Mass cytometry-based immune profiling of human Peyer's patches In Crohn’s disease
HUCK A. 1, RODRÍGUEZ SILLKE Y. 3, BOJARSKI C. 1, KUNKEL D. 3, STEINHOFF U. 2, SIEGMUND B. 1, GLAUBEN R. 1
1 Medical Department (Gastroenterology, Infectious Diseases, Rheumatology) Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Berlin, Germany; 2 Institute for Medical Microbiology and Hygiene, University of Marburg, Marburg, Germany; 3 Flow & Mass Cytometry Core Facility, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, Berlin, Germany
Background: Peyer’s Patches (PPs) play a crucial role in the development of oral tolerance due to a constant exposition to environmental factors like microbial or food antigens. Murine data indicate an activation and subsequent apoptosis of food-reactive CD4+ T-cells thus maintaining the healthy balance of the mucosal immune system. In inflammatory bowel diseases, this homeostasis is disturbed, which is indicated by an altered immune cell composition within PPs.
Methods: To study the composition of PPs in healthy individuals and Crohn’s disease (CD) patients, we collected human biopsies of a respective patient cohort in a prospective manner and performed multiplexed mass and flow cytometry for deep immunophenotyping on single cell suspensions. To evaluate the spatial distribution and cellular interactions of immune cell subsets, we used imaging mass cytometry (IMC) on FFPE samples.
Results: Flow cytometry analysis revealed a reduction of activated B cells and an increase of CD8+ effector memory T-cells in active Crohn’s disease. For CD4+ T-cells, total numbers were similar, but CD patients showed an increase of central memory and a reduction of effector memory T-cells. Furthermore, CD4+ T-cells of CD patients in PP revealed a significantly reduced apoptotic rate compared to healthy controls. As the IMC data of lymphoid follicles such as PPs presented several challenges regarding their highly condensed cellular structure, several improvements had to be implemented into our analysis pipeline including cell segmentation, cell type identification and sample integration.
Conclusions: Using an IMC analysis pipeline optimized towards the specific features of lymphoid follicles, we were able to resolve differences in immune cell interactions between lamina propria and Peyer’s Patch as well as differences of cell-cell interactions between Crohn’s disease patients and healthy controls.