Human alloreactive T cells rapidly produce TNF-α after TCR engagement. PBMCs from human donors were stimulated with (A) a mAb specific for human CD3 or (B-C) with either allogeneic or autologous CFSE-labeled PBMCs as described in “Materials and methods.” After 6 hours samples were stained for cell surface markers and for TNF-α and IFN-γ. For T cells stimulated with anti-CD3 mAb (A), samples were gated on CD3⁺ cells and then on either CD8⁺ or CD4⁺ cells and analyzed for their expression of TNF-α or IFN-γ. The values represent the percentage of T cells that produced TNF-α or IFN-γ. For T cells stimulated with CFSE-labeled PBMCs, samples were gated on the CFSE low cells that were CD3⁺ cells and either (B) CD8⁺ or (C) CD4⁺. The values represent the percentage of T cells that produced TNF-α. Samples were considered positive for TNF-α production if the values were more than 3 standard deviations over the mean of TNF-α produced by T cells from 12 donors after stimulation with autologous CFSE-labeled PBMCs (*0.09% for CD8 and 0.08% for CD4). This threshold value is indicated by the horizontal line on the graphs. The data are representative of 3 experiments with a total of 12 donors in 36 combinations.