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. 2021 Jan 12;10:614876. doi: 10.3389/fonc.2020.614876

Figure 3.

Figure 3

Pembrolizumab treatment is associated with spread of EBV/fLuc to the central nervous system. (A, B) Bioluminescence images showing representative EBV signal patterns in brains from mice infected with B95-8/fLuc (A) or with M81/fLuc (B) and treated with pembrolizumab (Pembro; most severely affected animals shown in each group), compared with non-treated controls (CTR). The bioluminescence signal intensities (photons/sec) are depicted by the color bar on the right side. (C, D) In-situ hybridization for detection of Epstein-Barr encoded RNA (EBER)-1 in brains of mice infected with B95-8/fLuc and treated with pembrolizumab. Observed patterns include invasion of tumor cells into the plexus choroideus (white inserts), as well as into periventricular brain parenchyma (red insert) and meningeal tumor spread (blue inserts). The green arrows indicate single tumor cells associated with blood vessels. (E) Comparison with three representative examples of human EBV-positive monomorphic transplantation-associated B-PTLD, with clinical manifestation as primary CNS lymphoma (PCNSL) revealed similar patterns of intraparenchymal (Case 01), perivascular (Case 02), and massive angiocentric/diffuse growth with meningeal spread (Case 03). (F) Control animals did not show any involvement of the plexus choroideus or periventricular brain areas. Only single EBER-positive cells in intravascular localization were detected (green arrows). (G–J) The immunophenotype of the neoplastic cells invading the brain of ICI-treated mice corresponded to the phenotype of the peripheral neoplastic population with variable CD30+, and constant CD20+ expression. The majority of neoplastic cells expressed EBNA2 but not LMP1 and were actively proliferating (CD20+/Ki67+). (G–H) and (I–J) Analyses of duplicate mice infected with B95-8/fLuc and M81/fLuc and treated with pembrolizumab, respectively. (K) Multiplexed image of two mouse brains from B95-8/fLuc infection model after pembrolizumab treatment confirmed co-localization of neoplastic cells (CD20+, white; CD30+, magenta) and T lymphocytes (CD8+ yellow; CD4+, green). Lymphocytes were actively proliferating (Ki67+, red), particularly CD8 (blue arrows). DAPI (blue) is used to mark cell nucleus. Bars correspond to 200 µm (full image) and 50 µm (inserts).