Figure 2. In vitro expanded CD8⁺CD28⁻ T cells suppress CD4⁺ T cells proliferation in a donor-specific manner.

CD8⁺ T cells from donor A were stimulated with APCs from donor B plus γc cytokines for 9 days, followed by enrichment of the CD8⁺CD28⁻ population as described in Materials and Methods . 5×10⁴ CFSE labeled purified responder CD4⁺ T cells (R) from donor A were stimulated with 5×10⁴ APCs from donor B (B-_APC) or from an indifferent donor (I-_APC, HLA-A, B and DR fully mismatched with donor B), or 1×10⁴ anti-CD3/CD28 coated Dynabeads in triplicates in 96-well plates. The in vitro expanded CD8⁺CD28⁻ T cells were added as putative suppressors (S) at S∶R ratios of 0.5∶1, 0.1∶1 and 0.02∶1 (with the cell number of “R” kept constant). Proliferation of the CD4⁺ cells was measured by CFSE dilution (A) or by ³H thymidine uptake (B). Data shown are representative of three independent experiments. * P = 0.002; ** P = 0.009; *** P = 0.008.