PHOSPHO FLOW CYTOMETRY: SINGLE-CELL SIGNALING NETWORKS IN NEXT-GENERATION DRUG DISCOVERY AND PATIENT STRATIFICATION - Flow Cytometry in Drug Discovery and Development - page 299

lpr mice to transduce cytokine signals, in particular an ablation of signal transduction
through Stat1 for several cytokines across many of the cell types [8]. In contrast, the
Stat6 response to IL-4 in B cells was completely preserved despite nearly complete
loss in all T cell subsets. IL-4 signaling is required for high-affinity antibody
responses, so this preservation of Stat6 signaling in B cells may contribute to
pathogenic autoantibody production.
Interestingly, not all signaling pathways showed decreased activity in the mouse
model. As an example, the Stat5 signaling response to IL-15 was higher in CD4 þ T
cells from the lpr mice than from the control mice. Surprisingly, this pathway was
downregulated in CD8 þ T cells from lpr mice and this response decreased further
during disease progression. Stat3 signal transduction varied greatly, with some
responses being greatly reduced as early as 10 weeks, while others such as the Stat3
response to IL-10 were preserved at all stages of SLE. These studies revealed the
complexity of SLE disease mechanism in a murine model and showed how broad
signaling profiling can yield a more complete picture of the immune signaling
network in the context of disease. By more clearly defining the perturbed signaling
network, we were able to identify many signaling pathways that potentially play
causative roles in the disease and may be excellent drug targets.
15.3.3 Signaling Ratios
There are a number of cytokines that have been shown to have tolerogenic actions
in one context and proinflammatory actions in another context. In addition, some
cytokines act on many different cell types, potentially inducing very different
functional responses in each (Figure 15.2). The contribution to the immune system
as a whole that comes from each of these cell types when stimulated by the cytokine is
determined, in part, by the different signaling responses activated by the cytokine. A
striking example is IFN a , which in CD4 þ or CD8 þ T cells sends an apoptotic signal
through Stat1, a weaker mitogenic signal through Stat3, and an even lesser signal
through Stat5 [9]. Under most conditions, the mitogenic signal through Stat3 is
overwhelmed by strong Stat1 activation. When the ratio of Stat3 activation to Stat1
activation reaches a high enough level, the cellular program induced by IFN a
switches from death to proliferation. There are a number of intracellular mechanisms,
including those acting through the SOCS family, that strongly and specifically inhibit
different Jak isoforms upstream of Stat pathways and shift the signaling ratio of a
number of cytokine receptors. An example of this shift in the signaling ratio was
observed in the aforementioned Stat1 and Stat3 responses to IL-6 in the T cell
populations of lpr mice.
15.3.4 Analysis of Human SLE Patient Samples
Given the findings with the mouse model, we hypothesized that similar changes in
signaling ratios would be proximal to an induction mechanism responsible for flare in
human patients. To clarify, wewere focusing not on signal transduction regulation that
correlated with static symptoms, but rather on signaling phenotypes that indicated