• The Korean Journal of Physiology and Pharmacology
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• v.16 no.4
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• pp.243-247
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• 2012

Recent studies suggest that immunization with autologous dendritic cells (DCs) results in protective immunity and rejection of established tumors in various human malignancies. The purpose of this study is to determine whether DCs are generated from peripheral blood mononuclear cells (PBMNs) by using cytokines such as F1t-3 ligand (FL), granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4, and TNF-${\alpha}$, and whether cytotoxic T cells activated against the thyroid cancer tissues by the DCs. Peripheral blood was obtained from 2 patients with thyroid cancer. DCs were established from PBMNs by culturing in the presence of FL, GM-CSF, IL-4, and TNF-${\alpha}$ for 14 days. At day 14, the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla, CD83, and CD86 were analyzed by immunofluorelescence microscopy. At day 18, DCs and T cells were incubated with thyroid cancer tissues or normal thyroid tissues for additional 4 days, respectively. DCs generated from the PBMNs showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed also. DCs and the CTLs were attached to the cancer tissues on scanning electron microscope. The DCs activated the CTLs, which able to specifically attack the thyroid cancer. This study provides morphologic evidence that the coculture of T cells/cancer tissues activated the T cells and differentiated CTLs. The CTLs tightly adhered to cancer tissues and lysed cancer tissues vigorously. Therefore DCs could be used as potential vaccines in the immunotherapy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.571-578
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• 2015

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-${\gamma}$, TNF-${\beta}$ and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-${\gamma}$, TNF-${\beta}$ and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

• Journal of applied mathematics & informatics
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• v.33 no.5_6
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• pp.559-578
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• 2015

Human Immune Deficiency Virus (or simply HIV) induces a persistent infection that leads to AIDS causing death in almost every infected individual. As HIV affects the immune system directly by attacking the CD4+ T cells, to exterminate the infection, the natural immune system produces virus-specific cytotoxic T lymphocytes(CTLs) that kills the infected CD4+ T cells. The reduced CD4+ T cell count produce reduced amount of cytokines to stimulate the production of CTLs to fight the invaders that weakens the body immunity succeeding to AIDS. In this paper, we introduce a mathematical model with discrete time-delay to represent this cell dynamics between CD4+ T cells and the CTLs under HIV infection. A modified functional form has been considered to describe the infection mechanism. Characteristics of the system are studied through mathematical analysis. Numerical simulations are carried out to illustrate the analytical findings.

• IMMUNE NETWORK
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• v.10 no.6
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• pp.206-211
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• 2010

Background: Dendritic cell (DC)-based tumor vaccine is an attractive modality for the treatment of colon cancer because it has been recurred and produced few side effects in patients. Secretory glycoprotein 90K has been found at elevated level in various cancer tissues and sera. We investigated to establish a more effective DC vaccine for the treatment of colon cancer in which the levels of 90K are elevated. Methods: We obtained the concentrated 90K from 293T cells stably expressing 90K. DCs were cultured from peripheral blood monocytes, and a DC vaccine pulsed with tumor lysate was compared with a DC vaccine pulsed with 90K. We measured the functional activity for CTLs by using IFN-${\gamma}$-enzyme linked immunoabsorbent spot (ELISPOT) assay. Results: DCs pulsed with tumor lysate+90K exhibited the enhanced T cell stimulation, polarization of $\ddot{i}$ T cell toward Th1. The CTLs generated by DCs pulsed with 90K efficiently lysed HCT116 cells. The results indicate that 90K-speicifc-CTLs can recognize 90K proteins naturally presented by colon cancer cells. Conclusion: Our study suggests that 90K-specific CTLs generated by 90K-pulsed DCs could be useful effector cells for immunotherapy in colon cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.611-616
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• 2014

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associated antigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whether transduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigen-specific cytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocyte-derived DCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated target of the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCs was measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry. DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag). Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulated by LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methyl thiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfully constructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83 than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstrated good stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumor effects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have an enhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.

• BMB Reports
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• v.45 no.7
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• pp.408-413
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• 2012

Almost all melanoma cells express at least one member of the MAGE-A antigen family, making the cytotoxic T cells (CTLs) epitopes with cross-immunizing potential in this family attractive candidates for the broad spectrum of anti-melanoma immunotherapy. In this study, four highly homologous peptides (P264: FLWGPRALA, P264I9: FLWGPRALI, P264V9: FLWGPRALV, and P264H8: FLWGPRAHA) from the MAGE-A antigens were selected by homologous alignment. All four peptides showed high binding affinity and stability to HLA-A$^*02:01$ molecules, and could prime CTL immune responses in human PBMCs and in HLA-A$^*02:01/K^b$ transgenic mice. CTLs elicited by the four epitope peptides could cross-lyse tumor cells expressing the mutual target antigens, except MAGE-A11 which was not tested. However, CTLs induced by P264V9 and P264I9 showed the strongest target cell lysis capabilities, suggesting both peptides may represent the common CTL epitopes shared by the eight MAGE-A antigens, which could induce more potent and broad-spectrum antitumor responses in immunotherapy.

