To develop efficacious tumor vaccines, potent antigen-presenting cells (APC) capable of displaying high levels of relevant peptides and co-stimulatory molecules are required to promote the cytotoxic T cell (CTL) immune response. Presently no human cell line capable of expressing large quantities of empty, loadable class I under physiological conditions is available. To provide a peptide acceptor cell line capable of presenting large amounts of relevant immunogenic peptide, we are developing human APC lines that can display MHC class I alleles of choice in an empty (peptide-free) configuration at the cell surface.To accomplish this goal: 1) Peptide loading-deficient B-lymphoblastoid lines were transfected with expression vectors encoding mutant endoplasmic reticulum chaparones (calnexin and calreticulin) that release class I molecules to the cell surface without bound peptide. 2) The B cell line was treated with a pH 3.3 citric acid solution to disassociate peptides from class I/β 2 microglobulin complexes at the cell surface. In peptide binding assays using 1 2 5 I-labeled high affinity peptides of known HLA binding motifs, B-lymphoblastoid cells treated by these two methodologies bound 2.5-fold and 3,000 fold more peptide, respectively, than those treated by current peptide pulsing protocols.The peptide-receptive empty class I molecules generated by these studies represent flexible peptide acceptors capable of displaying large quantities of tumor-specific peptides. The use of these cell lines in maximally stimulating expansion of patient-derived anti-tumor CD8+ cytolytic T-cells may provide a means for vaccination against malignancy.