The purpose of this study was to develop a method to highlight immune cells and cell clusters in histology tissue stained with single or multiple biomarkers. B6 mice were implanted (rear flank) with YUMM/YUMMER1.7 melanoma cells and treated with or without immunotherapy (PD-1, CTLA-4). Skin, lymph nodes, and spleens were formalin-fixed paraffin-embedded and stained by immunohistochemistry (IHC) or immunofluorescence (IF). Biomarkers for B cells (CD20), neutrophils (Ly6B.2), melanoma cells (GFP), helper T cells (CD4), and helper T cells activity (Granzyme B, IL-6, Ki-67, T-bet, Foxp3, RORγ(t)) were combined in single or multiple staining rounds. The TissueFAXS (TissueGnostics, Vienna, Austria) imaging system was used to acquire image tiles for analysis. Image processing and analysis was performed using StrataQuest software (TissueGnostics). An image stitching algorithm was used on the tiled images to reconstruct the whole image. Algorithms were created to align the stitched images, generate a composite image consisting of all biomarkers, isolate each cell in the composite image, and identify the positive cells in the composite image. Tissue cytometry coupled with backgating into the tissue images was used to visualize, quantitate, and validate the data. The results for IHC showed that B cell and neutrophil density was significantly increased during the natural host response in the time period post melanoma cell implantation corresponding to an adaptive immune response (days 7-19) versus immunosurveillance (days 0–6) (p<0.01, p<0.001). The density was further increased with immunotherapy treatment (p<0.01, p<0.05). For IF, using in silico analysis, we could identify three distinct clusters of CD4 expressing cell populations: intermediate, high, and mixed. Each CD4 cell cluster was composed of different combinations of biomarkers of CD4 activity. In summary, we developed a powerful method that utilized tissue cytometry to highlighting immune cells and immune cell clusters.

Profiling immune cells using tissue cytometry and immune cell clusters