Abstract
Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.
Highlights
Toll-like receptors (TLRs) have been widely studied since the discovery of Drosophila Toll in 1985 [1] and the description of its involvement in Drosophila antifungal immunity in 1996 [2]
Four well-characterized TLR complexes can be expressed on the cell surface: TLR2 as a heterodimer with TLR1 or TLR6, TLR4, which forms a complex with MD-2 and CD14, and TLR5, which forms homodimers
Contamination with TLR ligands is a major concern for the production of proteins for research and for protein-based therapeutics
Summary
Toll-like receptors (TLRs) have been widely studied since the discovery of Drosophila Toll in 1985 [1] and the description of its involvement in Drosophila antifungal immunity in 1996 [2]. TLRs are present on a variety of human cells, including cells of the innate and adaptive immune system, and they play an important role in the initiation of immune responses. The TLR2 construct was expressed in TLR non-responder cells and TLR2-expressing cells were sorted on a Sony SH800 cell sorter using an AlexaFluor647-coupled anti-human CD282 (TLR2) antibody (BD Biosciences). Single cell clones were established and a clone with high reactivity towards Fsl-1 (TLR2/6 ligand) was chosen as TLR2/6 reporter cell line. Cells were sorted for reactivity against Pam3CSK4 (TLR2/1 ligand) by sorting eGFP-expressing cells after activation with 100 nM Pam3CSK4 for 24 h on a Sony SH800 cell sorter. Single cell clones were established and a clone with high reactivity towards Fsl-1 (TLR2/6 ligand) and Pam3CSK4 (TLR2/1 ligand) was chosen as TLR2/1/6 reporter cell line
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