Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, RasGRP and SOS, catalyze Ras activation in lymphocytes. Binding of active Ras to SOS′ allosteric pocket markedly increases SOS′ activity establishing a positive feedback loop for SOS-mediated Ras activation. Integrating in silico and in vitro studies, we demonstrate that digital signaling in lymphocytes (cells are “on” or “off”) is predicated upon feedback regulation of SOS. SOS′ feedback loop leads to hysteresis in the dose-response curve, which can enable a capacity to sustain Ras activation as stimuli are withdrawn and exhibit “memory” of past encounters with antigen. Ras activation via RasGRP alone is analog (graded increase in amplitude with stimulus). We describe how complementary analog (RasGRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output. Numerous predictions regarding the impact of our findings on lymphocyte function and development are noted.

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B cell activation. However, the effects of antigen copy number, spacing, and affinity, as well as the dimensionality and rigidity of scaffold presentation on B cell activation remain poorly understood. Here, we displayed the clinical vaccine immunogen eOD-GT8, an engineered outer domain of the HIV-1 glycoprotein-120, on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B cell activation in vitro. We found that B cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40 nm viral-like nanoparticle. Increasing antigen spacing up to ~25–30 nm monotonically increases B cell receptor activation. Moreover, scaffold rigidity is essential for robust B cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B cell responses.

Summary The role of the immune synapse, and in particular the role of the central region of the synapse (the central supramolecular activation cluster or cSMAC), is controversial. One model suggests that the sole function of the cSMAC is to downregulate receptors and turn off signaling and that TCR signaling occurs only in the pSMAC. A second model suggests that the role of the cSMAC depends on antigen quality and can both enhance signaling and receptor downregulation. Here, we provide evidence that while at early time points signaling occurs mainly outside the cSMAC, at later time points signaling does occur in the cSMAC. Additionally, we find that cSMAC formation can increase the stimulatory potency of weak agonists for the TCR. Combined with previous studies showing that cSMAC formation decreases the stimulatory potency of strong agonists, our data support a model that posits that signaling and receptor degradation are linked in the cSMAC and that the balance between signaling and degradation in the synapse is determined by antigen quality.