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. 2022 Jun 27;12:889886. doi: 10.3389/fonc.2022.889886

Figure 5.

Figure 5

Multiplex immunofluorescence T lymphocytes panel. As on the activation panel, the T lymphocytes panel includes a universal biomarker for nuclear detection (DAPI) and cytokeratin for detection of epithelial cells (malignant cells), which is also helpful for compartmentalization of the image (tumor vs. stroma). T lymphocyte populations are detected using CD3+ for all lymphocytes, co-expressed CD8+ for cytotoxic T cells, and co-expressed FOXP3+ for regulatory T-cells. This panel includes granzyme B+ (GB) and CD45RO+ to identify activated T-cells and memory T-cells, respectively. (A) Composite image of TME from oral squamous cell carcinoma, showing all markers of a multiplex immunofluorescence “T lymphocytes phenotypes”, activated simultaneously. (B) Composite image showing CD45RO positive expression (yellow) of immune cells in the stromal compartment. (C) Composite image showing FOXP3 positive expression (green) of immune cells in the stromal compartment. (D) Composite image showing CD3 positive expression (red) of immune cells in the stromal compartment. (E) Composite image showing CD8 positive expression (pink) of immune cells in the stromal compartment. (F) Composite image showing GB positive expression (magenta) of immune cells in the stromal compartment. inForm ® image analysis software, Akoya bioscience (scale bar: 100 μm). Scanner Vectra Polaris. DAPI (blue-DAPI), cytokeratin (cyan-opal 620), CD3+ (red-opal 690), CD8+ (pink-opal 540), CD45RO+ (yellow-opal 520), FOXP3+ (green-opal 650), Granzyme B+ (magenta-opal 570).