DDX3Y encodes the H-Y antigen recognized by CTL clone 68H7-819. (A) Localization by Y-chromosome deletion mapping of the gene encoding the H-Y antigen recognized by CTL 68H7-819. The CTLs were tested for recognition of EBV-LCLs derived from males carrying Y chromosomes with constitutional deletions in a ⁵¹Cr release assay at E/T 10:1. EBV-LCLs were infected the day before the assay with a recombinant vaccinia virus carrying an HLA-B*2705 transgene. EBV-LCL WHT2996 is derived from an individual with a deletion encompassing genes DDX3Y and USP9Y.³⁸  WHT2780 is derived from an individual with a splice site deletion that results in 90% truncation of the USP9Y gene, indicated by an X.³⁸  WHY26 is from an individual with a chromosomal break in UTY. Arrows indicate the intact and aberrant segments of the Y chromosome. Y-chromosome landmarks include the boundaries of deletion intervals 5C and 5D, and selected sequence-tagged sites. + indicates lysis of 35% or more, and − indicates lysis of 4% or less. (B) Plasmids encoding DDX3Y minigenes were cotransfected into COS-7 cells with a plasmid encoding HLA-B*2705. On the following day, CTL 68H7-819 was added to the COS-7 transfectants, and IFN-γ release was measured in the supernatants by ELISA after 20 hours of coculture. (C) Epitope reconstitution assay to determine CTL 68H7-819 recognition of donor EBV-LCLs that had been pulsed for 30 minutes with the indicated synthetic peptides over the indicated range of concentrations; 4-hour ⁵¹Cr release assay, E/T 5:1. (D) Partial sequence alignment of the DDX3Y and DDX3X proteins spanning the region that includes the epitope recognized by CTL 68H7-819. Asterisks indicate disparate residues.