The extent of receptor clustering determines which kinases mediate B cell receptor signaling.

The responsiveness of mouse and human natural killer cells is inversely correlated with the abundance of the phosphatase SHP-1.

Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (Δ comE ) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro Δ comE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

Natural killer (NK) cells perform immunosurveillance of virally infected and transformed cells, and their activation depends on the balance between signaling by inhibitory and activating receptors. Cytokine receptor signaling can synergize with activating receptor signaling to induce NK cell activation. We investigated the interplay between the signaling pathways stimulated by the cytokine interleukin-2 (IL-2) and the activating receptor NKG2D in immature (CD56^bright) and mature (CD56^dim) subsets of human primary NK cells using mass cytometry experiments and in silico modeling. Our analysis revealed that IL-2 changed the abundances of several key proteins, including NKG2D and the phosphatase CD45. Furthermore, we found differences in correlations between protein abundances, which were associated with the maturation state of the NK cells. The mass cytometry measurements also revealed that the signaling kinetics of key protein abundances induced by NKG2D stimulation depended on the maturation state and the pretreatment condition of the NK cells. Our in silico model, which described the multidimensional data with coupled first-order reactions, predicted that the increase in CD45 abundance was a major enhancer of NKG2D-mediated activation in IL-2–treated CD56^bright NK cells but not in IL-2–treated CD56^dim NK cells. This dependence on CD45 was verified by measurement of CD107a mobilization to the NK cell surface (a marker of activation). Our mathematical framework can be used to glean mechanisms underlying synergistic signaling pathways in other activated immune cells.

Natural killer cells are controlled by peptide selective inhibitory receptors for MHC class I, including the killer cell immunoglobulin‐like receptors (KIRs). Despite having similar ligands, KIR2DL2 and KIR2DL3 confer different levels of protection to infectious disease. To investigate how changes in peptide repertoire may differentially affect NK cell reactivity, NK cells from KIR2DL2 and KIR2DL3 homozygous donors were tested for activity against different combinations of strong inhibitory (VAPWNSFAL), weak inhibitory (VAPWNSRAL), and antagonist peptide (VAPWNSDAL). KIR2DL3‐positive NK cells were more sensitive to changes in the peptide content of MHC class I than KIR2DL2‐positive NK cells. These differences were observed for the weakly inhibitory peptide VAPWNSRAL in single peptide and double peptide experiments (p < 0.01 and p < 0.03, respectively). More significant differences were observed in experiments using all three peptides (p < 0.0001). Mathematical modeling of the experimental data demonstrated that VAPWNSRAL was dominant over VAPWNSFAL in distinguishing KIR2DL3‐ from KIR2DL2‐positive donors. Donors with different KIR genotypes have different responses to changes in the peptide bound by MHC class I. Differences in the response to the peptide content of MHC class I may be one mechanism underlying the protective effects of different KIR genes against infectious disease.