Simultaneous monitoring of binding to and activation of tumor-specific T lymphocytes by peptide-MHC.
The recent advent of peptide-MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide-MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide-MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide-MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF gamma) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide-MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.
Cohen
Cyril J
CJ
Faculty of Biology, Technion-Israel Institute of Technology, Room 333, Technion City, 32000, Haifa, Israel.
Denkberg
Galit
G
Schiffenbauer
Yael S
YS
Segal
Dina
D
Trubniykov
Ella
E
Berke
Gideon
G
Reiter
Yoram
Y
D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008213:Lymphocyte Activation / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000276:immunology*; D008545:Melanoma / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D007371:Interferon-gamma