Iterative multiplex imaging cytometry for cross-species analyses of in situ tissue leukocytes

Traditional immunohistochemistry techniques are limited in the number of fluorescent detection channels, markers, and staining resolution, which restricts the ability to fully evaluate bulk tissue samples and biopsies. Recent technical advancements, such as co-detection by indexing and spatial proteomics, have enabled in situ visualization of tissues by combining multiplex assays, repetitive staining, and quantitative image analysis. These technologies can identify cellular co-expression, cellular spatial relationships, tissue heterogeneity, and detect low-abundance molecules, which are critical for basic immunology, disease evaluation, and therapeutic evaluation studies. However, these methods require specially conjugated antibodies and protocols to analyze highly multiplexed tissue imaging (HMTI) readouts. Here, we utilize imaging cytometry as a viable alternative that also enables individual cellular cytometry analyses in two-dimensional formats. This technique has been used to investigate human and mouse tissues but is underexplored in the translationally relevant rhesus macaque (RM) model. Here, we demonstrate the use of this platform to image RM placenta, jejunum, kidney, and liver stored in OCT, using commercially available fluorophore-conjugated antibodies to identify structural markers (cytokeratin and vimentin), pan-immune cells (CD45), T cells (CD3 and CD8), natural killer cells (NKG2A/C), monocytes (CD16) and macrophages (CD68 and CD163), without any customization. We compared these preclinical samples to human samples to emphasize the potential for cross-species translational analyses using this platform. The flexibility to perform multiple rounds of photobleaching and fluorophore-based staining, combined with the ability to compensate for autofluorescence, makes this technology extremely valuable for deciphering tissue architecture and the spatial distribution of immune cells. Furthermore, we leveraged the platform's ability to export data in HMTI format and flow cytometry standard format, compatible with other quantitative downstream analysis pipelines, to simultaneously visualize the spatial distribution of various cell populations.