A highly sensitive single-cell assay detects T-helper cell responses missed by conventional interleukin-2-based
Sherri Surman1, Julia L Hurwitz
1Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794, USA.
Insights
The conventional interleukin 2 (IL-2) assay for T-helper (Th) cell activity is less sensitive than a single-cell assay. The single-cell method detects responses missed by the conventional test, suggesting they are not interchangeable.
Area of Science:
- Immunology
- Cellular immunology
- Assay development
Background:
- Interleukin 2 (IL-2) assays are standard for studying T-helper (Th) cell activity.
- Conventional assays involve a two-step process: in vitro T(h) cell activation followed by IL-2-dependent indicator cell growth.
- A one-step, single-cell assay using reporter gene expression is available for mouse T(h) cell hybridomas.
Purpose of the Study:
- To compare the sensitivity and performance of conventional and single-cell IL-2 assays.
- To determine if the two assays yield comparable results for assessing T(h) cell activity.
- To evaluate the interchangeability of the conventional and single-cell IL-2 assays.
Main Methods:
- Side-by-side comparison of a conventional two-step IL-2 assay and a one-step single-cell IL-2 assay.
- Utilizing a reporter gene (beta-galactosidase) linked to an IL-2 enhancer element in the single-cell assay.
- Testing mouse T(h) cell hybridomas fused with the BWZ.36 parent line.
Main Results:
- A striking difference in sensitivity was observed between the two assays.
- The single-cell assay identified T(h) responses that were missed by the conventional assay.
- The conventional assay yielded negative results for responses detected by the single-cell assay.
Conclusions:
- The conventional and single-cell IL-2 assays are not interchangeable due to significant differences in sensitivity.
- Negative results from the conventional IL-2 assay should be considered preliminary.
- Further investigation using the more sensitive single-cell assay is recommended for comprehensive T(h) cell response assessment.
Abstract:
One of the most widely used methods for the study of T-helper (T(h)) cell activity is the interleukin 2 (IL-2) assay. Typically, this assay is a two-step process involving (a) the activation of T(h) cells in vitro and (b) the testing of IL-2-dependent indicator cells for growth in the presence of T(h) cell supernatants. The assay has served to quantify and characterize T(h) responses to a variety of unique pathogens and immunization regimens. A one-step, single-cell assay is also available for the testing of mouse T(h) cell hybridomas. In this assay, cells fused with the BWZ.36 parent line are scored for positive responses based on their expression of a beta-galactosidase gene linked to an IL-2 enhancer element. The experiments described in this report were designed to examine the conventional and single-cell assays in a side-by-side comparison. Results revealed a striking difference between the two assays. The single-cell assay proved to be extremely sensitive and identified T(h) responses that were altogether missed by the conventional test. Based on these results, we suggest that (i) the conventional and single-cell assays should not be used interchangeably, and (ii) negative results in the conventional IL-2 assay should be considered preliminary until the more sensitive, single-cell assay can be performed.
