Signaling by the Epstein–Barr virus LMP1 + + induces potent cytotoxic CD4 and CD8 responses

Il-Kyu Choia,b, Zhe Wanga,b, Qiang Kea,c, Min Honga,d, Yu Qiana,b, Xiujuan Zhaoa,e, Yuting Liuf, Hye-Jung Kimg, Jerome Ritza,b, Harvey Cantorg, Klaus Rajewskyh,1, Kai W. Wucherpfennigg, and Baochun Zhanga,b,g,1

aDepartment of Medical Oncology, Dana–Farber Institute, Boston, MA 02215; bDepartment of Medicine, Harvard Medical School, Boston, MA 02115; cDepartment of Diagnostics, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang 311121, China; dDepartment of Medical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China; eDepartment of Cell Biology, Tianjin Medical University, Tianjin 300070, China; fProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; gDepartment of and Virology, Dana–Farber Cancer Institute, Boston, MA 02215; and hImmune Regulation and Cancer, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany

Contributed by Klaus Rajewsky, December 9, 2017 (sent for review August 3, 2017; reviewed by Alan B. Rickinson and Susan L. Swain) The B-lymphotropic Epstein–Barr virus (EBV), pandemic in humans, pressive molecules that foster local immune privilege (9, 10). is rapidly controlled on initial infection by T cell surveillance; there- Apparently, host immune cells, particularly T cells, keep EBV- after, the virus establishes a lifelong latent infection in the host. If infected cells under constant surveillance, and EBV-driven ma- surveillance fails, fatal lymphoproliferation and lymphomagenesis + lignancies only arise when surveillance fails. ensue. The initial T cell response consists of predominantly CD8 + T cell responses against EBV-infected or transformed B cells cytotoxic T cells and a smaller expansion of CD4 cells. A major are believed to be directed at various latent gene products, and approach to treating EBV-associated lymphomas is adoptive trans- therefore a major approach for treating EBV-associated malig- fer of autologous or allogeneic T cells that are stimulated/expanded nancies has been adoptive transfer of EBV-specific T cells, which on EBV-transformed B cells. Strikingly, the clinical response corre- are generated by stimulating/expanding autologous or allogeneic lates with the frequency of CD4 cells in the infused T cells. Although T cells on EBV-transformed B-lymphoblastoid cell lines (LCLs) in vitro studies suggested that EBV-specific CD4 cells develop cyto- (11). The therapeutic T cells mostly comprise predominantly toxicity, they have not been comprehensively characterized and + CD8 cytotoxic T cells (CTLs), with variable frequencies of the molecular mechanism underlying their formation remains un- + known. Our recent work, using a transgenic approach in mice, has CD4 T cells. Strikingly, however, the results from a PTLD trial revealed a central role for the EBV signaling molecule LMP1 in im- showed that clinical responses significantly correlated with the mune surveillance and transformation of EBV-infected B cells. The frequency of CD4 cells in the infused T cells (12). Although the mouse model offers a unique tool for uncovering basic features of precise function of the adoptive CD4 cells in these PTLD EBV immunity. Here, we show that LMP1 expression in B cells in- duces potent cytotoxic CD4 and CD8 T cell responses, by enhancing Significance antigen presentation and costimulation by CD70, OX40 , and 4-1BB ligand. Our data further suggest that cytotoxic CD4 cells hold Epstein–Barr virus (EBV) drives human B cell proliferation and superior therapeutic value for LMP1 (EBV)-driven lymphomas. These transformation, but also potent T cell surveillance. When sur- findings provide insights into EBV immunity, demonstrating that veillance fails, EBV-driven malignancies arise. T cells can be LMP1 signaling alone is sufficient to induce a prominent cytotoxic stimulated/expanded on EBV-transformed B cells for adoptive CD4 response, and suggest strategies for immunotherapy in EBV- therapy. Clinical data point to the therapeutic importance of related and other . CD4 T cells, perhaps through direct cytotoxicity; the mechanism underlying such an activity remains unknown. Previous studies – + Epstein Barr virus | LMP1 | CD4 cytotoxic T cells | costimulatory ligand show that signaling by the EBV oncoprotein LMP1 enhances antigen presentation. Here, we show that LMP1+ B cells pro- pstein–Barr virus (EBV) is a potent tumorigenic virus with a vide costimulation through CD70 and OX40L to drive cytotoxic Enarrow tropism primarily for B cells but also epithelial cells CD4 (and CD8) differentiation. In a mouse model of LMP1 of primate origin. The virus infects and persists in >90% of hu- (EBV)-driven lymphoma, cytotoxic CD4 cells have superior an- mans worldwide (1). Primary EBV infection usually occurs during titumor activity. These findings provide a mechanism for the early childhood (beginning at ∼6 mo after birth) and is asymp- EBV-mediated cytotoxic CD4 response and suggest strategies tomatic (2–4); however, if the infection is delayed until adoles- for immunotherapy in EBV-related and other cancers. cence, it may manifest as a self-limiting lymphoproliferative disorder known as infectious mononucleosis (5). EBV-infected B Author contributions: I.-K.C., Z.W., H.-J.K., K.R., and B.Z. designed research; I.-K.C., Z.W., Q.K., M.H., Y.Q., and X.Z. performed research; I.-K.C., Y.L., K.R., and B.Z. analyzed data; cells are rapidly eliminated by the host , in which I.-K.C., J.R., H.C., K.R., K.W.W., and B.Z. wrote the paper; and B.Z. supervised research. T cells play a major role (6). However, in a minute fraction of B Reviewers: A.B.R., University of Birmingham; and S.L.S., University of Massachusetts cells, the virus acquires a latent state and persists for life (7). Medical School. Under conditions of immunosuppression or immune deficiency, Conflict of interest statement: I.-K.C., Z.W., and B.Z. have filed patent applications on latent EBV can reactivate and spread, resulting in the rapid ex- aspects of this research. pansion of infected B cells and their malignant transformation, as Published under the PNAS license. seen in pathologies such as posttransplant lymphoproliferative Data deposition: The microarray data have been deposited in the National Center for disorder (PTLD) and AIDS-associated B cell lymphoma (5). EBV Biotechnology Information (NCBI) Omnibus (GEO) database, https:// www.ncbi.nlm.nih.gov/geo (accession no. GSE108479). is also associated with other B cell malignancies that appear to 1To whom correspondence may be addressed. Email: [email protected] or have evaded immune surveillance via different strategies: Burkitt [email protected]. lymphoma cells express a severely limited number of EBV latent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genes (8), while Hodgkin lymphoma cells produce immunosup- 1073/pnas.1713607115/-/DCSupplemental.

