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

Abstract

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.

Conflict of interest statement

The authors declare competing financial interests: I.K.C., Z.W. and B.Z. are inventors on patent applications which cover parts of this work; Z.H. is a current employee of ElevateBio.

Figures

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+ Tregs 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 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 in Supplementary Information.

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+ Tregs) (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 cells,. Control d8, 8-day-old CD19-cre/+ mice; CL adult, 6–12-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 6–8-day-old CL mice. All mice are on the CB6F1 background.

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

a, Frequency of spontaneous GC B cells (CD19+FashighCD38low) analyzed by FACS in the mesenteric lymph nodes of the inducible LMP1-expressing ERT2-CL mice and littermate controls (ERT2-C) after tamoxifen treatment. Error bars denote 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.

Extended Data Fig. 4. LMP1 signaling 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 αCD40-activated B cells were prepared from B6 mice. All treated B cells were analyzed 2 days post-treatment. b, Numbers (mean) of CD4 cells recovered after 7-day co-culture with B cells expressing LMP1 or the signaling 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 b–c are from spleens of 2–3-month-old naive B6 mice.

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 (upper) or EphA2682–689 tetramer (EphA2-Tetrm) (lower). d, FACS analysis of splenic CD8 cells from the indicated mice stained with the indicated tetramers labeled 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 analyzed on day 5 after tamoxifen treatment. Mice used in a, c are on the CB6F1 background; in b, d on the B6 background.

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

LMP1 signaling 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 hematoxylin and eosin (H&E) 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 contracted. All mice are on the CB6F1 background. Scale bar, 1000 μm.

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

Ectopically expressing LMP1 in patient tumour B cells 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-tumoural 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.

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

a, Co-expression of CD69 and CD40L in effector/memory CD4 cells from two CLL patients after culturing 18 h with or without LMP1-transfected CLL cells (LMP1-CLL). Pt., patient. b, Co-expression of CD69 and CD40L in effector/memory CD4 cells from a CLL patient assessed after culturing 18 h alone, with untransfected CLL cells, LMP1TM1m- or LMP1-transfected CLL cells. c, d, Analysis of IFN-γ ELISPOT responses of CD4 cells pre-stimulated with LMP1-CLL (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 IFN-γ ELISPOT responses of the CD4 cells pre-stimulated with LMP1-CLL, against the indicated CLL TAA epitopes in the five CLL patients tested (Pt. 11 in c; Pts. 7, 8, 9 and 10 in Fig. 4f). +, positive ELISPOT response (see Methods); –, no response.

Fig. 1. T cells from CL mice recognize CD40-activated B cells lacking LMP1.

a, Left panel, in vitro cytotoxicity of CD4+ and CD8+ T cells from 6–8-day-old CL mice against B cells transduced to express LMP1 or its signaling-dead mutant (LMP1TM1m). B cells transduced with an empty vector or untransduced LPS-activated B cells (see Methods) served as controls. E:T ratio, effector-to-target cell ratio. Right panel, immunoblot of LMP1 and the mutant in the transduced B cells, with GAPDH as loading control. b, FACS analysis of MHC-I and -II levels on LMP1+ B cells and anti-CD40–activated WT B cells, compared to naive WT B cells. LMP1+ B cells were prepared by treating B cells from LMP1flSTOP mice with TAT-Cre in vitro. c, Cytotoxicity of CD4 and CD8 cells from 6–8-day-old CL mice against naive, αCD40-activated or LMP1+ B cells prepared as in b. d, Cytotoxicity of CD4 cells from 6–8-day-old CL mice against CD40-activated B cells, in the presence of Fas-Fc (to block FasL) and/or MHC-II blocking antibody, or isotype control antibodies. e, Proliferation of CD4 effector cells from Foxp3DTR/GFP;CL mice, co-cultured with CD40-activated B cells from WT or MHC-II–null CIITA–/– mice. All CL mice are on the CB6F1 background, and all B cells on B6 background except those in b and c (CB6F1). Statistics and reproducibility are in Supplementary Information.

Fig. 2. LMP1-induced cellular antigens known as TAAs are targets of T cells.

