Defining the range of immune signaling activity in multiple immune cell subsets and establishing an overall map of the immune cell signaling network in healthy individuals represents a critical first step in providing a baseline for the characterization of aberrant signaling responses and changes in the immune signaling network architecture that occur in diseases such as cancer and autoimmune disorders. Because the immune system consists of multiple interdependent cell types for which the behavior is mediated by complex intra- and intercellular regulatory networks, a comprehensive description of healthy immune function requires a systems-level approach capable of integrating information from multiple cell types, signaling pathways, and networks. In this study, we have used SCNP to perform a broad functional characterization of the healthy immune cell signaling network. As expected, many of the immunomodulators included in this study evoked cell type-specific responses (), highlighting the complexity of the regulation of biological function during immune responses. For a subset of the modulators and specific cell types investigated in this study, differential receptor expression and/or differential activation patterns have been previously reported. In instances in which such data are available, the cell-type specific signaling responses described in this paper are generally consistent with those reports (13
). However, to our knowledge, no prior published studies have measured in a quantitative fashion the simultaneous modulation of multiple signaling pathways by different immunomodulatory modulators within several immune cell subpopulations collected from such a large number of healthy individuals.
To gain insight into the connectivity of the immune cell signaling network, we mapped node-to-node correlations within and among each of the distinct immune cell subpopulations. A high-level analysis of this map revealed an abundance of positively correlated nodes, with a higher frequency of positive correlations for node-to-node pairs within the same immune cell subset than for pairs of nodes spanning different cell types (, Supplemental Fig. 3
). Importantly, this map can be compared with those generated using samples from patients with immune-based disorders to identify changes in the network architecture that occur under pathological conditions and can be applied to the analysis of samples obtained longitudinally from treated patients to monitor individual responses to therapeutics.
Aging is often accompanied by a deterioration of the immune system resulting in a higher susceptibility to infections and lower efficacy of vaccination in the elderly population (16
). Given the multitude of age-associated alterations in the function of the immune system, with some of the most profound occurring in T cells subsets (17
), we hypothesized that age may have an impact on the cell signaling responses measured in this study.
The results shown in this study demonstrate that some of the variation in healthy immune signaling responses can in fact be attributed to donor demographic characteristics such as age or race. Specifically, our analysis of the impact of age on immune signaling responses has revealed four individual signaling nodes with significant associations with age. Strikingly, all four of the individual age-associated immune signaling responses identified in this paper were within CD45RA+
T cells, a cell type that has been previously reported to undergo age-related functional changes such as reduced proliferation and cytokine production (17
The majority (three out of four) of the individual age-associated signaling nodes confirmed in the PCA analysis and with statistical significance in both training and test sets occurred within the CD45RA+
cytotoxic T cell subset, whereas only one of the four resided in the CD45RA+
Th cell subset. One of the most dramatic age-related changes in the cytotoxic T cell subset is a decrease in the frequency of CD45RA+
cytotoxic T cells with age (18
), and this was also observed in the samples analyzed in this study (data not shown). Additionally, we have observed an age-related decline in JAK-STAT signaling activity in the CD45RA+
cytotoxic T cell subset in response to multiple cytokines including IFN-α, IL-4, and IL-27 (). Signaling elicited by these cytokines is critical for cytotoxic T cell survival, proliferation, and differentiation (19
). Thus, the observed age-related decrease in responsiveness to these cytokines may underlie some of the functional changes within the cytotoxic T cell compartment (18
The single CD45RA+
Th cell age-associated signaling node was an increased IL-2–induced activation of Stat5 (). This signaling pathway is required for T cell proliferation and activation (24
), and both the production of IL-2 and the proliferation of naive Th cells have been shown to decrease with age (26
). The data reported in this paper suggest that the use of IL-2 may be an effective strategy for rescuing naive Th cell proliferation in the elderly and warrant further investigation.