E686–E695 | PNAS | Published online January 8, 2018 www.pnas.org/cgi/doi/10.1073/pnas.1713607115 Downloaded by guest on October 3, 2021 patients was unclear, several in vitro studies reported that –granzyme pathway, the main killing mechanism typi- PNAS PLUS CD4 cells generated on LCLs exhibited direct cytotoxicity (13– cally utilized by CD8 CTLs (23, 24), corroborating their similar 16). The molecular mechanism underlying these “cytotoxic” cytotoxic function. This notion is in agreement with our previous + CD4 responses remains a mystery. findings that LMP1 B cells remain well controlled in mice de- The understanding of the basic immune responses to EBV had ficient for Fas (17) but not in mice deficient for perforin (19). been limited by the paucity of small animal models, as the virus Overall, our data demonstrate that LMP1 expression by B cells cannot infect nonprimate organisms. Our previous work, initially induces potent cytotoxic CD4 and CD8 T cell-mediated immunity. aimed at studying the EBV oncoprotein latent membrane protein 1 (LMP1) in B cell transformation in a transgenic mouse model, Cytotoxic CD4 Cells Confer Potent Antitumor Immunity Against LMP1- unexpectedly led to the discovery of a novel role of LMP1 in in- Driven Lymphomas. Our previous work showed that, although ducing immune surveillance relevant to EBV infection (17). ∼50% of CD4 and CD8 cells in the BM of adult CL mice (re- flSTOP Specifically, we coupled a conditional LMP1 allele with a ferred to as “chronic stage” in this model system) maintain an + CD19-cre allele to target the expression of LMP1 to the B lineage, activated phenotype (CD69 ), the CD4 cells exhibited little cy- flSTOP starting at the pro-B stage; analysis of CD19-cre;LMP1 (CL) totoxicity in an in vitro killing assay, in contrast to CD8 cells from + mice revealed that LMP1 B cells were eliminated by T cells, as the same mice (17) (Fig. 2A). Nevertheless, when the CD4 cells + are EBV-infected B cells in humans. In the mice, T cell depletion were cotransferred with LMP1 lymphoma cells into immuno- resulted in rapid, fatal B cell proliferation and lymphomagenesis, deficient hosts, they exhibited superior antitumor activity relative resembling the EBV-driven malignancies seen in immunosup- to that of the CD8 cells; their antitumor activity remained intact pressed patients. These findings revealed a central role for LMP1 in the presence of blocking IFN-γ and TNF-α (17). in both the surveillance and transformation of EBV-infected B We therefore tested the possibility that the CD4 cells controlled cells in vivo (17). Additional studies, from us and others, in which the tumors by regaining cytotoxicity in vivo. Indeed, CD4 cells LMP1 was turned on initially in small populations of mature B that were recovered from the adoptive hosts at a point when cells in adult mice, corroborated the key role of LMP1 in inducing actively engaging in tumor control (Materials and Methods) dis- immune surveillance (18–20). played potent killing activity ex vivo (Fig. 2A), associated with The LMP1-based mouse models offer a unique tool for up-regulation of cytotoxic molecules perforin, GzmB, CD107a, uncovering basic features of EBV immunity. Previous studies and FasL, in contrast to the donor cells before transfer (Fig. 2B). + have shown that LMP1 signaling-mediated B cell immunoge- These data indicate that CD4 cells control LMP1 lymphomas nicity is a key to immune surveillance (17–20), and that the through direct cytotoxicity. + responding CD8 cells develop a typical antigen-specific cytotoxic The finding that, upon cotransfer with LMP1 lymphoma response (17, 19). However, the identity of the T cell antigens in cells, chronic-stage CD4 cells regain cytotoxicity and mediate the mouse model remain elusive and may include a wide range of superior antitumor activity relative to that of their CD8 coun- tumor-associated and self-antigens (17, 18, 21) (Discussion); in terparts, prompted us to test and compare these CD4 and humans, the T cell antigens would include peptides derived from CD8 cells for their therapeutic efficacy in a mouse model of −/− −/− a variety of EBV (6, 22). Other questions include the PTLD, namely TCRβ δ CL mice bearing aggressive LMP1- function/phenotype and formation of the antigen-specific CD4 driven primary lymphomas (17). Considering that the heavy tu- cells, which are known to be capable of exerting potent antitu- mor burden in these mice may establish an immunosuppressive mor activity in an IFN-γ– and TNF-α–independent manner, environment and thereby impede the expansion and function of + upon cotransfer with LMP1 lymphomas into immunodeficient adoptive T cells, we pretreated the mice with radiation therapy hosts (17). In this work, we focus on addressing these questions. (RT) to reduce the tumor burden and create a lymphopenic condition favorable for adoptive T cell expansion and function Results (25, 26), followed by transfer of a single dose (1 × 106 per re- Both CD4 and CD8 T Cells Develop Cytotoxic Responses to LMP1+ B cipient) of CD4 or CD8 cells. We found that RT alone moder- Cells. We investigated the detailed time course and nature of ately improved survival of tumor-bearing mice. The combination immune surveillance in CL mice. Analysis of the dynamics of with adoptive CD8 cells further prolonged mice survival, and + LMP1 B cell and T cell responses revealed a peak T cell re- CD4 cells displayed even stronger antitumor activity than the + + sponse against LMP1 B cells on days 6–8 after birth, followed CD8 cells (Fig. 2C). Thus, similar to the “T cell and LMP1 + by rapid elimination of LMP1 B cells (Fig. 1A); T cells con- lymphoma cotransfer system,” adoptive CD4 cells confer better + + tracted thereafter, but some CD4 and CD8 effectors (CD69 and tumor protection than CD8 cells in this primary LMP1 lym- + – CD44 CD62L ) persisted in the bone marrow (BM) (Fig. 1A) phoma model, presumably also through direct cytotoxicity. + (17) and continued to eliminate newly arising LMP1 B cells in the BM (the primary organ for B cell development). Accord- LMP1+ B Cells Suffice to Induce a Cytotoxic CD4 Response in Vitro. In + ingly, a small population of LMP1 B cells was detected in the line with our findings, other studies carried out in animal models BM, but not in the spleen, of adult mice (Fig. 1A). (25, 26) and in patients (27, 28) with various cancers unrelated to Particularly striking was the high level of cytotoxic activity by EBV highlight the potential therapeutic value of cytotoxic CD4 cells, which had similar cytotoxic function as CD8 cells. CD4 cells. However, the molecular mechanisms leading to cyto- CD4 and CD8 cells from the BM and spleen of day 6–8 CL mice toxic CD4 responses are incompletely understood. Unlike CD8 + displayed potent killing activity against LMP1 lymphoma cells cells, which inherently develop cytotoxic capacity upon priming [derived from T cell-deficient CL mice (17)] ex vivo, but not with antigens and various costimulatory signals, CD4 cells are B

