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

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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
Volume277
Issue number1-2
DOIs
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.

Funding

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.

FundersFunder number
Israel National Academy for Sciences and Humanities, Jerusalem, Israel
Israel Cancer Research Fund
Israel Science Foundation

    Keywords

    • CTL
    • Cancer
    • CellScan
    • Peptide-MHC tetramers

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