MULTIMODAL IMAGING UTILIZING MALDI IMS, IF, AUTOFLUORESCENCE, AND HISTOLOGICAL STAINING FOR THE INVESTIGATION OF LIPIDS INTEGRAL TO THE ADAPTIVE IMMUNE RESPONSE
Jones, Marissa A.
Adaptive immunity, the specified, targeted response of the immune system to pathogens or mutated cells, is integral to survival of many organisms. The malfunction of the adaptive immunity response in humans results in many diseases and disorders that affect over 23.5 million Americans. Adaptive immunity involves many organs within the lymphatic system, of which the spleen is the largest. Within the spleen, germinal centers are small structures in which B cells develop a specified response to a pathogen. Identifying the spatial distributions of biomolecules, specifically within the germinal centers, is crucial for understanding the integrated functions of these molecules. MALDI IMS enables untargeted molecular mapping of thousands of biomolecules directly from tissue sections and is ideal for studying the spatial distributions of thousands of biomolecules in a single experiment. Herein we addressed two significant technological challenges: 1) Distinguishing sample preparation artifacts from significant biological differences and 2) deriving biological context for spatial distributions of ion images through multimodal imaging of murine spleens. To address these challenges, I evaluated the effect of various sample preparation methods including fixation, freezing, and washing on lipid MS signal and tissue morphology. From this work, the optimal sample preparation strategy was selected to develop a multimodal imaging method that incorporated a genetically encoded fluorophore labeling splenic germinal centers in the same tissue imaged by MALDI IMS for lipid localization. Sixteen germinal-center-specific lipids were discovered, which led to the hypothesis that molecular programming of germinal center lymphocytes is tied to increased ether lipid synthesis and that these species are functionally important to adaptive immunity. A mouse model incorporating a genetic knockout of a peroxisomal enzyme that executes a late step in ether lipid synthesis reduces the level of ether lipids when compared to unmodified mice. Ether lipids were found to significantly affect germinal center size and density (p=0.02, p=0.02), indicating a connection between germinal centers and ether lipids. This work establishes a method for discovery of biomolecules that are integral to germinal center formation and function, to inform new paths of treatment for those who suffer from malfunctioning immune systems.