; Cells, Cultured; Female; Flow Cytometry; Galactosylceramides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Kaplan-Meier Estimate; Leukocytes, Mononuclear; Lung Neoplasms; Male; Middle Aged; Natural Killer T-Cells; Neoplasm Recurrence, Local

Human Valpha24 natural killer T (NKT) cells bearing an invariant Valpha24JalphaQ antigen receptor, the counterpart of murine Valpha14 NKT cells, are activated by a specific ligand, alpha-galactosylceramide (alphaGalCer, KRN7000), in a CD1d-dependent manner. I.v. administration of alphaGalCer-pulsed dendritic cells (DC) induces significant activation and expansion of Valpha14 NKT cells in the lung and resulting potent antitumor activities in mouse tumor metastatic models. We did a phase I dose escalation study with alphaGalCer-pulsed DCs in lung cancer patients.. Patients with advanced non-small cell lung cancer or recurrent lung cancer received i.v. injections of alphaGalCer-pulsed DCs (level 1: 5 x 10(7)/m(2); level 2: 2.5 x 10(8)/m(2); and level 3: 1 x 10(9)/m(2)) to test the safety, feasibility, and clinical response. Immunomonitoring was also done in all completed cases.. Eleven patients were enrolled in this study. No severe adverse events were observed during this study in any patient. After the first and second injection of alphaGalCer-pulsed DCs, dramatic increase in peripheral blood Valpha24 NKT cells was observed in one case and significant responses were seen in two cases receiving the level 3 dose. No patient was found to meet the criteria for partial or complete responses, whereas two cases in the level 3 group remained unchanged for more than a year with good quality of life.. In this clinical trial, alphaGalCer-pulsed DC administration was well tolerated and could be safely done even in patients with advanced disease.

Topics: Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Culture Techniques; Cell Proliferation; Dendritic Cells; Female; Galactosylceramides; Humans; Immunotherapy; Infusions, Intravenous; Killer Cells, Natural; Lung Neoplasms; Male; Middle Aged; Treatment Outcome

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; B7-H1 Antigen; Cell Line, Tumor; Female; Galactosylceramides; Immune Tolerance; Lung Neoplasms; Melanoma, Experimental; Mice; Paclitaxel; Skin Neoplasms; T-Lymphocytes, Cytotoxic

The recognition of α-galactosylceramide (αGC), a high-affinity CD1d antigen, by the invariant Natural Killer T (iNKT) lymphocytes results in potent immunostimulatory responses that have been exploited in advanced cancer patients. Therefore, to improve αGC biological activity, several studies vectorized this agonist in PLGA and/or PEG-based nanoparticles. Despite promising findings, these approaches require several steps, from organic solvent decontamination through extrusion in membrane systems. Using a nano spray dryer, we vectorized αGC into a cationic copolymer (dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate - DBM) in a single step process, free of organic solvent. This methodology allowed the production of stable αGC-vectorized nanoparticles (DBM + αGC) with a more potent biological activity than the free agonist. DBM nanoparticles improved in vivo αGC loading into the CD1d molecule and induced a higher frequency of IFN-γ-expressing iNKT cells. Consequently, mice treated with DBM + αGC presented higher levels of serum IFN-γ than those treated with free agonist. Also, vectorized nanoparticles improved αGC ability to control the growth of murine lung metastatic carcinoma. Thus, this is the first study showing that nano spray dryer technology is a simple and alternative approach to enhance iNKT responses.

Topics: Animals; Cell Line; Cytokines; Desiccation; Drug Carriers; Galactosylceramides; Killer Cells, Natural; Lung Neoplasms; Male; Melanoma, Experimental; Methacrylates; Mice, Inbred C57BL; Nanoparticles; Nanotechnology; Natural Killer T-Cells

Invariant natural killer T (iNKT) cells exhibit potent antitumor effects upon activation by recognizing a specific glycolipid antigen. We previously performed phase I-II clinical studies to utilize iNKT cells using α-galactosylceramide-pulsed dendritic cells and identified leukotriene B4 12-hydroxydehydrogenase (LTB4DH) as a biomarker highly expressed in T cells derived from non-small cell lung cancer (NSCLC) patients who showed prolonged survival in respond to the iNKT cell immunotherapy. Because LTB4DH expression correlated with prolonged survival of NSCLC patients, we considered LTB4DH to play a role in iNKT cell immunotherapy. We herein demonstrate that the overexpression of LTB4DH in CD4

