Volume 13, Issue 2, Pages 280-288 (February 2006) Transfection of Dendritic Cells with in Vitro-Transcribed CMV RNA Induces Polyclonal CD8+- and CD4+-Mediated CMV-Specific T Cell Responses  Annkristin Heine, Frank Grünebach, Tobias Holderried, Silke Appel, Markus M. Weck, Daniela Dörfel, Christian Sinzger, Peter Brossart  Molecular Therapy  Volume 13, Issue 2, Pages 280-288 (February 2006) DOI: 10.1016/j.ymthe.2005.08.019 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Induction of autologous CMV-specific CTL responses by DCs transfected with pp65 IVT. DCs generated from adherent PBMCs of CMV-positive healthy donors in the presence of GM-CSF, IL-4, and TNF-α were electroporated with pp65 IVT and then used as APCs for in vitro CTL induction. After 1 week of incubation with purified, autologous CD8+ T cells, T lymphocytes were assayed in IFN-γ ELISPOT assays using DCs pulsed with pp65-derived peptides or DCs electroporated with pp65 IVT as APCs. Additionally, unstimulated autologous T cells were analyzed in the assays. (A) IFN-γ ELISPOT assay of a CMV-positive HLA-A2+/HLA-A11+/HLA-B35+ donor. Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for corresponding negative control (DCs pulsed with irrelevant HIV peptide; DCs electroporated with irrelevant EGFP IVT). (B) IFN-γ ELISPOT assay of a CMV-positive HLA-A2+/HLA-A24+ donor. Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for corresponding negative control (DCs pulsed with irrelevant peptide E75; DCs electroporated with irrelevant MUC1 IVT). Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Immunodominance of HLA-B7 epitopes. DCs generated from adherent PBMCs of CMV-positive healthy donors in the presence of GM-CSF, IL-4, and TNF-α were electroporated with pp65 IVT and then used as APCs for in vitro CTL induction. After 1 week of incubation with purified, autologous CD8+ T cells, T lymphocytes were assayed in IFN-γ ELISPOT assays using DCs pulsed with pp65-derived peptides as APCs. Additionally, unstimulated autologous T cells were analyzed in the assays. (A) Representative IFN-γ ELISPOT assay of a HLA-A2+/HLA-B7+ donor. (B) Diagram of the IFN-γ ELISPOT assay shown in A. Values represent number of spots per 5 × 104 CD8+ T cells after subtraction of spots counted for negative control (DCs pulsed with E75 peptide). (C) IFN-γ ELISPOT assay of an HLA-A1+/HLA-A2+/HLA-B7+/HLA-B35 donor. Values represent number of spots per 5 × 104 CD8+ T cells after subtraction of spots counted for the negative control (DCs pulsed with E75 peptide). (D) IFN-γ ELISPOT assay of an HLA-A2+/HLA-A24+/HLA-B7+ donor. Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for the negative control (DCs pulsed with E75 peptide). Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 CTLs induced in vitro by pp65-IVT-transfected DCs are able to lyse CMV-expressing targets in a standard 51Cr-release assay. PBMCs of a CMV-positive HLA-A1+/HLA-A2+ donor were incubated with autologous pp65-IVT-transfected DCs and restimulated twice at weekly intervals. The cytotoxic activity of the induced CTLs was analyzed in a standard 51Cr-release assay using DCs pulsed with A1 or A2 peptide or DCs electroporated with pp65 IVT as targets. HIV-peptide-pulsed DCs as well as DCs transfected with the irrelevant MUC1 IVT served as negative controls. Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Induction of autologous CMV-specific CD4+-mediated T cell responses by DCs transfected with pp65 IVT. (A) DCs generated from adherent PBMCs of a CMV-positive healthy donor in the presence of GM-CSF, IL-4, and TNF-α were electroporated with pp65 IVT and cocultured with purified autologous CD4+ T lymphocytes. CD4+ T cells were analyzed in an IFN-γ ELISPOT assay using pp65-IVT- or MUC1-IVT-transfected DCs as APCs. Additionally, unstimulated autologous T cells were included in the assays. (B) Diagram of the IFN-γ ELISPOT assay shown in A. Values represent number of spots per 5 × 104 CD4+ T cells after subtraction of the negative control (DCs electroporated with MUC1 IVT). Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Induction of CD4+ T helper cell proliferation by DCs transfected with pp65 IVT. Purified CD4+ T cells of a CMV-positive donor were stimulated with autologous monocyte-derived DCs electroporated with pp65 IVT. Induced T lymphocytes were incubated for 5 days with autologous DCs electroporated with pp65 IVT as stimulators. Proliferation was measured by thymidine incorporation. Inhibition of HLA class I or class II was performed by incubating DCs for 1 h prior to the assay with anti-HLA class I or II antibodies. DCs electroporated with MUC1 IVT or pulsed with pp65-derived HLA class I-binding peptides or with irrelevant PADRE were utilized as controls. Values represent counts per minute (cpm) after incubation of 1 × 105 CD4+ T cells with 1 × 104 autologous stimulator cells. Data are expressed as the means ± SEM of quintuplicate wells. The results of one representative experiment of three are shown. Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Cotransfection of DCs with IVT coding for the antigens pp65 and IE1 leads to the induction of specific T lymphocytes that recognize peptides derived from both antigens. (A) DCs were generated from a CMV-positive HLA-A2+ donor in the presence of GM-CSF, IL-4, and TNF-α, electroporated with IE1 IVT, and used for CTL induction. IFN-γ secretion of the generated T lymphocytes was analyzed in an ELISPOT assay using IE1-derived HLA-A2-binding peptide antigens (IE1a-A2 and IE1b-A2). Values represent number of spots per 5 × 104 CD8+ T cells after subtraction of spots counted for negative control (DCs pulsed with Her-2/neu-derived peptide E75). (B) DCs generated from peripheral blood of a CMV-positive HLA-A2+/HLA-B7+ donor were cotransfected with IVT coding for the CMV antigens pp65 and IE1 and cocultured with autologous CD8+ T cells. The specificity of T cells was determined in an IFN-γ ELISPOT assay using pp65- and IE1-derived peptides. Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for negative control. Induced T lymphocytes were analyzed after 7 days of stimulation and compared to unstimulated autologous DCs. (C) DCs generated from the peripheral blood of a CMV-positive HLA-A2+/HLA-A24+/HLA-B35+ donor were cotransfected with a mixture of IVTs coding for the CMV antigens pp65 and IE1 and cocultured with autologous CD8+ T cells. IFN-γ ELISPOT assay using pp65- and IE1-derived peptides was performed to analyze the effector function and frequency of generated CTLs. Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for negative control. Induced T lymphocytes were analyzed after 7 days of stimulation and compared to unstimulated autologous CD8+ cells. Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Induction of CMV-specific cytotoxic T cells using DCs from a CMV-negative blood donor transfected with pp65 IVT. DCs were generated from CMV-negative donors in the presence of GM-CSF, IL-4, and TNF-α, electroporated with pp65 IVT, and used for CTL induction in vitro. After four restimulations, T lymphocytes were analyzed in IFN-γ ELISPOT assays (A and B). Values represent number of spots per 2 × 105 CD8+ T cells after subtraction of spots counted for the negative control (DCs + E75 peptide). (C) The cytotoxic activity of induced CTLs was analyzed after five restimulations in a standard 51Cr-release assay using DCs pulsed with pp65-derived peptides as APCs. DCs pulsed with irrelevant E75 peptide were utilized as negative control. Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 8 Induction of CMV-specific cytotoxic T cells using DCs from immunosuppressed blood donor transfected with pp65 IVT. DCs were generated from CMV-positive HLA-A2+ donor in the presence of GM-CSF, IL-4, and TNF-α, electroporated with pp65 IVT, and used for CTL induction in vitro. The cytotoxic activity of induced CTLs was analyzed after restimulation in a standard 51Cr-release assay using DCs electroporated with pp65 IVT or pulsed with pp65-derived peptides as target cells. DCs pulsed with irrelevant HIV peptide were utilized as negative control. Additionally human foreskin fibroblasts (HFF) infected with human CMV were included in the assay. Molecular Therapy 2006 13, 280-288DOI: (10.1016/j.ymthe.2005.08.019) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions