Dataset Information

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

ABSTRACT: Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity.Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-?, TNF-?, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period.The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-?- and IL-2-expressing T cells and less than 0.01% for TNF-?- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells.The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

SUBMITTER: Kagina BM

PROVIDER: S-EPMC4339399 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

Kagina Benjamin M BM Mansoor Nazma N Kpamegan Eloi P EP Penn-Nicholson Adam A Nemes Elisa E Smit Erica E Gelderbloem Sebastian S Soares Andreia P AP Abel Brian B Keyser Alana A Sidibana Mzwandile M Hughes Jane E JE Kaplan Gilla G Hussey Gregory D GD Hanekom Willem A WA Scriba Thomas J TJ

Journal of immunological methods 20141216

<h4>Background</h4>Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in t ...[more]

PMID: 25523923

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Project description:Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Project description:Cell-mediated responses to immunological stimuli are often localised in inflammatory sites and involve a number of cell types. These responses can be functionally characterised at the single-cell level on the basis of the types of cytokines expressed either in whole blood or PBMCs. The ability to measure antigen-specific cell responses at the single cell level is an important tool with a wide range of potential applications ranging from studies of disease pathogenesis to the evaluation of vaccines. A number of experiments were performed in this study in order to establish the optimal conditions for in vitro stimulation of cytokine production by T cells and monocytes in whole blood samples collected from healthy adult Malawian participants and the optimal staining conditions for various cytokine producing cells. Different stimulation methods and conditions, different culture tubes and incubators and different antibody labelling conditions were assessed in order to establish optimal conditions for detecting cytokine-producing cells in whole blood samples. The use of PMA plus Ionomycin produced highest cytokine-producing T cells whereas LPS was a better stimulant for cytokine producing monocytes. Stimulation of whole blood for 5 h was optimal for cytokine detection in T cells whereas 4 h was optimal for monocytes. BFA was found to be a better Golgi blocker than Monensin and the use of 15 ml Falcon-type polypropylene tubes while stationary resulted in the detection of the highest proportion of cytokine-producing cells. T cells were found to be producers of mainly TNF-α, IFN-γ and IL-2 whereas Monocytes were mainly producing TNF-α and IL-6. Anti-CD3-PerCP (used at a ratio of 1:25), anti-CD14-APC (used at a ratio of 1:50) and anti-cytokine-PE (used at a ratio of 1:12.5) resulted in the best results. The highest cytokine production monocytes were detected when 1 X FACS Lysing solution was used at a volume of 40X that of the whole blood sample compared to the other volumes. These optimal conditions are essential in determination of proportion of cytokine-producing cells using ICS in whole blood.

Dataset Information

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

Publications

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

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