Journal: Cells

Article Title: Spatial Distribution and Prognostic Value of T Cell Subtypes and Immune Biomarkers in p16-Negative HNSCC

doi: 10.3390/cells14110789

Figure Lengend Snippet: Multi color tissue cytometry for the detection of tumor and immune cells. ( A ) CD3, CD8, and FoxP3 were used as markers to classify T cells into cytotoxic T cells (CD3 + CD8 + FoxP3 − ), T helper cells (CD3 + CD8 − FoxP3 − ), and regulatory T reg (CD3 + CD8 − FoxP3 + ). CK was used to determine epithelial tumor cell nests. ( B – G ) Gating algorithm in StrataQuest to determine T cell subsets. Gates are represented by solid lines, whereas dashed lines indicate the corresponding gate in a different graph. Consistent colors are used to denote equivalent gates or cutoffs. ( B ) Nuclei were detected based on the DAPI staining. ( C , D ) Detection of T cells was based on high CD3 cytoplasmatic/membrane staining and low CD3 background staining. These parameters were used to create a gate including all T cells. ( E ) To distinguish between T helper cells and cytotoxic T cells the CD8 cytoplasmatic/membrane staining was used. ( F ) FoxP3 + T cells were detected using high FoxP3 nuclear staining and low or no FoxP3 cytoplasmatic/membrane staining. These parameters were used to create a gate including all FoxP3 + T cells. ( G ) Finally, the number of T reg (CD3 + FoxP3 + CD8 − ) was determined using the previous determined cutoff for CD8 cytoplasmatic/membrane staining. CK = Cytokeratin, CTL = cytotoxic T cell, DAPI = 4′,6-Diamidin-2-phenylindol, TC = T cell, T H cell = T helper cell, T reg = regulatory T cell.

Article Snippet: Semi-automated detection and quantification of T cell infiltration by immunofluorescence staining was performed by the StrataQuest software (version 5.0.1, TissueGnostics GmbH, Vienna, Austria) according to a defined algorithm ( B–G).

Techniques: Cytometry, Staining, Membrane