세포고사된 마우스 악성 흑색종주와 반응시킨 수지상세포의 T세포 자극 효과

Abstract

Dendritic cells(DCs) are antigen-presenting cells that induce T cell responses. DCs efficiently present antigens derived from apoptotic cells, stimulating class I-restricted CD8+ cytotoxic T lymphocytes. However, DCs that have ingested apoptotic cells may not become activated, therefore inducing T cell tolerance unless further DC maturation is promoted by additional maturation factors. In this study, the aim was to improve the methods for antigen preparation in DC immunotherapy. We postulated that apoptotic mouse melanoma cell would be a more efficient antigen with additional full DC maturation factors. To remove the supernatant which included late apoptotic and necrotic cells, floating cells were centrifuged and the pellet was obtained. When the pellet was stained with annexin-V/propidium iodide, more than 80% of the cells, which were previously treated with mitomycin-C for 48 hours, were early apoptotic cells. We also observed an increased expression of MHC class II, CD80 and CD 86 molecule after antigen pulsing with maturation factors(CD40 ligand and lipopolysaccride(LPS)). However, there were no definite differences in the expression of DC surface molecules, according to the method of preparation of tumor antigens. DCs pulsed with apoptotic cells, followed by activation with CD40 ligand and LPS, had enhanced T cell stimulatory activity compared to other experiment groups. When cultured DCs, which were pulsed with apoptotic cells, received simultaneous stimulation with CD40 ligand and LPS, interleukin(IL)-12 secretion was increased compared to the group stimulated with necrotic cells. These results reflect that DCs pulsed with apoptotic cells, with the supplement of additional maturation factors, induced enhanced T cell proliferation and secreted high levels of IL-12. It also suggests the utility of mitomycin-C induced apoptosis, supplemented with additional maturation factors, as a mean to generate efficient immunity for tumor in vivo.