1. Recovery of Varicella-Zoster Virus–Specific T Cell Immunity after T Cell–Depleted Allogeneic Transplantation Requires Symptomatic Virus Reactivation 
Eva Distler, Elke Schnürer, Eva Wagner, Charis von Auer, Bodo Plachter, Daniela Wehler, Christoph Huber, Karin Kolbe, Ralf Georg Meyer, Wolfgang Herr 
Biology of Blood and Marrow Transplantation 
Volume 14, Issue 12, Pages (December 2008)
DOI: /j.bbmt
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
VZV lysate is recognized by CD4+ memory T cells. A, PBMCs derived from VZV-seropositive healthy donor 1 were analyzed for reactivity to VZV-infected and uninfected Vero cell lysates in an IFN-γ ELISPOT assay. Lysates were added at titrated concentrations to the assay medium. Data are means of duplicate wells and are representative of 6 experiments in 3 different healthy virus carriers. B, CD4+ and CD8+ T cells were selected from PBMCs of VZV-seropositive healthy donor 2 and incubated with lysate-loaded autologous dendritic cells in ELISPOT plates. Dendritic cells were induced to mature after the addition of lysate preparations, which is a suitable strategy for promoting lysate-reactive CD8+ T cell responses [15]. C, ELISPOT reactivity to VZV lysate in PBMCs of seropositive healthy donor 3 was inhibited by monoclonal antibodies with blocking activity against HLA-DR, but not against HLA-class I. Data in panels B and C are means of triplicate wells and are representative of experiments in 5 different healthy immune donors.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Quantification of VZV-reactive T cells in healthy immune and nonimmune donors. Diagrams show results of IFN-γ ELISPOT reactivity to VZV lysate in PBMCs of 15 seropositive and 5 seronegative healthy donors. The frequency of VZV-reactive T cells/mL of peripheral blood was calculated from the SFC count per 5 × 105 plated PBMCs and the PBMC count/mL of peripheral blood. ELISPOT reactivity to PHA did not differ significantly among individual PBMC samples (data not shown). PB, peripheral blood.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Reconstitution of VZV-specific T cell immunity on posttransplantation herpes zoster. Patients 1 to 5 developed zoster during the first year after undergoing T cell–depleted allo-HSCT. Diagrams show the frequencies of VZV-reactive T cells in leukapheresis-derived PBMCs of stem cell donors before transplantation and in PBMCs of corresponding patients at various time points after transplantation. Results were obtained by IFN-γ ELISPOT assay using VZV lysate as the antigen source. Reactivity to uninfected control lysate was not found in any sample. Diagrams also include data of VZV-specific IgM (+, positive; -, negative) and VZV-specific IgG (mU/mL). Arrows indicate time points of zoster onset. SD, stem cell donor; DU, HLA-disparate unrelated; MU, HLA-matched unrelated; MS, HLA-matched sibling; Pat, patient; PB, peripheral blood.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
HLA restriction of VZV-reactive T cells during the first weeks after zoster. Shown is IFN-γ ELISPOT reactivity to VZV lysate in PBMCs of patients 1 and 5 isolated on days 14 and 35 after zoster onset, respectively. The assay medium contained mAbs for blocking HLA-class I or HLA-DR.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
Failure to reconstitute VZV-specific T cells in patients without zoster. Patients 6 to 17 did not develop zoster during the first year after T cell–depleted allo-HSCT. Diagrams show the frequency of VZV-reactive T cells in leukapheresis-derived PBMCs of stem cell donors before transplantation and in PBMCs of corresponding patients at different time points after transplantation. PBMCs of stem cell donors 12, 14, 16, and 17 were not available. Results were obtained by IFN-γ ELISPOT assay. For abbreviations, see Figure 3.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

7. Figure 6
Detection of VZV reactivity by proliferation assay. PBMCs isolated from patients prior to and after disease manifestation, as well as PBMCs from healthy immune and nonimmune donors (HD), were incubated for 6 days with 1 μg/mL of VZV-infected Vero cell lysate or uninfected control lysate, respectively. Medium and PHA (not shown) served as controls. T cell proliferation was measured by 3H-thymidine uptake for the last 18 hours of incubation on day 6. Prezoster PBMCs were derived at 2.75 months (patient 1), 0.75 month (patient 3), 4.5 months (patient 4), and 2.25 months (patient 5) after HSCT. Postzoster PBMCs were obtained at 4.75 months (patient 1), 3 months (patient 3), 6.25 months (patient 4), and months (patient 5) after HSCT. For correlation with ELISPOT data, see Figure 3.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions