Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.
The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.
He
X S
XS
Department of Medicine, Stanford University School of Medicine, California 94305-5187, USA. xiaosong@stanford.edu
Rehermann
B
B
Boisvert
J
J
Mumm
J
J
Maecker
H T
HT
Roederer
M
M
Wright
T L
TL
Maino
V C
VC
Davis
M M
MM
Greenberg
H B
HB
D000956:Antigens, Viral / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D016174:Hepacivirus / Q000276:immunology*; D006526:Hepatitis C / Q000276:immunology; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000502:physiology; D010455:Peptides / Q000378:metabolism; D010799:Phycoerythrin / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013194:Staining and Labeling
D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D010799:Phycoerythrin