Fig 2: T cell and microbial co-localization define distinct cellular neighborhoods in human pancreatic cancer(A) Study design for (B)–(D) (n = 185 for cold nests and n = 77 for hot nests pooled from 16 specimens).(B) Images of CD8 (yellow), CD68 (brown), tumor epithelium (teal, CK19), FOXP3 (purple), and nuclei (blue, hematoxylin). Scale bars, 1 mm (top) and 50 μm (bottom).(C) Quantification of CD8+ (left), FOXP3+ (middle), and CD68+ (right) cells.(D) Ratio of CD8+ cells to FOXP3+ cells.(E) Study design for (F) and (H)–(J).(F) Principal-component analysis (n = 5 for hot and cold tumor nests from 1 specimen).(G) 16S rRNA levels (n = 14 for cold and hot tumor nests from 3 patient specimens).(H and I) Heatmap (H) and enrichment score (I) for the relative expression of genes associated with response to bacterium (GO: 0009617) in hot and cold stroma.(J) Volcano plot of DEGs.(K) Multiplex immunohistochemistry images of CD8 (yellow), PIGR (purple), and nuclei (blue, hematoxylin).(L) Quantification of PIGR (n = 49 for cold nests and n = 46 for hot nests pooled from 7 specimens).(M) Multiplex immunohistochemistry images of CD8 (teal), CD20 (yellow), CD74 (purple), and nuclei (blue, hematoxylin).(N) Quantification of CD74 (n = 71 for cold nests and n = 45 for hot nests pooled from 10 specimens).(O) Summary of cell markers and proteins.For (K) and (M), scale bars, 2 mm (left) and 50 μm (right), and dashed lines indicate tumor epithelium. Statistical significance was calculated using a two-tailed Mann-Whitney test. Data are represented as violin plots (center line, median; top and bottom lines, upper and lower quartiles) and scatterplots (mean + SD or mean). PDAC, pancreatic ductal adenocarcinoma.