Human EHD2 encodes for a nonpolymorphic antigen recognized by CTL B5 in association with HLA-A*0301. (A) From MZ1257-RCC cells, cDNAs coding for full-length HLA-A*0301 or EHD2 were isolated and inserted into pcDNA3.1(+) expression vectors. They were cotransfected into antigen-negative 293T cells and were analyzed for recognition by CTL clone B5 in a 20-hour IFN-γ ELISPOT assay. Controls included 293T cells transfected with either HLA-A*0301 or EHD2 cDNAs alone, and MZ1257-RCC cells. Each bar represents the mean spot number of triplicates with 5000 CTLs seeded per well. (B) Genomic DNA was isolated from MZ1257-RCC and PBMCs of donor 860. From each sample, an EHD2 fragment containing the KLPNSVLGR epitope-coding region was amplified using specific primers. PCR products were sequenced using standard procedures.