Overall, the results reported in this study provide evidence of age-associated alterations in T cell cytokine signaling responses, with the most striking differences occurring specifically within the CD45RA+
cytotoxic T cell subset. Although age-associated differences in T cell signaling through the TCR have been widely reported (27
), relatively few studies have documented age-related differences in human T cell cytokine signaling (28
). Further, much of the work that has been conducted to examine associations between T cell cytokine signaling responses and age has been performed using isolated T cells with techniques such as Western blot analysis that allow for only population-level measurements of pathway activation. Analyses performed at the level of total T cells may fail to capture age-associated alterations specific to a given T cell subset as demonstrated in .
In this study, we have reported age-associated alterations within the CD45RA+
cytotoxic T cell subset, a subset that has been shown not only to consist of naive cells but also to contain a subpopulation of effector cells with cytolytic activity (29
). Future studies that use additional cell-surface markers, such as the lymph node homing receptor CCR7, are needed to assess whether there is an age-related difference in the composition of the CD45RA+
cytotoxic T cell subset. Should this be observed, with differential responses among the defined cell subpopulations, then the age-associated difference observed in this study may be driven by an age-related change in the composition of this subset.
We also explored differences in signaling between AAs and EAs, the two major ethnic groups with sufficient representation in this study cohort for statistical analysis. Because ethnic-related differences have been reported in the prevalence of autoimmune diseases such as systemic lupus erythematosus (30
) and multiple sclerosis (31
) and in response rates to immunotherapies such as IFN-α (10
), Benlysta/belimumab (11
), and stem cell transplantation (12
), we hypothesized that some of the variation in immune signaling responses may be attributable to racial differences among the study donors. Our assessment of race-associated signaling responses revealed that BCR-induced (anti-IgD) PI3K pathway activity was significantly higher in EAs than in AAs. Although BCR cross-linking can lead to the activation of multiple signaling pathways, BCR-mediated activation of the PI3K pathway has been shown to provide signaling critical for B cell survival (32
). Thus, the differences in PI3K pathway activity observed in this study may result in racial differences in B cell fate in response to BCR stimulation. Further dissection of BCR signaling in healthy donors is needed to assess whether other pathways activated by BCR stimulation, such as NF-κBand the MAPK pathway, show race-associated differences in response to IgD cross-linking. To our knowledge, no prior studies have reported a race-associated difference in BCR signaling pathway activation.
In this study, the mean age for the donor cohort was 49 y, with very few donors >65 y old. Future studies that include more elderly donors are needed to assess whether the age associations and race associations identified in this donor cohort are maintained in elderly donor populations (i.e., >65 y). A second limitation of this study was the relatively low proportion of female donors, which precluded an analysis of gender-based differences in immune signaling responses. These limitations can be addressed in future studies by applying SCNP to additional PBMC samples from healthy individuals with a wide range of demographic characteristics.
In conclusion, this work has demonstrated the utility of the SCNP technology in providing a systems-level description of immune signaling responses within interdependent immune cell subpopulations. Applying this approach to the characterization of immune cell signaling in a cohort of healthy donors allowed for the quantification of the range of signaling across donors and revealed tight ranges for the immune signaling responses measured, suggesting that the activation of these signaling nodes may be highly regulated in healthy individuals. Although intersubject differences in immune signaling responses were generally quite low, within the subset of nodes that displayed the most substantial interdonor variation, some of the variation in immune signaling pathway activation could be attributed to differences in demographic factors such as age or race. Future studies with independent sample sets will extend the approach taken in this study to investigate potential differences in immune signaling between genders, among additional ethnic populations, and across a wider age range. Future work will also analyze the degree of intradonor variation in immune signaling responses by applying SCNP to samples collected from healthy donors across multiple time points. Further, the breadth of immunobiology examined will be expanded by using additional surface markers and intracellular readouts to characterize an even higher number of immune cell subpopulations and immune signaling pathways. Overall, the healthy immune cell signaling network map generated in this study provides a reference for comparison with network maps generated under disease-associated conditions using samples from patients at baseline or over the course of therapeutic intervention to identify immune network restructuring that is thought to occur under therapeutic pressure and to guide therapeutic selection.