Real-time imaging defines the dynamics of TCR and T cell motility during early T cell activation in lymph nodes

Beemiller, Peter

Friedman, Rachel S.

Jacobelli, Jordan

Krummel, Matthew F.

Sorensen, Caitlin M.

English

PubMed

The real-time dynamics of the T cell receptor (TCR) reflect antigen detection and T cell signaling, providing valuable insight into the evolving events of the immune response. Despite considerable advances in studying TCR dynamics in simplified systems in vitro, live imaging of subcellular signaling complexes expressed at physiological densities in intact tissues has been challenging. In this study, we generated a transgenic mouse with a TCR fused to green fluorescent protein to provide insight into the early signaling events of the immune response. To enable imaging of TCR dynamics in naive T cells in the lymph node, we enhanced signal detection of the fluorescent TCR fusion protein and used volumetric masking with a second fluorophore to mark the T cells expressing the fluorescent TCR. These in vivo analyses and parallel experiments in vitro show minimal and transient incorporation of TCRs into a stable central supramolecular activating cluster (cSMAC) structure but strong evidence for rapid, antigen-dependent TCR internalization that was not contingent on T cell motility arrest or cSMAC formation. Short-lived antigen-independent TCR clustering was also occasionally observed. These in vivo observations demonstrate that varied TCR trafficking and cell arrest dynamics occur during early T cell activation

Friedman, Rachel S

Sorensen, Caitlin M

Krummel, Matthew F

eScholarship - University of California

Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

A single class II myosin modulates T cell motility and stopping, but not synapse formation.

and the ImarisXT Image Arithmetic function, the contribution of the CMTMR fluorescence in the green channel was removed using the equations determined. For example, for Fig. 4 (e and f), 2748 Real-time in vivo T cell receptor dynamics | Friedman et al.

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video recording software (IO Industries) was used for image acquisition

https://escholarship.org/uc/item/0kq7c181

Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics

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eScholarship - University of California