mart-1-antigen and fluorexon

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Cell Survival; Cholesterol; CpG Islands; Dendritic Cells; Drug Delivery Systems; Fatty Acids, Monounsaturated; Female; Fluoresceins; Fluorescent Dyes; gp100 Melanoma Antigen; Lipopeptides; Liposomes; Lung; Lung Neoplasms; Macrophages, Alveolar; MART-1 Antigen; Mice; Nanoparticles; Quaternary Ammonium Compounds; Tissue Distribution

It is well established that cytotoxic T lymphocytes (CTL) can kill target cells offering a very small number of specific peptide/MHC complexes (pMHC). It is also known that lethal hit delivery is a very rapid response that occurs within a few minutes after cell-cell contact. Whether cytotoxicity is efficient and rapid in the context of CTL interaction with target cells derived from solid tumours is still elusive. We addressed this question by visualizing the dynamics of human CTL interaction with melanoma cells and their efficiency in eliciting cytotoxicity. Our results show that in spite of CTL activation to lethal hit delivery, killing of melanoma cells is not efficient. Time-lapse microscopy experiments demonstrate that individual CTL rapidly polarize their lytic machinery towards target cells, yet the apoptotic process in melanoma cells is defective or 'delayed' as compared to conventional targets. These results indicate that although CTL activation to lethal hit delivery can be viewed as a 'digital' phenomenon rapidly triggered by a few ligands, melanoma cell annihilation is an 'analogue' response requiring multiple hits and prolonged contact time.

Topics: Apoptosis; Cell Proliferation; Cell Separation; Cytotoxicity, Immunologic; Flow Cytometry; Fluoresceins; Immunotherapy; Ligands; Major Histocompatibility Complex; MART-1 Antigen; Melanoma; Microscopy, Confocal; Peptides; T-Lymphocytes, Cytotoxic; Time Factors