The Critical Interaction Between Epstein-Barr Virus (EBV) Positive B-Cells and Tumor Associated Macrophages (TAMs)
Authors: Sato Ai , Yamakawa Natsuko , Okuyama Kazuki , Kotani Ai , Nakamura Naoya , Ando Kiyoshi 
Published: October 2019

Abstract
Introduction: EBV positive diffuse large B-cell lymphoma of the elderly (EBV positive DLBCL, elderly) has been newly categorized in 2008 WHO classification. The incidence is higher in Asian countries than Western countries. The prognosis of DLBCL has been improved by the introduction of rituximab, while EBV positive DLBCL remains unknown. Recently we reported that EBV positive DLBCL of the elderly did have quit inferior prognosis than EBV negative DLBCL. The mechanism lying under the inferiority has yet been elucidated. We hypothesize that tumor microenvironment plays a role in the mechanism, as EBV related lymphoma are usually accompanied with massive infiltration of non-tumor cells. We have previously found that tumor secreted small RNAs were selectively taken by macrophages and dramatically change the character into tumor supporting phenotype in the EBV positive lymphoma. Accordingly, we focus the interaction between tumor cells and macrophages in EBV positive lymphoma microenvironment.
Methods: We investigated the number of CD163-positive macrophage (CD163+M2) in the DLBCL specimen with and without EBV positivity. The effect of EBV positivity in the tumor cells on the macrophages infiltrated in the tumor were studied by use of the coculture system using human monocytic cell line (THP-1) and human Burkitt lymphoma cells lines (Akata) which has two subclones such as EBV positive and negative. PMA-induced macrophages from THP1 cells were cocultured with EBV positive or negative Akata, then the expression of the several cytokines such as TNF-a, IL-10, CXCL10, and VEGF were measured by real-time PCR. Finally, we tried to clarify the impact of the macrophages on tumor formation in vivo by using xeno-transplantation model. Hematopoietic humanized NOG mice were infected with EBV to induce EBV related lymphoproliferative disease (LPD). After the appearance of symptoms of the LPD such as body weight loss, mice were treated with Clodronate to deplete macrophages.
Result: The multivariate analysis for DLBCL patients demonstrated the statistically significant association between both high scores of CD163+M2 macrophages (CD163-positive cell > 20%) and EBV positivity, and poor prognosis (p = 0.0084, 0.0020, respectively.), which implies that EBV affected the quantity of CD163+M2 macrophages in the tumor microenvironment. Co-culture of THP1 with EBV positive lymphoma cells significantly upregulated CXCL10 and VEGF, when compared with EBV negative cells. (p = 0.0073, 0.0161, respectively.). (Figure 1) Most surprisingly, EBER positive B cells almost completely disappeared by macrophage depletion by Clodronate treatment (Figure 2). These results suggest that the macrophages in the EBV positive tumor microenvironment are crucial for survival of EBV+ tumor cells.
Conclusion: EBV status of the lymphoma cells affected on TAMs in the way such as up-regulation of CXCR10 and VGEF, angiogenetic factors which are presumed to support tumor survival. The in vivo depletion of macrophages by Clodronate also demonstrated that they were indispensable for EBV positive tumor cell survival.
Taken together, the interaction of EBV positive tumor cells and tumor associated macrophages is of crucial importance in the biology and formation of EBV positive lymphoma.
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.