Mature dendritic cells pulsed with freeze–thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4+ and CD8+ T lymphocyte responses by Wolfgang Herr, Elena Ranieri, Walter Olson, Hassane Zarour, Loreto Gesualdo, and Walter J. Storkus Blood Volume 96(5):1857-1864 September 1, 2000 ©2000 by American Society of Hematology

Freshly isolated T cells from EBV-seropositive healthy donor IP1 react against bulk antigenic formats prepared from autologous EBV B-LCL cells and presented by autologous DCs.EBV B-LCL–derived bulk antigens (for preparation, see “Materials and methods”) wer... Freshly isolated T cells from EBV-seropositive healthy donor IP1 react against bulk antigenic formats prepared from autologous EBV B-LCL cells and presented by autologous DCs.EBV B-LCL–derived bulk antigens (for preparation, see “Materials and methods”) were pulsed onto autologous immature DCs at a ratio of 100 tumor cell equivalents per DC and were screened for reactivity using CD4+ and CD8+ T-cell responders purified from the blood of donor IP1 (HLA-A2,32; B7,62; Cw3; DR4,15) in IFN-γ Elispot assays. Protein/peptide yields from 109 EBV B-LCL cells were in the range of the following: freeze–thaw lysates, 30 to 50 mg; TFA lysates, 10 to 30 mg; and eluted naturally presented peptides, 0.5 to 1 mg. Control wells contained T cells with untreated DCs. After a culture period of 20 hours, IFN-γ spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 105 CD4+ T lymphocytes or CD8+ T lymphocytes initially seeded per well. The numbers of antigen-reactive T cells per 105 T lymphocytes are calculated by subtraction of mean spot numbers induced by untreated DCs from mean spot numbers induced by antigen-loaded DCs (asterisks indicate significant results, ie, P < .05). No T-cell responses were observed for freeze–thaw and TFA lysate fractions smaller than 10 kd, acid-eluted HLA-A2 peptide fraction 3 kd or larger, and acid-eluted HLA-DR peptide fraction smaller than 3 kd. Spot production was not detected when T cells were incubated with EBV B-LCL–derived bulk antigens in the absence of DCs. Results were confirmed in 4 independent experiments. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

Autologous DCs pulsed with EBV B-LCL–derived freeze–thaw lysate induces EBV-specific CD4+T-cell responses in an antigen dose-dependent manner.CD4+ T cells directly isolated from blood lymphocytes of healthy anti-EBV–positive donor IP1 were seeded at 105cell... Autologous DCs pulsed with EBV B-LCL–derived freeze–thaw lysate induces EBV-specific CD4+T-cell responses in an antigen dose-dependent manner.CD4+ T cells directly isolated from blood lymphocytes of healthy anti-EBV–positive donor IP1 were seeded at 105cells per well and were tested for reactivity against freeze–thaw lysate fractions 10 kd or larger prepared from autologous LCL cells, B-, or T-cell blasts in IFN-γ Elispot assays. For antigen processing and presentation, autologous immature DCs were prepulsed with freeze–thaw cell lysates at the ratio of cell equivalents per DCs of 100:1, 10:1, or 1:1. Resulting spots were evaluated and presented as described in Figure 1. Results were confirmed in 3 independent experiments. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

Comparison of the ability of autologous monocytes and immature and mature DCs to stimulate CD4+ and CD8+ T-cell responses against EBV B-LCL freeze–thaw lysates in IFN-γ Elispot assays.CD4+ and CD8+ T cells were directly isolated from the blood of EBV-seropo... Comparison of the ability of autologous monocytes and immature and mature DCs to stimulate CD4+ and CD8+ T-cell responses against EBV B-LCL freeze–thaw lysates in IFN-γ Elispot assays.CD4+ and CD8+ T cells were directly isolated from the blood of EBV-seropositive healthy donor IP2 and were seeded at 105 cells per well. Autologous monocytes, immature DCs, or mature DCs were not pulsed or were pulsed with lysates derived from autologous EBV B-LCL cells, B-, or T-cell blasts (both 10 kd or larger) as indicated and were added to microwells containing T-cell responders. For maturation, immature DCs were treated on day 6 with TNF-α, IL-1β, IL-6, and PGE2 for 48 hours (see “Materials and methods”). Mature DC-Is were pulsed with lysate during the 48 hours of maturation from immature DC. Mature DC-IIs were first matured for 48 hours and then pulsed with the lysate for an additonal 48 hours prior to addition to Elispot wells. Resulting spots developed after 20-hour incubation were evaluated and presented as described in Figure 1. Each bar represents the mean spot number of triplicates ± SD with 105 CD4+ T lymphocytes or CD8+ T lymphocytes initially seeded per well. The data shown are from 1 representative experiment of 5 performed using donors IP1 and IP2. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

