How to Track T-Cell Antigen Receptors Using Imaris | Case Study

Immunology

Peter Beemiller, Jordan Jacobelli, Matthew F. Krummel, University of California - San Francisco

Peter Beemiller, Jordan Jacobelli and Matthew F. Krummel

T-cell activation is an important part of the immune response. It occurs when T-cell antigen receptors (TCRs) bind with antigen-presenting cells, forming what is known as an immunological synapse. Researchers at the University of California in San Francisco are using total internal fluorescence microscopy and Imaris image analysis software to learn more about the dynamic processes involved when immunological synapses form during T-cell motility.

During activation T-cell antigen receptors collect into microclusters, which are then reorganized into macromolecular domains as part of their regulation. The researchers illustrated how these domains, called supramolecular activation clusters (SMACs), are generated in motile T-cells. “This brings our understanding of the formation of these domains in line with in vivo imaging that shows that T cells are quite dynamic during their interactions with antigen presenting cells,” said Dr. Peter Beemiller, who led the research team.

The researchers used total internal reflection microscopy to image motile synapses formed during T-cell motility. The Imaris XT interface allowed them to work with data from other sources. They identified TCR microclusters outside of Imaris, and then transferred data on the location of the microclusters to Imaris for tracking. They could then switch between MATLAB and Imaris to analyze various track characteristics, such as time of formation.

“Imaris XT let us work with the data in Imaris and extended what we did beyond the rote routines that exist in all image analysis software,” Dr. Beemiller said. “This isn’t limited just to the primary image data, and isn’t limited to using Imaris as a one-way data server for other programs. Analyses from MATLAB can return data to Imaris, allowing us to quickly visualize the output of those routines in the context of our original data.”

The analysis revealed that TCR microclusters in motile synapses did not flow directly into SMACs. Instead, they moved toward an F-actin-poor ‘sink’ region. Inward microcluster flow often followed collapse of the leading edge, which suggested that actin depolymerization regulates microcluster flow and the formation of SMACs. The researchers continue to look for and study actin cytoskeletal proteins that control TCR signaling domain formation.

“By letting people get their data out of Imaris, the XT interface is a great way to make Imaris more valuable,” Dr. Beemiller said.

Research Paper: Integration of the movement of signaling microclusters with cellular motility in immunological synapses. Beemiller P, Jacobelli J, Krummel MF. Nat Immunol. 2012 Jul 1;13(8):787-95. doi: 10.1038/ni.2364.