A high-throughput assay using dysfunctional T cells for phenotypic screening immune checkpoint modulators.

Area of Science:

• Immunology
• Cell Biology
• Drug Discovery

Background:

• Prolonged stimulation during viral infections or cancer leads to dysfunctional effector T cells.
• Dysfunctional T cells exhibit reduced cytokine production and effector functions, impairing pathogen and cancer elimination.

Purpose of the Study:

• To establish a protocol for generating dysfunctional T cells from human peripheral blood mononuclear cells (PBMCs).
• To develop a fully automated, high-throughput assay for assessing immune checkpoint modulators.
• To identify compounds that can restore T cell function.

Main Methods:

• Dysfunctional T cells were generated by culturing PBMCs with interleukin-2 (IL-2) and Phaseolus Vulgaris Leucoagglutinin (PHA-L).
• Flow cytometry was used for cell population analysis and quantification.
• A high-throughput assay in 384-well format was developed involving compound pre-incubation, T cell stimulation (anti-CD3/anti-CD28), and measurement of IL-2 and interferon-γ (IFN-γ) via AlphaLISA, alongside a cytotoxicity assay.

Main Results:

• A validated protocol for generating dysfunctional T cells was established.
• A fully automated high-throughput assay was successfully developed and implemented.
• Out of 15 tested compounds, 2 demonstrated the ability to enhance IL-2 and IFN-γ production in dysfunctional T cells upon activation.

Conclusions:

• The developed assay provides a robust platform for screening immune checkpoint modulators.
• The identified compounds hold therapeutic potential for reversing T cell dysfunction in various disease contexts.
• This research facilitates the discovery of novel strategies to combat T cell exhaustion.