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Mechanism of EBV inducing anti-tumour immunity and its therapeutic use

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Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein–Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein–Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers.

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Fig. 1: T cells from CL mice recognize CD40-activated B cells lacking LMP1.
Fig. 2: LMP1-induced TAAs are targets of T cells.
Fig. 3: Ectopically expressing LMP1 in mouse tumour B cells enables generation of CD4 CTLs that target unmodified tumour B cells in vitro and in vivo.
Fig. 4: Ectopically expressing LMP1 in tumour B cells from patients with cancer enables generation of autologous CD4 CTLs targeting tumour antigens.

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Data availability

The microarray data have been deposited to the NCBI Gene Expression Omnibus (accession number GSE159854). All other data from the manuscript are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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  • 06 January 2021

    This Article was amended to correct the Peer review information, which was originally incorrect.


  1. Coulie, P. G., Van den Eynde, B. J., van der Bruggen, P. & Boon, T. Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy. Nat. Rev. Cancer 14, 135–146 (2014).

    Article  CAS  Google Scholar 

  2. Blankenstein, T., Coulie, P. G., Gilboa, E. & Jaffee, E. M. The determinants of tumour immunogenicity. Nat. Rev. Cancer 12, 307–313 (2012).

    Article  CAS  Google Scholar 

  3. Long, H. M. et al. CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein–Barr virus-transformed B cells. Blood 114, 807–815 (2009).

    Article  CAS  Google Scholar 

  4. Iheagwara, U. K. et al. Influenza virus infection elicits protective antibodies and T cells specific for host cell antigens also expressed as tumor-associated antigens: a new view of cancer immunosurveillance. Cancer Immunol. Res. 2, 263–273 (2014).

    Article  CAS  Google Scholar 

  5. Taylor, G. S., Long, H. M., Brooks, J. M., Rickinson, A. B. & Hislop, A. D. The immunology of Epstein–Barr virus-induced disease. Annu. Rev. Immunol. 33, 787–821 (2015).

    Article  CAS  Google Scholar 

  6. Zhang, B. et al. Immune surveillance and therapy of lymphomas driven by Epstein–Barr virus protein LMP1 in a mouse model. Cell 148, 739–751 (2012).

    Article  CAS  Google Scholar 

  7. Yasuda, T. et al. Studying Epstein–Barr virus pathologies and immune surveillance by reconstructing EBV infection in mice. Cold Spring Harb. Symp. Quant. Biol. 78, 259–263 (2013).

    Article  Google Scholar 

  8. Pearce, E. L. et al. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302, 1041–1043 (2003).

    Article  ADS  CAS  Google Scholar 

  9. Choi, I. K. et al. Signaling by the Epstein–Barr virus LMP1 protein induces potent cytotoxic CD4+ and CD8+ T cell responses. Proc. Natl Acad. Sci. USA 115, E686–E695 (2018).

    Article  CAS  Google Scholar 

  10. Smith, C. et al. Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation. Blood 113, 6148–6152 (2009).

    Article  CAS  Google Scholar 

  11. Acha-Orbea, H. & MacDonald, H. R. Superantigens of mouse mammary tumor virus. Annu. Rev. Immunol. 13, 459–486 (1995).

    Article  CAS  Google Scholar 

  12. Thorley-Lawson, D. A. Epstein–Barr virus: exploiting the immune system. Nat. Rev. Immunol. 1, 75–82 (2001).

    Article  CAS  Google Scholar 

  13. Schultze, J. L. et al. Follicular lymphomas can be induced to present alloantigen efficiently: a conceptual model to improve their tumor immunogenicity. Proc. Natl Acad. Sci. USA 92, 8200–8204 (1995).

    Article  ADS  CAS  Google Scholar 

  14. Kaye, K. M., Izumi, K. M. & Kieff, E. Epstein–Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. Proc. Natl Acad. Sci. USA 90, 9150–9154 (1993).

    Article  ADS  CAS  Google Scholar 

  15. Muranski, P. et al. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 112, 362–373 (2008).

    Article  CAS  Google Scholar 

  16. Brooks, A. et al. Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme. Proc. Natl Acad. Sci. USA 88, 3290–3294 (1991).

    Article  ADS  CAS  Google Scholar 

  17. Dengjel, J. et al. Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc. Natl Acad. Sci. USA 102, 7922–7927 (2005).

    Article  ADS  CAS  Google Scholar 

  18. Paludan, C. et al. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307, 593–596 (2005).

    Article  ADS  CAS  Google Scholar 

  19. Challa-Malladi, M. et al. Combined genetic inactivation of β2-microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma. Cancer Cell 20, 728–740 (2011).

    Article  CAS  Google Scholar 

  20. Roemer, M. G. M. et al. Major histocompatibility complex class II and programmed death ligand 1 expression predict outcome after programmed death 1 blockade in classic Hodgkin lymphoma. J. Clin. Oncol. 36, 942–950 (2018).

    Article  CAS  Google Scholar 

  21. Elpek, K. G., Lacelle, C., Singh, N. P., Yolcu, E. S. & Shirwan, H. CD4+CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model. J. Immunol. 178, 6840–6848 (2007).

    Article  CAS  Google Scholar 

  22. Kowalewski, D. J. et al. HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL). Proc. Natl Acad. Sci. USA 112, E166–E175 (2015).

    Article  CAS  Google Scholar 

  23. Bollard, C. M. et al. Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein–Barr virus latent membrane proteins. J. Clin. Oncol. 32, 798–808 (2014).

    Article  CAS  Google Scholar 

  24. Jayasooriya, S. et al. Early virological and immunological events in asymptomatic Epstein–Barr virus infection in African children. PLoS Pathog. 11, e1004746 (2015).

    Article  Google Scholar 

  25. Dunmire, S. K., Grimm, J. M., Schmeling, D. O., Balfour, H. H., Jr & Hogquist, K. A. The incubation period of primary Epstein–Barr virus infection: viral dynamics and immunologic events. PLoS Pathog. 11, e1005286 (2015).

    Article  Google Scholar 

  26. Haabeth, O. A. et al. How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules? Front. Immunol. 5, 174 (2014).

    Article  Google Scholar 

  27. Zhang, B. et al. An oncogenic role for alternative NF-κB signaling in DLBCL revealed upon deregulated BCL6 expression. Cell Rep. 11, 715–726 (2015).

    Article  CAS  Google Scholar 

  28. Koralov, S. B. et al. Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132, 860–874 (2008).

    Article  CAS  Google Scholar 

  29. He, L. et al. A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells. J. Immunol. Methods 304, 43–59 (2005).

    Article  ADS  CAS  Google Scholar 

  30. Cui, Y. et al. Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL. Blood 125, 2958–2967 (2015).

    Article  CAS  Google Scholar 

  31. Yamaguchi, S. et al. Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines. Cancer 110, 1469–1477 (2007).

    Article  CAS  Google Scholar 

  32. Porichis, F. et al. Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J. Virol. 88, 2508–2518 (2014).

    Article  Google Scholar 

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We thank the DFCI HN Flow Cytometry Core for assistance with the flow cytometry studies and cell sorting, the Pasquarello Tissue Bank in Hematologic Malignancies and the CLL Center Tissue Bank for patient samples, and the Dana-Farber/Harvard Cancer Center (DF/HCC) Specialized Histopathology Core for providing histology service. DF/HCC is supported in part by an NCI Cancer Center Support Grant (NIH 5P30CA06516). We thank D. P. Leahy and B. Beaulieu for administrative assistance and P. McCaffrey for valuable input. We are grateful to the NIH Tetramer Facility at Emory University for providing the tetramers. This work was supported in part by DFCI Faculty Startup Funds, Medical Oncology Translational Grant, and Claudia Adams Barr Award to B.Z.; the American Cancer Society Research Scholar Grant RSG-19-035-01-LIB to B.Z.; the Leukemia and Lymphoma Society grant TRP-6595-20 to B.Z.; the Wade F.B. Thompson/Cancer Research Institute CLIP Grant to B.Z.; the Claudia Adams Barr Program for Innovative Cancer Research to I.-K.C.; NIH grant P01CA206978-01 to J.R.B.

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Authors and Affiliations



I.-K.C. and B.Z. planned and coordinated the overall research; I.-K.C. and Z.W. designed and performed experiments and data analysis with Q.K., M.H. and D.W.P.; S.M.F., J.R.B. and J.R. provided the samples from patients with CLL; Z.H. provided technical advice; J.S. and I.G. characterized the samples from patients with CLL; H.-J.K., H.C., K.W.W., J.R.B. and J.R. provided scientific and experimental advice, and edited the manuscript; I.-K.C. and B.Z. interpreted the results and wrote the manuscript; B.Z. conceived and supervised the project.

Corresponding author

Correspondence to Baochun Zhang.

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Competing interests

I.-K.C., Z.W. and B.Z. are inventors on patent applications that cover parts of this work; Z.H. is a current employee of ElevateBio.

