Topology of multiple antigen expression in tonsillar lymphoid follicles and stroma analyzed by imaging mass cytometry.

Abstract

Abstract Investigation of co-distribution of 35 biomarkers simultaneously (major cell type and immune-oncology, stromal and tissue architecture markers, as well as cell proliferation and nuclear markers) in normal lymphoid sections using Imaging Mass Cytometry (Qing et al, Cytometry: Part A, 2017) is presented. Tonsil tissue revealed primary lymph follicles as dense clusters of lymphocytes expressing B-cell markers, surrounded by CD3, CD4, and CD8 T-cells. Secondary lymph follicles could be distinguished by proliferating B-cells with high Ki-67 expression. Strong expression of Bcl-6 was detected in germinal-center B-cells. Tonsillar FoxP3 CD8 T-cells exhibit a Treg phenotype with high CTLA-4 and CD45RO. CD68+ mac/mono were found in germinal centers and rarely in stroma. Squamous epithelium of crypts was beta-catenin positive and infiltrated by PD-L1+ T-cells, while PD-1+ cells were also found within the follicle centers. Images acquired on the Hyperion™ Imaging System (Fluidigm Inc.) were compared to immunofluorescence (IF) of sequential sections stained with the same antibodies (Ab) conjugated to fluorophores (FL). For direct comparison of IF and IMC, dual tagged Abs were created by attaching a metal tag first (Maxpar®, Fluidigm Inc.), then conjugating to FL using click chemistry. CD3 and CD19 were labeled with metals and FL, and tested in combination with the full panel on 8 μm frozen tonsil sections. CD3+ and CD19+ lymphs were identified by IF first, followed by IMC analysis. Results show that IMC is comparable to IF images and allows identification, characterization and localization of cell populations and tissue architecture in the absence of autofluorescence, photobleaching, with low background for acquisition of up to 50 targets.