Topological analysis reveals a PD-L1-associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma

Signaling between programmed cell death protein 1 (PD-1) and the PD-1 ligands (PD-L1, PD-L2) is essential for malignant Hodgkin Reed-Sternberg (HRS) cells to evade antitumor immunity in classical Hodgkin lymphoma (cHL). Copy number alterations of 9p24.1/ CD274(PD-L1)/PDCD1LG2 (PD-L2) contribute to robust PD-L1 and PD-L2 expression by HRS cells. PD-L1 is also expressed by nonmalignant tumor-associated macrophages (TAMs), but the relationships among PD-L1¹ HRS cells, PD-L1¹ TAMs, and PD-1¹ T cells remain undefined. We used multiplex immunofluorescence and digital image analysis to examine the topography of PD-L1¹ and PD-1¹ cells in the tumor microenvironment (TME) of cHL. We find that the majority of PD-L1 in the TME is expressed by the abundant PD-L1¹ TAMs, which physically colocalize with PD-L1¹ HRS cells in a microenvironmental niche. PD-L1¹ TAMs are enriched for contacts with T cells, and PD-L1¹ HRS cells are enriched for contacts with CD4¹ T cells, a subset of which are PD-1¹. Our data define a unique topology of cHL in which PD-L1¹ TAMs surround HRS cells and implicate CD4¹ T cells as a target of PD-1 blockade.