# Intravital imaging of the formation and resolution of MHC class II–positive T-cell activation niches

## TL;DR

The study introduces new fluorescent reporter strains to track immune cell interactions during inflammation in real time.

## Contribution

Two new spectrally distinct fluorescent reporter strains for MHC class II and CD8-positive T cells, enabling long-term intravital imaging.

## Key findings

- IEbeta-mAmetrine and CD8beta-LSSmOrange reporter strains allow simultaneous labeling of MHC class II and CD8-positive T cells.
- Sequential imaging of the same area over weeks revealed formation and resolution of T-cell activation niches.
- CXCL10-positive cell clusters and CD4 T cell accumulation were quantified during inflammatory responses.

## Abstract

This study describes the generation of two new fluorescent reporter strains that label MHC class II–positive cells and CD8-positive T cells and the use of the class II reporter to image the kinetics of an inflammatory response.

Intravital imaging has revealed many of the cellular interactions that regulate immune responses, but is limited by the number of cells that can be simultaneously identified and often restricted to analysis of a single time point. We have developed two new fluorescent reporter strains, IEbeta-mAmetrine and CD8beta-LSSmOrange, that faithfully label cells expressing MHC class II and CD8-positive conventional T cells, respectively. These fluorescent proteins are spectrally distinct from commonly used fluorescent proteins (GFP, YFP), and so, these mice can be used in combination with many previously created reporter mice. In addition, we established a protocol where we can sequentially image the same area of the ear dermis over several weeks without inducing additional inflammation. We applied these techniques to IEbeta-mAmetrine mouse co-expressing markers for CD11c, CXCL10, and CD4 T cells to quantify the formation of CXCL10-positive cell clusters, elaboration of different MHC class II–positive cells within these clusters, accumulation of CD4 T cells within these clusters, and the dissipation of these T-cell activation niches as the inflammatory response wanes.