Tumor-specific T cells which form clusters with dendritic cells and tumor cells and deliver macrophage-activating factors

T cells prepared from tumor (Meth A)-bearing mice were cocultured with homologous tumor cells and splenic dendritic cells to enrich tumor-specific T cells by the separation of clusters. T blasts generated from clusters were capable of inhibiting the in vivo tumor cell growth. The culture supernatant of clustering cells (CLSN) was effective in activating macrophages (M phi) to be cytostatic and cytocidal against tumor cells. Moreover, it was found that CLSN contains at least 3 distinct factors; one was identified as interferon-gamma (IFN-gamma), and the others are so far unidentified, but one acts synergistically with IFN-gamma, possibly as the second signal, and the other cooperates with lipopolysaccharide but not with IFN-gamma. We propose that the tumor-specific T cells secrete soluble mediators which cooperate with each other in M phi activation against tumor cells.     

Interferon-γ-producing Tumor Induces Host Tumor-specific T Cell Responses

We investigated the mechanism of host immune responses against two interferon-γ (IFN-γ) gene-transduced tumors, plasmacytoma MOPC104E(Muγ) and mammary cancer SC115(Kγ), which originally had weak immunogenicity. Both IFN-γ-producing tumor cells had reduced tumorigenicity and were rejected by syngeneic mice. The rejection was completely blocked by in vivo treatment with anti-CD8 or anti-IFN-γ monoclonal antibodies. While anti-CD4 monoclonal antibody also blocked the rejection of SC115(Kγ), it enhanced the initial tumor growth of MOPC104E(Muγ). Specific protection against subsequent challenge with the respective parental tumor cells was demonstrated in mice which rejected the IFN-γ-producing tumor cells. Cultured lymphocytes derived from immunized mouse spleens had cytotoxic T cell activity against parental tumor cells, as well as against cells that produced IFN-γ, These findings indicate that the antitumor effects are mediated by cytotoxic T cells and, partly, by helper T cells, and that locally secreted IFN-γ plays an important role in generating these effector cells.     

Cytokines Required for Induction of Histocompatibility Leukocyte Antigen-class I-restricted and Tumor-specific Cytotoxic T Lymphocytes by a SART1-derived Peptide

Although there have been several reports on peptides of human tumor-rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SARTI^690–698 peptide, which is capable of inducing HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen-presenting cells (APCs) with interferon (IFN)-γ, or to some extent with IFN-α, but not with any of the other cytokines tested, augmented the peptide-induced CTL activity in HLA-A24 heterozygotes, but not in HLA-A24 homozygotes. This IFN-γ -mediated augmentation was inhibited by either interleukin (IL)-4 or IL-10. IL-2 alone in culture, along with weekly stimulation by peptide-pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL-2-mediated activation of CTLs was inhibited by the addition of IFN-γ, IL-4, or IL-10 to the IL-2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) were required as APCs. These results indicate that IFN-γ and IL-2 are important in the activation of APCs and CTLs, respectively, while GM-CSF and IL-4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor-specific immunity, and also for the development of peptide-based specific immunotherapy.     

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.     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.     

Natural Human Interferon-γ Derived from Lipopolysaccharide-stimulated Human Myelomonocytic HBL-38 Cells

A human myelomonocytic cell line, HBL-38 cells, propagated in vivo , spontaneously produced interferon (IFN)-γ and IFN-α. Whereas hemmagglutinating virus of Japan (HVJ) enhanced the production of IFN-α, bacterial lipopolysaccharide (LPS) markedly enhanced the production of IFN-γ. LPS could be replaced with lipid A. Furthermore, the enahancement of production of IFN-γ by LPS was completely abolished by polymixin B. IFN-γ derived from LPS-stimulated HBL-38 cells was purified to homogeneity and characterized. The apparent molecular weight, subspecies composition, amino acid sequence and glycosylated sites were in agreement with those of the product of normal human peripheral blood lymphocytes (PBL). These results indicate that the myelomonocytic HBL-38 cells, not a T-cell line, can also produce IFN-γ identical to the product of normal human PBL.     

