(A–E) CD8⁺ Tregs were induced ex vivo from GCA patients and healthy controls as in Figure 1. (A) Expression of NOTCH1–4 receptor transcripts in CD8⁺ Tregs (RT-PCR, n = 11 patients). (B) Expression of NOTCH4 protein on control and patient-derived CD8⁺ Tregs (FACS, n = 11 samples each). (C) Transcripts for the NOTCH target gene HES5 (RT-PCR, n = 6 samples each). (D and E) Immunoblot analysis of the NOTCH4 intracellular domain (N4ICD) and HES5 in control and GCA CD8⁺ Tregs; β-actin served as loading control. N = 5 experiments. (F) Expression of NOTCH4 protein on CD8⁺CD39⁺CD26^– Tregs sorted from controls and patients determined by FACS. FMO, fluorescence minus one. Representative histograms and results from n = 8 samples. (G) Transcripts for the NOTCH target gene HES5 quantified by q-PCR in sorted CD8⁺CD39⁺CD26^– Tregs. Results from 10 patients and 10 controls. (H) Induced CD8⁺ Tregs were treated with DAPT or vehicle. Suppressive function was measured as in Figure 1A. Representative contour plots and results from 6 patients and 6 controls. (I) Induced CD8⁺ Tregs from GCA patients were transfected with NOTCH4 or control siRNA before suppressive function was measured as in Figure 1A. Representative contour plots. Results from 8 samples. (J) Induced CD8⁺ Tregs from healthy donors were transfected with an N4ICD-containing or control vector. Suppressive function was determined as in Figure 1A. Representative contour plots and results from 8 samples. (K and L) Induced CD8⁺ Tregs from GCA patients were transfected with NOTCH4 or control siRNA (K). Healthy CD8⁺ Tregs were transfected with a N4ICD-containing or control vector (L). Proliferation of CFSE-labeled CD4⁺ T cells was measured as in Figure 1C. Representative images from 6 experiments. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Mann-Whitney-Wilcoxon rank test (B–F), paired Mann-Whitney-Wilcoxon rank test (H and I), or 1-way ANOVA and post-ANOVA pairwise 2-group comparisons conducted with Tukey’s method (A and G).