against naive wild-type (WT) B cells (Fig. 1 ). Remarkably, CD4 multipotential (29). The strong polarization of CD4 cells toward a INFLAMMATION

cells isolated from day 6–8 CL mice expressed perforin, gran- cytotoxic phenotype in CL mice provides unique opportunities for IMMUNOLOGY AND zyme B (GzmB), and CD107a, at levels similar to those of the studying their induction. Because our previous work suggests that CD8 cells (Fig. 1 C and D). In addition, these cells expressed Fas LMP1 signaling makes B cells highly immunogenic, through en- + ligand (FasL) (Fig. 1 C and D). The CD4 T cell killing of LMP1 hanced antigen presentation and costimulation (17), we reasoned + lymphoma targets could be slightly reduced by blocking the that LMP1 B cells might function as antigen-presenting cells FasL–Fas apoptotic pathway, and more markedly suppressed by (APCs) to directly prime cytotoxic CD4 cells. To test this possi- blocking MHC-II recognition; blocking both FasL and MHC-II bility, we established an in vitro system, in which naive CD4 cells resulted in an additive effect (Fig. 1E). These data suggest were cocultured with LMP1-expressing B cells. To demonstrate the + that CD4 cells kill LMP1 B cells predominantly through the key role of LMP1 signaling in the induction of a T cell response, we

Choi et al. PNAS | Published online January 8, 2018 | E687 Downloaded by guest on October 3, 2021 + + Fig. 1. LMP1-expressing B cells trigger cytotoxic CD4 and CD8 T cell responses in vivo. (A) Dynamics of LMP1-expressing B cells (CD19 Fas ; Fas is induced by + + + LMP1 signaling and is thus used as a reporter for LMP1 expression in B cells) and CD4 and CD8 T cells (Upper), and activation status (CD69 ) of CD4 and CD8 cells (Lower), analyzed by FACS in the spleen (Spl) and BM of CL mice, compared with those in CD19-cre/+ littermate control (“C”) mice. The respective mean values of at least three mice of each genotype are plotted at each time point. CD4+, TCRβ+CD1dTetramer–CD4+; CD8+, TCRβ+CD8+.(B) Cytotoxicity of + splenic CD4 and CD8 T cells from day 6–8 CL mice was assessed on LMP1 lymphoma cells and naive wild-type (WT) control B cells. E:T ratio, effector-to-target + cell ratio. Data for LMP1 lymphoma targets are representative of five independent experiments; for naive B cell controls are representative of two in- dependent experiments. (C and D) FACS analysis of the indicated effector molecules in splenic total CD4 cells (C) and total CD8 cells (D) from day 6–8 CL mice, + compared with their counterparts from littermate control mice. For the CD4 analysis, Foxp3 Tregs are excluded. Representative FACS plots are shown in the Upper, and median fluorescence intensities (MFIs) in the Lower. Each symbol represents an individual mouse; bars show the respective mean values. (E)In vitro killing assay was performed with CD4 T cells from day 6–8 CL mice on LMP1+ lymphoma cells, in the presence of MHC-II blocking and/or Fas-Fc (to block FasL), or isotype control antibodies. Data are representative of two independent experiments using two different LMP1+ lymphoma cell lines. All

mice used in A–E are on a (C57BL/6 × BALB/c)F1 (CB6F1) background; the lymphoma cells are on a C57BL/6 × BALB/c mixed background; naive control B cells are from WT CB6F1 mice.

constructed an LMP1 mutant in which amino acids FWLY (38–41) potent cytotoxic CD4 response. We focused on identifying cos- + of transmembrane domain 1 (TM1) were changed to AALA (referred timulatory molecules that were expressed on LMP1 B cells and to as LMP1TM1m): this abolishes LMP1 clustering and signaling (30) able to induce the cytotoxic differentiation of CD4 cells. Re- (Fig. 3A) and presumably its immune-stimulatory function (31). cently, several studies described granzyme/perforin-featured cy- + We found that, after 7 d of coculture with LMP1 B cells in vitro totoxic CD4 T cells, whose differentiation is fully dependent on (without addition of any exogenous ), a sizable fraction the T-box transcription factor Eomesodermin (Eomes), but not of naive CD4 cells was activated/expanded; this effect depended on the Th1 polarizing T-bet (29, 32–34). Furthermore, systemic on LMP1 signaling in B cells, as CD4 cells failed to expand on activation of 4-1BB and/or OX40 costimulatory pathways (by LMP1TM1m-expressing B cells (Fig. 3B). Of note, the CD4 cells agonist antibodies) induces high levels of Eomes in antigen- + expanded on LMP1 B cells developed cytotoxicity toward their primed CD4 cells, which then drives their cytotoxic differentia- + specific targets—LMP1 B cells, but not lipopolysaccharide (LPS)- tion (32, 33, 35). Systemic CD27 activation also induces Eomes + + activated B cells (Fig. 3C). Thus, LMP1 B cells function as APCs expression in CD4 cells (33). Our data show that LMP1 B cells to directly prime a cytotoxic CD4 response. express greatly enhanced levels of 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), and CD70 (CD27 ligand), compared + LMP1 B Cells Express Costimulatory Ligands Associated with the with control B cells (Fig. 4 A and B). Proinflammatory , Cytotoxic Differentiation of T Cells. We next sought to uncover including IL27 and IL15, may also play a supportive role in cy- the molecular mechanisms via which LMP1 signaling induces a totoxic CD4 cell generation (33). However, with the exception of

