Tu135 - Deliniation of Secondary Lymphoid Organ Immune Landscaping with Multiplex Imaging Analysis (mIF)

Spiros Georgakis – Institute of Pathology, Department of Laboratory Medicine and Pathology; Cloe Brenna – Institute of Pathology, Department of Laboratory Medicine and Pathology; Craig Fenwick – Service of Immunology and Allergy, Department of Medicine, CHUV; Guiseppe Pantaleo – 2Service of Immunology and Allergy, Department of Medicine; Oliver Y. Chén – 3Department of Medicine and Department of Laboratory Medicine and Pathology, CHUV; Constantinos Petrovas – Institute of Pathology, Department of Laboratory Medicine and Pathology, CHUV

Abstract Text:

Background: what regulates the follicular/germinal center (GC) immune reactivity in human is not well understood. Imaging methodologies can greatly improve our understanding for the development of B cell responses.

Methods: FFPE tissues from four tonsils and two reactive LNs were used for analysis by mIF assays, i)(CD20/CD3/CD4/CD8/PD1/TIGIT/GITR/ICOS/CXCR3/CD57/Ki67/Bcl6/GATA3/Tcf1/Blimp1/GrzB/SYTO(, ii) (CD20/CD11c/CD163/CD68/FDC/CD123/MPO/CD31/CXCL13/IL21/IL10/SYTO) and iii) suppressor CD4 T cells (CD20/CD4/FOXP3/CD25/MCT1/MCT4/Helios/Neuropilin/IL10/SYTO). Imaging data were processed with HistoCytometry (FlowJo10) and computational modules for topological analysis.

Results: a high phenotypic heterogeneity of the follicular helper CD4 T cell (Tfh) was found among the tonsillar tissues as well as the LNs. FloxJo FlowSOM clustering analysis showed specific Tfh cell phenotypes with distinct localization across the follicular areas. The majority of FOXP3hiCD25hi/lo CD4 T cells (Tregs) were extrafollicular with a diverse -metabolism- profile. A lower frequency of monocytic subsets was found in LNs compared to tonsils in follicular and extrafollicular areas. Neighboring analysis also revealed differences between tonsils and LNs with respect to GC B and Tfh cell positioning.

Conclusion: we present an experimental pipeline allowing for a high dimension analysis of GC relevant immune cells in health. Application of such pipeline could provide clues for the interplay between cell types and possible association with B cell responses as well as to lead in novel diagnostic tools especially for certain lymphomas.