Integration of mass cytometry imaging and glyco-imaging to characterize B cell follicle dynamics 3505

Description The use of spatial mapping tools allows for a better understanding of tissue processes in pathophysiology. However, due to specific platform-to-platform incompatibility, protocols that maximize output from a single tissue section are lacking. Moreover, a central mechanism for regulating immunoglobulin (Ig) effector functions is through Fc glycosylation. Here, we present a workflow for the sequential imaging of the same tissue section using matrix assisted laser desorption ionization (MALDI) and multiplexed ion beam imaging (MIBI). We first profiled N-glycosylation on human lymphatic tissues to investigate how Ig Fc glycosylation is regulated within B cells. We stained the same tissue sections to quantify approximately 30 protein markers, targeting the single-cell composition of B cell follicles. After spatial registration, the imaging data was combined into a single dataset where each pixel retains bimodal information (N-glycans and MIBI probes) from the same tissue section. By using MIBI markers to guide the analysis throughout the follicular regions (i.e. dark/light zone, mantle zone), we studied the dynamic changes in mannosylation, fucosylation and sialylation across unique B cell populations in situ. Overall, these findings streamline a strategy that allows mass spectrometry imaging by MALDI, high-definition spatial proteomics by MIBI and H&E on the same tissue section. Moreover, it clarifies how Ig glycosylation is structurally organized during the follicular reaction.