Characterization of Cytotoxic Macrophages in a Pre-Clinical Model of Epstein-Barr Virus (EBV)-Driven Lymphoproliferative Disease

Epstein-Barr Virus (EBV) is an oncogenic herpes virus associated with the development of malignancies associated with poor outcomes. Current treatments lead to further immune suppression, increasing the risk of EBV reactivation and other opportunistic infections. Strategies aimed at reversing the immune-suppressive microenvironment are likely to result in more durable responses while avoiding deleterious effects of chemotherapy. It is well-established that macrophages play an important role in inhibiting the anti-tumor response as evidenced by poor prognosis associated with increasing tumor-associated macrophage (TAM) density or lower ratio of cytotoxic T lymphocytes. Here we describe a pre-clinical model showing the expansion of cytotoxic TAMs and potential mechanisms to prevent this immunosuppressive environment.

To assess soluble factors produced by the interaction of tumor on immune effector cells, we used a co-culture (CoCx) system of EBV-transformed human B cell lymphoblastoid cell lines (LCL) cultured with autologous peripheral blood mononuclear cells (PBMC). Serum-free medium collected after 48 hrs showed increased myeloid-associated factors which were absent from either the LCL or PBMC cultures, suggesting the interaction of tumor cells with immune effectors was leading to the production of chemokines known to attract myeloid cells. When purified autologous CD14+ monocytes were incubated in the presence of CoCx conditioned medium (CM) they generated proliferative foci that were absent in the LCL or PBMC CM alone. To better understand which cellular subsets were necessary for the release of these soluble factors, immune subsets were removed from PBMC prior to incubation with LCL or purified and incubated with LCL. We found that T cells, (CD8+ and CD4+), when incubated in the presence of LCL were necessary and sufficient to stimulate the proliferation of myeloid cells. CD56+, CD14+, or CD19+ subsets within the CM did not appear to contribute to the proliferation of purified myeloid cells.

We next used flow cytometry to characterize the monocyte-derived population that expanded when introduced to CoCx CM. We again isolated autologous CD14+ monocytes from PMBC and incubated them for 6 days with CoCx CM followed by immunophenotyping. The monocyte-derived population retained CD14 and CD11b in addition to showing pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR, and CD163 respectively), and PD-L1, indicating activation consistent with a TAM phenotype. To characterize the functional consequence of this cell population, CoCx CM-derived macrophages were incubated with autologous T cells that were either activated non-specifically with anti-CD3/C28 or left inactivated. Surprisingly, we noted a marked decrease in viable activated T cells (both CD4+ and CD8+) when exposed to conditioned macrophages. However, the inactivated T cell populations were only mildly effected. Together, with previously described experiments, this suggests a mechanism by which exposure of T cells to EBV+ lymphoblastic tumor lines results in cytokine release, polarizing monocytes to TAMs that, in turn, exhibit cytotoxic activity to tumor-specific T cells. The precise mechanism of cytotoxicity is under investigation.

We previously reported the eIF4A translational inhibitor silvestrol potentiated a potent immune-mediated anti-tumor response against EBV-driven lymphoma mainly by promoting expansion and activity of EBV-specific cytotoxic T cells. We next questioned if macrophages were important to this mechanism. We found that with silvestrol, when added to CoCx, the resultant CM did not drive the proliferation of TAMs, and Immunophenotype of CoCx showed silvestrol treatment to result in the ablation of CD14+ cells from culture. Interrogation by transcriptome analysis indicated a potential role of aryl hydrocarbon receptor (AhR). Protein analysis by western blotting indicated an increase of AhR in isolated monocytes when exposed to CoCx CM, which was abrogated by addition of silvestrol. Together these results suggest AhR may play a role in the polarization of macrophages within the tumor microenvironment leading to elimination of tumor-specific T cells. While full mechanistic characterization is ongoing, these preliminary results provide a potentially novel approach to modulate tumor specific immunity in this challenging group of EBV-related malignancies.