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Application of the ELISPOT assay to the characterization of CD8+ responses to Epstein-Barr virus antigens by Jie Yang, Victor M. Lemas, Ian W. Flinn, Chris.

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Presentation on theme: "Application of the ELISPOT assay to the characterization of CD8+ responses to Epstein-Barr virus antigens by Jie Yang, Victor M. Lemas, Ian W. Flinn, Chris."— Presentation transcript:

1 Application of the ELISPOT assay to the characterization of CD8+ responses to Epstein-Barr virus antigens by Jie Yang, Victor M. Lemas, Ian W. Flinn, Chris Krone, and Richard F. Ambinder Blood Volume 95(1): January 1, 2000 ©2000 by American Society of Hematology

2 Cellular sources of IFN-γ secretion in response to autologous LCLs
Cellular sources of IFN-γ secretion in response to autologous LCLs.PBMCs from a healthy EBV-seropositive donor FT were sequentially fractionated into CD8+, CD4+, CD56+, and CD8−CD4−CD56−cell populations using CD8 and CD4 Dynal-beads and CD56 Microbeads. Cellular sources of IFN-γ secretion in response to autologous LCLs.PBMCs from a healthy EBV-seropositive donor FT were sequentially fractionated into CD8+, CD4+, CD56+, and CD8−CD4−CD56−cell populations using CD8 and CD4 Dynal-beads and CD56 Microbeads. Each cell population was incubated with autologous LCL stimulators for the detection of IFN-γ secretion in an ELISPOT assay. Each bar represents the mean ± SD of triplicate wells. The numbers of spots/1 × 106 cells of the respective cell population are shown on the Y axis. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

3 Frequency of LCL-responsive CD8+ cells
Frequency of LCL-responsive CD8+ cells.Isolated CD8+ cells from 13 healthy EBV-seropositive and 3 seronegative donors were incubated with autologous LCL stimulators in ELISPOT assays. Frequency of LCL-responsive CD8+ cells.Isolated CD8+ cells from 13 healthy EBV-seropositive and 3 seronegative donors were incubated with autologous LCL stimulators in ELISPOT assays. Each bar represents the mean ± SD as determined in 2 separate experiments; data from each experiment represent the average of triplicate wells. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

4 Frequency of LMP2 peptide-responsive CD8+ cells
Frequency of LMP2 peptide-responsive CD8+ cells.PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 μg/mL of the LMP2 peptide (residues ) in ELISPOT assays. Frequency of LMP2 peptide-responsive CD8+ cells.PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 μg/mL of the LMP2 peptide (residues ) in ELISPOT assays. Each bar represents the mean ± SD as determined in 2 separate experiments; data from each experiment represent the average of more than 12 wells. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

5 Frequency of BMLF1 peptide-responsive CD8+ cells
Frequency of BMLF1 peptide-responsive CD8+ cells.PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 μg/mL of the BMLF1 peptide (residues ) in ELISPOT assays. Frequency of BMLF1 peptide-responsive CD8+ cells.PBMC from 13 healthy EBV-seropositive donors, 11 HLA-A2 and 2 non-HLA-A2, were incubated with 10 μg/mL of the BMLF1 peptide (residues ) in ELISPOT assays. Each bar represents the mean ± SD as determined in 2 separate experiments; data from each experiment represent the average of triplicate wells. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

6 Increases in the number of EBV-specific CD8+ cells over the course of polyclonal CTL line generation.EBV-specific polyclonal CTL lines from HLA-A2 donors ES (A), SB (B), MS (C), and TZ (D) were established. Increases in the number of EBV-specific CD8+ cells over the course of polyclonal CTL line generation.EBV-specific polyclonal CTL lines from HLA-A2 donors ES (A), SB (B), MS (C), and TZ (D) were established. Cells were harvested on day 28 and cryopreserved. On the day of the ELISPOT assay, aliquots of CTL lines were thawed and incubated with stimulators in multiscreen HA plate. Stimulators for ELISPOT assay included autologous LCLs (1 × 105/well), and autologous PBMCs (1 × 105/well) in medium containing the LMP2 or BMLF1 peptide (10 μg/mL, final concentration). The fold-increase in the number of LCL-responsive (filled diamonds), LMP2 peptide-responsive (open circles), and BMLF1 peptide-responsive CD8+ cells (open triangles) is indicated to the right of the day 28 data point. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

7 Positive correlation between the number of IFN-γ-secreting cells and cytolytic activity in CTL lines.EBV-specific CTL lines from 4 HLA-A2 donors were generated by weekly restimulation with irradiated autologous LCLs for 2 to 4 weeks. Positive correlation between the number of IFN-γ-secreting cells and cytolytic activity in CTL lines.EBV-specific CTL lines from 4 HLA-A2 donors were generated by weekly restimulation with irradiated autologous LCLs for 2 to 4 weeks. The CTL lines were then assayed as responders/effectors in a paired ELISPOT/51Cr-release assay. Stimulators for the ELISPOT assay or targets for 51Cr-release assay included autologous LCLs for both the ELISPOT and 51Cr-release assays (filled diamonds), autologous PBMCs in medium containing peptide (LMP2 or BMLF1 peptide) as stimulators for ELISPOT assays, and autologous PHA blasts in medium containing peptide as targets for 51Cr-release assay (open diamonds). Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

8 Application of the ELISPOT assay to the monitoring of changes in the number of LCL-responsive CD8+ cells following adoptive CTL infusion.Two patients with PTLD received EBV-specific polyclonal CTL infusion from partially matched donors. Application of the ELISPOT assay to the monitoring of changes in the number of LCL-responsive CD8+ cells following adoptive CTL infusion.Two patients with PTLD received EBV-specific polyclonal CTL infusion from partially matched donors. The HLA types of patients and donors were: patient 1 (A1, 24, B7, 8), donor for patient 1 (A1, 33, B8, 17); patient 2 (A2, 32, B13, 27), donor for patient 2 (A2, 3, B7, 27). Blood was drawn immediately before and at the indicated time points after infusion. CD8+ cells were used as responders in ELISPOT assays with the patient's LCLs as stimulators. Each bar represents the mean ± SD of triplicate wells. Jie Yang et al. Blood 2000;95: ©2000 by American Society of Hematology

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