Topics: Alcohol Oxidoreductases; Carcinoma, Non-Small-Cell Lung; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Dendritic Cells; Dinoprostone; Galactosylceramides; Gene Expression Regulation, Neoplastic; Granzymes; Humans; Immunotherapy; Interferon-gamma; K562 Cells; Lung Neoplasms; Natural Killer T-Cells; Perforin; Primary Cell Culture; RNA, Messenger; Signal Transduction; Survival Analysis

The potent antitumor effect of α-galactosylceramide (α-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic α-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained. To optimize α-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of α-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble α-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 α-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase α-GalCer solubility in aqueous solution without limiting its antitumor properties.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Carriers; Galactosylceramides; Liposomes; Lung Neoplasms; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Natural Killer T-Cells

Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.

Topics: Animals; Antigens, Ly; CD4 Antigens; Cell Differentiation; Cytokines; DNA-Binding Proteins; Galactosylceramides; Gene Deletion; Gene Knock-In Techniques; Interferon-gamma; Lung Neoplasms; Melanoma, Experimental; Mice; Natural Killer T-Cells; NK Cell Lectin-Like Receptor Subfamily B; Transcription Factors

For the application of invariant natural killer T (iNKT) cells in cancer therapy, the CD40-CD40L interaction is indispensable in administering alpha-galactosylceramide (αGalCer). We hypothesized that CD40 plays an important role in dendritic cells (DC) pulsed with αGalCer (DCGs) in the treatment of lung metastases.. Wild-type (WT) and CD40(-/-) mice were treated with DCGs isolated from WT or CD40(-/-) mice in a B16F10 lung metastases model and NK and NKT cell activity in lungs and the spleen were examined.. DCG treatment improved WT mice survival but CD40(-/-) hosts received no survival benefit. Conversely, attenuation of a therapeutic effect in mice treated with CD40(-/-) DCGs was not observed. The functional activities of NK and NKT cells in DCG-treated CD40(-/-) mice were partially suppressed.. Host CD40 is essential for DCG treatment to have a therapeutic effect on B16F10 lung metastases.

Topics: Animals; CD40 Antigens; Dendritic Cells; Female; Galactosylceramides; Immunotherapy; Interferon-gamma; Interleukin-12; Killer Cells, Natural; Lung Neoplasms; Melanoma, Experimental; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Neoplasm Transplantation

α-galactosylceramide (GalCer), which is a natural killer T (NKT) cell ligand, has been reported to exert therapeutic effects against cancer in humans and mice. Toll-like receptor (TLR) agonists systemically or locally boost antitumor efficacy in mouse cancer models. In our previous study, the co-administration of GalCer and a TLR agonist synergistically enhanced interferon-γ (IFN-γ) production in mouse splenocytes in vitro and in vivo. The increased IFN-γ production promoted a tumor antigen-specific Th1 response. Therefore, co-treatment with GalCer and a TLR agonist is expected to exert an enhanced antitumor effect. In the present study, we examined the effect of GalCer and lipopolysaccharide (LPS) combination therapy in a mouse lung-metastasis model. GalCer and LPS combination therapy markedly decreased the number of lung metastatic tumor nodes. Co-treatment with GalCer and LPS enhanced the mRNA expression of CXCL9 and CXCL10 in mediastinal lymph nodes (MLNs) and increased the number of CD8+ cells in the MLNs. Furthermore, the depletion of CD8+ T cells canceled the antitumor effect of GalCer and LPS combination therapy. Thus, GalCer and LPS combination therapy significantly enhanced tumor antigen-specific immune responses and suppressed tumor growth in a mouse lung-metastasis model.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CXCL10; Chemokine CXCL9; Disease Models, Animal; Flow Cytometry; Galactosylceramides; Interferon-gamma; Ligands; Lipopolysaccharides; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Spleen; Toll-Like Receptors

