High Resolution Studies of Immune Cell Signaling

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In 2014, we continued to make progress in the following projects: Pre-existing nature of T Cell Receptor (TCR) microclusters, TCR signaling in response to low potency ligands and cross talk among common gamma chain family of cytokine receptors. TCR signaling events that contribute to discrimination between self and foreign peptide-loaded Major Histocompatibility Complexes (pMHC) occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These preexisting microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters LAT and Grb2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could in part explain the rapid kinetics with which TCR signal transduction occurs. This work has been published in the Journal of Immunology. We asked the question whether TCR signaling in response to low-potency ligands is qualitatively different from agonist stimulation. To address this question we used a Fos-GFP transgenic mouse as a read out of TCR signaling and identified doses of agonist and low potency ligands that gave rise to similar amount of Fos induction and analyzed TCR proximal signaling events, such as calcium fluxes, Map kinase activation, PLCg1 phosphorylation, LAT phosphorylation, Zap70 phosphorylation and CD3zeta phosphorylation. We did not find evidence for a qualitatively different TCR signaling pathway in response low potency ligand stimulation. These studies led to the observation that there exists a metal ion dependent negative feedback acting on TCR proximal signaling events that is specific to CD28 engagement by CD80. We are following up on these observations and a manuscript describing these results will soon be submitted. Cytokines of the gamma chain family (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) signal through receptor complexes using cytokine-specific alpha chains in combination with the common gamma chain. Given its shared nature, we wondered whether the gamma chain could be limiting when required simultaneously by multiple cytokine bound receptors. Using quantitative flow cytometric analysis, we found that the gamma chain is outnumbered by cytokine specific private chains on the surface of nave T cells by 5:1. We further found that signaling via common gamma chain cytokines is very sensitive requiring between 1 and 10 private chains occupied, suggesting that not many gamma chains are required for signal transduction. The limiting nature of the gamma chain was revealed when it was found that pre-incubating cells with IL-7 prevents efficient signaling via IL-4 and IL-21 receptors. The extent of IL-7 mediated cross-inhibition of IL-4 and IL-21 responses correlated with the ratio of IL-7R to the gamm chain. We ruled out specific endocytosis of gamma chain and induction of STAT specific phosphatases as mechanisms responsible for cross-inhibition. T cells derived from mice carrying a knock-in mutation in IL7-receptor (Y449F) do not cause STAT5 phosphorylation or PI3-kinase activation in response to IL-7, yet, in these T cells, IL-7 pre-incubation inhibited IL-4 responses. These results suggest that IL-7 induced redistribution of gamma chains limits access to it by other gamma chain cytokine receptors. We are in the process of submitting this manuscript. We have an extensive on going collaboration with the lab of Martin Meier-Schellersheim. His group is developing computational models of signaling via the common gamma chain family of cytokine receptors. They have developed new computational tools that allow them to scan multiple parameters in the model and map them on to kinetics and dose response data. These models are providing us with valuable insights in to the mechanism by which IL-7 mediates its cross-inhibitory effect on other gamma chain family members. In another on going collaboration, we are investigating the mechanisms of chemotaxis in dictyostelium. For this purpose, we have developed a high-speed confocal imaging system. We are using this to map receptor distribution and signaling outcome in response to chemo-attractant in real time. We have an on going collaboration with the labs of Drs. Laurent Limozin and Kheya Sengupta at the Mediterranean University at Marseille, France, to study the topography of the immune synapse using reflection interference contrast microscopy. We have studied the effect of ligand mobility on the spreading characteristics of T cells. A manuscript describing these results is under review at the Biophysical journal. As part of a collaborative effort within the Laboratory of Systems Biology to study Toll Like Receptor (TLR) signaling, in collaboration with Iain Fraser's group we have begun to image the cellular response to single and multiple TLR stimulations in multiple cell lines. Imaging the molecular players downstream of TLR2 and TLR4 stimulation is giving valuable insights into the intracellular signaling events.