E688 | www.pnas.org/cgi/doi/10.1073/pnas.1713607115 Choi et al. Downloaded by guest on October 3, 2021 T cells, high levels of Eomes and GzmB were expressed in a PNAS PLUS major population of CD4 cells in day 6–8 CL mice (Fig. 4E). Systemic 4-1BB activation is known to result in selective ex- pression of Eomes, without T-bet expression (33), while simul- taneous activation of 4-1BB and OX40 induces both Eomes and + T-bet in CD4 cells (32). Because LMP1 B cells express ligands for both pathways, we also examined T-bet expression in the CD4 cells: analysis of Eomes and T-bet expression by CD4 cells from CL mice revealed three populations of effector cells— + – + + – + Eomes T-bet , Eomes T-bet , and Eomes T-bet —in sharp contrast to CD4 cells from control littermate mice (Fig. 4F). Furthermore, CD4 cells from CL mice expressed GzmB and/or IFN-γ, in contrast to those from the control mice (Fig. 4F). GzmB expression depends on Eomes (32, 33), while IFN-γ is mainly driven by T-bet (29); thus, our FACS analyses revealed three subtypes of effector CD4 cells in CL mice: (i) Eomes/ GzmB-featured cytotoxic cells (similar to those described in ref. 33); (ii) T-bet/IFN-γ–expressing Th1 cells (29); and (iii)a population that displayed features of both the cells described in (i) and (ii) (these cells were similar to the “cytotoxic CD4 Th1 cells”; described in ref. 32). CD4 cells from CL mice exhibited no expression of GATA3 or RORγt(Fig. S2), in- dicating no commitment toward the Th2 or Th17 subsets (29). The costimulation pathways may similarly affect CD8 cells (32, 33), but in contrast to their CD4 counterparts, the CD8 cells in day 6–8 CL mice developed into a single, nearly uniform pop- + + + + ulation, that was Eomes T-bet GzmB IFN-γ (Fig. 4G). Overall, these findings indicate that the CD4 (and CD8) T cells skew toward an Eomes-programmed cytotoxic phenotype, + perhaps due to costimulation by LMP1 B cells through the 4-1BB, OX40, and/or CD27 pathways.

LMP1+ B Cells Drive Eomes-Programmed Cytotoxic CD4 and CD8 Responses via Costimulation by CD70, OX40L, and 4-1BBL. To ad- + Fig. 2. Cytotoxic CD4 cells confer potent antitumor immunity against LMP1- dress the roles of 4-1BBL, OX40L, and CD70 in the LMP1 B driven lymphomas in vivo. (A) Upon adoptive transfer, CD4 T cells regain cy- + cell-driven cytotoxic CD4 (and CD8) T cell generation, we totoxicity against LMP1 lymphoma cells. Cytotoxicity of primary CD4 and CD8 employed a similar in vitro system as described in Fig. 3B, T cells from adult (day 42–84) CL mice BM, or the CD4 cells after cotransfer + + −/−γ −/− namely the coculture of T cells and LMP1 B cells. Our data with LMP1 lymphoma cells into Rag2 c recipients (adoptive CD4 cells; + CL B see the scheme and Materials and Methods for details), was measured by in show that, similar to ex vivo LMP1 B cells from mice (Fig. 4 ), + vitro killing assay using LMP1 lymphoma cells as targets. Data are pooled from two independent experiments, with adoptive CD4 cells analyzed at E:T ratios of 2:1 and 10:1 in one experiment and 2:1 and 15:1 in another. (B)FACS analysis of the indicated effector molecules in the adoptive CD4 cells (as in A), compared with primary CD4 cells from adult CL mice BM (chronic stage) and spleens (negative control). Representative FACS plots are shown in the Upper and MFI fold changes in adoptive CD4 cells relative to CD4 cells in adult CL mice BM are shown in the Lower. Data are pooled from two independent experiments (three mice per group). (C) Therapeutic efficacies of adoptive CD4 and CD8 cells in mice bearing aggressive LMP1-driven primary lympho- − − − − mas. TCRβ / δ / CL mice at 8 wk of age were treated with 500 rad of radi- ation therapy (RT), followed 1 d later by transfer (i.v. injection) of the indicated T cells isolated from CL mice (1 × 106 cells per recipient), or left untreated, and then monitored for survival. Survival curves were compared using the log- test. CL mice used in A–C are on a CB6F1 background; −/− −/− TCRβ δ CL mice are on a C57BL/6 × BALB/c mixed background. Fig. 3. LMP1 signaling makes B cells suffice to induce cytotoxic CD4 response in vitro. (A) Fluorescence microscopy imaging of B cells expressing LMP1-GFP fusion or LMP1TM1m-GFP fusion. Note that WT LMP1 aggregates into large the gene for the IL27 subunit β, the other cytokine genes were complexes on the cell membrane, while the mutant LMP1TM1m loses its ability + only marginally, if at all, induced in LMP1 B cells (Fig. S1). to aggregate. (B) Numbers (mean ± SEM) of CD4 cells recovered after cocul- turing for 7 d with B cells expressing LMP1 or LMP1TM1m. The cell culture was INFLAMMATION We examined the levels of the corresponding costimulatory re- 6 begun with 1.5 × 10 purified CD4 T cells in triplicate wells of 12-well plates. IMMUNOLOGY AND ceptors on T cells as a sign of their functional interaction with the + (C) CD4 cells expanded on LMP1-B cells were assessed for cytotoxicity against respective ligands on LMP1 B cells. In accord with their known B cells transduced with the MSCV-LMP1-IRES-GFP retrovirus, which contained patterns of expression following activation by their respective ligands, GFP+ (LMP1-B cells) and GFP– cells (not successfully transduced cells and thus 4-1BB and OX40 were up-regulated, while CD27 was down-regulated representing LPS-activated B cells; Materials and Methods; these cells served on CD4 and CD8 effectors in day 6–8 CL mice, compared with naive as controls). Data in A and B are representative of two to four independent C D experiments; data in C are pooled from two independent experiments, in one CD4 and CD8 cells in littermate control mice (Fig. 4 and )(36). of which the killing assay was performed at all of the indicated E:T ratios and Consistent with the plausible roles of 4-1BB and OX40 (and in another at a single E:T ratio of 50:1. In B and C, B and T cells are from 2- to CD27) pathways in inducing the Eomes–Granzyme program in 3-mo-old naive B6 mice spleens.

