In this study, we demonstrate that ChipCytometry is a viable method for single-cell suspension cytometry and analysis, with the additional benefit of allowing phenotyping in a spatial context using tissue sections. Both supervised and unsupervised analysis approaches identified MAIT cells in single-cell suspensions, confirming a phenotype similar to that of blood-derived MAIT cells. We were able to show that PD1 +CD4 + T cells are comprised of CXCR5 +BCL6 high follicular Th cells and CXCR5 −BCL6 mid pre–follicular Th cells. Using single-cell suspension ChipCytometry, we further dissected human tonsillar T cell subsets via unsupervised clustering analysis as well as supervised traditional manual gating. Additionally, we were able to identify the rare tonsillar T cell subsets, mucosal-associated invariant T (MAIT) and γδ-T cells, within tonsil tissue. Using ChipCytometry of tissue sections, we successfully demonstrated the architecture of human palatine tonsils, including the B and T cell zones, and characterized subcompartments such as the B cell mantle and germinal center zone, as well as intrafollicular PD1-expressing CD4 + T cells. In this study, we explored the feasibility of using ChipCytometry to identify and phenotype cell subsets, including rare cell types, using a combination of tissue sections and single-cell suspensions. Images are acquired by iterative cycles of immunostaining with fluorescently labeled Abs, followed by photobleaching, which allows the accumulation of multiple markers on a single sample. ChipCytometry is a multiplex imaging method that can be used to analyze either cell suspensions or tissue sections.

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