Real-Time Discrimination of Immune Cell Function and Fate

Beyond Immune Cell Characterization
Brian P. Dranka, Ph.D. , Luke Dimasi
The immune system is continually surveilling the body—looking for pathogens, xenobiotics, and other non-self signals. The various cell types which constitute the immune system are, thus, incredibly dynamic and capable of upregulating the processes that go into handling these insults on a time scale of minutes to hours. Many cell types in this system are capable of activation to secrete cytokines, rapidly proliferate, or otherwise communicate to surrounding cells that there is a pathogen to consider. Upon clearance of the pathogen, the cell population must then contract in a controlled manner. Furthermore, in some cell populations (e.g., T cells) a subset of the cells are retained as long-lived memory cells to protect and prime the system for future insults. To enable this dynamic function, immune cells integrate internal cellular signals, cues from the tissue microenvironment, and cellular metabolic activity.
The dynamic nature of immune-cell function poses a unique challenge to the analysis of these cell types. Many of the typical methods that are used to quantify immune-cell function are in fact not “functional” assays at all. Typically, researchers utilize snapshot or point-in-time methods to interrogate whether the cells are activated. Alternatively, many scientists screen for marker expression to characterize which cell subpopulations are present. For example, expression of the CD69 surface antigen is frequently used as a marker of T-cell activation. Notably, this approach is most relevant for defining or quantifying a subpopulation of cells used in an experiment. Surface markers used to identify activated cells often require many hours to become present on the cell surface. While these antigens are often used to inform experiments, they don’t provide the temporal discrimination to see activation kinetics or allow researchers to modulate immune functionality in real time.
Recent advances in the field of immunology identify cell metabolism as a critical regulator of immune-cell function. Indeed, changes in cellular metabolism are not only permissive for altered cell function but are, in fact, sufficient to cause these changes. Metabolic reprogramming occurs in the order of minutes to allow changes in cell function, providing a novel and unique marker for functional analysis. Agilent Seahorse XF technology is at the forefront of the field of immunometabolism, enabling researchers to measure cellular metabolic activity acutely and continuously, as cells are stimulated in a real-time manner. Label-free sensor technology coupled with integrated drug/compound injection ports and real-time interrogation provide a powerful platform to quantify and modulate the dynamic function of immune cells in a manner that is complementary to traditional point-in-time analysis methods.
To illustrate the principles described above, we present three vignettes of immune-cell activation, each of which highlight a unique aspect or need for researchers attempting to uncover the points of regulation in their cell models. SeaHorse

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