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Topics: Animals; Antigens, Ly; Autoantibodies; Cell Transformation, Neoplastic; Chemokines, CXC; Chronic Disease; Colitis; Disease Models, Animal; Female; G(M1) Ganglioside; Galactosylceramides; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Depletion; Melanoma, Experimental; Mice; Mice, Transgenic; Neoplasms; NK Cell Lectin-Like Receptor Subfamily B; Tumor Burden

Alpha-garactosylceramide (GalCer) has been shown to have anti-tumor effect in the basic research and clinical studies. However, anti-tumor effect of GalCer is limited. The administration of GalCer increases the production of IFN-γ which is involved in the suppression of tumor growth. On the other hand, the enhancement of IFN-γ production increases immunosuppressive factors such as nitric oxide. This suppressive action might impair the anti-tumor effect of GalCer. In the present study, we evaluated the anti-tumor effect of GalCer in the absence of inducible nitric oxide synthase (iNOS). In lung metastatic model, the number of tumor nodules in the lung of iNOS-KO mice treated with GalCer was significantly reduced compared with that of WT mice treated with GalCer. Moreover, L-NAME, which is the inhibitor for iNOS, enhanced the anti-tumor effect of GalCer in lung metastatic model. The frequency of CD8+ cells in bronchoalveolar lavage fluid increased in iNOS-KO mice treated with GalCer. The administration of GalCer increased the frequency of myeloid-derived suppressor cells (MDSCs) in the lung from tumor-bearing WT mice, but the increase of MDSCs in the lung was not induced in iNOS-KO mice. The subcutaneous tumor experiments revealed that the administration of GalCer in the absence of iNOS expression significantly enhanced the induction of tumor antigen-specific response. Finally, our results indicated that the inhibition of iNOS expression could enhance the therapeutic efficacy of GalCer via the increase of tumor antigen-specific immune response and the suppression of MDSCs.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Bronchoalveolar Lavage Fluid; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytokines; Enzyme Inhibitors; Galactosylceramides; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Time Factors

Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Administration, Sublingual; Animals; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Dendritic Cells; Female; Galactosylceramides; Immunity, Humoral; Lung Neoplasms; Melanoma, Experimental; Mice, Inbred C57BL; Mouth Mucosa; Natural Killer T-Cells; Neoplasm Transplantation; Ovalbumin; Vaccination

We aimed to investigate whether allogeneic dendritic cells pulsed with alpha-galactosylceramide (DCG) treatment induces activation of Natural Killer T (NKT) cells.. C57BL/6 and BALB/c mice were injected with syngeneic and allogeneic DCG. We then examined NK and NKT cell activity in the lung and spleen and antitumor effect in various lung tumor metastatic models.. While splenic NKT activity was suppressed after allogeneic DCG treatment, allogeneic DCG treatment induced similar antitumor effect as well as lung NKT and NK cell activity compared to syngeneic DCG treatment. Furthermore, allogeneic DCG treatment prolonged survival in B16F10, LLC, Pan02, MethA, and CT26 tumor models in two strains of mice.. This allogeneic DCG treatment could be substituted for syngeneic DCG treatment. The results obtained in the present study suggest that allogeneic DCG treatment may provide a new approach in cancer immunotherapy with NKT cells.

Topics: Animals; Colonic Neoplasms; Female; Galactosylceramides; Interferon-gamma; Lung Neoplasms; Lymphocyte Activation; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Natural Killer T-Cells

Alpha-galactosylceramide (αGC), a lipid antigen present on CD1d molecules, is predicted to have clinical applications as a new class of adjuvant, because αGC strongly activates natural killer T (NKT) cells which produce large amounts of IFN-γ. Here, we incorporated αGC into stearylated octaarginine-modified liposomes (R8-Lip), our original delivery system developed for vaccines, and investigated the effect of nanoparticulation. Unexpectedly, the systemic administered R8-Lip incorporating αGC (αGC/R8-Lip) failed to improve the immune responses mediated by αGC compared with soluble αGC in vivo, although αGC/R8-Lip drastically enhanced αGC presentation on CD1d in antigen presenting cells in vitro. Thus, we optimized the αGC/R8-Lip in vivo to overcome this inverse correlation. In optimization in vivo, we found that size control of liposome and R8-modification were critical for enhancing the production of IFN-γ. The optimization led to the accumulation of αGC/R8-Lip in the spleen and a positive therapeutic effect against highly malignant B16 melanoma cells. The optimized αGC/R8-Lip also enhanced αGC presentation on CD1d in antigen presenting cells and resulted in an expansion in the population of NKT cells. Herein, we show that R8-Lip is a potent delivery system, and size control and R8-modification in liposomal construction are promising techniques for achieving systemic αGC therapy.

