Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. In the past year we have published studies that focus on different aspects of LAT-mediated signaling. The first can be viewed as a continuation of efforts to bring cutting-edge microscopic techniques to the study of T cell activation. Previously we demonstrated that LAT-based complexes known as microclusters are the site of molecular complex formation occurring with T cell activation. Others later questioned whether these microclusters are the site where signaling downstream of the TCR occurs or whether signaling initiates at intracellular vesicles. Several years ago we addressed this question using a chimeric form of LAT that we could track using antibody-binding methods. We concluded that signaling began at microclusters, but some thought the controversy was not settled, as we had used a partially artificial system. To definitively resolve this question, we were able to use state-of-the-art lattice light sheet microscopy to visualize LAT microcluster formation from the initiation of TCR activation. This microscope provides rapid imaging of the entire live cell with little phototoxicity. We observed that LAT microclusters form rapidly at the site of activation. Vesicles containing LAT arrive 0.5-1 minute later. We confirmed these results by using a specialized form of electron microscopy (focused ion beam-scanning electron microscopy-FIBS-SEM). With this technique we saw no vesicles at the site of activation initially- they were elsewhere in the cell. At later times the vesicles had been recruited to the plasma membrane. A number of other interesting observations were a part of this study. We found that vesicles containing LAT move on microtubules between microclusters. These vesicles appear to partially and transiently fuse upon interaction with the microcluster. These interactions produce a flare-like enhancement of LAT signal. Proof that these flares represent fusion events and that LAT is delivered to microclusters during these interactions are questions under investigation. We have been collaborating with Dr. Nick Restifo of the Surgery Branch for several years. He and his colleagues have been working on a model in which the E3 ubiquitin ligase, Cish, is deleted in T cells. They had found that such T cells have an increased response to antigen in various assays. Moreover, T cells that are specific for a tumor antigen in a particular model system more readily clear the tumor. They turned to us to help them determine the critical target of Cish that would persist in the absence of Cish. We proved that the enzyme phospholipase-Cgamma1, a critical LAT-binding protein was one such target molecule. This study was published in 2015. In the past year we completed a published a follow-up study in which we map the interaction between Cish and its phospholipase substrate. We found that the Cish SH2 domain is critical to binding and Cish-mediated degradation of phospholipase-Cgamma1. This publication and the 2015 studies provide important information on the Cish mechanism as Dr. Restifo and his colleagues prepare to target this molecule in tumor patients' T cells in the clinic.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010304-20
Application #
9779609
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Balagopalan, Lakshmi; Yi, Jason; Nguyen, Tiffany et al. (2018) Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation. Nat Commun 9:2013
Guittard, Geoffrey; Gallardo, Devorah L; Li, Wenmei et al. (2017) Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice. Front Immunol 8:343
Yi, Jason C; Samelson, Lawrence E (2016) Microvilli set the stage for T-cell activation. Proc Natl Acad Sci U S A 113:11061-11062
Palmer, Douglas C; Guittard, Geoffrey C; Franco, Zulmarie et al. (2015) Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J Exp Med 212:2095-113
Hui, King Lam; Balagopalan, Lakshmi; Samelson, Lawrence E et al. (2015) Cytoskeletal forces during signaling activation in Jurkat T-cells. Mol Biol Cell 26:685-95
Rodriguez-Peña, A B; Gomez-Rodriguez, J; Kortum, R L et al. (2015) Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules. Gene Ther :
Rouquette-Jazdanian, Alexandre K; Kortum, Robert L; Li, Wenmei et al. (2015) miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways. PLoS One 10:e0131823
Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi et al. (2015) Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration. Eur J Immunol 45:2389-95
Kunii, Naoki; Zhao, Yangbing; Jiang, Shuguang et al. (2013) Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation. Hum Gene Ther 24:27-37
Sommers, Connie L; Rouquette-Jazdanian, Alexandre K; Robles, Ana I et al. (2013) miRNA signature of mouse helper T cell hyper-proliferation. PLoS One 8:e66709

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