FIG. 4.

Impact of AdE1-LMPpoly-mediated expansion on T-cell polyfunctionality. (A and B) Cytokine profiles of AdE1-LMPpoly-expanded T cells. T cells from cultures stimulated with AdE1-LMPpoly were incubated with EBNA1 or LMP-1/2 epitopes in the presence of brefeldin A. IFN-γ, TNF, and MIP1β expression by CD8⁺ T cells was assessed using intracellular cytokine assay. (A) Percentages of CD8⁺ T cells from individual donors producing each cytokine combination. (B) Mean percentages of specific cells producing 1, 2, or 3 cytokines. (C) Degranulation of AdE1-LMPpoly-stimulated T cells. Cultured T cells were incubated with EBNA1 or LMP-1/2 epitopes in the presence of monensin and anti-CD107α. Data represent the frequency of T cells that produce CD107α or IFN-γ in response to LMP or EBNA1 CTL epitopes. (D) Effect of recombinant Gal-1 on the proliferation of AdE1-LMPpoly-expanded T cells. AdE1-LMPpoly-expanded T cells were preincubated with Gal-1 at 5 μg/ml (or mock treated) and stimulated with LMP-1/2 CTL epitopes. IFN-γ expression by CD8⁺ T cells was assessed using intracellular cytokine assay. Data represent the comparative proliferation in PBMC from individual donors with and without Gal-1 treatment. (E) AdE1-LMPpoly-stimulated T cells were stimulated with autologous LCL at responder-to-stimulator ratios of 10:1, 20:1, and 40:1 in the presence of brefeldin A for 12 h. Data represent the percentages of IFN-γ-producing, MHC peptide pentamer-specific T cells following incubation with a panel of HLA-matched LCLs.