Topics: Animals; Antigen-Presenting Cells; Antigens, CD1d; Antineoplastic Agents; Cell Line; Cell Line, Tumor; Female; Galactosylceramides; Interferon-gamma; Liposomes; Liver; Lung; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nanoparticles; Natural Killer T-Cells; Oligopeptides; Spleen

Natural killer dendritic cells (NKDCs) possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT) cells is required for the anti-tumor immune responses that are elicited by α -galactosylceramide ( α -GC) in mice. The rapid and strong expression of interferon- γ by NKDCs after α -GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α -GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α -GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α -GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.

Topics: Animals; Antigens, Neoplasm; Cell Transformation, Neoplastic; Cytokines; Dendritic Cells; Galactosylceramides; Immunity, Cellular; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred C57BL

CD1d-restricted natural killer T (iNKT) cells have been shown to provide adjuvant activity against cancer by producing interferon (IFN)-γ. However, the role of invariant NKT (iNKT) cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the antitumor effect of iNKT cells in the tumor microenvironment by using an intrathoracic murine malignant pleural mesothelioma model that we had previously developed and to provide pleural effusion as a good surrogate of the tumor microenvironment. We found that the number of iNKT cells increased dramatically in the pleural effusion after intrathoracic tumor cell injection at an earlier phase compared with accumulation of CD8 T cells. These iNKT cells showed increased expression of CD25 and increased ratio of cells positive for IFN-γ intracellular staining. iNKT cells sorted from pleural effusion of tumor burden mice produced larger amount of IFN-γ compared with naive mice. Mice pretreated in vivo with anti-CD1d-blocking Ab showed increased amount of pleural effusion and decreased ratio of total and effector-type CD8 T cells as well as decreased intracellular IFN-γ expression of CD8T-cell in the pleural effusion. In vivo administration of α-galactosylceramide (α-GalCer) showed prolonged survival associated with less pleural effusion and increased ratio of IFN-γ-positive iNKT cells and CD8 T cells in the pleural effusion. Therefore, this study suggests that iNKT cells accumulating in the tumor microenvironment play an antitumor effect by producing IFN-γ and enhancing subsequent CD8 T-cell response. Furthermore, in vivo administration of α-GalCer could suppress mesothelioma growth by activating iNKT cells.

Topics: Animals; Antibodies, Blocking; Antigens, CD1d; CD8-Positive T-Lymphocytes; Cell Growth Processes; Cell Line, Tumor; Female; Galactosylceramides; Interferon-gamma; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Mice; Mice, Inbred BALB C; Natural Killer T-Cells; Neoplasm Transplantation; Pleural Effusion, Malignant; Pleural Neoplasms; Tumor Burden; Tumor Microenvironment

Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have proven fruitful using the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). In these different models, the effects of α-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell, B cell, or conventional CD4(+) and CD8(+) T cell activation/regulation as well as immunomodulatory cytokine production. In this study, we showed that γδ T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells (cytokine production and cytotoxic properties) and were required to achieve an optimal α-GalCer-induced immune response. Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the α-GalCer-induced IFN-γ production by γδ T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting their bystander effect on γδ T cells. In line with this, γδ T cells failed to directly recognize the CD1d/α-GalCer complex. We also provided evidence that γδ T cells increase their cytotoxic properties after α-GalCer injection, resulting in an increase in killing of tumor cell targets. Moreover, using cancer models, we demonstrated that γδ T cells were required for an optimal α-GalCer-mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with α-GalCer enhanced the adaptive immune response elicited by OVA, and this effect was strongly mediated by γδ T cells. We conclude that γδ T cells amplify the innate and acquired response to α-GalCer, with possibly important outcomes for the therapeutic effects of this compound.

