Research Paper
The interferon gamma secretion assay: a reliable tool to study interferon gamma production at the single cell level

https://doi.org/10.1016/j.jim.2004.01.001Get rights and content

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 IFNγ Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNγ 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 IFNγ+ labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNγ-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNγ-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 IFNγ secretion. These results clearly demonstrate that the IFNγ+ 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 IFNγ+ secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.

Introduction

Following appropriate antigen-specific stimulation, lymphocytes rapidly express and secrete cytokines. These cytokines can be measured in the culture supernatant using Elisa, but this method, although very useful for certain applications, lacks the capacity to identify the number and the phenotype of the IFNγ-secreting cells. Techniques to investigate cytokine production at a cellular level (single-cell analysis) include Enzyme-linked immunospot (Elispot), intracellular cytokine staining (IC), in situ hybridization (mRNA) and cytokine secretion assay. The two flow cytometric assays, IC staining and cytokine secretion assay, are able to identify the phenotype of the cytokine-secreting cell. In this paper we thoroughly analyzed and validated the cytokine secretion assay, which not only allows for the identification but also the physical isolation of viable cytokine-secreting cells.

The cytokine secretion assay was developed in the mid-1990s as a method to analyze and isolate single lymphoid cells based on the molecules they secreted (Manz et al., 1995). Cells are induced to secrete cytokines by stimulation with a lectin or a recall antigen. The cytokine is retained on the secreting cell by a bispecific antibody molecule consisting of a conjugated pair of monoclonal antibodies. One antigen-binding site binds to a cell surface molecule, e.g. CD45 present on all leukocytes, while the other binding site recognizes the cytokine studied (e.g. IFNγ). The immobilized cytokine can then be revealed by adding a detection reagent which is a phycoerythrin-labeled anti-cytokine antibody. The phenotype of the cytokine-secreting cell can be determined by labeling selected surface markers with appropriate monoclonal antibodies (e.g. anti-CD3, anti-CD4, etc.). To physically isolate the cytokine-secreting cells from the cell suspension an anti-phycoerythrin antibody conjugated to superparamagnetic particles is added Assenmacher et al., 1998, Brosterhus et al., 1999, Oelke et al., 2000.

In this paper we describe the validation of the IFNγ Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany) without physical isolation or enrichment of the cytokine-producing cells. When performed according to the manufacturer's instructions and adhering to a series of quality measures, the ISA proves to be a very reliable assay. This method clearly identifies different IFNγ-secretion patterns which vary according to the stimulus used and the subject studied. To further demonstrate the applicability of the ISA-technology in vaccine-research, we analyzed the IFNγ-secretion pattern after HBsAg-stimulation in PBMC from several subjects before and after HBsAg vaccine administration.

Section snippets

Isolation and freezing of human peripheral blood mononuclear cells (PBMC)

Buffy coats (BC) were obtained from the Blood Transfusion Center, Red Cross, Ghent, Belgium. PBMC were prepared by standard Ficoll-Isopaque (Lymphoprep™, Nycomed Pharma AS, Oslo, Norway) density gradient centrifugation, washed twice in Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Invitrogen, Carlsbad, CA, USA) and frozen (liquid N2, 3×107 PBMC/cryotube) in freezing solution (10% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO) in Fetal Calf Serum (FCS; Invitrogen)). After thawing,

Calculation-methods for the ISA

The flow cytometric analysis of the IFNγ Secretion Assay has already been described in detail in the Materials and Methods section, and shown in Fig. 1. The lymphocyte gate (R1) for this analysis is based on dead cell-and monocyte-exclusion (PI/anti-CD14-PerCP vs. FSC-Height). In this gate, 105 lymphocytes were counted and analyzed for IFNγ secretion and phenotype. Three different approaches were used to express the IFNγ-producing fraction. Table 1 gives an overview of these

Discussion

Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracytoplasmic cytokine staining, cytokine secretion assay, etc.) have been designed. In this paper we present the results of a thorough validation of the IFNγ Secretion Assay (ISA, Miltenyi Biotec). In this assay the secreted IFNγ is bound to the cell surface, then stained as an artificial surface molecule and analyzed by flow-cytometry. For the

Acknowledgements

The authors are grateful to Dr. B. Riegler and Dr. M Assenmacher from Miltenyi Biotec for their scientific and technical support in performing the IFNγ Secretion Assay. We thank E. Krijnen, A. Van de Putte and Y. Gybels for excellent technical support. We thank Dr B. Vandekerckhove from the Blood Transfusion Center of Oost-Vlaanderen (BTC) for supplying buffy coats. We thank Eurocetus (The Netherlands) for the kind gift of recombinant IL2.

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