Tumor-associated-antigens or osteosarcoma cell line lysates: two efficient methods for in vitro generation of CTLs with special regard to MHC-I restriction.

The expression of tumor associated antigens (TAA) in osteosarcoma cell lines allowed us to design an in vitro model for the generation of TAA-specific CTLs. Since the MHC-I-restriction of these peptides represents the major obstacle to clinical applications, we studied a second method for the generation of CTLs starting from osteosarcoma cell line lysates and PBMCs of HLA-I compatible healthy donors. TAA-specific CTLs showed high and homogeneous cytotoxic response against each peptide; high levels of IFN-γ were released by osteosarcoma cell line lysate specific-CTLs in response to the osteosarcoma cell line they were activated for. The MHC-I dependent osteosarcoma cell line lysate-specific CTLs activity was proved by the indifference against the HLA-I-negative erytroleukaemia cell line K562 and by the absence of IFN-γ production with the addition of HLA-class I blocking antibodies. These two methods may be considered the model for the autologous setting in the context of immunotherapeutic approaches for osteosarcoma patients.