Figure 3.

TCR activation is essential and sufficient to sensitize S2B5 cells to CD8⁺ Treg cells. (A) CD4⁺ target cell (S2B5) viability was measured by the MTS assay after 2 day incubation with CD8⁺ Treg cells (1E2) under various conditions. S2B5 cells co-incubated with 1E2 cells without anti-CD3 activation (1E2, S2B5); anti-CD3 pre-activated S2B5 co-incubated with 1E2 cells (1E2, S2B5 αCD3); S2B5 co-incubated with anti-CD3 pre-activated 1E2 cells (1E2 αCD3, S2B5); anti-CD3 pre-activated S2B5 co-incubated with anti-CD3 pre-activated 1E2 cells (1E2 αCD3, S2B5 αCD3); and S2B5 cells co-incubated with 1E2 cells in the presence of αCD3 antibodies (1E2, S2B5, αCD3 present). (B) CD4⁺ target cell (S2B5) viability was measured by flow cytometry after 4 h incubation with CD8⁺ Treg cells under various conditions. S2B5 cells were incubated with CD8⁺ Treg-cell clone (1E2) or CD8⁺ non-Treg-cell clone (1D1) without αCD3 pre-activation, and αCD3 antibody pre-activated S2B5 cells were incubated with 1E2 (1E2, aCD3) or 1D1 cells (1D1, aCD3). The results are shown as mean±SD of data pooled from three independent experiments. (C) CD4⁺ target cells (S2B5) were incubated with 1E2 cells at E/T ratio of 1:1 without anti-CD3 activation (−), with anti-CD3 preactivation of S2B5 cells (Pre-incubation) and in the presence of anti-CD3 antibodies (+). Apoptosis of S2B5 cells was monitored by flow cytometry after 4 h incubation. The results are shown as mean±SD of data pooled from three independent experiments. p-Values were obtained via unpaired, two-tailed t-test.