Choi et al. PNAS | Published online January 8, 2018 | E689 Downloaded by guest on October 3, 2021 + Fig. 4. LMP1 B cells express high levels of costimulatory ligands associated with the cytotoxic differentiation of T cells. (A) Relative transcript levels of the indicated costimulatory and coinhibitory molecules in LMP1+ B cells compared with control B cells. Splenic B cells from LMP1flSTOP/YFPflSTOP and YFPflSTOP/+ mice were treated with TAT-Cre to generate LMP1+ B cells and YFP control B cells; 2 d posttreatment, reporter (YFP)+ cells were sorted for array analysis. + (B) FACS analysis of the indicated costimulatory ligands in LMP1 B cells from day 6–8 CL mice spleens, compared with splenic B cells from littermate control (ctr) mice. Representative FACS plots (Upper) and MFI (Lower) are shown. Each symbol represents an individual mouse; bars show the respective mean values. (C and D) FACS analysis of the indicated costimulatory receptors in splenic total CD4 cells (C) and total CD8 cells (D) from day 6–8 CL mice, compared with their + – – counterparts from littermate control mice. For the CD4 analysis, Foxp3 Tregs are excluded. “Neg,” CD19 TCRβ splenocytes from the littermate control mice (used as negative control in the CD27 staining). Representative FACS plots are shown in the Upper and MFI in the Lower. Each symbol represents an individual mouse; bars show the respective mean values. (E) FACS analysis of Eomes and GzmB expression in CD4 T cells from day 6–8 CL mice and littermate controls. GzmB levels in Eomes+ CD4 cells from CL mice (blue line) were compared with those in total CD4 cells from control mice (shaded gray) and are shown on the + + Right. For these analyses, Foxp3 Tregs are excluded. (F) FACS analysis of Eomes vs. T-bet (Upper) and GzmB vs. IFN-γ (Lower) in CD4 T cells (excluding Foxp3 Tregs) from day 6–8 CL mice and littermate controls. The frequencies (mean ± SEM) of indicated populations are shown within the gates. (G) FACS analysis of Eomes vs. T-bet (Upper) and GzmB vs. IFN-γ (Lower) in CD8 T cells from day 6–8 CL mice and littermate controls. Data in E–G are representative of three mice per group; all mice used in A–G are on a CB6F1 background.

+ LMP1 B cells generated in vitro (by retroviral transduction of Fig. S4A). With regard to CD8 cells, blocking OX40L and + LPS-activated B cells) express highly elevated levels of 4-1BBL, CD70 each reduced the frequency and number of the Eomes OX40L, and CD70, compared with vector control B cells; the up- population, to an extent similar to that seen with CD4 cells; regulation of these costimulatory ligands is tied to LMP1 signaling, however, 4-1BBL blockade also reduced the frequency and sig- + as it is not seen in LMP1TM1m-B cells (Fig. 5A). In the in vitro nificantly decreased the number of Eomes CD8 cells (Fig. 5 D + culture system, we observed that, when cocultured with LMP1 B and E and Fig. S4B), in sharp contrast to the lack of effect seen cells (but not LMP1TM1m-B cells), CD4 cells gave rise to an overt with the CD4 cells. Furthermore, blocking all three costimulatory + Eomes population on day 7 (Fig. S3), while CD8 cells readily ligands altogether almost completely abrogated the generation of + + differentiated into Eomes by day 3 (see below). Using blocking Eomes CD8 cells (Fig. 5 D and E and Fig. S4B). Together, these + antibodies in culture, we found that 4-1BBL blockade did not alter results demonstrate that LMP1 B cells drive the differentiation + the fraction of CD4 cells with the Eomes phenotype (Fig. 5B)or and expansion of Eomes-programmed CD4 CTLs via CD70- and + the absolute number of Eomes CD4 cells (Fig. 5C); OX40L OX40L-mediated costimulation, and of CD8 CTLs via CD70, + blockade led to a slight reduction in the fraction of Eomes cells OX40L, as well as 4-1BBL. CD70 has a pronounced role in the but a significant decrease in their number; and CD70 blockade generation of both types of CTLs. caused an even more severe reduction of the fraction and total + number of Eomes CD4 cells (Fig. 5 B and C and Fig. S4A). Discussion Blocking both OX40L and CD70 together nearly completely ab- Previous studies in genetic mouse models have indicated a key + rogated the generation of Eomes CD4 cells (Fig. 5 B and C and role for LMP1 in inducing immune surveillance of EBV-infected

E690 | www.pnas.org/cgi/doi/10.1073/pnas.1713607115 Choi et al. Downloaded by guest on October 3, 2021 PNAS PLUS

+ Fig. 5. LMP1 B cells drive CD4 and CD8 CTLs through CD70, OX40 ligand, and 4-1BB ligand. (A) FACS analysis of the indicated costimulatory ligands in the various B cells. Unstained vector-transduced B cells served as negative control. Representative FACS plots and MFI of three replicate wells from one of two independent experiments are shown. Bars show the respective mean values. (B) FACS analysis of Eomes expression in CD4 cells (excluding Foxp3+ Tregs), either freshly isolated from naive B6 mice (ex vivo), or after coculturing for 7 d with LMP1-B cells, in the presence of the indicated blocking antibodies, or + corresponding isotype controls. Representative data from one of triplicate wells are shown, with the frequency of Eomes cells in the gate. Note that αOX40L and αCD70 share the same isotype control (Isotype2); for OX40L and CD70 double blocking, the “Isotypes” means Isotype2 used at a 2× concentration. + + (C) Numbers (mean ± SEM) of Eomes CD4 cells (excluding Foxp3 Tregs) recovered from culture wells treated with the indicated blocking antibodies, relative to those recovered from corresponding isotype control-treated wells. (D) FACS analysis of Eomes expression in CD8 cells either freshly isolated from naive B6 mice, or after coculturing for 3 d with LMP1-B cells in the presence of the indicated blocking antibodies or corresponding isotype controls. Representative data from one of triplicate wells are shown, with the frequency of Eomes+ cells in the gate. For 4-1BBL, OX40L, and CD70 triple blocking (3 Abs), the Isotypes + means Isotype1 used at a 1× concentration plus Isotype2 used at a 2× concentration. (E) Numbers (mean ± SEM) of Eomes CD8 cells recovered from culture wells treated with the indicated blocking antibodies, relative to those recovered from corresponding isotype control-treated wells. Data in A–E are repre- sentative of two independent experiments, using splenic B and T cells from 2- to 3-mo-old naive B6 mice. INFLAMMATION IMMUNOLOGY AND

B cells, by showing that LMP1 expression makes B cells highly timulatory ligands—CD70, OX40L, and 4-1BBL—that drive immunogenic, leading to their efficient elimination by T cells Eomes-programmed cytotoxic CD4 and CD8 T cell differentia- (17–20), similar to EBV-infected B cells in humans. LMP1 ex- tion (see schematic in Fig. 6); and that cytotoxic CD4 cells can pression in mouse and human B cells is known to up-regulate confer potent antitumor immunity against LMP1-driven lym- MHC class I and class II molecules on the cell surface and en- phomas (Fig. 2) (17). Understanding the key role of LMP1 in hance antigen presentation (17, 31, 37). In the present work, we these processes could provide a powerful tool for developing show that LMP1 signaling in B cells up-regulates several cos- cancer immunotherapies.

