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Multiparametric cytometry for exploration of complex cellular dynamics
Françoise Gondois-Rey1, Samuel Granjeaud, Suong Le Thi Kieu
1Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, France. francoise.gondois-rey@inserm.fr
Insights
Polychromatic cytometry reveals innate immune responses to influenza virus in human cells. This method tracks activation markers in dendritic cells and NK cells, uncovering complex cellular interactions.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Polychromatic cytometry has advanced human immunology research.
- Previous studies focused on rare cell populations.
- This study explores complex innate immune systems using polychromatic cytometry.
Purpose of the Study:
- Investigate the dynamics of complex cellular systems in innate immunity.
- Utilize polychromatic cytometry to study peripheral blood mononuclear cells (PBMC) stimulated with live influenza virus.
- Analyze the time course of PBMC activation, including dendritic cells (DC), monocytes, and NK cells.
Main Methods:
- Developed 12-color panels for polychromatic cytometry.
- Simultaneously measured intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein.
- Performed kinetic experiments using low cell numbers and reduced reagents, coupled with bioinformatic tools for data analysis.
Main Results:
- Reproducibly determined activation markers in DC and NK cells.
- Revealed insights into indirect DC-mediated NK cell activation.
- Generated dynamic clustering maps for visualizing PBMC activation events and cellular interactions.
Conclusions:
- Polychromatic cytometry is a powerful tool for exploring complex cellular systems in immunology and virology.
- The study provides new information on the coordination of innate immune pathways and cellular interactions during viral infection.
- This approach enables discoveries in previously unexplored complex cell systems.
Abstract:
The development of polychromatic cytometry has contributed to significant progress in the field of human immunology. Although numerous functional studies of rare cell populations have been performed using this technology, here we used polychromatic cytometry to explore the dynamics of complex cellular systems implicated in innate immunity. We used PBMC stimulated with live influenza virus as an experimental model. We studied the time course of activation of PBMC, which contain DC, monocytes, and NK cells, all of which are, in addition to their innate immune properties, susceptible to Flu infection. We developed 12 color panels to investigate intracellular expression of IFN-α, TNF-α, IL-12, IL-6, IFN-γ, CD107, and influenza virus nucleoprotein simultaneously in these cell populations. These panels allowed reproducible determination of activation markers induced in DC after their direct exposure to various stimulations or in NK cells by indirect DC-mediated activation within the complex cellular environment. The ability to use a low number of cells and reduced quantities of reagents permitted us to perform kinetic experiments. The power of polychromatic cytometry associated with bioinformatic tools allowed us to analyze the multiple functional data generated as dynamic clustering maps. These maps present a readily understandable view of activation events induced in different populations of PBMC. In addition, it reveals new information on the coordination of the complex pathways induced and on the cellular interactions that sustained indirect DC-mediated NK cell activation. Our work shows that polychromatic cytometry is a tool for discoveries in unexplored complex cell systems, at the crossroads of immunology and virology. © 2012 International Society for Advancement of Cytometry.
