Work

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

Year: 2018

Type: article

Abstract: T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

Source: Journal of Visualized Experiments

DOI: 10.3791/56655

Authors: Elisa Sanchez, Morgan Huse

Institutions: Memorial Sloan Kettering Cancer Center, Cornell University, Weill Cornell Medicine

Language: English

Topic: T-cell and B-cell Immunology

Subfield: Immunology

Field: Immunology and Microbiology

Domain: Life Sciences

Open Access status: green

Funders: Memorial Sloan-Kettering Cancer Center, National Institutes of Health, National Cancer Institute, National Institute of Allergy and Infectious Diseases

Awards: G2051006376, G289723095, G9000003249, Cancer Center Support Grant, Mechanoregulation of cytotoxic lymphocyte function