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Peer review information Nature thanks Paul J. Farrell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 LMP1+ B cells drive CD4+ T cell differentiation into EOMES-programed granzyme/perforin-dependent cytotoxic effectors.

a, FACS analysis of EOMES, granzyme B and perforin expression in CD4 cells, from mice with CD4-specific Eomes knockout (Cd4-cre;EomesF/F) or with normal levels of EOMES (Cd4-cre), primed in vitro by LMP1+ B cells. Granzyme B and perforin levels in EOMES+ CD4 cells from Cd4-cre mice (P3) were compared with those in EOMES CD4 cells from the same mice (P2) or Cd4-cre;EomesF/F mice (P1), and are shown on the right. For these analyses, FOXP3+ Treg cells were excluded. b, c, Killing activity of EOMES-null CD4 cells (from Cd4-cre;EomesF/F mice) or perforin-null CD4 cells (from Prf1–/– mice) in comparison with wide-type (WT) CD4 cells, primed as in a, against LMP1+ lymphoma cell targets. E:T ratio, effector-to-target cell ratio. All mice are on the B6 background. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 2 CD4 and CD8 cells mount a polyclonal response to LMP1+ B cells.

a, b, Analysis of TCR Vβ repertoire on CD8 cells (a) and CD4 cells (excluding FOXP3+ Treg cells) (b) from spleen (Spl) or bone marrow (BM) of control or CL mice, using a panel of monoclonal antibodies for the indicated Vβ chains. These antibodies collectively detected 85–95% of TCRs in all the samples. The majority of CD4 and CD8 cells in the spleen and BM of 8-day-old CL mice and BM of adult CL mice were CD44+CD62L effector/memory cells6,9. Control d8, 8-day-old Cd19-cre/+ mice; CL adult, 6- to 12-week-old CL mice. In a, b, data are shown as mean ± s.e.m. c, In vitro killing of LMP1+ lymphoma cells by the indicated CD4 subsets from 6- to 8-day-old CL mice. All mice are on the CB6F1 background. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 3 Transient reduction of germinal centre (GC) B cells around the time of peak T cell response.

a, Frequency of spontaneous GC B cells (CD19+FAShighCD38low) analysed by FACS in the mesenteric lymph nodes of the inducible LMP1-expressing ERT2-CL mice and littermate controls (ERT2-C) after tamoxifen treatment. Data are mean ± s.e.m. b, c, Numbers (b) and representative FACS plots (c) of GC B cells from the indicated mice 7 days post-tamoxifen treatment as in a. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 4 LMP1 signalling and CD40 activation in B cells lead to differential expression of costimulatory ligands.

a, Relative transcript levels of the indicated costimulatory molecules in LMP1+ B cells compared with CD40-activated B cells and control B cells. Splenic B cells from LMP1flSTOP/YFPflSTOP and YFPflSTOP/+ mice (both on the CB6F1 background) were treated with TAT-Cre to generate LMP1+ B cells and YFP control B cells; naive B cells (ex vivo) and anti-CD40-activated B cells were prepared from B6 mice. All treated B cells were analysed 2 days post-treatment. b, Numbers (mean) of CD4 cells recovered after 7-day co-culture with B cells expressing LMP1 or the signalling dead mutant LMP1TM1m, or pre-activated with anti-CD40 antibody. Purified CD4 cells (1.5 × 106) were cultured with irradiated B cells as indicated at 1:1 ratio in duplicate wells of 12-well plates. No exogenous cytokine was added. c, EOMES expression in CD4 cells co-cultured with the indicated B cells as in b. Ex vivo CD4 cells served as control. B and T cells in bc are from spleens of 2- to 3-month-old naive B6 mice. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 5 Specificity analysis of CD8 cells in ERT2-CL mice using MHC-I tetramers.

a, Representative FACS analysis of splenic CD8 cells from control mice (ERT2-C) or mice expressing inducible LMP1 (ERT2-CL) stained with survivin20–28 tetramer (Surv-Tetrm) versus an irrelevant control tetramer (H-2Db loaded with the LCMV GP33–41 epitope peptide (LCMV GP-Tetrm)). b, Validation of the LCMV GP-tetramer by staining splenic CD8 cells from LCMV (clone 13)-infected mice at day 8 post-infection, versus uninfected control mice. c, Representative FACS analysis of splenic CD8 cells from the indicated mice stained with anti-CD44 and Surv-Tetrm (top) or EPHA2682–689 tetramer (EPHA2-Tetrm) (bottom). d, FACS analysis of splenic CD8 cells from the indicated mice stained with the indicated tetramers labelled with PE and APC. Representative FACS plots are shown on the left, and summary data on the right. Each circle represents one mouse; bars show mean ± s.e.m. All ERT2-CL and littermate control mice were analysed on day 5 after tamoxifen treatment. Mice used in a, c are on the CB6F1 background; in b, d on the B6 background. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 6 Schematic view of how LMP1 signalling in B cells induces cytotoxic CD4 and CD8 T cell responses to TAAs.

