A new strategy for tumor antigen discovery based on in vitro priming of naive T cells with dendritic cells.
We describe a method for discovery of new tumor antigens that uses dendritic cells (DCs) as antigen-presenting cells to prime autologous naive CD4+ and CD8+ T cells from healthy donors against tumor proteins and peptides. For the identification of HLA class I-restricted tumor antigens, peptides were extracted from tumor HLA class I molecules, fractionated by reverse phase-high performance liquid chromatography, and loaded onto in vitro-generated DCs to prime naïve CD8+ T cells. Our results show that we were able to prime naive CD8+ T cells in vitro to several peptide fractions and generate specificity for the tumor. Electrospray ionization mass spectrometry was used to confirm that these fractions contained peptides derived from MHC class I molecules, and the primed CD8+ T cells were used to further analyze the immunostimulatory peptide fractions. For the identification of HLA class II-restricted tumor antigens, we fractionated tumor protein extracts using reverse phase-high performance liquid chromatography and loaded individual fractions onto DCs to prime naive CD4+ T cells. Our results show that we were also able to prime naive CD4+ T cells to several protein fractions and generate specificity for the tumor. These results illustrate the potential of this method to identify new immunostimulatory MHC class I- and class II-restricted tumor antigens.
Kao
H
H
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
Amoscato
A A
AA
Ciborowski
P
P
Finn
O J
OJ
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003713:Dendritic Cells / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005805:Genes, MHC Class I; D006680:HLA Antigens / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D010455:Peptides / Q000737:chemistry; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013347:Subcellular Fractions / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D006680:HLA Antigens; D010455:Peptides