Enhancement of antiproliferative activity of interferons by RNA interference-mediated silencing of SOCS gene expression in tumor cells

The suppressor of cytokine signaling (SOCS) proteins, negative regulators of interferon (IFN)-induced signaling pathways, is involved in IFN resistance of tumor cells. To improve the growth inhibitory effect of IFN-β and IFN-γ on a murine melanoma cell line, B16-BL6, and a murine colon carcinoma cell line, Colon26 cells, SOCS-1 and SOCS-3 gene expression in tumor cells was downregulated by transfection of plasmid DNA expressing short hairpin RNA targeting one of these genes (pshSOCS-1 and pshSOCS-3, respectively). Transfection of pshSOCS-1 significantly increased the antiproliferative effect of IFN-γ on B16-BL6 cells. However, any other combinations of plasmids and IFN had little effect on the growth of B16-BL6 cells. In addition, transfection of pshSOCS-1 and pshSOCS-3 produced little improvement in the effect of IFN on Colon26 cells. To understand the mechanism underlining these findings, the level of SOCS gene expression was measured by real time polymerase chain reaction. Addition of IFN-γ greatly increased the SOCS-1 mRNA expression in B16-BL6 cells. Taking into account the synergistic effect of pshSOCS-1 and IFN-γ on the growth of B16-BL6 cells, these findings suggest that IFN-γ-induced high SOCS-1 gene expression in B16-BL6 cells significantly interferes with the antiproliferative effect of IFN-γ. These results indicate that silencing SOCS gene expression can be an effective strategy to enhance the antitumor effect of IFN under conditions in which the SOCS gene expression is upregulated by IFN. ( Cancer Sci 2008; 99: 1650–1655)     

Enhancing Effect of an Inhibitor of Nitric Oxide Synthesis on Bacillus Calmette-Guérin-induced Macrophage Cytotoxicity against Murine Bladder Cancer Cell Line MBT-2 in vitro

We studied the effect of an inhibitor of nitric oxide (NO) synthesis, N^G-monomethyl-L-arginine (LNMMA), on the Bacillus Calmette-Guérin (BCG)-induced antitumor activity of murine peritoneal exudate cells (PEC) against murine bladder cancer cell line MBT-2 in vitro. L-NMMA enhanced BCG-induced cytotoxic activity of PEC, as well as interferon (IFN)-γ and tumor necrosis factor (TNF)-α production. The L-NMMA-induced enhancement was due to the prolonged survival of BCG in macrophages, because no enhancement of cytotoxicity was observed and neither IFN-γ nor TNF-α production was significantly enhanced by killed BCG. Anti-TNF-α antibody (Ab) and anti-IFN-γAb reduced the L-NMMA-induced enhancement of the cytotoxicity. The depletion of T cells from PEC reduced the production of both IFN-γ and TNF-α, as well as the enhancement of cytotoxicity induced by viable BCG plus L-NMMA. These results suggest that L-NMMA has an enhancing effect on BCG-induced macrophage cytotoxicity and the enhancement is partially mediated by T cells and their soluble products. Accordingly, NO inhibitor should be a valuable adjunct to BCG immunotherapy for bladder cancer.     

Murine Asialo GM1+CD8+ T Cells as Novel Interleukin-12-responsive Killer T Cell Precursors

Freshly isolated CD8⁺ T cells, but not CD4⁺ T cells, contained 20–30% of asialo GM1⁺ (ASGM1⁺) T cells which were distinct from ASGM1⁺NK1.1⁺ natural killer cells. This novel ASGM1⁺CD8⁺ T cell subpopulation showed a strong proliferative response to interlenkin-12 (IL-12) in the presence of IL-2. Culture of ASGM1⁺CD8⁺ T cells with IL-12 plus IL-2 allowed the generation of anomalous killer T cells concomitantly with the accumulation of cytolytic molecules. Moreover, ASGM1⁺CD8⁺ T cells produced high levels of interferon-γ (IFN-γ), but not IL-4, upon stimulation with IL-12 plus IL-2. Such immune responses were not observed in ASGM1⁻ CD8⁺ T cell snbpopulations constituting the majority of CD8⁺ T cells. These results demonstrated that ASGM1⁺CD8⁺ T cells are a novel subpopulation of IL-12-responsive and IFN-γ-producing killer T cell precursors.     

