Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

This work was funded by Swiss National Foundation grant 31003A_135649 (to JVS), Sinergia grant CR23I3_156234 and CRSII3_141918 (to JSh and JVS), EC FP7 Marie Curie RG grant 276702 (to JVS), and a Novartis Research (NRG-2011-0341) grant (to AO). JA was supported by Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science. JR acknowledges support from the EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and MINECO grant FIS2013-41802-R MESO-IMAGING. JSw and JSh acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. The SPIM setup was financed by a grant of the Pierre Mercier Foundation.