Distances from PD-1⁺CD4⁺and PD-1⁺CD8⁺T cells to the nearest PD-L1⁺TAMs. (A) Representative multiplex IF image (20× resolution; case N10) showing staining for CD4 (cyan), PD-1 (yellow), CD68 (magenta), and PD-L1 (green). (B) Cellular phenotype map of image shown in A depicting the locations of PD-1⁺ CD4⁺ T cells (green dots), PD-L1⁺ TAMs (purple dots), PD-L1⁻ TAMs (pink dots), and undefined cells (gray dots). (C) The mean distances (microns) and standard errors for all 20 study tumors, divided into mean distance from PD-1⁺ CD4⁺ T cells to the nearest PD-L1⁺ TAMs (blue) and mean distance from PD-1⁺ CD4⁺ T cells to the nearest PD-L1⁻ TAMs (red). Tumors are ordered by the distance between PD-1⁺ CD4⁺ T cells and PD-L1⁻ TAMs, from highest to lowest. P value (.004) was calculated by paired Student t test. (D) Representative multiplex IF image (20× resolution; case P6) showing staining for CD8 (red), PD-1 (yellow), CD68 (magenta), and PD-L1 (green). (E) Cellular phenotype map of image shown in D depicting the locations of PD-1⁺ CD8⁺ T cells (red dots), PD-L1⁺ TAMs (purple dots), PD-L1⁻ TAMs (pink dots), and undefined cells (gray dots). (F) The mean distances (microns) and standard error for all 20 study tumors, divided into mean distance from PD-1⁺ CD8⁺ T cells to the nearest PD-L1⁺ TAMs (blue) and mean distance from PD-1⁺ CD8⁺ T cells to the nearest PD-L1⁻ TAMs (red). Tumors are ordered by the distance from PD-1⁺ CD8⁺ T cells to the nearest PD-L1⁻ TAMs, from highest to lowest. P value (.005) was calculated by paired Student t test.