Figure 4. Suppression by the in vitro expanded CD8⁺CD28⁻ T cells is contact dependent but IFN-γ or TGF-β independent.

Suppression assays were set up as described in Figure 2 at an S∶R ratio of 0.5∶1. Proliferation of the CD4⁺ cells was measured by CFSE dilution. A, Transwell assays: the lower chambers of 24-well transwell plates were plated with CFSE-labeled responder cells (3×10⁵ of R) and stimulator cells (3×10⁵ of B-_APC), and the in vitro expanded CD8⁺CD28⁻ T cells were added either in the lower chamber to allow cell-cell contact or in the upper chamber to prevent cell-cell contact. Data shown are representative of three independent experiments. B, Control suppression without any blocking antibodies. C, Suppression in the presence of anti-human IFN-γ antibody or its isotype control antibody at concentrations of 1 µg/ml, 10 µg/ml and 100 µg/ml. D, Suppression in the presence of anti-human TGF-β antibody or its isotype control antibody at concentrations of 1 µg/ml, 10 µg/ml and 100 µg/ml. Data shown for B and C are representative of three independent experiments. Abbreviations used for A–D: “R”: CD4⁺ responder cells from donor A; “B-_APC”: APC stimulators from donor B; “S”: the in vitro expanded CD8⁺CD28⁻ T cells.