CTLA-4 is the predominant checkpoint on T cells in cHL, which are frequently negative for either PD-1 or LAG-3 and are often in direct contact with HRS cells. (A-B) Representative case of cHL (CHL5) including full field view (panel A) following cell segmentation and phenotyping (panel B) [purple, HRS; blue, B cell; green, CTLA-4–positive T cell; orange, LAG-3–positive T cell; yellow, PD-1–positive T cell; red, T-cell positive for multiple checkpoints; gray, triple-negative T cell; dark gray, “other” cell]. (C) Pooled cell counts (135 298 T cells) across all analyzed cases (n = 18) reveals that CTLA-4–positive cells (46 981) outnumber PD-1–positive (26 256) and LAG-3–positive (22 061) cells (58 349 T cells were negative for CTLA-4, PD-1, and LAG-3). (D) Focal view of a representative case (CHL5) showing examples of isolated CTLA-4–positive cells (single arrow) and PD-1/LAG-3 double-positive cells (double arrow). (E) Cell segmentation and phenotype map corresponding to panel D. Filled cell borders denote cells in direct contact with HRS cells, whereas skeletal cell borders denote cells not in contact with HRS cells [purple, HRS; blue, B cell; green, CTLA-4–positive T cell; orange, LAG-3–positive T cell; yellow, PD-1–positive T cell; red, T-cell positive for multiple checkpoints; gray, triple-negative T cell; dark gray, “other” cell]. (F) CTLA-4–positive cells (median, 28%; range, 8%-73%) comprise a greater fraction of T cells in direct contact with HRS cells than do PD-1–positive (median, 9%; range, 3%-66%) or LAG-3–positive (median, 14%; range, 0%-36%) cells [CTLA-4 vs PD-1, P = .09; CTLA-4 vs LAG-3, P = .02].