Topics: Adaptive Immunity; Animals; Antigens, CD1d; Carcinoma, Lewis Lung; Cytotoxicity, Immunologic; Galactosylceramides; Immunity, Innate; Interleukin-12; Interleukin-18; Killer Cells, Natural; Liver; Lung Neoplasms; Lymphocyte Activation; Lymphoma, B-Cell; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Binding; Receptors, Antigen, T-Cell, gamma-delta; Spleen; T-Lymphocytes

Vitamin A compounds are promising for cancer prevention and reducing risk of recurrence. Herein we have evaluated the combination of all-trans-retinoic acid (RA), a vitamin A metabolite, and alpha-galactosylceramide (αGalCer), a lipid immune activator, in Balb/C mice inoculated with syngeneic 4T1 breast tumor cells on reduction in breast tumor growth and lung metastasis. In Balb/c inoculated with the syngenic 4T1 primary tumor, and administered dendritic cells treated with RA + αGalCer, the size of the primary tumor and the number of lung metastatic foci were reduced. When 4T1 cells were introduced into the circulation as a model of hematogenous spread of tumor cells and RA and αCalCer were administered directly to mice without dendritic cells, lung metastatic foci were reduced 70% (P < 0.05), whereas each agent alone resulted in an intermediate decrease. Concomitantly, the expression of matrix metalloproteinases (MMP), membrane type-1 (MT1)-MMP and MMP3, were reduced by RA + αGalCer in lung. MMP3 protein was also reduced in plasma and culture supernatants from RA + αGalCer-treated 4T1 cells. Together, our results provide new evidence that a nutritional-immunological combination of RA + αGalCer may be promising for preventing or slowing the growth of metastatic foci, and suggest reduced MMP production as a possible mechanism.

Topics: Animals; Cell Proliferation; Female; Galactosylceramides; Lung Neoplasms; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 14; Matrix Metalloproteinase 3; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Tretinoin

Both invariant natural killer T (NK T) cells and CD4(+)CD25(+) T regulatory cells (T(regs)) regulate the immune system to maintain homeostasis. In a tumour setting, NK T cells activated by alpha-galactosylceramide (alpha-GalCer) execute anti-tumour activity by secreting cytokines. By contrast, T(regs) intrinsically suppress antigen-specific immune responses and are often found to be elevated in tumour patients. In this study, we have shown that T(regs) regulate NK T cell function negatively in vitro, suggesting a direct interaction between these cell types. In a murine mammary tumour model, we demonstrated that administration of either alpha-GalCer or anti-CD25 antibody alone markedly suppressed tumour formation and pulmonary metastasis, and resulted in an increase in the survival rate up to 44% (from a baseline of 0%). When treatments were combined, depletion of T(regs) boosted the anti-tumour effect of alpha-GalCer, and the survival rate jumped to 85%. Our results imply a potential application of combining T(reg) cell depletion with alpha-GalCer to stimulate NK T cells for cancer therapy.

Topics: Animals; Antibodies, Monoclonal; Dendritic Cells; Disease Models, Animal; Female; Galactosylceramides; Immunity, Cellular; Interferon-gamma; Interleukin-2 Receptor alpha Subunit; Lung Neoplasms; Lymphocyte Activation; Lymphocyte Depletion; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Natural Killer T-Cells; Survival Rate; T-Lymphocytes, Regulatory

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

Topics: Animals; Antigens, CD1d; Cell Line, Tumor; Cytotoxicity Tests, Immunologic; Epitopes, T-Lymphocyte; Fas Ligand Protein; fas Receptor; Galactosylceramides; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Natural Killer T-Cells; Up-Regulation

