Am J Clin Exp Immunol 2012;1(1):1-11.

Original Article
Inter-donor variation in cell subset specific immune signaling responses in
healthy individuals

Diane M Longo, Brent Louie, Ena Wang, Zoltan Pos, Francesco M Marincola, Rachael E Hawtin, Alessandra Cesano

Nodality, South San Francisco, CA 94080; Infectious Disease and Immunogenetics Section, Department of Transfusion
Medicine, Clinical Center, and Center for Human Immunology, National Institutes of Health, Bethesda, MD 20892, USA;
Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, H-1089, Hungary

Received April 3, 2012; accepted April 21, 2012; Epub April 24, 2012; Published June 30, 2012

Abstract: Single cell network profiling (SCNP) is a multi-parameter flow cytometry based approach that allows for the
simultaneous interrogation of intracellular signaling pathways in multiple cell subpopulations within heterogeneous
tissues, without the need for individual cell subset isolation. Thus, the technology is extremely well-suited for
characterizing the multitude of interconnected signaling pathways and immune cell subpopulations that regulate the
function of the immune system. Recently, SCNP was applied to generate a functional map of the healthy human immune
cell signaling network by profiling immune signaling pathways downstream of 12 immunomodulators in 7 distinct
immune cell subsets within peripheral blood mononuclear cells (PBMCs) from 60 healthy donors. In the study reported
here, the degree of inter-donor variation in the magnitude of the immune signaling responses was analyzed. The highest
inter-donor differences in immune signaling pathway activity occurred following perturbation of the immune signaling
network, rather than in basal signaling. When examining the full panel of immune signaling responses, as one may
expect, the overall degree of inter-donor variation was positively correlated (r = 0.727) with the magnitude of node
response (i.e. a larger median signaling response was associated with greater inter-donor variation). However, when
examining the degree of heterogeneity across cell subpopulations for individual signaling nodes, cell subset specificity
in the degree of inter-donor variation was observed for several nodes. For such nodes, relatively weak correlations
between inter-donor variation and the magnitude of the response were observed. Further, within the phenotypically
distinct subpopulations, a fraction of the immune signaling responses had bimodal response profiles in which (a) only a
portion of the cells had elevated phospho-protein levels following modulation and (b) the proportion of responsive cells
varied by donor. These data exemplify the application of SCNP to provide a detailed characterization of inter-donor
variation in immune signaling pathway activation in a healthy donor cohort. This dataset provides a basis for identifying
cell subpopulation specific immune signaling abnormalities in cancer and immune-mediated diseases. Building upon
these data in future studies may help inform on disease etiology, maintenance and therapeutic selection.

Keywords: Signal transduction, multi-parameter flow cytometry, systems immunology

Address all correspondence to:
Dr. Diane M. Longo
Nodality, 170 Harbor Way, Suite 200
South San Francisco, CA 94080, USA.
Tel: (650) 827-8043; Fax: (650) 827-8001
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