Interferon-γ-inducing Factor Gene Transfection into Lewis Lung Carcinoma Cells Reduces Tumorigenicity in vivo

To investigate the immunoregulatory effect of murine mterferon-γ-inducing factor (mIGIF), we transfected Lewis lung carcinoma (IXC) cells with a mammalian expression vector containing the mIGIF complementary DNA. The culture medium of the transfectant cells stimulated interferon-γ (IFN-γ) production by spleen cells in vitro in the presence of anti-CD3 antibody and markedly potentiated the effect of interleukin-12 (IL-12) on IFN-γ production by spleen cells. mIGIF transfectant cells showed reduction of tumorigenicity and induction of an in vivo immimo-protective effect against the parental LLC cells. To examine the combined effect of systemic administration of recomhinant IL-12 (rIL-12) and local mIGIF on the tumorigenicity, mice were challenged with LLC or transfectant cells on day 0, and the tumor-bearing mice were injected with 50 ng of rIL-12 intraperitoneally from day 7 to 11. Systemic rIL-12 showed an anti-tumor effect. However, mIGIF gene expression did not potentiate this effect of systemic rIL-12 in vivo.     

Allogeneic tumor-dendritic cell fusion vaccines for generation of broad prostate cancer T-cell responses

Antigen-loaded dendritic cells (DC) are considered potent stimulators of protective immunity. In some studies, DC hybridized with tumor cells have shown promising antitumor responses in mice as well as in humans. A practical drawback of this approach is the limited availability of autologous tumor samples. We investigated the possibility of hybridizing allogeneic prostate cancer cells with human-monocyte-derived DC, and thereby combine the wide repertoire of antigens from the tumor cells with the costimulatory features of the autologous antigen-presenting cells. Three tumor cell lines were used for hybridization using polyethylene glycol (PEG). We show that all three cell lines formed hybrids with DC to the same extent and without significant loss of viability. Restimulation of autologous T cells with these hybrids resulted in generation of tumor-specific IFNγ-producing T cells with all three tumor cell lines. Similar tumor specificity could not be obtained if T cells were stimulated using a mixture of non-PEG-fused tumor cells and DC. Moreover, these T cells were capable of specific recognition of other tumor cells of prostate cancer origin and autologous DC pulsed with lysate from these prostate cancer cell lines, while a panel of unrelated EBV-transformed B cell lines were not recognized. These results are strongly indicative of the true tumor specificity of these T cells. Our results suggest that DC hybridized with allogeneic prostate cancer cell lines are potent stimulators of a broad prostate-tumor-specific response.     

Suppression by Interleukin 4 of Activation of Human Blood Monocytes to the Tumoricidal State

The effect of interleukin 4 (IL-4) on expression of antitumor activity of blood monocytes purified by counter-flow centrifugal elutriation from healthy donors was examined. The blood monocytes were incubated for 24 h in medium with lipopolysaccharide, interferon γ (IFN-γ) or desmethyl muramyl dipeptide (norMDP) or with IFN-γ and norMDP in the presence of IL-4, and then their tumoricidal activity was assayed by measuring ¹²⁵IUdR release from human melanoma (A375) cells. Irrespective of activation stimulus, addition of IL-4 to cultures of monocytes and activators resulted in dose-dependent suppression of the tumoricidal activity of monocytes against parent A375 melanoma cells and the variant cells, A375-R resistant to IL-1 and tumor necrosis factor α. IL-4 suppressed the early induction phase of monocyte activation. Rabbit anti-IL-4 antisera completely blocked the IL-4-mediated suppression of monocyte activation to the tumoricidal state. These findings suggest that IL-4 is important in vivo in down-regulation of anti-tumor expression of monocytes.     

