In situ imaging of pulmonary invariant Natural Killer T cells in precision cut lung slices.

Invariant Natural Killer T (iNKT) cells are key players in bridging innate and adaptive immunity, recognizing lipid antigens presented by CD1d molecules. Flow cytometry has tremendously contributed to the study of iNKT cell biology through the use of CD1d tetramers. While this technique was pivotal to uncover the anti-tumor potential of iNKT cells, spatial characteristics are difficult to assess as effective tools are lacking. Here, we describe a method for detecting iNKT cells in mouse lung tissue by employing Precision Cut Lung Slices (PCLS) alongside CD1d tetramer staining and fluorescence imaging. This technique maintains lung architecture while facilitating the spatial localization of iNKT cells within the pulmonary milieu. The accuracy of our imaging approach was validated with critical negative controls, including unloaded CD1d tetramer staining and iNKT cell-deficient (Traj18-/-) mice. Co-staining with anti-CD3 confirmed the specificity of the CD1d tetramer signal and validated iNKT cell detection. This method provides a valuable tool to map iNKT cell localization and proximity to cells and histological structures, offering a novel angle to study their biology in lungs.