• IMMUNE NETWORK
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• v.11 no.3
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• pp.182-189
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• 2011

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.96-102
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• 2003

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccines induce very potent systemic anti-tumor immunity in preclinical and clinical models. Our previous phase I clinical trial in patients with metastatic renal cell carcinoma (RCC) has demonstrated both immune cell infiltration at vaccine sites and T cell-mediated delayed-type hypersensitivity (DTH) response to whole tumor cell vaccines. Methods: To investigate the immune responses to autologous genetically- modified tumor cell vaccines, tumor-specific $CD8^+$ T cell lines were generated from peripheral blood lymphocytes (PBL) of a RCC patient 1.24 by repeated in vitro stimulation with either B7.1-transduced autologous RCC tumor cells or B7.1-transduced autologous tumor cells treated with interferon gamma ($IFN{\gamma}$), and cloned by limiting dilution. Results: Among several RCC-specific cytotoxic T lymphocytes (CTLs), a $CD4^+/CD8^+$ double positive T cell clone (17/A2) appeared to recognize $IFN{\gamma}$-treated autologous RCC restricted by HLA-B39. The 17/A2 also recognized other HLA-B39 positive RCC tumor cells after $IFN{\gamma}$ treatment. Conclusion: These results demonstrate that autologous RCC vaccination successfully generates the tumor-specific CTL 17/A2, and suggest that the presentation and recognition of the tumor antigen by the 17/A2 might be upregulated by $IFN{\gamma}$.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.22
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• pp.9667-9672
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• 2014

Background: Pancreatic adenocarcinoma is a malignant gastrointestinal cancer with significant morbidity and mortality. Despite severe side effects of chemotherapy, the use of immunotherapy combined with chemotherapy has emerged as a common clinical treatment. In this study, we investigated the efficacy of the combined doxorubicin and interferon-${\alpha}$ (IFN-${\alpha}$) therapy on murine pancreatic cancer Panc02 cells in vitro and in vivo and underlying mechanisms. Materials and Methods: A Panc02-bearing mouse model was established to determine whether doxorubicin and interferon-${\alpha}$ (IFN-${\alpha}$) could effectively inhibit tumor growth in vivo. Cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) was evaluated using a standard LDH release assay. To evaluate the relevance of NK cells and CD8 T cells to the combination therapy-mediated anti-tumor effects, they were depleted in tumor-bearing mice by injecting anti-asialo-GM-1 antibodies or anti-CD8 antibodies, respectively. Finally, the influence of doxorubicin+interferon-${\alpha}$ (IFN-${\alpha}$) on the ligands of NK and T cells was assessed by flow cytometry. Results: The combination therapy group demonstrated a significant inhibition of growth of Panc02 in vivo, resulting from activated cytotoxicity of NK cells and CTLs. Depleting CD8 T cells or NK cells reduced the anticancer effects mediated by immunochemotherapy. Furthermore, the doxorubicin+IFN-a treatment increased the expression of major histocompatibility complex class I (MHC I) and NKG2D ligands on Panc02 cells, suggesting that the combined therapy may be a potential strategy for enhancing immunogenicity of tumors. All these data indicate that the combination therapy using doxorubicin and interferon-${\alpha}$ (IFN-${\alpha}$) may be a potential strategy for treating pancreatic adenocarcinoma.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2003.06a
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• pp.55-62
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• 2003

The mucosal immune system provides a first line of defense against invasion of infectious agents via inhalation, ingestion and sexual contact. For the induction of protective immunity at these invasion sites, one must consider the use of the CMIS, which interconnects inductive tissues, including PP and NALT, and effector tissues of the intestinal, respiratory and genitourinary tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant or use of mucosal antigen delivery vehicle has been shown to be essential. When vaccine antigen is administered via oral or nasal route, antigen-specific Th 1 and Th2 cells, cytotoxic T lymphocytes(CTLs) and IgA B cell responses are effectively induced by the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium(FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying DCs for the generation of antigen-specific T cells and IgA committed B cells. These antigen-specific lymphocytes are then home to the distant mucosal effector tissues for the induction of antigen-specific humoral(e.g., IgA) and cell-mediated (e.g., CTL and Th1) immune responses in order to form the first line of defense. Elucidation of the molecular/cellular characteristics of the immunological sequence of mucosal immune response beginning from the antigen sampling and processing/presentation by M cells and mucosal DCs followed by the effector phase with antigen-specific lymphocytes will greatly facilitate the design of a new generation of effective mucosal antigen-specific lymphocytes will greatly facilitate the design of a new generation of a new generation of effective mucosal adjuvants and of a vaccine deliver vehicle that maximizes the use of the CMIS.