Tetramer staining and functional analyses of HY-specific T-cell clones. Three representative examples of tetramer staining by FACS, cytotoxicity, and proliferation of the T-cell clones isolated. (A) HY^A2 tetramer^pos T-cell clone lysed the male natural ligand (Male EBV) and HY peptide (SMCY) pulsed female EBV-LCL (F EBV) at different E:T ratios. The CTL clone proliferates on this stimulation measured by 3H thymidine incorporation. (B) Recognition of the HY peptide-pulsed female EBV-LCL only. (C) HY^A2tetramer^pos T-cell clone without HY-specific recognition. Note that none of the clones lysed the unloaded female EBV-LCL or HLA-A2/HX (SMCX) peptide-pulsed female EBV-LCL, but did proliferate on aspecific stimulation with 1% PHA or 25 U/mL IL-2.