Unsupervised mass cytometry analysis of peripheral blood T cell signatures in patients with irAE-n and controls. (A) Overlay t-distributed stochastic neighbor embedding (t-SNE) maps of all patients with irAE-n and controls, each plot represents the expression of the indicated marker. One dot represents one cell. Heat colors show overall expression levels (red, high expression; dark blue, low or no expression). (B) 2D single-cell t-SNE maps with 50 metaclusters in patients with irAE-n and controls. Analysis for differential cluster abundance using significance analysis of microarrays (SAM) reveals differences in 3 metaclusters (cluster 2, cluster 18, cluster 21; printed in bold). (C) Heatmap of 37 different T cell markers illustrates individual expression levels for the 3 differential clusters 2, 18, and 21. Colour intensity shows overall expression levels (dark = high expression, bright = low or no expression). (D) Inspection of marker expression and plotted frequencies demonstrate a decrease of CD4⁺ CD127⁺ T cells (cluster 2) and CD4⁺ CXCR⁺ CD127⁺ EM1 T cells (cluster 18) as well as an increase of CD8⁺ effector memory type 1 (EM1) T cells (cluster 21) in patients with irAE-n compared to controls. Tukey’s box plots depict median (horizontal bar), mean (cross), interquartile range (hinges), and whiskers (fences). Outliers are represented as dots. * = P value ≤ .05, *** = P ≤ .001. CTLA-4, cytotoxic T-lymphocyte-associated protein 4; FoxP3, forkhead-Box-Protein P3; ICOS, inducible T-cell costimulatory; KLRG-1, killer cell lectin-like receptor subfamily G member 1; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PD-L2, programmed death-ligand 2; Tbet, T-box expressed in T cells; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TIM-3, T-cell immunoglobulin and mucin-domain containing-3.