Our previous report indicated that carbon ion beam irradiation upregulated membrane-associated immunogenic molecules, underlining the potential clinical application of radioimmunotherapy. The antimetastatic efficacy of local combination therapy of carbon ion radiotherapy and immunotherapy was examined by use of an in vivo murine model.. Tumors of mouse squamous cell carcinoma (NR-S1) cells inoculated in the legs of C3H/HeSlc mice were locally irradiated with a single 6-Gy dose of carbon ions (290 MeV/nucleon, 6-cm spread-out Bragg peak). Thirty-six hours after irradiation, α-galactosylceramide-pulsed dendritic cells (DCs) were injected into the leg tumor. We investigated the effects on distant lung metastases by counting the numbers of lung tumor colonies, making pathologic observations, and assessing immunohistochemistry.. The mice with no treatment (control) presented with 168 ± 53.8 metastatic nodules in the lungs, whereas the mice that received the combination therapy of carbon ion irradiation and DCs presented with 2.6 ± 1.9 (P = 0.009) at 2 weeks after irradiation. Immunohistochemistry showed that intracellular adhesion molecule 1, which activates DCs, increased from 6 h to 36 h after irradiation in the local tumors of the carbon ion-irradiated group. The expression of S100A8 in lung tissue, a marker of the lung pre-metastatic phase, was decreased only in the group with a combination of carbon ions and DCs.. The combination of carbon ion radiotherapy with the injection of α-galactosylceramide-pulsed DCs into the primary tumor effectively inhibited distant lung metastases.

Topics: Animals; Calgranulin A; Carbon Radioisotopes; Carcinoma, Squamous Cell; Combined Modality Therapy; Dendritic Cells; Galactosylceramides; Intercellular Adhesion Molecule-1; Lung Neoplasms; Mice; Mice, Inbred C3H; Models, Animal; Neoplasm Proteins; Radioimmunotherapy; Radiotherapy Dosage; Xenograft Model Antitumor Assays

Valpha24NKT cells are lymphocytes expressing both T-cell antigen receptors and NK-cell antigen receptors on their cell surface and are involved in tumor immunity. They exert their antitumor effects after being activated by a specific ligand, alpha-galactosyl ceramide (alpha-GalCer). Malignant pleural effusion, a frequently occurring complication in patients with lung cancer, contains numerous lymphocytes. In the present study, we examined the presence and functions of Valpha24NKT cells in the lymphocytes in pleural effusion in vitro. The subjects were 13 untreated patients with primary lung cancer, who suffered malignant pleural effusion as a complication and who were treated between April 2004 and October 2007 at our hospital. Mononuclear cells were separated from the malignant pleural effusion and incubated with alpha-GalCer and IL-12. The production of IFN-gamma and IL-4 after incubation and the proportion of the Valpha24NKT cells before and after incubation were determined and compared. In the group cultured with alpha-GalCer alone, no significant increase in IFN-gamma production was observed in comparison with the control group. In the group cultured with alpha-GalCer+IL-12, IFN-gamma production increased significantly in comparison with the control group, and the proportion of Valpha24NKT cells increased after incubation. IL-4 production was very much lower than IFN-gamma production. Valpha24NKT cells were present in malignant pleural effusion in patients with lung cancer, but IFN-gamma production did not increase after addition of alpha-GalCer alone. The Valpha24NKT cells were activated by alpha-GalCer in the presence of IL-12. The Valpha24NKT cells in malignant pleural effusion were not activated by alpha-GalCer alone, suggesting that Valpha24NKT cell function is attenuated in malignant pleural effusion.

Topics: Adult; Aged; Antigens, CD; CTLA-4 Antigen; Female; Galactosylceramides; Humans; Interferon-gamma; Interleukin-12; Interleukin-2 Receptor alpha Subunit; Interleukin-4; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Male; Middle Aged; Pleural Effusion, Malignant; Receptors, Antigen, T-Cell, alpha-beta

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Antineoplastic Agents; Cytotoxicity, Immunologic; Dendritic Cells; Female; Galactosylceramides; Immunoglobulin Fragments; Interferon-gamma; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Receptor, ErbB-2; Recombinant Fusion Proteins

