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Proliferation assay amplification by IL-2 in model primary and recall antigen systems
Amy S M Kennell, Keith G Gould, Myer R Salaman1
1Department of Immunology, Imperial College School of Medicine, London, UK. m.salaman@imperial.ac.uk.
Insights
Adding Interleukin-2 (IL-2) late in culture enhances the detection of T lymphocyte proliferation to antigens. This method improves the sensitivity of T cell assays, making weak responses more observable.
Area of Science:
- Immunology
- Cellular Biology
Background:
- Detecting weak T lymphocyte proliferation to antigens is challenging in standard peripheral blood mononuclear cell (PBMC) cultures.
- Interleukin-2 (IL-2) is a cytokine known to influence T cell responses.
Purpose of the Study:
- To evaluate the effectiveness of IL-2 in amplifying T lymphocyte proliferative responses.
- To determine the optimal timing for IL-2 addition to enhance antigen-specific T cell assays.
Main Methods:
- PBMCs from healthy donors were cultured with keyhole limpet hemocyanin (KLH) or tuberculin PPD.
- IL-2 was added either at the start of culture or on day five.
- Tritiated thymidine incorporation was used to measure proliferation at day eight.
Main Results:
- Early IL-2 addition caused high proliferation, even without antigen.
- Late IL-2 addition (day five) enabled clear detection of KLH-induced T cell proliferation.
- Late IL-2 also boosted responses to PPD, LPS, and poly(I:C) but not PWM.
Conclusions:
- Late addition of IL-2 significantly increases the sensitivity of T lymphocyte proliferation assays.
- This strategy allows for the reliable observation of low-level T cell responses to specific antigens.
Background:
It can be difficult to register a weak proliferative response of T lymphocytes to an antigen, particularly in a simple culture system of peripheral blood mononuclear cells (PBMC). Here we assess the usefulness of the cytokine IL-2 in amplifying such a response.
Methods:
PBMC from healthy donors were cultured in the presence or absence of keyhole limpet haemocyanin (KLH), an antigen to which people have not been previously exposed. IL-2 was added from the beginning or on the fifth day of culture. Proliferation was determined by incorporation of tritiated thymidine at eight days. The recall antigen, tuberculin PPD, provided a positive control.
Results:
IL-2 added at the beginning of culture can induce extremely high levels of proliferation even in the absence of antigen. However, when added on the fifth day it allowed the clear observation of a proliferative response to KLH that was barely detectable in its absence. Added late it was similarly able to boost low responses to PPD and to the mitogens lipopolysaccharide and poly(I:C), but it had no such effect with pokeweed mitogen.
Conclusions:
IL-2 added late in culture is highly effective in increasing the sensitivity of T lymphocyte proliferative assays.
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