Successful Induction of Tumor-specific Cytotoxic T Lymphocytes from Patients with Non-small Cell Lung Cancer Using CD80-transfected Autologous Tumor Cells

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80-transfected tumor cells as stimulators of the in vitro induction of autologous tumor-specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non-small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80-transfected tumor cells (CD80-AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non-transfected tumor cells (AT). AT-stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80-AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT-stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)-class I-restricted cytokine production in response to AT, while the MHC-class I-restricted responses were found in CD80-AT-stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.     

Generation and Characterization of Lymphokine-activated Killer Cells against Fresh Human Leukemia Cells

Lymphokine-activated killer (LAK) cells generated from 15 acute leukemia patients in remission showed significant levels of cytotoxicity against Daudi 1A4, a natural killer-resistant cell line. This indicates that lymphocytes of leukemia patients in remission could respond to interleukin-2 to generate conventional LAK cells. However, LAK cells caused lysis of autologous leukemia cells at considerably lower levels in seven out of the 15 patients, with the exception of one case (48.6% cytolysis). None of the remaining eight patients exhibited LAK activity against autologous leukemia cells. On the other hand, patients' LAK could lyse allogeneic leukemia cells including those resistant to autologous LAK. Thus, patients' LAK seem not to be defective in lysis of leukemia cells. In the cold target competition analysis, the binding of patients' LAK to leukemia cells could be inhibited by autologous and allogeneic leukemia cell competitors, implying that almost all leukemia cells could be recognized by patients' LAK. Most LAK cells from normal donors showed significant lysis of allogeneic leukemia cells, but some leukemia cells were found to be resistant to lysis. LAK cells against both leukemia cells and Daudi 1A4 were phenotypically heterogenous, and were predominantly observed in the T3⁻ fraction in the precursor phase. In the effector phase, whereas LAK activity against leukemia cells was also predominantly shown in the T cell-depleted fraction, similar levels of LAK activity against Daudi 1A4 were found in both the T cell-depleted and -enriched fractions.     

Histocompatibility Leukocyte Antigen-A2402-restricted Cytotoxic T Lymphocytes Recognizing Adenocarcinoma in Tumor-infiltrating Lymphocytes of Patients with Colon Cancer

To cast light on T cell-mediated specific immunity at the tumor site of colon cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) from colon cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted cytotoxicity against adenocarcinoma. IL 2-activated TIL from all four HLA-A24 patients examined lysed HLA-A2402+ adenocarcinomas, but not HLA-A2402 tumors. Those of two of the four cases also lysed HLA-A2402⁺ squamous cell carcinomas. CD8⁺ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2402⁺ adenocarcinomas were established from one CTL line. This CTL line produced IFN-γ upon recognition of an HLA-A2402^- adenocarcinoma transfected with HLA-A2402 cDNA. These results suggest the presence of HLA-A2402-restricted CTL recognizing adenocarcinoma at the tumor site of colon cancer. Furthermore, HLA-A31-restricted CTL activity was found in IL-2-activated TIL from one of two HLA-A31⁺ patients, suggesting the existence of HLA-class I-restricted CTL involving an allele other than A24     

Requirements of adherent cells for activating Lyt-1+2- T cells as well as for functioning as antitumor effectors activated by factor(s) from Lyt-1+2- T cells

The mechanism by which tumor-specific Lyt-1+2- T cells exhibit their antitumor effect in collaboration with an adherent cell population was investigated with the use of a double diffusion chamber. The double diffusion chamber was prepared by separating the two chambers from each other by a Millipore membrane and implanted in the peritoneal cavity of C3H/He mice. When one chamber contained normal C3H/He spleen cells plus syngeneic viable MH134 hepatoma cells and the other contained normal C3H/He spleen cells plus syngeneic viable X5563 plasmacytoma cells, tumor cells in both chambers continued to proliferate. In contrast, the injection of spleen cells immunized to the MH134 tumor into one (the first) chamber containing MH134 tumor cells not only resulted in the growth inhibition of MH134 tumor cells, but also exhibited an appreciable inhibitory effect on the growth of X5563 tumor cells admixed with normal spleen cells in the other (second) chamber. The growth inhibition of X5563 tumor cells in the second chamber was mediated by Lyt-1+2- T cells specific for MH134 tumor cells admixed in the first chamber. Such tumor-specific Lyt-1+2- T cell function was dependent on the existence of adherent cells in the first chamber, and adherent cells in the second chamber were also required for the X5563 growth inhibition. In addition, when the second chamber containing adherent cells, instead of the connection to the first chamber, was provided with macrophage-activating factor (MAF), X5563 growth inhibition was also observed. These results indicate that adherent cells are required for activating tumor-specific Lyt-1+2- T cells and for functioning as nonspecific antitumor effector cells activated by factor(s) such as MAF from Lyt-1+2- T cells.     

