These considerations point to a key challenge in improving our understanding of signal integration in mammalian cells—the development of enhanced experimental design practices that address the importance of cellular context, physiological conditions of stimulation, and minimal perturbation of cellular content and molecular structure. Although it is clear that immune cell signaling pathways are connected by physical and functional interactions, how do we more accurately assess the physiological relevance of these connections? Most published studies are characterized by one or all of the following experimental strategy elements: acute stimulation, (near to) saturating ligand concentration, overexpressed components, and/or use of a heterologous cell system. Because physiological stimuli are typically subsaturating with respect to available receptor pools, often transient or pulsatile in nature, and have consequences that are highly susceptible to the effects of cellular composition43
, the experimental methods in common use raise the obvious question of the extent to which the results reflect the behavior of the cell under more physiologic conditions versus the outcome of extreme perturbations.
To a large extent, this experimental design paradigm has been driven by practical necessity. Such unphysiological conditions are often required to elicit a consistently measurable response from cells that invest heavily in dampening and negative regulation, a characteristic that may be an even greater factor in immortalized cell lines. Although this is helpful in generating robust data that are convincing in publications, there is great value to developing and using more sensitive multiplex methodologies with a broad range of readouts that permit experimentation under more realistic conditions of cell stimulation. Emerging sensor-based assays using 384- and 1,536-well microplates to facilitate genome-wide perturbation screens44
and the use of microfabricated devices to permit single cell measurements with high resolution45
will also allow an increase in the breadth and dynamic range of measurements involving non-transformed cells available in limited number. Application of such methods to network analysis will permit an increase in sensitivity, analysis at the single-cell level with time resolution, and an increased number of tested ligand concentrations and ligand combinations, which together should markedly enhance the likelihood that we will observe events relevant to physiology. The next step in the evolution of such analyses will be making the same sets of observations in vivo
, using imaging methods that permit observation of cells interacting within the tissue environment and involving physiologic amounts of mediators in that natural setting46,47
Assessment of cellular responses to combined rather than single stimuli is also a generally neglected but vitally important experimental design parameter in efforts to unravel complex signaling events48
. For example, there is growing evidence that cytokine receptors activate non–Jak-STAT signaling effectors (reviewed by Bezbradica and Medzhitov32
), but little is known about how this influences activation of these effectors through their canonical pathways when they become activated by non-cytokine stimuli also present in the cell's environment. Also, pathogens represent a complex stimulus, invariably activating several pathways. These stimuli often combine to produce a synergistic response that is not predictable from the output evoked by an individual canonical pathway downstream of a single receptor.
Careful modeling of quantitative data would also be valuable for explaining observed phenomena. For example, Ghoreschi, Laurence and O'Shea34
discuss the unexpected success of broad-spectrum kinase inhibitors such as staurosporine for treatment of various malignancies, achieved even though these drugs target a substantial proportion of the entire kinome. This success may relate to differences in network flux between normal and transformed cells. Normal cells may be capable of tolerating, or recovering from, inhibition of large portion of their cellular kinases. Tumor cells, by contrast, may require more kinase-driven pathways operating close to their maximum capacity to maintain the transformed state, and they may thus be more susceptible to a partial reduction in signaling capacity through multiple routes. A comprehensive recent assessment of the kinase dependencies of many cell types shows notable heterogeneity in kinase requirements49
, pointing to a need for caution in predicting the physiological effects of kinase inhibitors. This also highlights the possibility, discussed in the review34
, that combination therapies targeting multiple kinases are likely to be more fruitful, considering the interconnected nature of at least portions of the signaling network. Dual perturbation increases the likelihood that the drug treatment will inhibit signal flow (because of the branched nature of the signaling network) and lead to a significant effect, and it also may reduce the incidence of drug resistance as the network is perturbed at multiple points. The potential of this strategy has led to the recent development of screens specifically designed to identify dual-target inhibitors50
On a related note, Saveliev and Tybulewicz33
describe the value of partial loss-of-function mutants in assessing the various functions of the ZAP70 tyrosine kinase in T cell antigen receptor (TCR) signaling, demonstrating that different amounts of kinase activity are required for different aspects of T cell development. These latter studies emphasize two key points. First is the importance of quantitative parameter recording. The contribution of ZAP70 is not a digital phenomenon, but instead resembles a rheostat in its control of signaling output from the TCR complex. Second, partial loss-of-function can provide important quantitative insights into signal flow, emphasizing the value of RNA-mediated interference (RNAi) technology—especially viral short hairpin RNA (shRNA) expression systems that permit the conditional knockdown of targets and reexpression of mutant proteins even in moderately intractable hematopoietic cells51–53
—as a complement to knockout and knock-in strategies.
Finally, on the qualitative side of the equation, the more sophisticated gene perturbation technologies discussed by Saveliev and Tybulewicz33
hold particular promise for improving our ability to identify the normal physiologic function of a protein and its domains: the selective ablation of a single property in a multifunctional signaling protein will more accurately describe its function within a network than the complete removal of this protein from the cell by classical knockout methods. The findings emerging from such research highlight the value of therapies that combine inhibitors of catalytic function with inhibitors of protein-protein interactions.