High-dimensional analysis of the aging immune system: verification of age-associated differences in immune signaling responses in healthy donors

Abstract

Background: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors.

Methods: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)].

Results: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets.

Conclusions: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.

Figure 1

Distributions of donor ages. Histograms of donors’ ages in the previously published (training) donor cohort (A) and in the current study (test) donor cohort (B).

Figure 2

Diagram of the immune signaling pathways and cell subsets profiled in the current study cohort.

Figure 3

Verified age-associated immune signaling responses. Boxplots for Young and Elderly donors are shown for the 9 age-associated immune signaling responses that were independently verified in the current study cohort using the Gatekeeper strategy. The boxplots were constructed using the 1^st quartile, the median, and the 3^rd quartile. The whiskers represent the lowest and highest data points within 1.5 IQR (interquartile range). The circles represent data points for individual donors.

Figure 4

Age-associated immune signaling responses in the current study cohort. Wilcoxon (two-sided) test statistics were calculated for each node in each cell subpopulation. Responses that differed significantly between Young and Elderly donors are indicated with purple (lower responses in Elderly than Young) and green (higher responses in Elderly than Young) with the color intensity corresponding to the magnitude of the p value. Asterisks indicate the 9 responses verified using the Gatekeeper approach. The number sign indicates the pre-specified response significantly associated with age that was not verified by the Gatekeeper approach. Solid gray boxes represent nodes that were responsive in the given cell subset but did not associate with age (p > 0.05). Solid white boxes represent nodes that did not show a response in the corresponding cell subset.