IFN-γ REGULATES Fas/FasL EXPRESSION IN CHOLANGIO CARCINOMA CELLS

Objective: To study the regulation of Fas receptor (Fas)/Fas ligand (FasL) expression by IFN-γ in cholangiocarcinoma cell lines. Methods: We studied the expressions of Fas and FasL gene in the human cholangiocarcinoma cell line QBC939 by RT-PCR, Western blot, and immunohistochemistry and their apoptosis effects on human T cell line Jurkat cell. At the same time, we investigated the regulative effect of IFN-γ on them. Results: Fas, FasL mRNA and protein were expressed by cholangiocarcinoma cells. We also found IFN-γ could up-regulate the expressions of these two genes. However, IFN-γ could also down-regulate the ability of them to make Jurkat cells apoptotic. With the increasing of dosage and time, the effect was enhanced. Conclusion: IFN-γ could regulate the expression of Fas and FasL in cholangiocarcinoma cells, and might influence the ability of cholangiocarcinoma to regulate immune escape. This study provides new theoretical basis for immunological therapy of cholangiocarcinoma.     

Delayed growth of EL4 lymphoma in SR-A-deficient mice is due to upregulation of nitric oxide and interferon-γ production by tumor-associated macrophages

Class A scavenger receptors (SR-A, CD204) are highly expressed in tumor-associated macrophages (TAM). To investigate the function of SR-A in TAM, wild-type and SR-A-deficient (SR-A^−/−) mice were injected with EL4 cells. Although these groups of mice did not differ in the numbers of infiltrating macrophages and lymphocytes and in neovascularization, SR-A^−/− mice had delayed growth of EL4 tumors. Expression of inducible nitric oxide (NO) synthase and interferon (IFN)-γ mRNA increased significantly in tumor tissues from SR-A^−/− mice. Engulfment of necrotic EL4 cells induced upregulation of NO and IFN-γ production by cultured macrophages, and production of NO and IFN-γ increased in SR-A^−/− macrophages in vitro . IFN-β production by cultured macrophages was also elevated in SR-A^−/− macrophages in vitro . These results suggested that the antitumor activity of macrophages increased in SR-A^−/− mice because of upregulation of NO and IFN-γ production. These data indicate an important role of SR-A in regulating TAM function by inhibiting toll-like receptor (TLR)4–IFN-β signaling. ( Cancer Sci 2009); 00: 000–000)     

Dendritic Cells Pulsed with Total Tumor RNA for Activation NK-like T Cells Against Glioblastoma Multiforme

Summary Dendritic cells (DCs) are potent antigen presenting cells and play critical role in T cell-mediated immunity. DCs have been shown to induce strong anti-tumor responses both in vitro and in vivo. Their efficacies in tumor therapy are being investigated in clinical trials. Previous evidence has shown that these DCs enhance the cytotoxicity of NK cells. We generated NK-like T cells (CD3⁺CD56⁺), a novel type of effector cells differentiated from normal lymphocyte, which is now being used for adoptive immunotherapy in clinical trials. This study aimed to elucidate the effects of NK-like T cells after co-culturing with DCs against tumor cells. The result revealed that tumor-derived RNA-pulsed DCs can enhance the immune responses of NK-like T cells against glioblastoma multiforme cell line but these effector cells did not appear to have the cytotoxic effect against normal cells (human umbilical vein endothelial cells (HUVEC) and fibroblasts) in vitro. This study may be beneficial for the development of new immunologic effector cells for using in adoptive immunotherapy for glioblastoma multiforme in the future.