A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions

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.