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A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2−

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Presentation on theme: "A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2−"— Presentation transcript:

1 A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2− donors  Aaron E. Foster, Kenneth F. Bradstock, Uluhan Sili, Marina Marangolo, Cliona M. Rooney, David J. Gottlieb  Biology of Blood and Marrow Transplantation  Volume 10, Issue 11, Pages (November 2004) DOI: /j.bbmt Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

2 Figure 1 Transduction of DCs with Ad5pp65GFP. DCs were incubated on day 5 with Ad5pp65GFP and analyzed on day 7 for the expression of GFP. DCs were gated by high forward and side scatter (A) and analyzed by comparing CD11c and GFP expression on nontransduced DCs (B) and Ad5pp65GFP-transduced DCs (C). Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

3 Figure 2 Autologous mixed lymphocyte reaction with CMV-loaded DCs. DCs were analyzed for the ability to stimulate CMV-specific proliferation in a CMV-seropositive donor. (A) Proliferation of PBMCs stimulated with DCs that were mock-transduced, or transduced with Ad5GFP only or Ad5pp65GFP encoding pp65, and then used in a 5-day [3H]thymidine proliferation assay in quadruplicate. (B) Proliferation of PBMCs stimulated with DCs incubated with CMV lysate was compared with DCs loaded with control antigen and mock-loaded DCs. PBMCs and DCs were also compared for proliferation alone. *P < .05 against DCs loaded with control antigen. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

4 Figure 3 Tetramer expansion after stimulation with different CMV antigen types. The frequency of CD8+ tetramer+ T cells was compared after PBMCs from a CMV-seropositive donor were stimulated with DCs loaded with NLV peptide, Ad5pp65GFP, or CMV lysate. Unstimulated PBMCs are shown on the left, and CMV-specific T-cell cultures are shown at day 7 and 14 for their respective antigen types. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

5 Figure 4 Expansion of CMV-specific T cells from a single HLA-A2+ donor after stimulation with CMV-loaded DCs. Total cells (A) and total tetramer+ CD8+ T cells (B) were enumerated after 21 days in culture from HLA-A2+ donors (n = 3; donors 1, 2, and 5) after stimulation with DCs loaded with Ad5pp65GFP (•), CMV lysate (■), or NLV peptide (▴). Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

6 Figure 5 Cytotoxicity of CMV-specific T-cell cultures against peptide-pulsed T2 target cells. (A) The cytotoxicity of CMV-specific T-cell cultures generated by DCs loaded with NLV peptide (triangles), Ad5pp65GFP (squares), and CMV lysate (circles) was tested against that of chromium-labeled T2 cells loaded with NLV peptide (closed symbols) or the irrelevant peptide VLQ (open symbols). (B) CMV-specific T-cell cultures stimulated with CMV lysate (•), Ad5pp65GFP (■), or NLV peptide (▴) were tested against T2 cells loaded with the CMV IE1 peptide VLE. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

7 Figure 6 TCR Vβ analysis of CD4+ CMV-specific T cells. To assess whether CD4+ T cells expanded in response to CMV antigen stimulation, the CD4+ TCR Vβ frequency was analyzed on 2 donors (donor 1 and 2; panel 1 and 2, respectively) before CMV antigen stimulation and after 2 stimulations with DCs loaded with either Ad5pp65GFP or CMV lysate. Hashed boxes indicate expansion of similar CD4+ TCR Vβ regions between stimulating antigens. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

8 Figure 7 Production of IFN-γ by sorted CD4+ CMV-specific T cells. CD4+ CMV-specific T cells from donor 1 were sorted by flow cytometry and plated in microtiter plates in triplicate. DCs either loaded with CMV lysate transduced with Ad5pp65GFP or mock-loaded were then used to stimulate the T cells. IFN-γ production in response to stimulation was then measured by enzyme-linked immunosorbent assay. Both CMV lysate-stimulated and Ad5pp65GFP-stimulated T-cell cultures contained CD4+ T cells capable of recognizing CMV antigens. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

9 Figure 8 Specificity of HLA-A2− CMV-specific T cells generated from Ad5pp65GFP-loaded and CMV lysate-loaded DCs. T-cell cultures from an HLA-A2− donor (donor 7) were assayed for CMV specificity by cytotoxicity and IFN-γ production by comparing the response to stimulation by DCs loaded with Ad5pp65GFP, CMV lysate, or control DCs. (A) Ad5pp65GFP-generated CMV-specific T cells lyse DC target cells transduced with Ad5pp65GFP (▴) and CMV lysate-pulsed DCs (■) but do not recognize Ad5GFP (□) or mock-pulsed DC (▵) target cells. (B) CMV lysate-generated CMV-specific T cells lyse both DC target cells transduced with Ad5pp65GFP (▴) and CMV lysate-pulsed DCs (■) but do not kill Ad5GFP (□) or mock-pulsed DC (▵) target cells. (C) CMV-specific T cells derived from an HLA-B35 donor (donor 6) after stimulation with Ad5pp65GFP, CMV lysate, or VFP (HLA-B35-restricted pp65 peptide) were tested for cytotoxicity against autologous chromium-labeled DC target cells pulsed with either VFP or irrelevant control peptide at a ratio of 50:1 effector/target cells. Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt ) Copyright © 2004 American Society for Blood and Marrow Transplantation Terms and Conditions

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