Granulocyte–Macrophage Colony-Stimulating Factor Gene Transfer to Dendritic Cells or Epidermal Cells Augments Their Antigen-Presenting Function Including Induction of Anti-Tumor Immunity  Hiroaki Ozawa, Wanhong Ding, Hideshi Torii, Junichi Hosoi, Kristina Seiffert, Kristina Campton, Richard D. Granstein  Journal of Investigative Dermatology  Volume 113, Issue 6, Pages 999-1005 (December 1999) DOI: 10.1046/j.1523-1747.1999.00769.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ad-vector-mediated gene transfer and expression of βgal or GFP in XS 106, XS52–4D, and BMDC in vitro. (A) βgal; (B) GFP; (A-a, A-b, A-c, B-a) XS 106; (A-d, A-e, A-f, B-b) XS52–4D; (A-g, A-h, A-i, B-c) BMDC. Cells were infected in vitro with an Adβgal, AdGFP, or AdNull at moi of 100 for 2 h. Twenty-four hours later, βgal or GFP expression was quantitated by flow cytometry. Cells were stained with propidium iodide to facilitate exclusion of dead cells. For all the panels, the y-axis reflects cell number and the x-axis reflects log fluorescence intensity. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ad-vector-mediated gene transfer and expression of GFP to epidermal cells in vitro. Epidermal cells from BALB/C mice were infected with AdGFP at moi of 20 for 2 h. Twenty-four hours later, GFP expression was quantitated by flow cytometry. Epidermal cells were stained with phycoerythrin conjugated anti-I-Ad MoAb, followed by propidium iodide to facilitate discrimination among live and dead cells. The y-axis reflects log fluorescence intensity of anti-I-Ad MoAb conjugated with phycoerythrin and the x-axis reflects log fluorescein intensity of GFP. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ad-GM-CSF augmented the secretion of GM-CSF from XS106, XS52–4D, BMDC, and epidermal cells. (a) XS106; (b) XS52–4D; (c) BMDC; (d) epidermal cells. Cells infected in vitro with AdGM-CSF, AdNull (moi of 100 for XS cells and BMDC or at moi of 20 for epidermal cells), or untreated cells were cultured 48 h at concentration of 2 × 105 XS cells or BMDC per ml or 2 × 106 epidermal cells per ml. The culture supernatants were assayed for murine GM-CSF by enzyme-linked immunosorbent assay. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 XS cells infected with AdGM-CSF show prolonged dendrites. XS106 (a–c) or XS52–4D (d–f) were cultured 72 h after infection with AdGM-CSF (a, d), AdNull (b, e) at moi of 100, or without infection (c, f). Morphologic changes were assessed using in inverted microscope. Scale bar: 200 μm. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 XS cells infected with AdGM-CSF show augmented expression of I-A and CD86, but not CD54 or CD80. XS106 (a–d) or XS52–4D (e–h) cells were cultured for 72 h after Ad-vector-mediated gene transfer at moi of 100. AdGM-CSF-treated (thick histograms) or AdNull-treated (medium histograms), or untreated cells (thin histogram for XS106 only) were stained with FITC-conjugated anti-I-Ak MoAb (a), anti-I-Ad MoAb (e), anti-CD54 MoAb (b, f), anti-CD80 MoAb (c, g), or anti-CD86 MoAb (d, h) and analyzed by flow cytometry. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 XS cells, BMDC, or epidermal cells infected with AdGM-CSF show increased allostimulatory capacity. XS106 (a), XS52–4D (b), BMDC (c), or epidermal cells (d) infected with AdNull, AdGM-CSF (moi of 100 for XS cells and BMDC and 20 for epidermal cells), or untreated were cultured for 24 h in CM. These cells were washed, irradiated (4000 rads for XS cells and 2000 rads for BMDC or epidermal cells, respectively) and cultured with nylon wool-enriched splenic T cells. For experiments with XS52-4D cells, BMDC, or epidermal cells as stimulators, responding T cells were from C3H mice (H-2k). For experiments using XS106 cells as stimulators, responding T cells were from BALB/C mice (H-2d). After 5 d of culture, all wells were pulsed with 1 μCi per well of [3H]thymidine for an additional 24 h of culture. Then, cells were harvested and [3H]thymidine incorporation was measured. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Epidermal cells infected with AdGM-CSF induce anti-tumor immunity. (a) Twenty-four hours after infection with AdGM-CSF or AdNull, CAF1 or C57BL/6 epidermal cells were pulsed with a soluble extract of the S1509a spindle cell tumor (H-2a) as a source of TAA, washed three times, and 2 × 105 cells were injected subcutaneously into each of five naïve CAF1 mice. Primings were repeated weekly two times and 1 wk after the last immunization each mouse was challenged subcutaneously with 2 × 106 living S1509a cells. Tumor growth was scored over time. Control mice were immunized with cells not pulsed with TAA. Group A, AdGM-CSF + TAA; group B, AdGM-CSF-TAA; group C, AdNull + TAA; group D, AdNull-TAA. Data points represent mean values. A versus B, C, or D: p < 0.01. (b) Deletion of I-A+ cells prevents induction of anti-tumor immunity by AdGM-CSF-infected epidermal cells. MHC class II positive Langerhans cells were deleted from epidermal cells by treatment with anti-I-Ad MoAb and complement. Epidermal cells were then infected with AdGM-CSF or AdNull and 24 h later pulsed with a soluble extract of the S1509a spindle cell tumor (H-2a) as a source of TAA, washed three times, and 2 × 105 cells were injected subcutaneously into each of five naïve CAF1 mice. Positive control mice were immunized with AdGM-CSF-infected cells that had not undergone I-A deletion prior to TAA pulsing. Group A, AdGM-CSF + TAA; group B, I-A-deleted; AdGM-CSF + TAA; group C, AdNull + TAA. A versus B or C: p < 0.01. (c) MHC restriction of epidermal cell-induced anti-tumor immunity. Mean tumor growth over time in mice that received three consecutive immunizations at weekly intervals syngeneic or allogeneic epidermal cells treated with AdGM-CSF that were pulsed with TAA and used for immunization. Group A, syngeneic epidermal cells + AdGM-CSF + TAA; group B, syngeneic epidermal cell AdGM-CSF–TAA; group C, allogeneic epidermal cells + AdGM-CSF + TAA; group D, allogeneic epidermal cells + AdGM-CSF-TAA. A versus B, C, or D: p < 0.01. Journal of Investigative Dermatology 1999 113, 999-1005DOI: (10.1046/j.1523-1747.1999.00769.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions