EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors
Robert E. White, … , Christian Münz, Martin J. Allday
Robert E. White, … , Christian Münz, Martin J. Allday
Published March 12, 2012
Citation Information: J Clin Invest. 2012;122(4):1487-1502. https://doi.org/10.1172/JCI58092.
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EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors

Abstract

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc–/– mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell–chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell–mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

Authors

Robert E. White, Patrick C. Rämer, Kikkeri N. Naresh, Sonja Meixlsperger, Laurie Pinaud, Cliona Rooney, Barbara Savoldo, Rita Coutinho, Csaba Bödör, John Gribben, Hazem A. Ibrahim, Mark Bower, Jamie P. Nourse, Maher K. Gandhi, Jaap Middeldorp, Fathima Z. Cader, Paul Murray, Christian Münz, Martin J. Allday

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Figure 7

EBNA3BKO-transformed tumor cells produce and secrete reduced amounts of CXCL10 and attract fewer EBV-specific T cells.

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EBNA3BKO-transformed tumor cells produce and secrete reduced amounts of ...
(A) CXCL10 mRNA levels, determined by qPCR, normalized to GAPDH expression, and expressed relative to CXCL10 expression of wtBAC-transformed tumor cells. (B) CXCL10 protein levels, determined in culture supernatants of EBV-transformed tumor cells by ELISA after 24 hours of culture with or without added IFN-γ. (C) Migration of the EBV-specific CD8+ T cell clone MS.B11 toward tumor cell–conditioned supernatants, assessed by Transwell migration assay. For EBNA3BKO-transformed tumor lines and patient PTLD LCLs, CXCL10 was supplemented as indicated to reach concentrations present in wtBAC- and EBNA3Brev-conditioned supernatants. (A–C) Shown is 1 representative of 2 experiments with 2 wtBAC, 3 EBNA3Brev, 7 EBNA3BKO, and 2 patient PTLD LCLs (TRL1-post and TRL595). (D and E) Killing of CXCL10-reexpressing EBNA3BKO LCLs by an autologous T cells line was tested in vivo. LCLs were transduced with a CXCL10-expressing lentivirus or control virus. In vivo killing assays were performed after labeling of LCLs with PKH26 and high or low concentration of CFSE. (D) Example of FACS gating for the experiment, and representative histograms for tumor cell composition before and after injection in the presence or absence of autologous T cells. Percentages denote CXCL10+ and CXCL10– cells as a fraction of total tumor cells. (E) Summary of 3 experiments performed as described in D. (A–E) Data are mean ± SD.