Generation and validation of artificial antigen-presenting cells. The K562 cell line was modified using retroviral vectors to stably express transgenic HLA-A*02, CD80, CD40L, and OX40L. (A) The expression of the transgenic molecules in modified (K562/aAPC; filled profiles) and nontransduced cells (bold lines). Isotype controls are illustrated as thin lines. To assess functionality, K562/aAPCs loaded with either NLV or CLG peptides were used to reactivate NLV- or CLG-specific CTLs, respectively, from PBMC of CMV- or EBV-seropositive HLA-A*02 healthy donors. (B) Tetramer staining illustrates the enrichment of expanded CTLs in NLV and CLG T-cell precursors (right panels) after 28 days of culture, compared with PBMCs before ex vivo culture (left panels). (C) Frequency of CTLs responding to NLV (■) or CLG peptides () as assessed by IFNγ ELIspot assay in a representative donor after 3 stimulations with aAPCs loaded with the specific peptides. IFNγ production in response to an irrelevant peptide (ELA peptide) was negligible. (D) Cytotoxic activity of these CTLs using a standard ⁵¹Cr release assay at an effector:target ratio of 20:1. Target cells were autologous PHA blasts loaded either with NLV or CLG-peptides, and autologous EBV lymphoblastoid cell lines (LCL) transduced with an adenoviral vector encoding the full protein of either pp65³³  or LMP-2.³⁴  Specific killing was MHC-restricted because it was inhibited by preincubation of PHA blasts with class I MHC blocking antibodies, but not with isotype. Shown is 1 representative experiment of 5 donors. NT indicates not tested.