IN-VITRO PRIMING OF CYTOTOXIC T-LYMPHOCYTES AGAINST POORLY IMMUNOGENIC EPITOPES BY ENGINEERED ANTIGEN-PRESENTING CELLS

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented b y major histocompatibility complex class I (MHC-I) molecules on the su rface of target cells. Optimal induction of CD8(+) CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stim ulatory molecules on antigen-presenting cells (APC). The antigen-proce ssing defective mutant cell line RMA-S, when cultured at low temperatu re, expresses high amounts of MHC-I molecules that do not contain endo genously derived peptides. These ''empty'' MHC-I molecules can be stab ilized by addition of MHC-binding peptides. RMA-S cultured at low temp eratures with selected peptides have been used for in vitro CTL induct ion with conflicting results. RMA-S cells do not express detectable am ounts of B7 co-stimulatory molecule. This could explain their unpredic table efficiency as APC. We have evaluated whether RMA-S cells; stably transfected with cDNA encoding for the human B7.1 molecule could prov ide effective co-stimulation for CD8(+) T Iymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and som etimes unique efficiency for in vitro primary CTL induction, even when ''sub-optimal'' antigen peptides were used. Most importantly, RMA-S/B 7 cells pulsed with naturally processed peptides extracted from the po orly immunogenic B16 melanoma cells were able to prime CD8(+) cells ag ainst B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without pri or in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy o f MHC-I by single peptide epitopes in CTL priming.