a, Relative transcript levels of genes encoding known TAAs in LMP1+ and CD40-activated B cells compared to control B cells (see Methods). b, Immunoblots of Survivin and EphA2 in the indicated B cells. c, d, MHC-I tetramer (Tetrm) staining of CD8 cells specific for H-2Db-restricted Survivin20–28 (Surv) epitope or H-2Kb-restricted EphA2682–689 epitope from spleens of 6-day-old CL mice (c) or ERT2-CL mice 5 days post-tamoxifen treatment (d), compared to the respective littermate controls (C, CD19-cre/+; ERT2-C, CD19-creERT2/+). Left, representative FACS plots; right, summary data. Each circle represents one mouse. e, Dynamics of Surv-Tetrm+ and EphA2-Tetrm+ CD8 cells (upper) and LMP1+ B cells (lower, CD19+Fas+; Fas is used as a surrogate marker for LMP1 expression) analyzed by FACS in spleens of ERT2-CL mice and littermate controls over time after tamoxifen treatment. f, Proliferation of CD4 cells primed in vitro by LMP1+ B cells, in response to 775 that expresses or lacks MHC-II (upper) or 773 in the presence of MHC-II blocking antibody or isotype control (lower). g, Left panel, analysis of MHC-II (I-Ab) expression in 1019 and its CIITA–/– subline. Right panel, immunoblotting of Trp1 or Trp1-Flag in 1019 untransfected or transfected with Trp1-Flag mRNA. B16-F10 melanoma cells served as positive control for Trp1 expression. h, CD69 expression on naive CD4 cells from Trp1 mice after co-culturing 18 h with 1019 (MHC-II+) or the CIITA–/– (MHC-II–) subline transfected with Trp1 mRNA, or untransfected 1019 cells. i, Upper panel, scheme of strategy for assessing Trp1 MHC-II presentation pathways (see Methods). Lower panel, CD69 expression in CD4 cells of the indicated cultures. j, Cytotoxicity of Trp1-specific CD4 cells primed for 8 days in vitro by Trp1-transfected 1019 cells, against Trp1-transfected or untransfected 1019 cells. Error bars denote mean ± s.e.m. All mice and cells are on the B6 background, except those in c–e (CB6F1).

Fig. 3. Ectopically expressing LMP1 in murine tumour B cells enables generation of CD4 CTLs that target unmodified tumour B cells in vitro and in vivo.

a, FACS analysis of MHC-II and the indicated costimulatory ligands in A20 cells transduced with retroviral vectors expressing LMP1 or LMP1TM1m, or an empty vector. Unstained vector-transduced cells served as negative control. b, Numbers of CD4 cells recovered after co-culturing naive CD4 cells (1 × 106) with LMP1- or LMP1TM1m-transduced A20 cells for 6 days. c, Eomes levels in CD4 cells primed by LMP1-A20 cells as in b, compared to naive CD4 cells. Foxp3+ Tregs were excluded. d, Cytotoxicity of primed CD4 cells and naive CD4 cells against the indicated target cells. e, Analysis of MHC-II expression in A20 and A20 CIITA–/– cells. f, Cytotoxicity of primed CD4 cells against A20 versus A20 CIITA–/– cells. g, Schematic diagram of the ACT protocol (see Methods). TBI, preconditioning total body irradiation. h, Upper panel, mean tumour volumes in the indicated groups of mice treated as in g. Lower panel, representative pictures taken on day 27 of tumours in mice treated with the indicated CD4 cells. i, Numbers (per gram of tumour) of intra-tumoural adoptive CD4 cells (excluding Foxp3+ Tregs) recovered from A20-bearing mice (CD45.2+) receiving the indicated CD4 cells (CD45.1+), determined by FACS 8 days after adoptive transfer. j, Granzyme B and Perforin levels in intra-tumoural CD45.1+ adoptive CD4 cells recovered from CD4 CTL-treated mice as in i, compared to naive CD4 cells from normal mice. k, PD-1 expression on intra-tumoural adoptive CD4 cells as in j. l, Schematic diagram of the ACT protocol combined with PD-1 blockade (see Methods). m, Tumour volumes (left; each line represents an individual tumour) and survival (right) of A20-bearing mice treated as in l. Error bars denote mean ± s.e.m. All mice are on the BALB/c background.

Fig. 4. Ectopically expressing LMP1 in patient tumour B cells enables generation of autologous CD4 CTLs targeting tumour antigens.

a, FACS analysis of transfection efficiency in patient CLL cells electroporated with GFP or LMP1 mRNA, assessed 2 days post-electroporation. Fas is used as a surrogate marker for LMP1 expression. b, FACS analysis of HLA-II and the indicated costimulatory ligands in LMP1-transfected versus untransfected CLL cells. Unstained CLL cells served as negative control in HLA-II analysis. c, Mean fluorescence intensities (MFI) of CD70 and OX40L in LMP1-transfected versus untransfected (Untr) CLL cells, assayed as in b. Each circle represents one patient. d, Cytotoxicity of autologous CD4 cells stimulated by LMP1-transfected CLL cells, against parental CLL cells at an E:T ratio of 25:1 in the presence of HLA-II blocking or isotype control antibody. Pt., patient. Unstimulated CD4 cells had no killing activity (data not shown). e, Co-expression of CD69 and CD40L on effector/memory CD4 cells from CLL patients after culturing 18 h with or without LMP1-transfected CLL cells (LMP1-CLL). Representative FACS plots are shown on the left and summary data on the right. f, IFN-γ ELISPOT responses of CD4 cells pre-stimulated with LMP1-CLL, against autologous dendritic cells pulsed with individual (CTSH185–198) or pooled (TFRC198–210 plus TFRC680–693 or VAMP244–60 plus VAMP250–66) epitope peptides from the selected CLL TAAs. In Pt. 10, the assay was performed with or without HLA-II blocking antibody. An irrelevant HIV p24164–181 peptide served 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. +, positive ELISPOT response (see Methods).

Methods references