Journal:

Article Title: Dendritic Cells Infected with Poxviruses Encoding MART-1/Melan A Sensitize T Lymphocytes In Vitro

doi:

Figure Lengend Snippet: Representative example of phenotype analysis of dendritic cells (DC) by fluorescence-activated cell sorter analysis. Surface phenotype of peripheral blood mononuclear cells (PBMC); adherent cells (AC) prepared on 75-cm2 flasks after removal of nonadherent cells after a 3-h incubation with human recombinant interleukin-3 (hrIL-3) (200 IU/ml) at 37°C; and DC cultured with hrIL-4 (2,000 IU/ml) and human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) (2,000 IU/ml) at day 5. Cell fluorescence is indicated by solid histograms and the control staining with isotype-matched IgG in blank histograms.

Article Snippet: Recombinant vaccinia and fowlpox viruses encoding MART-1 (rV-MART-1 and rF-MART-1) were provided by Therion Biologies (Cambridge, MA, U.S.A.).

Techniques: Fluorescence, Incubation, Recombinant, Cell Culture, Control, Staining

Journal:

Article Title: Dendritic Cells Infected with Poxviruses Encoding MART-1/Melan A Sensitize T Lymphocytes In Vitro

doi:

Figure Lengend Snippet: Morphologic appearance of dendritic cells (DC). Vital stain with calcein AM of DC after 5 days in culture with human recombinant interleukin-4 (hrIL-4) (2,000 IU/ml) and human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) (2,000 IU/ml) (A). Viral expression of MART-1 in the same DC by immunofluorescence (B). DC were incubated with rV-MART-1 for 1 h at 37°C at 10:1 multiples of infection. After 12 h, DC were stained with anti-MART-1 mAb (42). No staining or fluorescence was detected with anti-gp100 monoclonal antibody (mAb) (HMB45) as the negative controls. Conversely, DC infected with the rV-gp100 gene were not stained by anti-MART-1 mAb but were brightly stained with HMB 45.

Article Snippet: Recombinant vaccinia and fowlpox viruses encoding MART-1 (rV-MART-1 and rF-MART-1) were provided by Therion Biologies (Cambridge, MA, U.S.A.).

Techniques: Staining, Recombinant, Expressing, Immunofluorescence, Incubation, Infection, Fluorescence

Journal:

Article Title: Dendritic Cells Infected with Poxviruses Encoding MART-1/Melan A Sensitize T Lymphocytes In Vitro

doi:

Figure Lengend Snippet: Cytotoxic activity (calcein AM release) of anti-MART-1 cytotoxic T lymphocytes (CTL) induced with recombinant virus-infected dendritic cells (DC). Anti-MART-1 CTL were induced by a primary in vitro stimulation with DC infected with rV-MART-1 followed 7 days later with a second stimulation with DC infected with rF-MART-1 virus (rV-MART-1 = left two panels). By reversing the sequence of the viral vectors used for stimulation, anti-MART-1 CTL were also induced by a primary stimulation with DC infected with rF-MART-1 virus followed 7 days later by a second stimulation with DC infected with rV-MART-1 virus (rF-MART-1 = right two panels). Recognition of the epitope MART-127-35 was tested by pulsing T2 cells with 1 μg/ml MART-127-35 (■) or 1 μg/ml irrelevant G9-209-2M (□). Recognition of naturally processed MART-1 epitopes was tested against the 624.38 (▲) HLA-A*0201+/MART-1+ and the 624.28 (△), HLA-A*0201-/MART-1 + melanoma clones.

Article Snippet: Recombinant vaccinia and fowlpox viruses encoding MART-1 (rV-MART-1 and rF-MART-1) were provided by Therion Biologies (Cambridge, MA, U.S.A.).

Techniques: Activity Assay, Recombinant, Virus, Infection, In Vitro, Sequencing, Clone Assay

Journal: The Journal of Experimental Medicine

Article Title: Dendritic Cells Initiate Immune Control of Epstein-Barr Virus Transformation of B Lymphocytes In Vitro

doi: 10.1084/jem.20030646

Figure Lengend Snippet: DCs are required for priming naive T cells to EBV antigens. T cells and EBV-infected B cells from EBV-seronegative (A–D) and -seropositive (E and F) donors were cultured in the presence (B + DC + T) or the absence (B + T) of mature DCs for 12–14 d. Results shown have the vector control, vvTK − , subtracted from the specific response, where vvTK − responses were always <20 spots. A response was considered meaningful if it was at least twice that of the negative control (vvTK − ) as well as 10 spots greater than the vvTK − background and is indicated with an asterisk. (G) The seronegative B + DC + T cultures were also tested against autologous LCLs and the EBV-negative HD cell line HD-MY-Z by IFNγ ELISPOT. (H) Two sources of EBNA1 were compared for loading of DCs, recombinant vaccinia viruses (vv) leading to the expression of Gly-Ala–deficient EBNA1 (vvE1) or recombinant EBNA1 protein (rE1). DCs loaded with rPCNA (rP) or infected with vvTK − were used as controls. The responses in A, G (donor 3), and H (donor 2) are representative of four experiments with the same seronegative donor.

Article Snippet: Mature DCs were infected with recombinant vaccinia vectors (vvTK − ) as a negative control or vaccinia vectors expressing EBV latency (EBNA1, 2, 3A, 3B, 3C, LMP1, and LMP2A) and lytic (BMLF1) antigens at a multiplicity of infection of 2 for 1 h at 37°C (provided by M. Kurilla, Dupont Pharmaceuticals Corp., Wilmington, NC).

Techniques: Infection, Cell Culture, Plasmid Preparation, Control, Negative Control, Enzyme-linked Immunospot, Recombinant, Expressing