Simultaneous monitoring of binding to and activation of tumor-specific T lymphocytes by peptide-MHC

Cyril J. Cohen, Galit Denkberg, Yael S. Schiffenbauer, Dina Segal, Ella Trubniykov, Gideon Berke, Yoram Reiter

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1 Scopus citations


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 γ) 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.

Original languageEnglish
Pages (from-to)39-52
Number of pages14
JournalJournal of Immunological Methods
Issue number1-2
StatePublished - 1 Jun 2003
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported in part by a research grant from the Israel Science Foundation (ISF) administered by the Israel National Academy for Sciences and Humanities, Jerusalem, Israel and a Research Career Development Award (RCDA) by the Israel Cancer Research Foundation, New York.


  • CTL
  • Cancer
  • CellScan
  • Peptide-MHC tetramers


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