The interferon gamma secretion assay: a reliable tool to study interferon gamma production at the single cell level
I Desombere1, P Meuleman, H Rigole
1Center for Vaccinology, Department Clinical Biology, Microbiology and Immunology, Ghent University and Hospital, De Pintelaan 185, 9000 Ghent, Belgium. isabelle@desombere@ugent.be
Insights
The Interferon-gamma (IFNγ) Secretion Assay (ISA) offers a reliable method for detecting antigen-specific T cells. This validated assay reveals diverse IFNγ-producing cell patterns, crucial for vaccine research.
Area of Science:
- Immunology
- Cellular Biology
- Biotechnology
Background:
- Single-cell analyses detect antigen-specific T cells via cytokine production.
- Existing methods include elispot and intracellular cytokine staining.
- The Interferon-gamma (IFNγ) Secretion Assay (ISA) is a novel flow cytometry-based technique.
Purpose of the Study:
- To validate the Interferon-gamma (IFNγ) Secretion Assay (ISA) for reproducibility and reliability.
- To analyze the cellular subsets contributing to IFNγ production after VZAg-stimulation.
- To assess the utility of ISA in vaccine research using HBsAg-stimulation.
Main Methods:
- Validation of the IFNγ Secretion Assay (ISA) using five quality criteria.
- Flow cytometry analysis of cell surface-bound IFNγ.
- Stimulation of peripheral blood mononuclear cells (PBMCs) with VZAg and HBsAg.
Main Results:
- ISA demonstrated high reliability with low intra-assay (<5%) and inter-assay (<20%) variability.
- Recovery experiments showed nearly 100% detection of IFNγ-producing cells.
- Three distinct IFNγ secretion patterns were identified, varying in T cell and NK cell contributions.
- Individual differences in IFNγ(+) subset distribution were observed post-VZAg-stimulation.
- ISA successfully profiled IFNγ secretion patterns in HBsAg-vaccinated individuals.
Conclusions:
- The validated ISA is a reliable and reproducible assay for detecting antigen-specific immune responses.
- ISA reveals significant individual variability in IFNγ-producing cell subsets.
- ISA is a valuable tool for vaccine research, enabling detailed analysis of immune responses.
Abstract:
Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgamma Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgamma is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNgamma(+) labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNgamma-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNgamma-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3(+)CD56(+) and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60-70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNgamma secretion. These results clearly demonstrate that the IFNgamma(+) subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNgamma(+) secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.
