(B-70) Deciphering the immune microenvironment and the stromal network in lymphoid tissues using highly multiplexed, spatially resolved imaging mass cytometry.

Introduction:: The secondary lymphoid organs (SLOs) are characterized by their rich immune microenvironments as they contain functionally specialized phenotypes of B and T lymphocytes and form the adaptive immune response¹. Within the secondary lymphoid organs, germinal centers (GCs) are the specialized structures for the B cells’ development and differentiation. GGs also have an intricate stromal network that maintains chemokine gradients, transports antigens, and recruits lymphocytes. Despite the significance of the SLOs and their diverse immune regulatory systems, the positional interactions among different cellular phenotypes in the SLOs remain poorly understood. The highly multiplexed, single-cell imaging mass cytometry (IMC) technique was used to fill in this by profiling 25 markers in tonsil, lymph node, and intestinal tissues. Phenotypic identification is often a daunting task in imaging datasets due to the dense cellular organization of the tissue and the developmentally similar immune cell phenotypes. Herein, we developed SpatialVisPheno which is a highly modular computational pipeline to profile cell types in high-content imaging datasets using two approaches. The first approach is manual gating using the widely used FlowJo software and the second approach is the semi-supervised gating which relies on iterative clustering and manual annotation. Using these approaches, immune phenotypes across the SLO tissues were profiled and the spatial neighborhoods between them were quantified. Further, SpatialVizPheno enabled extracellular matrix (ECM)-driven analysis of the follicles in the lymphoid tissues

Materials and Methods:: Formalin-Fixed Paraffin-Embedded (FFPE) human tissue blocks from tonsil, lymph node, and intestinal Peyer’s patches samples were attained from a third-party vendor (Biomax, US). They were then sectioned into slices with a thickness of 5-µm and baked at 60^oC for 2 hours before staining. The tissues were then deparaffinized using xylene and hydrated using descending concentrations of ethanol. Heat-induced epitope retrieval (HIER) was then performed under basic conditions (pH=9) for 15 minutes. The samples were then stained with the metal-conjugated antibody cocktail mix overnight at 4^oC followed by a nucleus counter-strain using an intercalator for 30 minutes at room temperature. The tissues were then left to dry at room temperature for 20 minutes before performing the imaging using the Hyperion Imaging System².

Results, Conclusions, and Discussions:: IMC was used to profile the immune microenvironment of germinal centers in the SLOs (Fig. 1a). Using the gating approaches, lymph nodes were found to have the highest density of B-cells while the intestinal tissues had the highest density of regulatory T-cells, and follicular helper T-cells (Fig. 1b). The orientations of the follicles were analyzed based on the direction of the highest collagen expression and the shape of the follicles. Collagen-enriched regions were found to be associated with the organization of the light zone and the dark zone orientation in both tonsil and lymph node tissues, but not in intestinal tissues (Fig. 1c-d).
In sum, the high-parameter, single-cell IMC data compared the immune microenvironment in different models of lymphoid tissues in relation to the immune cell density, their spatial interaction, and the association of the ECM network in follicle organization.


Acknowledgements (Optional): :

References (Optional): : 1-Ruddle, N. H. & Akirav, E. M. Secondary lymphoid organs: responding to genetic and environmental cues in ontogeny and the immune response. J. Immunol. Baltim. Md 1950 183, 2205–2212 (2009).

2-Allam, M. et al. Spatially visualized single-cell pathology of highly multiplexed protein profiles in health and disease. Commun. Biol. 4, 632 (2021).