Development of a clinical assay to evaluate toll-like receptor function

Area of Science:

• Immunology
• Genetics

Background:

• Toll-like receptors (TLRs) are crucial for innate immunity, recognizing pathogens and initiating immune responses.
• Genetic defects in TLRs are linked to recurrent or severe infections, necessitating diagnostic tools.

Purpose of the Study:

• To develop a reproducible assay for evaluating Toll-like receptor (TLR) function in patients with recurrent infections.
• To identify potential genetic defects affecting TLR signaling pathways.

Main Methods:

• Peripheral blood mononuclear cells (PBMCs) were used to assess TLR function.
• Tumor necrosis factor (TNF) production was measured via ELISA after stimulation with various TLR ligands (TLRs 1/2, 2/6, 3, 4, 5, 6, 7, and 9).
• Assay variables including anticoagulants, media, incubation time, and storage conditions were optimized.

Main Results:

• Optimized assay conditions established mean responses to TLR ligands in healthy donors.
• Patients with IKBKG gene mutations (NEMO deficiency) showed significantly reduced TNF production across all tested TLR ligands.
• The assay demonstrated sensitivity to genetic defects impacting TLR signaling.

Conclusions:

• The developed TLR function assay is reproducible and can be optimized for clinical use.
• This assay can aid in screening patients with primary immunodeficiencies affecting TLR signaling.
• Identifying defects in TLR function is critical for diagnosing and managing recurrent infections.