Fig. 4
Multiplex immunofluorescence studies demonstrate that PD-1 is expressed on multiple cell types in MCC, including CD8+ cells, CD4+ cells, Treg (CD4 + FoxP3+), CD20+ B-cells, and even sometimes on tumor cells. a Representative photomicrograph of multiplex panel (CD8, yellow; CD68, magenta; FoxP3, red; NSE (tumor), orange; PD-1, cyan; PD-L1, green and DAPI) from a responder in the cohort of patients treated with anti-PD-1. Higher magnification photomicrograph shows that while there is a significant proportion of PD-1+/CD8+ cells (arrow), there are also PD-1+ cells that are CD8- (arrowhead), and CD8+ cells that are PD-1- (asterisk). Left and right panels: 200× and 400× original magnification, respectively. b A second multiplex panel (PD-1, cyan; CD8, yellow; CD4, magenta; FoxP3, red; CD20, white; NSE (tumor), orange, and DAPI) was applied to archival MCC specimens to further characterize cell types expressing PD-1. Left panel: Representative photomicrograph showing host-tumor interface, 200× original magnification. Upper-right panel (1): Cell types expressing PD-1 include CD4 + FoxP3+ cells (arrow) and PD-1 + FoxP3- (arrowhead) cells, 400× original magnification. Lower-right panel (2): CD20+ B-cells (arrowhead) were also noted to express PD-1. (only CD20 and PD-1 channels are shown in the inset, 400× original magnfication). c In one case, low-level, constitutive PD-1 expression on nearly every tumor cell was observed (arrowhead). High levels of PD-1 expression were also seen on TIL (arrow). d PD-1+ cell densities across n = 16 tumor specimens show that PD-1 expression on multiple cell types is observed across different levels of inflammation. The virus status of each specimen is displayed below each specimen number