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Published on: October 2, 2015
Characterization of the murine immunological signaling network with phosphospecific flow cytometry
Peter O Krutzik1, Matthew B Hale, Garry P Nolan
1Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305, USA.
Insights
Immune cell responses are regulated by intracellular signals, cell-cell interactions, and the environment. In vitro studies may not fully capture in vivo immune cell behavior due to context-dependent signaling.
Area of Science:
- Immunology
- Cellular Signaling
- Systems Biology
Background:
- The immune system operates through three interconnected tiers: intracellular signaling, cell-cell interactions, and the microenvironment.
- Understanding immune cell function requires analyzing these tiers simultaneously to grasp cohesive responses against foreign antigens.
- Existing in vitro models may not fully recapitulate the complexity of in vivo immune cell regulation.
Purpose of the Study:
- To analyze immune cell signaling networks across multiple tiers using phosphospecific flow cytometry.
- To compare immune cell responses to cytokines and LPS in vitro versus in vivo conditions.
- To investigate how the external environment influences immune cell signaling specificity and responsiveness.
Main Methods:
- Utilized phosphospecific flow cytometry to profile single-cell phosphoprotein responses.
- Analyzed B cells, T cells, and myeloid cells stimulated with cytokines (IFN-gamma, GM-CSF, IL-2, IL-10) and LPS.
- Investigated key signaling pathways including Jak-Stat and MAPK.
Main Results:
- Specific stimuli induced distinct phosphorylation patterns in defined immune cell subsets.
- In vivo stimulation revealed that cell type and signaling pathway specificity are heavily influenced by the external environment.
- Immune cells cultured ex vivo showed altered responsiveness, cytokine sensitivity, and phosphorylation kinetics compared to in vivo.
Conclusions:
- Immune regulation is fundamentally context-dependent, highlighting the importance of the in vivo microenvironment.
- In vitro culture systems can lead to altered immune cell behavior, necessitating careful interpretation of findings.
- Simultaneous analysis of multiple immune network tiers provides critical insights into context-dependent immune regulation.
Abstract:
The immune system is a multitiered network that at the first level uses changes to intracellular signaling proteins to commit cells to determined fates. At the second tier, cells interact with one another via specifically expressed surface receptors and their cognate signaling molecules. At the third level, the local environments of immune cells change the outcomes of intracellular signaling pathways and thereby the role of cells during immune challenge. The interplay among these three tiers allows the distinct cell types of the immune system to respond cohesively to eliminate foreign Ags. In this study, using phosphospecific flow cytometry, we analyze elements of these network tiers by generating profiles of single-cell phosphoprotein responses in B cells, T cells, and myeloid cells to a number of mechanistically and clinically relevant cytokines (IFN-gamma, GM-CSF, IL-2, and IL-10) as well as LPS at key regulatory interfaces (Jak-Stat and MAPK pathways). The stimuli typically induced phosphorylation of specific signaling pathways and exerted their effects on distinct subsets of immune cells. However, upon comparison of stimulation in vitro and in vivo, we noted that signaling pathway specificity and cell type specificity were influenced strongly by the external environment. When taken from the in vivo environment, certain cell subsets became hypo- or hyper-responsive, showed profound differences in sensitivity to cytokine levels, or displayed altered phosphorylation kinetics. Thus, simultaneous analysis of the three tiers of the immune system network illustrates the principles by which immune regulation is context dependent and how in vitro culture systems compare with the in vivo environment.
