CTLA-4–positive T cells are enriched in cHL. (A-B) Representative mIF image of cHL (CHL9) [inset: CTLA-4–positive cells in contact with PAX5-positive HRS cells] and the cell segmentation and phenotype map for the same image (panel B) [purple, HRS; green, CTLA-4–positive/FOXP3-negative; red, CTLA-4–negative/FOXP3-positive; yellow, CTLA-4–positive/FOXP3-positive; dark gray, “other” cell]. (C-D) mIF image of interfollicular zone from representative RLT [inset: rare CTLA-4–positive/FOXP3-positive cells] and the cell segmentation and phenotype map for the same image (panel D) [green, CTLA-4–positive/FOXP3-negative; red, CTLA-4–negative/FOXP3-positive; yellow, CTLA-4–positive/FOXP3-positive; dark gray, “other” cell]. (E) The densities of CTLA-4–positive/CD4-positive (median, 1243 cells/mm² vs 135 cells/mm²; P = .0004) and CTLA-4–positive/CD8-positive (median, 113 cells/mm² vs 22 cells/mm²; P = .02) cells are greater in cHL than in RLTs. Each dot corresponds to the results of an individual case. (F) CTLA-4–positive fraction of total T cells (CD4-positive plus CD8-positive cells) is greater in cHL (32%; 6%-87%) compared with RLTs (2%; 0.4%-5%) [cHL vs RLT, P = .0002]. (G) The density of CTLA-4–positive/CD4-positive/FOXP3-positive cells is greater in cHL relative to RLT (median, 260 cells/mm² vs 14 cells/mm²; P = .0032), whereas there is no significant difference in the density of CD8-positive/CTLA-4–positive/FOXP3-positive cells between groups (median, 11 cells/mm² vs 9 cells/mm²; P > .05). Each dot corresponds to the results of an individual case. (H) FOXP3-positive fraction of total CTLA-4–positive T cells in cHL (25%; 0.07%-66%) and RLTs (31%; 0.15%-68%) [cHL vs RLT, P > .05]. Epstein-Barr virus (EBV)-positive cases are marked by asterisks. DAPI, 4′,6-diamidino-2-phenylindole; RLN, reactive lymph node; TS, tonsil.