Mature DCs cross-present LCL-derived epitopes derived from freeze–thaw lysates to freshly isolated CD4+and CD8+ T cells.Immature DCs generated from donors IP1 and IP2 were loaded with lysate fractions 10 kd or larger prepared from either donors' EBV B-LCL o... Mature DCs cross-present LCL-derived epitopes derived from freeze–thaw lysates to freshly isolated CD4+and CD8+ T cells.Immature DCs generated from donors IP1 and IP2 were loaded with lysate fractions 10 kd or larger prepared from either donors' EBV B-LCL or B-cell/T-cell blasts. After maturation was induced (see “Materials and methods”), DCs were added to freshly isolated autologous CD4+ and CD8+ T cells in 20-hour IFN-γ Elispot assays. Resulting spots were developed and counted as described in Figure 1. Each bar represents the mean spot number of triplicates ± SD per 105 CD4+ T lymphocytes (▪) or CD8+ T lymphocytes (░) initially seeded per well. Calculation of lysate-responsive T-cell frequencies were performed as outlined in Figure 1. Results were confirmed in 4 independent experiments. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

CD4+ T cells reactive against autologous EBV B-LCL cells may be efficiently induced by repeated stimulations with LCL lysate-pulsed mature autologous DCs.CD4+ T cells freshly isolated from EBV-seropositive donor IP1 were stimulated on a weekly regimen (days... CD4+ T cells reactive against autologous EBV B-LCL cells may be efficiently induced by repeated stimulations with LCL lysate-pulsed mature autologous DCs.CD4+ T cells freshly isolated from EBV-seropositive donor IP1 were stimulated on a weekly regimen (days 0, 7, and 14) with autologous mature DCs prepulsed with freeze–thaw lysate (10 kd or larger) prepared from autologous EBV B-LCL. Freshly isolated (day 0) CD4+ T cells and T lymphocyte responders harvested on day 21 of culture (both seeded in triplicates at 105 and 104 cells per well) were analyzed in IFN-γ and IL-5 Elispot assays. T-cell reactivity was screened against intact autologous EBV B-LCL (●); against autologous mature DCs preloaded with freeze–thaw lysates (10 kd or larger) isolated from autologous EBV B-LCL (○), B-cell blasts (▾), or T-cell blasts (▪); and against autologous mature DCs pulsed with naturally processed peptides acid-eluted from affinity-purified HLA-DR complexes of the autologous EBV B-LCL (■). Resulting spots were developed and evaluated as described in Figure 1. Spot production observed in microwells where CD4+ (responder) lymphocytes were seeded with the autologous EBV B-LCL or with mature DCs loaded with the EBV B-LCL lysate was completely blocked by the addition of the anti-HLA-DR (class II) antibody L243 (100 μg/mL) but not by the anti-HLA class I antibody W6/32 (100 μg/mL). Results were confirmed in 2 independent experiments. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

Autologous mature DCs pulsed with EBV B-LCL cells versus freeze–thaw lysates stimulate anti-EBV CD8+ CTL in vitro.CD8+ T cells were purified from the blood of EBV-seropositive donor IP2 and were then repetitively stimulated on a weekly basis (days 0, 7, and... Autologous mature DCs pulsed with EBV B-LCL cells versus freeze–thaw lysates stimulate anti-EBV CD8+ CTL in vitro.CD8+ T cells were purified from the blood of EBV-seropositive donor IP2 and were then repetitively stimulated on a weekly basis (days 0, 7, and 14) with autologous intact EBV B-LCL cells (●) or with autologous mature DCs preloaded with freeze–thaw lysates (10 kd or larger) prepared from autologous EBV B-LCL (○), autologous T-cell blasts (■), or allogeneic EBV-B LCL of donor IP1 (▾). On day 23 of culture, responder lymphocytes were harvested and were tested in a 6-hour 51Cr release assay at the indicated effector-to-target ratios for cytolytic activity against autologous EBV B-LCL (A) or autologous T-cell blasts (B) in the presence of a 20-fold excess of nonlabeled K562 competitors. For all responder lymphocyte cultures, lysis of labeled K562 in the presence of a 20-fold excess of nonlabeled K562 was below 5% at all effector-to-target ratios evaluated (not shown). The data depicted are from 1 representative experiment of 3 performed. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology

Autologous mature DCs pulsed with autologous or allogeneic EBV B-LCL freeze–thaw lysates stimulate anti-EBV CD8+ CTL in vitro.Purified CD8+ lymphocytes from EBV-seropositive donor IP3 were stimulated with autologous DCs loaded with freeze–thaw lysates prepa... Autologous mature DCs pulsed with autologous or allogeneic EBV B-LCL freeze–thaw lysates stimulate anti-EBV CD8+ CTL in vitro.Purified CD8+ lymphocytes from EBV-seropositive donor IP3 were stimulated with autologous DCs loaded with freeze–thaw lysates prepared from autologous IP3 EBV B-LCL (A) or allogeneic IP2 EBV B-LCL (B) as described in “Materials and methods” and Figure 6. Six-hour51Cr-release assays were performed on day 24 (10 days after restimulation on day 14). K562 cells were not added as cold-target inhibitors because T-cell specific lysis of K562 was less than 5% at all effector-to-target ratios (data not shown). The percent specific lysis is reported against IP1 EBV B-LCL (▪), IP2 EBV B-LCL (▿), IP3 EBV B-LCL (●), IP3 EBV B-LCL in the presence of blocking mAb directed against MHC class I (W6/32, ○) or MHC class II (L243, ▾) molecules, or IP3 T blasts (■). Donors IP1, IP2, and IP3 are completely mismatched for HLA class I. Wolfgang Herr et al. Blood 2000;96:1857-1864 ©2000 by American Society of Hematology