In 2013, we made progress in the following projects: Pre-existing nature of T Cell Receptor (TCR) microclusters, TCR signaling in response to low potency ligands, role of RhoH in TCR signaling 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 un-stimulated cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These preexisting microclusters are stabilized by low-potency ligand engagement, including positively selecting ligands, and the density of stabilized TCR microclusters increases with ligand potency without a change in the number of TCRs in a microcluster. In characterizing their composition, we found that such microclusters exclude the phosphatase CD45, and contain the signaling adapters LAT and Grb2. Signaling through these microclusters in response to high concentrations of low-potency ligands results in low-grade signals. The utilization of preexisting microclusters by pMHC for TCR triggering is a novel mechanism that may explain the sensitivity and fidelity exhibited by the TCR. This work has been submitted and is under review. The current paradigm of TCR ligand discrimination is based on the kinetic proofreading model, which assumes a singular and sequential signaling cascade downstream of the TCR. To test this assumption, we identified doses of the agonist and a low potency TCR ligand that induced the same amount of the immediate early gene, Fos. Analysis of TCR proximal signals revealed that calcium fluxes in response to these doses of the agonist and low potency ligand were also identical;however, the high dose of the low potency ligand led to higher MAP kinase responses. ERK activation in response to the agonist ligand was calcium independent while it was calcium dependent in response to the low potency ligand. Total internal reflection fluorescence microscopy demonstrated lack of significant Zap70 kinase recruitment to TCR microclusters engaging low potency ligand, leading us to propose the lack of Zap70 involvement. Reducing Zap70 expression in T cells specifically affected calcium fluxes in response to the agonist. The existence of a Zap70 independent pathway downstream of TCR stimulation by low potency ligand suggests that ligand discrimination is accompanied by structural changes in the TCR complex that causes induction of distinct signaling pathways downstream of the two types of ligands. We continued our studies exploring the role of RhoH in TCR signaling. We find that RhoH localizes to TCR microclusters. RhoH is a protein that has been shown to interact with both Zap70 and the kinase Lck. The amount of RhoH present in TCR microclusters is correlated with the amount of Zap70 in TCR microclusters, allowing us to hypothesize that perhaps RhoH is stabilized in microclusters by Zap70 binding. Anke Knauf, a postdoctoral fellow working on this project developed a new algorithm to detect TCR microclusters, reducing the amount of time required to analyze the images. 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 signals. Using quantitative flow cytometric analysis, we quantified cell surface receptor expression, STAT phosphorylation dose-responses and kinetics, and crosstalk among cytokine-induced signals. We found that, even though gamma chain would be limiting if each private chain were pre-associated with it, signal-induced recruitment of gamma chain to ligated private chains would not lead to a shortage of gamma chain for physiological cytokine concentrations. We observed strikingly lower EC50 values for STAT phosphorylation than would be expected from the receptor binding curves. The kinetics of signaling through these receptors was relatively slow, taking several minutes to reach saturation. IL-7 pre-exposure led to cross inhibition of IL-4 and IL-21 responses at receptor occupancies where gamma chain would not be limiting. An assay was designed to determine the fraction of each STAT protein phosphorylated in response to IL-7, IL-4 and IL-21. This assay revealed that IL-7 exerted the maximum negative regulation on STAT phosphorylation that correlated with its ability to cause cross-inhibition. Additionally, over expressing the gamma chain relieved the IL-7 mediated cross-inhibition of IL-4 and IL-21 responses. 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. 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 cell surface distribution of TLR2 and TLR4 in mouse macrophage cell lines.
|Crites, Travis J; Chen, Lirong; Varma, Rajat (2012) A TIRF microscopy technique for real-time, simultaneous imaging of the TCR and its associated signaling proteins. J Vis Exp :|
|Vardhana, Santosha; Choudhuri, Kaushik; Varma, Rajat et al. (2010) Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32:531-40|
|Padhan, Kartika; Varma, Rajat (2010) Immunological synapse: a multi-protein signalling cellular apparatus for controlling gene expression. Immunology 129:322-8|
|Ishihara, Satoru; Varma, Rajat; Schwartz, Ronald H (2010) A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions. Nucleic Acids Res 38:e124|
|Srivastava, Shekhar; Di, Lie; Zhdanova, Olga et al. (2009) The class II phosphatidylinositol 3 kinase C2beta is required for the activation of the K+ channel KCa3.1 and CD4 T-cells. Mol Biol Cell 20:3783-91|
|Beal, Allison M; Anikeeva, Nadia; Varma, Rajat et al. (2008) Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL. J Immunol 181:4815-24|
|Varma, Rajat (2008) TCR triggering by the pMHC complex: valency, affinity, and dynamics. Sci Signal 1:pe21|