Choi et al. PNAS | Published online January 8, 2018 | E691 Downloaded by guest on October 3, 2021 TAAs. An involvement of multiple LMP1-induced cellular an- tigens (TAAs) could perhaps explain the unusually rapid and potent T cell responses in the mouse model. The identification of such TAAs warrants further investigation. With regard to T cell costimulation, in agreement with our findings, Izawa et al. (45) recently reported that expression of CD70 is highly up-regulated in EBV-LCLs derived from healthy donors. Here, we have used a unique in vitro culture system, coupled with antibody-mediated blockade, to unravel the roles of the three costimulatory ligands, CD70, OX40L, and 4-1BBL, in + the differentiation and expansion of LMP1 B cell-driven cyto- toxic T cells. In this effort, we monitored the Eomes-featured cytotoxic CD4 and CD8 responses; during the preparation of this manuscript, a new, Eomes-low/negative, subset of cytotoxic CD4 cells was described, which can be generated during certain virus infections in mice and phenotypically marked by expression + of NKG2C/E (46); however, such NKG2C/E cells were barely seen in our in vitro system (Fig. S6). Our data document that Fig. 6. Schematic view of how LMP1 signaling in B cells induces cytotoxic CD70 and OX40L contribute to the generation of Eomes- CD4 and CD8 T cell responses. LMP1 signaling in B cells leads to enhanced programmed CD4 and CD8 CTLs, and that 4-1BBL contrib- antigen presentation (MHCs) and strong costimulation through CD70, utes to the generation of CD8, but not CD4, CTLs. Among the OX40L, and 4-1BBL, and thus drives CD4 and CD8 differen- tiation. See text for details. three costimulatory ligands, CD70 apparently plays the dominant role in both CD4 and CD8 CTL generation. This is in line with the recent discovery of a critical role of the CD70–CD27 cos- LMP1 expression appears critical for the antigen-presenting timulatory pathway in EBV immunity: immune control of EBV function of EBV-transformed B cells. Ectopic expression of infection is compromised in patients with defective CD70 or LMP1 in mouse and human B cells leads to highly up-regulated CD27, and culminates in EBV-associated lymphoproliferation MHC class I and class II molecules on the cell surface and en- and lymphomagenesis (45, 47–50). To our knowledge, there are hanced presentation of endogenously expressed antigens (17, 31, no reports in the literature of OX40 or 4-1BB signaling defi- 37). LMP1 expression displays a cyclic pattern and varies over ciencies associated with EBV-related diseases. The human study 100-fold in individual cells of a LCL culture (38, 39), and its highlights the key role of CD70 (on EBV-B cells) in the ex- expression level correlates with HLA-I expression and antigen- pansion of CD8 CTLs (45), while our data indicate that CD70 is presenting function (38). Such variation is due to transcription also critical for priming CD8 CTLs and for generating CD4 regulation through as-yet-unknown mechanisms (39). In our CTLs. Overall, our findings explain why the CD4 T cells that are mouse model and the retrovirus-mediated expression system, stimulated/expanded on autologous LCLs often skew toward a LMP1 is driven by constitutive promoters; although its expres- cytotoxic phenotype (13–16). sion leads to marked up-regulation of MHC-I (17), there appears The observation that 4-1BBL is not involved in the generation no obvious correlation between the levels of LMP1 and MHC-I, of CD4 CTLs appears to contradict a report by Curran et al. in contrast to the clear correlation observed in LCLs (Fig. S5). (33), which showed that administration of 4-1BB agonist anti- + Of note, the transgenic B cells (LMP1 lymphoma cells) appear body in mice induces a CD4 CTL response. However, their to express comparable levels of LMP1 as the HLA-Ilow LCL findings were based on systemic activation of the 4-1BB pathway, cells, yet they are highly immunogenic (17), while the latter are raising the possibility that the observed cytotoxic CD4 differen- presumably of low immunogenicity (38). Perhaps other EBV tiation was the result of an indirect effect of 4-1BB activation in gene products interfere with antigen processing/presentation in myeloid APCs. Another factor that may explain the discrepancy LCLs (40), thereby dampening the potency of LMP1 (only between their results and ours is a different level of 4-1BB ac- LMP1hi LCL cells are highly immunogenic). tivation in the two systems. Whereas in humans the T cell antigens are known to comprise EBV-LCLs have been widely used as APCs to generate EBV- peptides derived from various EBV proteins, their identity re- specific T cells for adoptive therapy in EBV-associated lym- mains elusive in the mouse model. Our previous work failed to phomas (11). Apparently, LMP1 signaling underlies the APC reveal LMP1-derived T cell epitopes, but that does not exclude function of LCLs, by enhancing antigen presentation (17, 31, 37, their existence (17). However, considering that (i) LMP1 ex- 38) and costimulation for cytotoxic CD4 and CD8 responses (the pression up-regulates MHC class I and class II and enhances present work). The T cells expanded on LCLs usually consist presentation of endogenously expressed antigens (17, 31, 37, 38); of predominantly CD8 CTLs, with variable frequencies of + and (ii) CD4 and CD8 CTLs from CL mice lyse LMP1 B cells, CD4 cells (presumably mostly CD4 CTLs), yet Haque et al. (12) but not resting B cells (Fig. 1B) or WT B cells activated by LPS reported that, in a PTLD trial, clinical responses strongly cor- (through a pathway irrelevant to LMP1 signaling; Fig. 3C), we related with the frequency of CD4 cells in the infused T cells. + speculate that the T cell targets might include LMP1-induced Our results in T cell and LMP1 lymphoma cotransfer experi- cellular antigens. Because LMP1 is the key oncoprotein for ments and in the mouse model of PTLD (Fig. 2) (17) further EBV-driven tumorigenesis (41), the cellular antigens that are underscore the potency of cytotoxic CD4 cells. Whether other induced by LMP1 and recognized by T cells would be tumor- properties of CD4 cells, beyond the cytotoxic activity per se, associated antigens (TAAs) belonging to the subgroup of make them superior to CD8 cells in tumor protection remains “overexpression antigens” (21). In agreement with this notion, unclear. Nevertheless, our results suggest that increasing the several recent studies show that stimulation by autologous EBV- abundance of CD4 CTLs in therapeutic T cells, or using solely LCLs can induce human cytotoxic CD4 cells that display HLA- CD4 CTLs, may lead to a better therapeutic efficacy in future II–restricted recognition of a broad range of EBV-negative B trials. Toward this aim, CD4 and CD8 CTLs could be generated and T cell lymphomas (these clones do not recognize any known separately on LCLs and then mixed at a desired ratio for EBV latent antigens) (42–44), implying recognition of certain adoptive transfer.

E692 | www.pnas.org/cgi/doi/10.1073/pnas.1713607115 Choi et al. Downloaded by guest on October 3, 2021 Cytotoxic CD4 cells also hold great potential for treating non– sorting (FACS) was performed using a FACSAria II (BD Biosciences). In all PNAS PLUS EBV-related cancers, as demonstrated in preclinical animal T cell-sorting experiments, the CD1d tetramer (NIH Tetramer Facility) was models (25, 26) and in patients (27, 28). However, these antigen- employed to exclude natural killer T cells. specific CD4 cells were either produced by a TCR transgene approach or spontaneously arose in cancer patients, and a gen- Retroviral Constructs and Transduction. LMP1 cDNA was cloned into the MSCV-IRES-GFP or MSCV-Puro retroviral vector to generate MSCV-LMP1-IRES- eral approach for rapid generation of tumor antigen-specific GFP or MSCV-LMP1-Puro. To generate a retrovirus expressing the signaling- CD4 CTLs is so far unavailable. Although such CD4 CTLs defective LMP1 mutant LMP1TM1m, amino acids FWLY (38–41) of the trans- may be generated using LCLs as an APC system (the desired membrane domain 1 (TM1) of LMP1 were altered to AALA by QuikChange antigens may be supplied through ectopic expression or peptide site-directed mutagenesis (Stratagene), and the resultant mutant was cloned loading), their frequencies would be limited by competition from into the MSCV-IRES-GFP or MSCV-Puro retroviral vector. CD43-depleted (by EBV-specific CD4 cells (6, 22). Perhaps for this reason, Hunder using anti-CD43 microbeads from Miltenyi Biotec) splenic B cells were acti- et al. (51) used LCLs only to stimulate/expand preselected tumor vated in vitro by 20 μg/mL LPS (Sigma) for 24 h, infected with retroviruses, antigen-specific CD4 cells for melanoma therapy. In this context, and continually cultured in the presence of LPS. For B cells transduced with GFP-carrying retroviruses, at 48 or 72 h postinfection the cells were exten- LMP1-B cells may provide an urgently needed advance as an + APC for generating CD4 CTLs (along with CD8 CTLs) against sively washed and then used in downstream experiments (GFP indicates successfully transduced cells). For B cells transduced with Puro-carrying ret- desired antigens for immunotherapy in EBV-unrelated cancers. roviruses, at 24 h postinfection the cells were selected with puromycin (6 μg/mL; Here, the competition from LMP1-specific T cells would be Sigma) for 18 h, followed by extensive wash and recovery in fresh medium minor, as LMP1 protein is rarely targeted by human T cells (22). for 1 d before using in downstream experiments. Another advantage in using LMP1-B cells as APCs lies in the brevity of the T cell production protocol (totaling about 11 d for LMP1 Localization Analysis. cDNA for LMP1 or LMP1TM1m was each subcloned preparation of LMP1-B cells and subsequent generation of into the pCAG-GFP vector (#11150; Addgene) to obtain C-terminally GFP-tagged antigen-specific T cells), in sharp contrast to the lengthy (2– constructs. The resultant plasmids (pCAG-LMP1-GFP, pCAG-LMP1TM1m-GFP, or 3 mo) LCL-based protocol (11). Furthermore, one may specu- vector control pCAG-GFP) were then electroporated into mouse lymphoma late that LMP1 can be ectopically expressed in malignant B cells B cells (line 775) (52). Twenty-four hours after electroporation, cells were and thereby make them present endogenous antigens (such as counterstained with the DNA-specific fluorescent dye Hoechst 33342 (blue; TAAs and neoantigens) and simultaneously provide cos- Sigma) and imaged with fluorescence microscopy. timulatory signals for eliciting CD4 and CD8 CTL responses. In Vitro Killing Assay. Various target cells were labeled with CellTrace Violet This strategy may hold promise for T cell-based therapy in EBV- (Invitrogen) before use. CD4 and CD8 T cells were purified from the BM or unrelated B cell lymphomas/leukemia. spleen of mice by FACS. The T cells were then cocultured with 2 or 4 × 103 Taken together, our findings provide insight into EBV im- target cells at different effector/target ratios for 4 h in 96-well round- munity, suggesting that EBV polarizes CD4 T cells toward a bottomed plates, followed by active -3 staining (BD Biosciences) cytotoxic response via LMP1-induced costimulatory ligands, (17, 53). For the blocking assay, target cells were preincubated with anti- CD70 and OX40L. This suggests strategies for immunotherapy MHCII (IA/IE) blocking antibody (M5/114.15.2) or with isotype control rat in EBV-related and other cancers. IgG2b (both at 10 μg/mL; Biolegend) for 20 min at 37 °C, whereas the CD4 T cells were preincubated with the Fas-ligand neutralizing fusion protein Materials and Methods rmFas-Fc or with isotype control human IgG1 (both at 10 μg/mL; R&D Sys- − − − − Mice. C57BL/6J (B6), CD19-cre, TCRβ / , TCRδ / , and YFPflSTOP (all on a tems) under the same conditions. In all killing assays, effector/target mix- × B6 background) and BALB/c were obtained from The Jackson Laboratory. tures in 96-well plates were spun down at 8 g for 2 min before the − − − − − − − − Rag2 / common γ chain / (Rag2 / γc / ) mice were bred in our mouse col- incubation at 37 °C; cultures were stained for CD4 or CD8 (to exclude ef- flSTOP fector cells) and analyzed for active Caspase-3 levels in CellTrace-labeled ony or purchased from Taconic. The LMP1 allele on a BALB/c back- + + ground has been described (17). Homozygous CD19-cre mice were crossed target cells. Active Caspase-3 CellTrace cells represent apoptotic target = with LMP1flSTOP heterozygous mice on BALB/c background to generate cells. Percentage (%) specific killing % apoptotic target cells in cultures flSTOP with both effectors and targets − % apoptotic target cells in cultures with CD19-cre;LMP1 and CD19-cre control mice on a (C57BL/6 × BALB/c)F1 background. All mice were bred and maintained in the animal facilities at targets alone. the Dana–Farber Cancer Institute (DFCI), under specific pathogen-free con- −/− −/− ditions. All animal experiments were conducted per protocols approved by Adoptive Cell Transfer. In one set of experiments, Rag2 γc mice (lacking B, 5 + the DFCI Institutional Animal Care and Use Committee. T, and natural killer cells) were transferred (i.v.) with 2 × 10 LMP1 lym- phoma cells (line 966) (17), and 2 d later with 1 × 106 CD4 T cells sorted from – Generation of EBV-LCLs. LCLs were established by infection of human primary the BM of adult (day 42 84) CL mice. Splenic cells were isolated from re- cipients 10 d after the tumor transfer (our preliminary histological obser- B cells with the EBV strain B95.8, as previously described (44). The cells were + maintained in RPMI medium 1640 (Gibco) supplemented with 10% FBS vation suggested CD4 cells actively controlling LMP1 tumor cells at this (Sigma) and 2 mM glutamine (Gibco). The human blood samples (from health point) and processed for CD4 cell sorting for in vitro killing assay and FACS donors) were obtained per DFCI IRB-approved protocol from the Brigham and analysis of cytotoxic molecules in the CD4 cells. In another set of experiments Women’s Hospital Specimen Bank. These deidentified specimens were con- to test the therapeutic effect of adoptive T cells, CD4 or CD8 T cells were – × 6 sidered discarded tissues that did not require informed consent. sorted from the BM of adult (day 42 84) CL mice, and 1 10 cells of each − − − − were transferred (i.v.) into TCRβ / δ / CL mice at 8 wk of age [at the time + Flow Cytometry. Lymphoid single-cell suspensions were stained with the point they usually have clonal LMP1 lymphomas (17)]. One day before T cell following monoclonal antibodies specific for mouse CD4 (L3T4), CD8 (53-6.7), transfer, the CD4 and CD8 T cell treatment groups and a control group re- CD19 (1D3), CD27 (LG.3A10), CD43 (S7), CD69 (H1.2F3), CD70 (FR70), 4-1BB ceived 500 rad of total body irradiation (RT). (17B5), 4-1BBL (TKS-1), OX40 (OX-86), OX40L (RM134L), Fas (Jo2), TCRβ (H57- flSTOP/+ 597), IFN-γ (XMG1.2), GzmB (NGZB), Perforin (eBioOMAK-D), CD107a (1D4B), Gene Expression Profiling. B cells were isolated from the spleens of YFP flSTOP flSTOP INFLAMMATION FasL (MFL3), TRAIL (N2B2), Foxp3 (FJK-16s), Eomes (Dan11mag), T-bet (4B10), and LMP1 /YFP mice, by CD43 depletion using magnetic-activated IMMUNOLOGY AND GATA-3 (TWAJ), RORγt (Q31-378), NKG2A/C/E (20d5), NKG2A (16a11), H2-Kb cell sorting (Miltenyi Biotec); the cells were treated with TAT-Cre as previously + (AF6-88.5), human CD19 (H1B19), and HLA-ABC (W6/32) from BD Biosciences, described (54). Two days posttreatment, reporter (YFP) cells were sorted, and Biolegend, or eBioscience. Topro3 (Invitrogen) or eFluor 506 (eBioscience) total RNA was extracted with TRIzol reagent (Invitrogen) according to man- staining was used to exclude dead cells. Intracellular staining for GzmB, ufacturer’s specifications and analyzed on GeneChip Mouse Gene 2.0 ST arrays perforin, Foxp3, Eomes, T-bet, GATA-3, and RORγt was done with the (Affymetrix) at the Molecular Biology Core Facility at DFCI. Foxp3 staining buffer set (eBioscience). Intracellular staining for GzmB and IFN-γ was conducted using the Intracellular Fixation/Permeabilization Buffer Generation of Cytotoxic CD4 T Cells on LMP1-Expressing B Cells in Vitro. Sorted (eBioscience). All samples were acquired on a FACSCanto II (BD Biosciences) CD4 T cells from the spleens of naive B6 mice were plated in 12-well plates at + + and analyzed by FlowJo software (Tree Star). Fluorescence-activated cell 1.5 × 106 per well with irradiated (500 rad) LMP1 or LMP1TM1m B cells at a

