Fig. 5

HPVnegVSCC are infiltrated with highly activated CD4⁺ and CD8⁺ effector/memory T cells. PBMC of healthy controls (n = 11) as well as PBMC (n = 29) and freshly dissociated tumor-derived TIL (n = 12) of HPVnegVSCC patients were analyzed by 13-parameter flow cytometry analysis. a Hierarchical Stochastical Neighbor Embedding (HSNE) clusters (left) and density plots (right) visualizing the high-dimensional flow cytometry data in two dimensions for the collective total CD3⁺ T cells for indicated groups. The identified cell subsets are identified in the cluster plots by the different colors. b CITRUS automatic discovery of stratifying biological signatures within tumor and blood samples visualizes 10 distinctive populations of CD8⁺ and CD4⁺ T cells the total CD3⁺ immune population. Every cell population represented by a node is divided on basis of median level of expression of a differently expressed marker into two new nodes (cellular subsets) going from the center (all cells) to the periphery of the plot. c The distribution of CD4⁺ and CD8⁺ T-cell frequencies (mean ± SEM) within the total CD3⁺ T cell population is depicted for healthy control and VSCC PBMC and tumors. d Scatter plots with bars displaying frequencies of CD8⁺ (# 1 to 5; top panel) and CD4⁺ (# 6 to 10; bottom panel) T cell populations are given as % of CD8⁺ and CD4⁺ cells. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001)