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Updated: Sep 27, 2025
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions
David Barrett1, Meghan Wyatt1, Haim Bar2
1Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, United States.
Abstract:
The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.
Insights
Researchers developed a new assay to find drugs that can overcome tumor microenvironment-induced immune suppression. The assay screens for compounds that interfere with cyclic AMP (cAMP)-mediated suppression of T cell receptor (TCR) signaling, a key pathway in solid tumors.
Area of Science:
- Immunology
- Molecular Biology
- Drug Discovery
Background:
- The solid tumor microenvironment (TME) creates an immunosuppressive environment that hinders anti-tumor immune responses.
- Elevated cyclic AMP (cAMP) levels within the TME suppress T cell receptor (TCR) signaling, a critical pathway for T cell activation and function.
- Targeting the cAMP-mediated suppression pathway presents a potential strategy to enhance anti-tumor immunity.
Purpose of the Study:
- To develop and validate a phenotypic assay for screening small molecules that can counteract cAMP-mediated suppression of T cell signaling.
- To identify compounds that interfere with the pathway where elevated cAMP suppresses T cell receptor (TCR) signaling in the tumor microenvironment.
- To optimize assay conditions for high-throughput screening and assess assay performance characteristics.
Main Methods:
- Development of a phenotypic assay using TALL-104 human leukemic cytotoxic T lymphocytes stimulated with anti-CD3 antibodies.
- Measurement of lytic granule exocytosis via lysosome associated membrane protein 1 (LAMP-1) using flow cytometry.
- Screening of compound libraries, including epigenetic regulatory proteins and the Prestwick Compound Library, using 96-well and automated 384-well assay formats.
Main Results:
- The assay successfully detected compounds that restored T cell function, with phorbol ester treatment confirming its ability to identify active agents.
- Screening of an epigenetic compound collection and the Prestwick Library did not yield confirmed hits that prevented cAMP-mediated suppression.
- Analysis of assay performance indicated that using percent positive cells for quantification resulted in a higher Z' value compared to mean fluorescence intensity.
Conclusions:
- The developed phenotypic assay is a viable tool for screening compounds targeting the cAMP-mediated suppression pathway in T cells.
- Despite screening large compound libraries, no small molecules were identified that could overcome the suppressive effects of elevated cAMP on T cell signaling in this assay.
- Optimization of signal quantification methods, such as using percent positive cells, can improve assay performance and Z' values for drug discovery efforts.
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