Figure 5. Cytotoxicity does not contribute to the suppression of proliferation by the in vitro expanded CD8⁺CD28⁻ T cells.

A CFSE based cytotoxicity assay was set up as follows: target cells were the original priming APCs (for generating the CD8⁺CD28⁻ T cells) labeled with a high concentration (2.0 µM) of CFSE (B-_APC-CFSE^high), or APCs from an HLA-A, -B, and -DR mismatched indifferent individual labeled with a low concentration (0.2 µM) of CFSE (I-_APC-CFSE^low). A 1∶1 mixture of I-_APC-CFSE^low and B-_APC-CFSE^high cells were plated either by themselves (left panels), or with the same number of control (CD8⁺CD28⁺, middle panels) or putative (CD8⁺CD28⁻, right panels) effector cells in 96 U-bottom plates in triplicates. At 24, 72, and 120 hr of culture, cells were collected and analyzed by FACS to enumerate CFSE^low and CFSE^high cells. The ratio of cell numbers of I-_APC-CFSE^low over B-_APC-CFSE^high was calculated as “R” for all time points. An increase in the value of R indicates specific killing of B-_APC-CFSE^high cells by the effector cells, whereas a stable value of R at 1.0 indicates no specific killing of B-_APC-CFSE^high cells by the effector cells. Data shown are representative of two independent experiments.