In 2011, we made progress in the following projects: TCR signaling in response to partial agonists, Fos induction in T cells, cross talk among common gamma chain family of cytokine receptors in T cells and developing a method of tuning diffusion coefficients of proteins in glass supported lipid bilayers. Additionally we initiated a new project on studying the role of RhoH in TCR triggering. We have an interest in understanding the signaling pathways activated downstream of TCR in response to low potency MHC-peptide complexes. Based on our previous work we have observed that there are a class of TCR ligands that generate Ras signals in the absence of calcium signals. We have now further biochemically determined that these low potency ligands lead to the phosphorylation of Erk albeit at low levels. Our hypothesis to explain this observation is that these Ras signals are generated by recruitment of Grb2-Sos module to the TCR complex in microclusters without recruitment or phosphorylation of LAT. We have now made several observations to support our hypothesis. We have visualized the recruitment of GFP tagged LAT, Zap70 and Grb2 transfected in in-vitro activated AND TCR transgenic T cells using Total Internal Reflection Fluorescence microscopy (TIRFM) of cells interacting with glass supported lipid bilayers containing lipid anchored peptide-MHC complexes, ICAM-1 and CD80. We find that the low potency ligands do not recruit LAT to the TCR microclusters and only poorly recruit Zap-70. We are currently refining our results to understand the nature of recruitment of Grb2 and working towards expressing Sos in T cells which has been a significant challenge. We want to understand the relationship between TCR engagement events at the cell surface and activation of transcription factors in the nucleus. For these experiments we have obtained transgenic mice that express a fusion protein between the transcription factor Fos and GFP under the control of the Fos promoter. Fos is not expressed in T cells in the basal state and is induced upon signaling. We have crossed these mice to AND TCR transgenic mice and have begun to study the induction of Fos using single cell imaging in response to ligands of varying strengths. We find that Fos is induced within 20 minutes of interaction with antigen and continues to accumulate for three hours after which the expression plateaus presumably due to degradation of Fos. Given that it takes about 6-7 minutes for GFP to mature, it is likely that the kinetics of Fos induction are even faster. Fos expression is controlled by Ras dependent MAPK and Calcium dependent CamK-Creb pathway in various cell types. Active Erk is known to phosphorylate members of the ternary complex factors, Elk-1, Sap-1 and Net which cause the transcription of Fos by binding SRE element in the Fos promoter. On the other hand active CREB binds to the CRE element in the fos promoter. Calcium and MAPK signaling occurs downstream of TCR, however, how strength of TCR signaling affects the efficacy of these signaling pathway as well as their relative activity is not known. We find that Fos induction occurs with distinct kinetics depending on the strength of TCR stimulation. The kinetics of Fos induction is more rapid in response to ligands that cause calcium signaling. MAPK inhibitors have a differential effect on Fos induction depending on the strength of TCR stimulation. Weaker affinity ligands are more affected by p38 inhibitors than the higher affinity ligands. We are currently exploring the contribution of the CREB pathway in Fos induction. The common gamma chain family of cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) shares the gamma chain for signaling. Since many receptors are expressed on T cells at the same time, it is not clear how these receptors share the gamma chain and if it ever is limiting for signaling. Binding curves that reflect receptor occupancy and dose response curve representing functional response are rarely aligned in biological systems. For example, in the hormone receptor system, the EC-50 of biological response occurs at receptor occupancy less than 50%, a phenomenon known as the Strickland effect. We have explored the relationship between receptor occupancy and STAT phosphorylation for the common-gamma-chain family of cytokine receptors in nave mouse T cells. We find that EC50 for STAT phosphorylation occurs at receptor occupancy of less than 3%. To explain this, we invoke a serial triggering mechanism, where in a few cytokine bound receptors cause phosphorylation of multiple STAT molecules by sequentially engaging many gamma chains. Consistent with this proposal we find that the kinetics of STAT phosphorylation are slow, requiring at least 10 minutes to saturate. We further postulate that STAT phosphorylation reaches saturation at low receptor occupancy at least due to two mechanisms;one, the creation of transiently inactive SOCS-1 bound gamma chain molecules which limit the availability of gamma chains harboring Jak3 molecules that can be activated and two, the activation of phosphatases that negatively regulate signaling via Jak kinases. A consequence of this postulate is the observation of cross-inhibition among IL-4, IL-7 and IL-21 when cells are stimulated sequentially with these sets of cytokines. We are collaborating with the lab of Dr. Martin Meier-Schellersheim who is using detailed computer simulations of the signaling cascades downstream of the cytokine receptors to explore the plausibility of this model and analyze the contributions of membrane-bound and cytoplasmic reactions to the overall response kinetics. Diffusion of MHC molecules in the plasma membrane of antigen presenting cells affects how they trigger TCRs. To study this phenomenon using glass supported bilayers, we are trying to develop a system where we can tune the diffusion coefficient of molecules in the bilayer. If transmembrane anchored molecules are incorporated in bilayers, their cytoplasmic tail interacts with glass and gets stuck and hence they don't diffuse. We are exploring the possibility that if we incorporate a cytoplasmic tail deleted protein, then it would have reduced interaction with glass and hence may diffuse slower than a lipid anchored protein. We could then modulate the thickness of the bilayer using different lipids and thereby tune the diffusion coefficients of the incorporated proteins. We first made several truncated CD80 molecules;however, we found that they ended up becoming GPI anchored when expressed in CHO cells. Using bioinformatics software that would predict whether a protein sequence is likely to be GPI linked or not we found that the transmembrane domain of CD28 when truncated is not likely to be GPI-anchored. Using this we have generated several CD80 molecules containing the TM domain of CD28 of differing length and expressed them in CHO cells. These molecules were not GPI anchored and trafficked to the cell surface. We could purify them and incorporate them in glass supported lipid bilayers and observed that they were mobile. We are now characterizing their diffusion coefficient. RhoH is a member of the Rho family of GTPases, but is unusual in that it does not have a GTPase activity, but instead has an ITAM like motif which binds Zap70. We have initiated a project to understand what role it plays in TCR triggering. Does it help bring Zap70 to the membrane as is currently thought? RhoH is also implicated in negatively regulating Rac activity by competing for phosphoinositide lipids. How do these two functions of RhoH play out in TCR triggering? This project is in collaboration with the lab of Dr. Martin Meier-Schellersheim who will incorporate these observations in the context of a mathematical model. We have hired a joint postdoc for this project.
