T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell–based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals. @*Materials and Methods@#Antigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). @*Results@#T cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization. @*Conclusion@#These results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

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.

Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.

Association of Killer Cell Ig-like Receptor (KIR) with an Adaptor Protein Shc

BACKGROUND: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. METHODS: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. RESULTS: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T cell lysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. CONCLUSION: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.

Activation of B cells using Schneider 2 cells expressing CD40 ligand for the enhancement of antigen presentation in vitro

CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.

A Case of Pregnancy in Patient with Pulmonary Lymphangioleiomyomatosis / 대한산부인과학회잡지

Pulmonary lymphangioleiomyomatosis is a rare disorder of unknown cause and characterised by hamartomatous proliferation of smooth muscle occurring in women of reproductive age exclusively. It causes dyspnea, recurrent pneumothorax, chylothorax, hemoptysis and respiratory failure eventually. Chest radiographs show diffuse interstitial infiltrates and cysts of uniform size, and pulmonary function tests often show airflow limitation with increase in residual volume. Hormonal factors are thought to play a role because it generally affects premenopausal women, but there is no definite treatment yet. We present an unusual case of pulmonary lymphangioleiomyomatosis during normal pregnancy with a review of literature.

Efficacy and Safety of Concurrent Chemoradiotherapy of Paclitaxel and Carboplatin as Adjuvant Therapy after Primary Surgery in High-risk Cervical Cancer / 대한산부인과학회잡지

OBJECTIVE: To evaluate the efficacy and safety of concurrent chemotherapy of paclitaxel and carboplatin with standard pelvic radiotherapy as adjuvant therapy after primary surgery in high-risk cervical cancer. METHODS: Twenty-eight patients with FIGO stage IB1-IIB cervical cancer who received adjuvant concurrent chemoradiotherapy with paclitaxel and carboplatin from February 2000 to November 2001 were analyzed retrospectively in this study. Adjuvant chemoradiotherapy was done if there were lymph node involvement or at least 2 positive findings among following risk factors; lymphovascular space invasion, full- thickness involvement of cervix and tumor size larger than 4 cm in diameter. Two cycles of paclitaxel 135 mg/m2, followed by carboplatin with AUC of 4.5 were administered intravenously with an interval of at least 4 weeks. The radiotherapy was initiated concurrently at the first day of chemotherapy. The therapeutic results were evaluated by pelvic examination, Pap smear, SCCA (Squamous cell carcinoma antigen) and computed tomography (CT). The toxicities of the treatment were evaluated and graded by NCI-CTC version 2.0. RESULTS: Total 56 cycles of paclitaxel/carboplatin chemotherapy with concomitant pelvic radiotherapy was delivered. None of the patients had a progressive or recurrent disease during the follow-up period ranging from 6 to 33 months (median: 12.5 months). Neutropenia was the most common and concerned toxicity. Fifteen cases of grade 3 and 4 neutropenia (26.8%) were observed. Non-hematologic toxicities were mild and mainly related to neurologic or gastrointestinal symptoms. Eight cases of grade 1 and 2 neurotoxicity were observed (14.3%). CONCLUSION: The adjuvant chemoradiotherapy with paclitaxel and carboplatin seems to be effective and well-tolerated for the treatment of high risk group cervical cancer after primary surgical therapy. But a large randomized study with longer duration of follow-up is needed to justify this conclusion.

Suppression of Tumor Formation and Induction of Natural Killer Cell Activity in BALB/c Nude Mice by Human B7-1 (CD80) Gene Transfer Subcutaneously Injected with Human Hepatocellular Carcinoma Cells (Huh-7) / 대한간학회지

BACKGROUND/AIMS: Immunogene therapy is extensively studied for a therapeutic modality of various cancers. This study was conducted to investigate the efficacy of immunogene therapy using the T-cell costimulatory molecule and human B7-1 (CD80, hB7-1) in an in vivo human hepatocellular carcinoma (HCC) model. METHODS: The stable HCC cell line expressing hB7-1 gene was established using retroviral vector (Huh-7/hB7-1). Of fourteen BALB/c nude mice, 7 were subcutaneously injected with 2 X 10(6) Huh-7/hB7-1 cells, while the other 7 were injected with 2 X 10(6) Huh-7/mock cells as a control group. After the injection, the mice were observed weekly for three months for subcutaneous tumor formation. Assay for natural killer (NK) cell cytotoxicity and serum IFN-gamma was performed at 1 and 2 weeks after inoculation. RESULTS: In BALB/c nude mice inoculated with Huh-7/hB7-1 cells, no tumor growth was observed. BALB/c nude mice inoculated with Huh-7/hB7-1 cells showed significantly increased NK cell activities of splenocytes compared with those with Huh-7/mock cells. Serum IFN-gamma was not measurable at 1 week, but significantly increased at 2 weeks after inoculation to the level of 470 pg/ml in BALB/c nude mice with Huh-7/mock cells and 521 pg/ml in BALB/c nude mice with Huh-7/hB7-1. CONCLUSIONS: Our results demonstrate the in vivo anti-tumor immunity and NK cell activation by transfer of hB7-1 gene into human HCC in xenogeneic BALB/c nude mice model. This approach may provide a tool for the development of immunogene therapies against human malignant tumors.

