Diane Longo

A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.

Many cellular stress-responsive signaling systems exhibit highly dynamic behavior with oscillatory features mediated by delayed negative feedback loops. What remains unclear is whether oscillatory behavior is the basis for a signaling code based on frequency modulation (FM) or whether the negative feedback control modules have evolved to fulfill other functional requirements. Here, we use experimentally calibrated computational models to interrogate the negative feedback loops that regulate the dynamic activity of the transcription factor NF-κB. Linear stability analysis of the model shows that oscillatory frequency is a hard-wired feature of the primary negative feedback loop and not a function of the stimulus, thus arguing against an FM signaling code. Instead, our modeling studies suggest that the two feedback loops may be tuned to provide for rapid activation and inactivation capabilities for transient input signals of a wide range of durations; by minimizing late phase oscillations response durations may be fine-tuned in a graded rather than quantized manner. Further, in the presence of molecular noise the dual delayed negative feedback system minimizes stochastic excursions of the output to produce a robust NF-κB response.