Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis.
CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.
Wilson
R L
RL
Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, Iowa City, IA 52242, USA.
White
D W
DW
Harty
J T
JT
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001427:Bacterial Toxins; D001483:Base Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D019556:COS Cells; D002461:Cell Line, Transformed; D004269:DNA, Bacterial; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005057:Eukaryotic Cells; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006183:H-2 Antigens / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D006460:Hemolysin Proteins; D064532:Histocompatibility Antigen H-2D; D006801:Humans; D008285:Major Histocompatibility Complex; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
C078516:60 kDa protein, Listeria monocytogenes; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D018984:Epitopes, T-Lymphocyte; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D064532:Histocompatibility Antigen H-2D; D001616:beta-Galactosidase; C038705:hlyA protein, Listeria monocytogenes