Dr. Wetzel serves as the Scientific Director of the CEHS Molecular Histology and Fluorescence Imaging Core.
CD4+ T lymphocytes recognize antigenic peptide fragments presented on the surface of antigen presenting cells (APC) by major histocompatiblility complex (MHC) class II proteins. Triggering of the T cell antigen receptor (TCR) by binding to the MHC:peptide ligand induces dramatic morphological changes as the T cell flattens against the APC and increases contact area forming stable T-APC conjugates. This initial antigen recognition is followed by large-scale spatial and temporal molecular rearrangements of plasma membrane proteins and intracellular signaling molecules. These rearrangements lead to the formation of an ordered structure at the T-APC interface termed the immunological synapse. The synapse is the site of sustained T cell signaling as well as the site for delivery of T cell effector functions.
While we use a variety of techniques to examine these questions including flow cytometry, animal experiments, biochemical and molecular biology approaches, the main focus of our research is live cell, 3D imaging.
Work in our lab makes use of fibroblasts transfected with a GFP-tagged MHC:peptide covalent complex as surrogate antigen presenting cells. We also use dendritic cells derived from a transgenic mouse expressing the same GFP-tagged MHC:peptide complex under the control of the MHC class II promoter.
We are using these systems to examine the mechanism and biological significance of intercellular transfer of molecules from APC to T cells (termed trogocytosis). We have previously shown that upon dissociation from APC, T cells capture MHC:peptide molecules from the immunological synapse and imaging data suggests that these molecules continue to signal to the T cell. Our working hypothesis is that this controls an ongoing immune response by continuous cell autonomous signaling and by intercellular interactions in T cell – T cell antigen presentation. This T-T presentation may potentiate an immune response and/or may play an important role in peripheral tolerance, as antigen presentation by T cells results in anergy induction in vivo in some experimental systems.
We are employing some new, cutting-edge techniques to examine sustained signaling within the T cells after trogocytosis including fusing Kaede to various signaling molecule such as NFkB, ZAP-70, NFAT, and PKC Theta and following their intracellular movements after transfer.
Another project in the lab is characterizing the immunological synapses formed by cells rendered anergic by recognizing antigen on costimulation deficient APC. We have observed that known negative regulators of TCR signaling, c-Cbl and Cbl-b, are preferentially recruited to the immunological synapse formed by anergic cells. We have also reported that converting a negative signal from the small G-protein Rap1 by expression of B-Raf prevents anergy and the immunological synapses formed are phenotypically normal. We are performing live cell imaging experiments to characterize potential differences that occur in synapse formation and molecular recruitment to the synapse during the induction phase of anergy.
Finally, we are examining the impact on Atrazine on the formation of the immunological synapse and the nature of the subsequent immune response. Atrazine is a very widely applied herbicide that the EPA estimates has contaminated 70% of the ground water in the US. It has been linked to sex bias and birth defects in amphibians and in mammals to cancer, immune developmental defects and modulation of immune cell effector functions. We are examining whether Atrazine alters dynamics of synapse formation and/or molecular components. We are also examining the impact of Atrazine on survival, prliferation and phenotype of lymphocytes and antigen presenting cells in viro and in vivo.
Doherty, M., Osborne, D.G., Browning, D.B., Parker, D.C. Wetzel, S.A. (2010) Anergic CD4+ T cells form mature immunological synapses with enhanced accumulation of c-Cbl and Cbl-b. Journal of Immunology 184:3598-3608. PMCID: PMC2843782.
Osborne, D.G. and Wetzel, S.A. (2012) Trogocytosis results in sustained intracellular signaling in CD4+ T cells. Journal of Immunology 189(10).
Thueson, L.E; Browning, D.B., Wetzel S.A. (2013) In vitro exposure to the common water contaminant Atrazine inhibits CD4+ T cell activation and increases frequency of Foxp3+ Regulatory T cells. Journal of Immunology (in revision)
Wolf, S., Hayes, T.L.; Wetzel, S.A. (2013) TCR affinity for antigenic peptide determines the efficiency of trogocytosis. Journal of Immunology (In preparation)
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