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

<p>Suppression assays were set up as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028948#pone-0028948-g002" target="_blank">Figure 2</a> at an S∶R ratio of 0.5∶1. Proliferation of the CD4⁺ cells was measured by CFSE dilution. <i>A</i>, 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 <i>in vitro</i> 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. <i>B</i>, Control suppression without any blocking antibodies. <i>C</i>, 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. <i>D</i>, 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 <i>B</i> and <i>C</i> are representative of three independent experiments. Abbreviations used for <i>A</i>–<i>D</i>: “R”: CD4⁺ responder cells from donor A; “B-_APC”: APC stimulators from donor B; “S”: the <i>in vitro</i> expanded CD8⁺CD28⁻ T cells.