Flow cytometric analysis, cytokine expression, mixed lymphocyte reaction, and cytokine secretion. (A) Representative flow analysis of 1 experiment repeated 5 times that shows cell composition and subsets from start to finish of preselection, post–CD45RA depletion, and post–CD8⁺ enrichment. Cells were stained for expression of CD45RA, CD45RO, and CD62L, and plots are shown for CD4⁺ and CD8⁺ gated cells at each step of the processing procedure. The surface phenotype of the CD45RA^–CD8⁺-enriched cells is consistent with the phenotypic CD8⁺ T_EM subset being predominantly CD45RA^–CD45RO⁺CD62L^–. The T_EM cells ranged from 81.7% to 98.1% of the final cell composition. (B) Flow cytometric analysis of cytokine expression by enriched cell subsets. Cells were activated by ionomycin and phorbol myristate acetate, treated with monensin, and stained for expression of CD45, CD4, CD8, and CD45RA. Cells were fixed, permeabilized, and stained for INF-γ, and IL-2. Plots show comparison of IL-2 and IFN-γ expression in CD8⁺CD45RA^–, CD8⁺CD45RA⁺, and CD4⁺CD45RA^– cells as indicated. (C) Proliferation of CD8⁺CD45R^– and CD4⁺ cells activated by co-culture with or without irradiated allogeneic stimulators and assessed by ³H-thymidine uptake during the last 24 hours of culture. Mean and standard deviations are shown (n = 4). (D) Cytokine secretion assessment in CD8⁺CD45RA^– and CD4⁺ cells activated by co-culture with or without irradiated allogeneic stimulators. Supernatants from 7-day cultures were analyzed by flow cytometry using cytokine bead arrays for INF-γ, IL-2, and TNF-α (n = 4). Means with standard deviations are shown. Teff, T effector cells.