Identifying and Targeting Cytotoxic Tumor-Associated Macrophages in Epstein-Barr Virus-Driven Lymphoproliferative Disease
Authors: Patton John T. , Mitchell Monica L , Pan Li , Kinghorn A. Douglas , Grever Michael R. , Lucas David M. , Baiocchi Robert A. 
Published: October 2019

Abstract
Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with the development of a range of malignant B-cell lymphoproliferative diseases that are associated with immune suppression, aggressive clinical courses, and poor outcomes. Current treatment options typically lead to further immune suppression, increasing the risk of EBV reactivation and other potentially lethal opportunistic infections. These complications make it essential to identify novel therapeutic approaches targeting mechanisms of immune evasion while promoting tumor immune surveillance.
Immune evasion of tumors is supported by aberrant ligand and cytokine expression within the microenvironment, and tumor-associated macrophages (TAM) play an important role in inhibiting anti-tumor immune responses. In particular, TAMs are known to suppress T cell activation, and increased TAM density and ratio to cytotoxic T lymphocytes (CTL) in the tumor microenvironment are negative prognostic indicators.
Here we demonstrate the use of an in vitro co-culture (CoCx) system in which EBV-transformed lymphoblastoid cell lines (LCL) are cultured with autologous peripheral blood mononuclear cells (PBMC). Media from these CoCx collected after 48 hrs., showed elevated levels of the monocyte/macrophage associated cytokines and chemokines MCP, IL-8, IP-10, MIG, GRO and MMP-9, which were absent in media from LCL or PBMC cultures. Furthermore, LCL+PBMC CoCx showed a notable outgrowth of a CD14+ monocyte/macrophage population. To analyze the direct effect of these secreted factors, conditioned media was collected from cultures of PBMC, LCL or CoCx and added to purified autologous monocytes. MTS assays at 7 days showed pronounced proliferation of monocytes in CoCx conditioned media relative to monocytes in PBMC or LCL conditioned media. Microscopy indicated that monocytes cultured with CoCx conditioned media formed non-adherent, proliferating colonies, whereas monocytes incubated in unconditioned, PBMC conditioned, or LCL conditioned media remained adherent. Depletion of CD8+ lymphocytes (but not CD4+, CD19+, CD56+ or CD14+ subsets) from CoCx led to a decrease in proliferation of monocytes, indicating a role of CTL in this phenomenon. This monocyte-derived cell population retained monocyte markers CD14 and CD11b and showed pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR and CD163, respectively), consistent with a TAM phenotype. Furthermore, the T cell inhibitory molecule PD-L1 was prominently expressed on these cells. This phenotype was confirmed to be the same as for the CD14+ cells that expanded from PBMC cultured with LCL. The functional activity of these macrophages on T cells was assessed by incubating autologous T cells alone or with macrophages derived from CoCx conditioned media for 24 hrs. Quantitative flow cytometric analysis showed a moderate decrease in CD4+ T helper cells and CD8+ CTL in the CoCx, compared to T cells cultured alone. However, if T cells were first activated (anti-CD3/CD28), the total numbers of viable CD3+ cells in the CoCx dropped 10-fold compared with activated T cells cultured alone. We previously reported that the eIF4A-specific translation initiation inhibitor silvestrol elicits marked anti-tumor activity against EBV-driven lymphoma. This occurs both by direct cytotoxicity to EBV-transformed B cells and by facilitating the activation, proliferation and cytotoxic activity of anti-tumor immune subsets including EBV-specific CTL. Here, the addition of 10nM silvestrol into CoCx blocked the release of monocyte/macrophage-associated cytokines, and silvestrol-treated CoCx conditioned medium did not drive proliferation or colony expansion of TAM-like macrophages. This finding indicates that selective targeting of the eIF4F translation initiation complex, which includes eIF4A, may block TAM formation in the setting of EBV-driven lymphoma.
In summary, we have identified an immune evasion mechanism by which exposure of normal PBMC to EBV+ lymphoma cells results in expansion of a TAM-like macrophage population with potent cytotoxic activity against T cells. Low-dose silvestrol treatment abrogates the outgrowth of this TAM-like population and allows expansion of EBV-specific CTL. This finding provides an entirely new approach to modulate the immune response in this challenging group of EBV-related diseases.

Disclosures
No relevant conflicts of interest to declare.