Figure 2
Figure 2. Differential recognition of endogenously processed CMV epitopes. (A-B) PBMCs from CMV-seropositive donors were incubated in the presence of monensin and anti-CD107a with a panel of TB40E-bac–infected fibroblast cell lines at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody and IFN-γ expression was assessed using an intracellular cytokine assay. Data represent the proportion of MHC-multimer+ cells expressing CD107a (A) or IFN-γ (B) after incubation with HLA-matched fibroblasts either uninfected or infected for 24 or 48 hours. (C-D) CMV-specific T cells were incubated in the presence of monensin and anti-CD107a with a panel of TB40E-bac–infected fibroblast cell lines at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody, and IFN-γ expression was assessed using an intracellular cytokine assay. Data represent the mean ± SEM from 3 independent experiments of the proportion of antigen-specific cells expressing CD107a (C) or IFN-γ (D) relative to peptide pulsed controls. (E) CMV-specific T cells were stimulated in the presence of monensin and anti-CD107a with a panel of HLA-matched paraformaldehyde-fixed LCLs infected with recombinant Vaccinia virus encoding either IE-1 or pp65 at a responder to stimulator ratio of 5:1. Cells were incubated with MHC-peptide pentamers and anti-CD8 antibody. Data represent the mean ± SEM of the proportion of antigen-specific cells generating IFN-γ. (F) CMV-specific T cells were incubated in the presence of monensin and anti-CD107a with the HLA A1 A2 B8-positive JuSt fibroblast cell lines infected overnight at a responder to stimulator ratio of 5:1 with AdCMVpoly. Data represent the mean ± SEM of the proportion of antigen-specific cells generating IFN-γ. (G) PBMCs from HLA B7 CMV-seropositive donors were incubated for 5 hours with the peptide epitopes RPHERNGFTVL (RPH) and TPRVTGGGAM (TPR), labeled with anti-CD8, and then assessed for intracellular expression of IFN-γ, T-bet, and Eomes. Data represent the mean ± SEM of the proportion of RPH or TPR-specific T cells displaying a T-betloEomeshi, T-betintEomeshi, or T-bethiEomeshi/lo phenotype. (H-I) RPH-, TPR-, and NLV-specific T cells were incubated in the presence of monensin and anti-CD107a with a TB40E-bac-infected HLA A2 B7-positive fibroblast cell line at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody, and IFN-γ expression was assessed using an intracellular cytokine assay.

Differential recognition of endogenously processed CMV epitopes. (A-B) PBMCs from CMV-seropositive donors were incubated in the presence of monensin and anti-CD107a with a panel of TB40E-bac–infected fibroblast cell lines at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody and IFN-γ expression was assessed using an intracellular cytokine assay. Data represent the proportion of MHC-multimer+ cells expressing CD107a (A) or IFN-γ (B) after incubation with HLA-matched fibroblasts either uninfected or infected for 24 or 48 hours. (C-D) CMV-specific T cells were incubated in the presence of monensin and anti-CD107a with a panel of TB40E-bac–infected fibroblast cell lines at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody, and IFN-γ expression was assessed using an intracellular cytokine assay. Data represent the mean ± SEM from 3 independent experiments of the proportion of antigen-specific cells expressing CD107a (C) or IFN-γ (D) relative to peptide pulsed controls. (E) CMV-specific T cells were stimulated in the presence of monensin and anti-CD107a with a panel of HLA-matched paraformaldehyde-fixed LCLs infected with recombinant Vaccinia virus encoding either IE-1 or pp65 at a responder to stimulator ratio of 5:1. Cells were incubated with MHC-peptide pentamers and anti-CD8 antibody. Data represent the mean ± SEM of the proportion of antigen-specific cells generating IFN-γ. (F) CMV-specific T cells were incubated in the presence of monensin and anti-CD107a with the HLA A1 A2 B8-positive JuSt fibroblast cell lines infected overnight at a responder to stimulator ratio of 5:1 with AdCMVpoly. Data represent the mean ± SEM of the proportion of antigen-specific cells generating IFN-γ. (G) PBMCs from HLA B7 CMV-seropositive donors were incubated for 5 hours with the peptide epitopes RPHERNGFTVL (RPH) and TPRVTGGGAM (TPR), labeled with anti-CD8, and then assessed for intracellular expression of IFN-γ, T-bet, and Eomes. Data represent the mean ± SEM of the proportion of RPH or TPR-specific T cells displaying a T-betloEomeshi, T-betintEomeshi, or T-bethiEomeshi/lo phenotype. (H-I) RPH-, TPR-, and NLV-specific T cells were incubated in the presence of monensin and anti-CD107a with a TB40E-bac-infected HLA A2 B7-positive fibroblast cell line at a responder to stimulator ratio of 5:1. Cells were then incubated with MHC-peptide multimers and anti-CD8 antibody, and IFN-γ expression was assessed using an intracellular cytokine assay.

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