Single cell network profiling (SCNP) assay allows for the identification of heterogeneity in the activation of multiple signaling pathways in distinct human B cell subsets (174.12)

Abstract The specific functions of phenotypically defined B cell subsets in the regulation of protective immune responses and the pathogenesis of immune-mediated diseases is incompletely understood. SCNP is a multi-parametric flow cytometry based approach that simultaneously measures intracellular signaling in multiple cell subsets in heterogeneous tissues. In this study, SCNP was applied to examine 34 signaling nodes (paired modulator/intracellular readout) in B cell subsets (CD27-IgM+IgD+ naïve B cells, total CD27+ memory B cells, CD27+IgM-IgD- switched memory B cells, CD27+IgM+IgD+ IgM memory B cells) in peripheral blood mononuclear cells from 6 healthy donors. Signaling responses in B cell subsets were masked when the total B cell population was analyzed. Specifically, a higher responsiveness was observed in the memory B cell subset relative to the naïve; 14 signaling nodes showed statistically significant differences between these subsets (p<.05, paired Wilcoxon test) of which 12 displayed stronger activation in response to CD40L, SDF1α, PMA, IFNα, IFNγ, IL10 and IL27 modulation in the memory B cells. 11 signaling nodes differed significantly in IgM memory vs. switched memory B cells (p<.05, paired Wilcoxon test); 7/11 displayed stronger activation in response to CpG, R848, thapsigargin, IFNγ and IL10 modulation in IgM memory B cells. An increased understanding of B cell subset signaling may allow for the development of more effective B cell targeted therapies.