Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation.
Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA. mbetts@mail.nih.gov
Michael R
MR
Betts
Jason M
JM
Brenchley
David A
DA
Price
Stephen C
SC
De Rosa
Daniel C
DC
Douek
Mario
M
Roederer
Richard A
RA
Koup
D015703:Antigens, CD / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D015550:Cell Degranulation; D003602:Cytotoxicity, Immunologic; D004768:Enterotoxins / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000032:analysis; D054353:Perforin; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D052899:Pore Forming Cytotoxic Proteins; D012680:Sensitivity and Specificity; D060149:Tetraspanin 30
D015703:Antigens, CD; C554586:CD63 protein, human; D004768:Enterotoxins; D015395:Histocompatibility Antigens Class I; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D060149:Tetraspanin 30; D054353:Perforin; C031818:enterotoxin B, staphylococcal; D007371:Interferon-gamma