Human V alpha 24 natural killer T (NKT) cells bearing an invariant V alpha 24 J alpha Q antigen receptor, the counterpart of murine V alpha 14 NKT cells, are activated by a specific ligand, alpha-galactosylceramide (alpha GalCer; KRN 7000), in a CD 1 d-dependent manner. Previous findings showed that alpha GalCer-pulsed dendritic cells (DCs) exerted a strong antitumor activity in the mouse tumor metastatic models, and intravenous administration of alpha GalCer-pulsed DCs led to V alpha 14 NKT cell expansion in the lung. With these results, we performed a phase I dose escalation study of alpha GalCer-pulsed DCs treatment in patients with lung cancer. Patients with advanced non-small cell lung cancer or recurrent lung cancer received intravenous injection of alpha GalCer pulsed dendritic cell immune therapy to test the safety, feasibility, and clinical response. Immunomonitoring was also performed in all completed cases. Eleven patients were enrolled in this study, None of whom experienced severe adverse events. Peripheral blood V alpha 24 NKT cells dramatically increased after the first and second injection of alpha GalCer pulsed DCs in one patient of level 3. The clinical trial of alpha GalCer-pulsed DCs administration is well tolerated,and this therapy has been carried out safely. To obtain more conclusive findings about immune responses and antitumor responses, a phase I-II study with greater numbers of patients is ongoing.

Topics: Animals; Antineoplastic Agents; Cell Culture Techniques; Dendritic Cells; Galactosylceramides; Humans; Immunotherapy; Killer Cells, Natural; Lung Neoplasms; Mice

Interaction of alpha-galactosylceramide (alpha-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by alpha-GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of alpha-GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN-gamma and IL-4 as well as cell proliferation in response to alpha-GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4+ mononuclear cells incubated with alpha-GalCer and CD11c+ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN-gamma and IL-4 than did those incubated with alpha-GalCer and CD11c+ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for alpha-GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4+ T cells by DCs.

Topics: Animals; Antigens, CD1; Antigens, CD1d; CD11c Antigen; Cytotoxicity, Immunologic; Dendritic Cells; Galactosylceramides; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Immunologic; src Homology Domains; T-Lymphocyte Subsets; Th1 Cells; Th2 Cells

We showed previously that NKT cell-deficient TCR Jalpha18(-/-) mice are more susceptible to methylcholanthrene (MCA)-induced sarcomas, and that normal tumor surveillance can be restored by adoptive transfer of WT liver-derived NKT cells. Liver-derived NKT cells were used in these studies because of their relative abundance in this organ, and it was assumed that they were representative of NKT cells from other sites. We compared NKT cells from liver, thymus, and spleen for their ability to mediate rejection of the sarcoma cell line (MCA-1) in vivo, and found that this was a specialized function of liver-derived NKT cells. Furthermore, when CD4(+) and CD4(-) liver-derived NKT cells were administered separately, MCA-1 rejection was mediated primarily by the CD4(-) fraction. Very similar results were achieved using the B16F10 melanoma metastasis model, which requires NKT cell stimulation with alpha-galactosylceramide. The impaired ability of thymus-derived NKT cells was due, in part, to their production of IL-4, because tumor immunity was clearly enhanced after transfer of IL-4-deficient thymus-derived NKT cells. This is the first study to demonstrate the existence of functionally distinct NKT cell subsets in vivo and may shed light on the long-appreciated paradox that NKT cells function as immunosuppressive cells in some disease models, whereas they promote cell-mediated immunity in others.

Topics: Adoptive Transfer; Animals; CD4 Antigens; Cell Line, Tumor; Galactosylceramides; Immunity, Cellular; Interleukin-4; Killer Cells, Natural; Liver; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Sarcoma, Experimental; T-Lymphocyte Subsets; Thymus Gland

The activation of human Valpha24+Vbeta11+natural killer T cells (NKT) cells (Valpha24 NKT cells) induces effective antitumor responses with secondary immune effects through activation of conventional T cells and natural killer cells. In this study, we attempted to analyze the characteristics of human NKT cells in lung cancer patients. Valpha24 NKT cells stimulated with alpha-GalCer from healthy volunteers exhibited direct cytotoxic activity against two (RERF-LC-OK and PC-3) of seven human lung cancer cell lines studied. Cytotoxicity by Valpha24 NKT cells against human lung cancer cells was dependent on the perforin pathway and independent of Fas/FasL pathway. Intracellular adhesion molecule (ICAM)-1 expression on tumor cells was clearly associated with the cytotoxicity of Valpha24 NKT cells. On the other hand, the proportion of Valpha24 NKT cells in the patients with lung cancer was lower than that in the healthy volunteers. Furthermore, the proliferative response of Valpha24 NKT cells to alpha-GalCer was significantly lower in the peripheral blood mononuclear cells in the patients with lung cancer. Addition of granulocyte colony-stimulating factor moderately restored the low proliferative response of Valpha24 NKT cells in the patients with lung cancer, however the percentage by which the response was restored in these patients was still lower than the natural response in healthy volunteers. These results suggest that Valpha24 NKT cells may play a pivotal role or the antitumor response in lung cancer.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, CD; Antigens, CD1; Antigens, CD1d; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cytokines; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Female; Galactosylceramides; Humans; Killer Cells, Natural; Leukocytes, Mononuclear; Lung Neoplasms; Lymphocyte Activation; Lymphocyte Subsets; Male; Membrane Glycoproteins; Middle Aged; Perforin; Pore Forming Cytotoxic Proteins; Receptors, Antigen, T-Cell, alpha-beta

