Abstract: The present invention provides methods for generating antigen-reactive cytotoxic T cells in vitro comprising culturing immune cells and antigenic cells that have at least one MHC allele in common (and preferably, are syngeneic), in which the antigenic cells have been treated according to the methods of the invention. The antigenic cells are treated by subjecting them to osmotic shock followed by irradiation. As a result, a subset of T cells are activated and mature into antigen-reactive cytotoxic T cells. The effectiveness of the procedure may be enhanced by repeated restimulations and/or the addition of heat shock protein-peptide complexes. Methods and compositions are also disclosed for the treatment and prevention in a subject of cancer or infectious disease comprising administering to the subject matched cytotoxic T cells that are generated in vitro by the present methods.