Induction of CEA-specific Cytotoxic T Lymphocytes by Murine Dendritic Cells Expressing CEA

BACKGROUND: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. METHODS: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard 51Cr-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-gamma ELISPOT. RESULTS: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of IFN-gamma secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. CONCLUSION: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

Enhanced Induction of CEA Specific Tumor Immunity by TatCEA Fusion Protein

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.

Induction of Cellular Immune Response by Dendritic Cells Pulsed with Glioma Apoptotic Bodies

OBJECTIVE: The aim of this study is to verify the hypothesis that human dendritic cells(DCs) can process antigens from glioma cell apoptotic bodies and induce antigen-specific effector T cells. METHODS: DCs generated in the presence of granulocyte macrophage-colony stimulating factor(GM-CSF) and interleukin-4(IL-4) from peripheral blood mononuclear cells(PBMCs) of healthy donors with human leucocyte antigen(HLA) A*0201 were cultured for 7 days. Glioma apoptotic bodies(GABs) from T98G glioblastoma cells following 18 hour-actinomycin D treatment were co-incubated with DCs for 3 days. CD8 T cells isolated from peripheral blood of same donors were cultured in media containing IL-2 and were stimulated by GAB-pulsed DCs three times at a weekly interval. The interferon-gamma(IFN-gamma), a cytokine related to cytotoxicity, concentrations of cell culture supernates were measured by enzyme immunoassay technique. RESULTS: Induced DCs had DC's own phenotypic characteristics such as highly expressed major histocompatibility complex(MHC) class II, CD1a and CD86 molecules. They also had high endocytotic activity. Preteatment of T98G glioma cells with actinomycin D resulted in 53% of cells undergoing apoptosis. IFN-gamma production of effector T cells stimulated by GAB-pulsed DCs was significantly higher than that of T cells stimulated by non-pulsed DCs. CONCLUSION: Naive CD8 T cells can be activated by human GAB-pulsed DCs to become antigen-specific effector T cells. Using GABs as a antigen source may be a novel approach in future DC-based immunotherapeutic trials for malignant glioma.

Antitunor effect of carcinoma cells transduced with herpes simplex virus - thymidine kinase by gancyclovir and radiation

BACKGROUND: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. METHODS: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. RESULTS: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X 10(6) CFU/ml . After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. CONCLUSIONS: The result s suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.

The Use of Canine Bone-Marrow Stromal Cell Line, DO64, as Feeder Cells for The Efficient Cloning of Human B Lymphoblastoid Cell Lines ( B-LCLs ) / 대한면역학회지

Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and a tool for the establishment of human B lymphoblastoid cell lines (B-LCLs), which have proven useful for several human immunologic applications. B-LCLs serve as efficient antibody-producing cells and antigen-presenting cells. In spite of these advantages, the cloning efficiency of B-LCLs is less than 1%. In order to generate clones of B-LCLs, we cultured B-LCLs with and without canine stromal cell line, DO64, as feeder cell which was immortalized by transduction of retrovirus encoding E6 and E7 of the human papilloma virus type 16 (HPV-16), which was defined to produce various cytokines including stem cell factor (SCF) and interleukin- 6 (IL-6). After 3 weeks of B-LCLs cultured with DO64, 8.3% and 37.5% in 1 cell and 3 cells per well were efficiently cloned, respectively. There was no significant effect on growing of 8-LCLs without DO64 cells and on high concentration of FBS. The cloning efficiency of B-LCLs transduced by retrovirus cultured with and without DO64 cells was 4.2% and 0% in 3 cells per well, respectively, while that of stable transfectant 33.3% and 8.3% in 1 cell per well, respectively. Our results suggest that the use of DO64 cells as feeder cells might permit the cloning of B-LCLs. This efficient cloning of B-LCLs could be used for the convenient source of autologous antigen-presenting cells expressing foreign antigen for the study of human immune responses in vitro, and for a variety of additional purposes, such as the production of human monoclonal antibodies.

Efficient Production of Retroviruses Encoding Human Costimulatory Molecule, B7 - 1 ( CD80 ) / 대한면역학회지

No abstract available.