LMP1 signalling in B cells induces massive cellular gene expression. This leads to (1) upregulation of cellular machinery involved in antigen processing and presentation, (2) upregulation of costimulatory ligands (CD70, OX40L and others), and (3) overexpression of many cellular antigens known as TAAs. Presentation of the LMP1-induced cellular antigens/TAAs and simultaneous costimulation through CD70 and OX40L drive cytotoxic CD4 and CD8 T cell responses.

Extended Data Fig. 7 No discernible pathological changes in non-lymphoid tissues of LMP1 mice after contraction of the T cell response.

Representative haematoxylin and eosin staining of liver, kidney, pancreas and intestine sections from control (C) and CL mice at 6–7 weeks after birth, at which time the T cell response against LMP1+ B cells has contracted9. All mice are on the CB6F1 background. Scale bar, 1,000 μm. Statistics and reproducibility are presented in the Supplementary Information.

Extended Data Fig. 8 Schematic of the proposed LMP1-based CD4 CTL therapeutic strategy.

Ectopically expressing LMP1 in tumour B cells from patients with cancer will (1) enhance presentation of endogenous antigens, such as TAAs and neoantigens, on MHC-II, and (2) provide costimulation through CD70 and OX40L, thereby eliciting CD4 CTLs against these tumour antigens. CD4 CTLs generated in this fashion will mediate cytotoxicity to unmodified tumour B cells that express the same antigens.

Extended Data Fig. 9 Characterization of CD4 CTLs primed by LMP1-transduced A20 cells.

a, Cytotoxicity of CD4 CTLs primed by LMP1-A20 cells against the B cell lymphoma line BCL1 at an E:T ratio of 50:1, in the presence of MHC-II blocking antibody or isotype control antibody. b, Representative FACS analysis of intra-tumoral CD45.1+ adoptive CD4 cells (excluding FOXP3+) recovered from A20-bearing mice (CD45.2+) treated as in Fig. 3g–i. All mice are on the BALB/c background. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Extended Data Fig. 10 Reactivity analysis of autologous CD4 cells before and after stimulation with LMP1-transfected tumour B cells from patients with cancer.

a, Co-expression of CD69 and CD40L in effector/memory CD4 cells from two patients with CLL after culture for 18 h with or without LMP1-transfected CLL (LMP1-CLL) cells. Pt., patient. b, Co-expression of CD69 and CD40L in effector/memory CD4 cells from a patient with CLL assessed after culture for 18 h alone, with untransfected CLL cells, LMP1TM1m- or LMP1-transfected CLL cells. c, d, Analysis of IFNγ ELISPOT responses of CD4 cells pre-stimulated with LMP1-CLL cells (c) or unstimulated (ex vivo) CD4 cells (d), against individual (CTSH185–198) or pooled (TFRC198–210 plus TFRC680–693 or VAMP244–60 plus VAMP250–66) epitope peptides from the selected CLL TAAs pulsed on autologous dendritic cells. PMA- and ionomycin-stimulated CD4 cells served as positive control; an irrelevant HIV p24164–181 peptide as negative control. Numbers of spot-forming cells (SFC) in individual wells and their mean value per initial seeding number of CD4 cells are presented on the y axis; representative ELISPOT images below the x axis. e, Summary of IFNγ ELISPOT responses of the CD4 cells pre-stimulated with LMP1-CLL cells, against the indicated CLL TAA epitopes in the five patients with CLL tested (patient 11 in c; patients 7, 8, 9 and 10 in Fig. 4f). +, positive ELISPOT response (Methods); –, no response. Statistics and reproducibility are presented in the Supplementary Information.

Source data

Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Statistics and reproducibility, and Supplementary Figures 1-3. Supplementary Figure 1 contains source images for western blot data. Supplementary Figure 2 contains gating strategies for cell analysis by flow cytometry. Supplementary Figure 3 contains gating strategies for cell sorting.

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Choi, IK., Wang, Z., Ke, Q. et al. Mechanism of EBV inducing anti-tumour immunity and its therapeutic use. Nature 590, 157–162 (2021).

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