Activation status of CD8+ T cells. Activation status of CD8+ T cells. Dotted lines indicate clusters of tumor epithelial cells, yellow arrows mark double-positive T cells, and white stars mark epithelial cells positive for the marker of interest. A–D, Immunofluorescence analysis of granzyme B (A) and Ki67 (C) expression in CD8+ T cells. SMA staining was used to mark the myoepithelial cell layer in DCIS. Images are a montage of nine fields captured from one area of the tissue. Graphs depict the frequencies of GZMB+ (B) and Ki67+ (D) CD8+ T cells in multiple regions of ten samples per group. Error bars, SEM. Scale bars, 50 μm. P values are calculated using two-tailed t test. E–H, Immunofluorescence analysis of GZMB (E) and Ki67 (G) expression in CD8+ T cells in matched DCIS and locally recurrent IDC samples. Graphs depict the frequencies of GZMB+ (F) and Ki67+ (H) CD8+ T cells in multiple regions. Error bars, SEM. Scale bars, 50 μm. P values are calculated using paired two-tailed t test. I, Box plot depicting the Shannon index of TCR clonotype diversity in normal breast tissues, DCIS, and IDC. Significance of the difference between tissue types was calculated using Wilcoxon rank sum test. Carlos R. Gil Del Alcazar et al. Cancer Discov 2017;7:1098-1115 ©2017 by American Association for Cancer Research