Endotoxin Contamination can distort T Cell Assay Results
March 13, 2025
Dr. Lilian Schoefer
News
News
Endotoxins, the hydrophobic portion of bacterial lipopolysaccharides (LPS), are a powerful immune modulator that can impact the outcomes of T cell assays. Also known as Lipid A, endotoxins are found in the outer membrane of gram-negative bacteria, and as a result, they are ubiquitous in the environment—present in the air, water, and on our skin. Our immune system responds strongly to LPS in our body, by activating immune cells through Toll-like receptor 4 (TLR4) and triggering potent inflammatory responses.[1] These responses are designed to help the body eliminate invading bacteria. However, this strong immune activation can also influence immunological studies, leading to false-positive results in T cell assays.[2]
For this reason, it is critical to control endotoxin contamination in peptide synthesis. At peptides&elephants, we take this seriously: Since 2016, we have continuously tested our peptides and peptide pools for endotoxin contamination through a certified laboratory—and each time, the results have come back negative.
How Endotoxins Affect T Cell Assays
Endotoxins are known to affect several immune cells, including monocytes, macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes.[3] They can also enhance the proliferation and cytokine production of T lymphocytes. As a result, endotoxins can introduce unwanted effects into T cell assays. Here are some of the primary ways endotoxins can distort assay outcomes:
- Non-Specific Immune Cell Activation:
By activating monocytic cells such as macrophages and dendritic cells via TLR4 signaling [4], endotoxins induce a cytokine-rich environment. This can lead to indirect activation of T cells, independent of the antigen or peptide being tested. This non-specific activation can result in false-positive results and obscure the true T cell response. - Altered Cytokine Release:
Endotoxins can promote the production of cytokines like Interleukin-1 (IL-1) and Tumor Necrosis Factor-alpha (TNF-α) by antigen-presenting cells.[5] These cytokines can mask the actual, antigen-specific T cell response. - Compromised Cell Viability:
High endotoxin concentrations can trigger apoptosis or stress responses in T cells and other immune cells.[6] This can result in inaccurate assessments of T cell function or viability, potentially leading to misinterpretation of assay results.
Endotoxins are not only a concern in T cell assays but can also significantly influence studies on cancer immunotherapy. High endotoxin levels can alter the metabolic activity of cancer cells, promoting proliferation and invasion, potentially skewing results on the effectiveness of new therapies. Moreover, endotoxins can drastically alter the efficacy of macrophage-modulating therapies.
The Importance of Endotoxin-Free Reagents
To ensure the accuracy of immunological studies, endotoxin-free peptides are essential. Endotoxins, due to their hydrophobic nature, readily bind to materials such as plastic laboratory utensils (e.g., beakers, stirring rods), making it easy for contamination to spread across laboratory equipment. This means that endotoxins can persist on lab tools, and even regular sterilization methods are not sufficient to eliminate them.
It is crucial for laboratories to actively control endotoxin contamination and use reagents that are guaranteed to be endotoxin-free. This helps maintain the integrity of T cell assays and ensures that the results reflect the true immune response rather than being influenced by external contaminants.
Endotoxins can have a significant and potentially misleading impact on T cell assays, primarily through non-specific immune activation. To ensure reliable, reproducible results in immunological studies, it is essential to use endotoxin-free peptides and reagents. At peptides&elephants, we are committed to providing high-quality peptides that are free from endotoxin contamination, ensuring that your assays yield precise and trustworthy results.
Literature
- Kim, HJ., Kim, H., Lee, JH. et al. Toll-like receptor 4 (TLR4): new insight immune and aging. Immun Ageing 20, 67 (2023). https://doi.org/10.1186/s12979-023-00383-3]
- Costa JP, Jesus S, Colaço M, Duarte A, Soares E, Borges O. Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies. Vaccine. 2023 May 26;41(23):3481-3485. doi: 10.1016/j.vaccine.2023.04.063.
- Ulmer, A J et al. “Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS).” Toxicology vol. 152,1-3 (2000): 37-45. doi:10.1016/s0300-483x(00)00290-0
- Li Y, Boraschi D. Endotoxin contamination: a key element in the interpretation of nanosafety studies. Nanomedicine (Lond). 2016 Feb;11(3):269-87. doi: 10.2217/nnm.15.196. Epub 2016 Jan 20. Erratum in: Nanomedicine (Lond). 2016 Mar;11(6):739. PMID: 26787429.
- Fujihara, Mitsuhiro et al. “Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex.” Pharmacology & therapeutics vol. 100,2 (2003): 171-94. doi:10.1016/j.pharmthera.2003.08.003
- Rathinam, Vijay A K et al. “Innate immunity to intracellular LPS.” Nature immunology vol. 20,5 (2019): 527-533. doi:10.1038/s41590-019-0368-3