alpha-Galactosylceramide (alphaGalCer) stimulates NKT cells and has antitumor activity in mice. Murine NKT cells may directly kill tumor cells and induce NK cell cytotoxicity, but the mechanisms are not well defined. Newly developed human CD1d/alphaGalCer tetrameric complexes were used to obtain highly purified human alphaGalCer-reactive NKT cell lines (>99%), and the mechanisms of NKT cell cytotoxicity and activation of NK cells were investigated. Human NKT cells were cytotoxic against CD1d(-) neuroblastoma cells only when they were rendered CD1d(+) by transfection and pulsed with alphaGalCer. Four other CD1d(-) tumor cell lines of diverse origin were resistant to NKT cells, whereas Jurkat and U937 leukemia cell lines, which are constitutively CD1d(+), were killed. Killing of the latter was greatly augmented in the presence of alphaGalCer. Upon human CD1d/alphaGalCer recognition, NKT cells induced potent cytotoxicity of NK cells against CD1d(-) neuroblastoma cell lines that were not killed directly by NKT cells. NK cell activation depended upon NKT cell production of IL-2, and was enhanced by secretion of IFN-gamma. These data demonstrate that cytotoxicity of human NKT cells can be CD1d and ligand dependent, and that TCR-stimulated NKT cells produce IL-2 that is required to induce NK cell cytotoxicity. Thus, NKT cells can mediate potent antitumor activity both directly by targeting CD1d and indirectly by activating NK cells.

Topics: Adjuvants, Immunologic; Adult; Animals; Antigens, CD1; Antigens, CD1d; Antineoplastic Agents; Carcinoma, Small Cell; Cell Line; Cytokines; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Galactosylceramides; HeLa Cells; HL-60 Cells; Humans; Immunomagnetic Separation; Immunophenotyping; Interferon-gamma; Interleukin-2; Jurkat Cells; Killer Cells, Natural; Ligands; Lung Neoplasms; Lymphocyte Activation; Melanoma; Mice; Neuroblastoma; Receptors, Antigen, T-Cell; Recombinant Proteins; T-Lymphocyte Subsets; Transfection; Tumor Cells, Cultured; U937 Cells

Liver metastasis of primary tumor is a clinically major problem. KRN7000, an alpha-galactosylceramide, significantly augments natural killer (NK) activity of spleen cells and shows strong antitumor activity in mice with lung metastasis of melanoma B16 cells. To test whether KRN7000 has an antitumor activity in mice with hepatic metastasis of tumors, we examined the effect of KRN7000 on NK activity of hepatic mononuclear cells (MNC) and the antitumor activity in mice with liver metastasis of EL-4 cells. The in vivo administration of KRN7000 significantly augmented NK activity of hepatic MNC and inhibited tumor growth of EL-4 cells in the liver more markedly than chemotherapeutic agents, leading to a relatively high rate of cured mice. In addition, it appeared that the KRN7000 treatment is effective in mice with established EL-4 tumors. Moreover, we found that KRN7000 can produce significant amounts of interleukin 2 (IL-2), IL-4, IL-12, and interferon-gamma in a dose-dependent manner. These results suggest that KRN7000 will be useful for the treatment of cancer liver metastasis.

Topics: Animals; Antineoplastic Agents; Cytokines; Female; Galactosylceramides; Interferon-gamma; Interleukin-12; Interleukin-2; Interleukin-4; Liver Neoplasms, Experimental; Lung Neoplasms; Melanoma; Mice