title: T Cell Receptor Activation of NF-κB in Effector T Cells: Visualizing Signaling Events Within and Beyond the Cytoplasmic Domain of the Immunological Synapse.
abstract: The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells. Additionally, in effector T cells, much signaling occurs at cytoplasmic locations distant from the IS. Visualization of these cytoplasmic signaling complexes has provided key insights into the complex and dynamic regulation of NF-κB signal transduction in effector T cells. In this chapter, we provide in-depth protocols for activating and preparing effector T cells for fluorescence imaging, as well as a discussion of the effective application of distinct imaging methodologies, including confocal and super-resolution microscopy and imaging flow cytometry.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, 20814, USA.|Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, 20817, USA.
authors.forename: Maria K
authors.initials: MK
authors.lastname: Traver
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, 20814, USA.|Department of Internal Medicine, University of Toledo Medical Center, Toledo, OH, 43614, USA.
authors.forename: Suman
authors.initials: S
authors.lastname: Paul
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, 20814, USA. brian.schaefer@usuhs.edu.|Center for Neuroscience and Regenerative Medicine, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD, 20814, USA. brian.schaefer@usuhs.edu.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Schaefer
mesh_terms: D000818:Animals; D005434:Flow Cytometry / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D016328:NF-kappa B / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell
keywords: Bcl10; Confocal microscopy; Imaging flow cytometry; NF-κB; POLKADOTS; Retroviruses; Signal transduction; Super-resolution microscopy; T cell receptor; T lymphocyte