Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The expression of 6Ckine, IFNγ and 6Ckine/IFNγ fusion protein in HepG2 cells mediated by adenovirus vector (L, Ad-LacZ; C, Ad-6Ckine; I, Ad-IFNγ; F, Ad-6Ckine/IFNγ; M, marker; +, positive control; N, mock-transfection). The HepG2 cells were transfected with recombinant adenoviruses at 10 MOI or mock-transfection (N) for 48 h, and then the expression and biological activity of 6Ckine, IFNγ and 6Ckine/IFNγ were assessed. a RT-PCR analysis for 6Ckine, IFNγ and 6Ckine/IFNγ mRNA. β-actin was used as an internal control (6Ckine 405 bp, IFNγ 501 bp, 6Ckine/IFNγ 849 bp, T-bet 450 bp, and β-actin 245 bp). b Immunofluorescent staining (×200). Red fluorescence indicated positive cells. c Western blot analysis. d Interferon activity was analyzed following the protocol from NICPBP. e 6Ckine function was detected by microchemotaxis assay

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Expressing, Plasmid Preparation, Marker, Positive Control, Transfection, Recombinant, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Fluorescence, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: The influences of recombinant adenoviruses infection on the endocytosis of DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, Non-transfected DCs. The percentage of FITC-Dextran uptake by the DCs was assessed with FACS analysis after 24 h of adenoviruses infection. The data represent three separate experiments. P > 0.05 when compared among all groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Recombinant, Infection, Transfection

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Chemokines secretion of DCs and the recruiting function to T cells. a and b DCs were pulsed with HepG2 cell lysates, and incubated at 37°C for 24 h. Supernatants from each DC group were harvested, and then 6Ckine (a) and RANTES (b) concentration was measured by the specific ELISA assay. The amounts of chemokines from five separate experiments were shown in nanograms per million DCs 48 h after transfection. c Microchemotaxis assay. The above-mentioned culture supernatant was collected, and the chemotaxis activity to naïve T cells was analyzed by a Transwells® apparatus. RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μl of recombinant human 6Ckine protein was used as positive or negative control individually. Supernatant pre-incubated with recombinant human anti-6Ckine monoclonal antibody was used for chemotaxis blocking assay (results represent three independent experiments). *P < 0.05 when compared with other groups; **P < 0.05 compared with medium control; ***P < 0.05 compared with Ad-6Ckine/IFNγ-DCs and Ad-6Ckine-DCs, P > 0.05 compared with Ad-IFNγ-DCs Ad-LacZ-DCs and NTDCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Chemotaxis Assay, Activity Assay, Recombinant, Negative Control, Blocking Assay, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytokine secretion of gene modified-DCs (C, Ad-6Ckine-DCs; I, Ad-IFNγ-DCs; F, Ad-6Ckine/IFNγ-DCs; L, Ad-LacZ-DCs; N, non-transfected-DCs). DCs were pulsed with 100 μg/ml of HepG2 cell lysates, and incubated for 24 h. The supernatant from each DC group was harvested, and then the level of IFNγ (a), IL-12p70 (b) and IL-10 (c) was detected by LiquidChip assay, while TNFα (d) were measured by ELISA. The values referring to the cytokine production were obtained from the average of five separate experiments. *P < 0.01 when compared with Ad-LacZ-DCs and non-transfected-DCs

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Modification, Transfection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Ad-6Ckine/IFNγ-DCs promote T cell activation. a Autologous T cell reaction. Following inactivation with mitomycin, antigen-loaded DCs were co-cultured with autologous T cells at a DC:T ratio of 1:20 for 96 h. 1 μCi/well of 3H-TdR was pulsed for 16 h. The values of counts per minute (cpm) were measured with a LS6500 liquid scintillation counter. Data were expressed as the stimulating indices (SIs). The results were obtained from five independent experiments. b IL-2 production of T cells. The IL-2 concentrations in the supernatants of co-cultured DCs and T cells were measured by ELISA. The results were obtained from six independent experiments. c The T-bet expression in T cells. The T-bet mRNA levels in T cells co-incubated with DCs were measured by semi-quantitative RT-PCR. The data were obtained from five separate experiments. d One of the representive experiments of RT-PCR. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs; **P < 0.05 when compared with any of other four groups

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro

doi: 10.1007/s00262-007-0327-y

Figure Lengend Snippet: Cytotoxic function of T cells stimulated with Ad-6Ckine/IFNγ-DCs (C, Ad-6Ckine-DC group; I, Ad-IFNγ-DC group; F, Ad-6Ckine/IFNγ-DC group; L, Ad-LacZ-DC group; N, NTDC group). Autologous T cells were co-cultured with antigen-pulsed DCs at a DC:T ratio of 1:20 for 5 days. The cytotoxicity of activated T cells was analyzed with a CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA), HepG2 or LoVo cells were used as the target cells (E:T = 20:1 or 50:1). The data were obtained from five separate experiments. *P < 0.05 when compared with Ad-LacZ-DCs and NTDCs groups; **P < 0.05 when compared with other four groups, respectively

Article Snippet: The RPMI 1640 containing 5% human AB serum with or without 0.1 ng/μL of recombinant human 6Ckine protein (R&D Systems, USA) was used as positive or negative controls, respectively.

Techniques: Cell Culture, Cytotoxicity Assay