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

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.