Toll-Like Receptor Function Testing

Last Literature Review: April 2022 Last Update:
  • Use to assist in diagnosis of innate immunodeficiencies when genetic defects of the innate immune system are suspected in individuals negative for other immunodeficiencies (eg, no detectable abnormality of antibody function, complement activity, neutrophil function, or cell-mediated immunity)
  • This test does not measure the function of toll-like receptor 3 (TLR3); molecular testing is the preferred method for detection of defects in TLR3.

Toll-like receptors (TLRs) enable innate immunity to prevent infection. Immunodeficiencies associated with impaired innate immunity (nuclear factor kappa B signaling) include disruptions to signaling pathways for interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK4) and myeloid differentiation primary response protein 88 (MYD88) and may be associated with recurrent pyogenic bacterial infections. IRAK4 deficiency and MYD88 deficiency are rare so they should not be considered unless more common immunodeficiencies are ruled out. After screening for more common immunodeficiencies (eg, by assessing immunoglobulin levels, complement testing, cell-mediated immunity testing, and neutrophil function testing), consider TLR functional testing to determine TLR function and identify a possible molecular defect in the innate immune system that is related to TLR function.

Test Information

TLRs are tested independently by stimulation with TLR-specific ligands in a peripheral blood mononuclear cell (PBMC) culture. Mononuclear cells isolated from anticoagulated whole blood are incubated with the TLR ligands described in the table below.

TLR Target Ligand


Pam3CSK4 synthetic bacterial lipopeptide


Zymosan cell wall particles from Saccharomyces cerevisiae


Lipopolysaccharide (LPS), ultrapure, from Salmonella minnesota variant R595


Flagellin from Salmonella typhimurium


CL097 derivative of imidazoquinoline compound R848

PBMC production of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) are measured by multiplex bead assay for TLR1, 2, and 4-8.

Test Interpretation


Interpretation that compares an individual’s results to a client’s normal control, as well as the laboratory control, will be provided.

Result Finding Clinical Significance


Lack of or marginal response to specific TLR ligands

Suggests a possible molecular defect in the innate immune system related to TLR function or other components of the signaling pathway, such as IRAK4 or MYD88


Normal cytokine responses

Suggests normal TLR function


  • Results should be interpreted in conjunction with the individual’s clinical status
  • Defects in IRAK4 and MYD88 result in compromised TLR signaling.
    • Exception is endosomal TLR4, which is IRAK4 and MYD88 independent