Choi et al. PNAS | Published online January 8, 2018 | E693 Downloaded by guest on October 3, 2021 1:1 ratio. Five days later, the CD4 T cells were restimulated with 0.75 × 106 of Immunoblot Analysis. Protein extraction and immunoblotting were per- the respective B cells for an additional 2 d. All cells were cultured in RPMI formed as previously described (17), with the following primary antibodies: 1640 medium (Gibco) supplemented with 10% FBS (Sigma), 100 IU/mL anti-LMP1 (clone S12; gift from Dr. Fred Wang, Brigham and Women’s –β penicillin (Gibco), 10 mM Hepes (Corning), 1× nonessential amino acids Hospital, Boston) and anti -Actin (clone 4C4; Yurogen Biosystems). (Corning), 1 mM sodium pyruvate (Gibco), and 50 μM β-mercaptoethanol (Sigma), without addition of any growth factors or cytokines. Statistical Analysis. Statistical significance was determined by unpaired two-tailed Student’s t test, except where indicated; a value of P < 0.05 was + considered significant (ns, not significant; *P < 0.05, **P < 0.01, ***P < Blockade of Costimulatory Ligands During LMP1 B Cell-Driven Cytotoxic T Cell 0.001, and ****P < 0.0001). Survival curves were compared using the log- Production. Irradiated LMP1-expressing B cells were preincubated with rank test. blocking antibodies against CD70 (FR70, rat IgG2b), OX40L (RM134L, rat IgG2b), and/or 4-1BBL (TKS-1, rat IgG2a), or the corresponding isotype con- ACKNOWLEDGMENTS. We thank D. W. Paul for technical assistance, the 6 trols (all at 10 μg/mL; Biolegend), for 50 min at 37 °C. Splenic CD4 (1 × 10 )or DFCI Hematologic Neoplasia Flow Cytometry Core for excellent assistance CD8 cells (0.5 × 106) sorted from naive B6 mice were subsequently cocultured with the flow cytometry studies and cell sorting, Dr. B. E. Gewurz for with the pretreated LMP1+ B cells at a 1:1 ratio in 24-well plates. The CD8 the EBV (B95.8) virus, D. P. Leahy for administrative assistance, and Dr. M. Shipp for critical reading of the manuscript. We are grateful to the T cells were harvested for FACS analysis after 3 d of coculture, whereas the NIH Tetramer Facility at Emory University for providing the PE-conjugated 6 CD4 T cells were restimulated at day 5 with 0.5 × 10 of the respective B cells mCD1d-PBS57 tetramer. This work was supported by DFCI Faculty Startup for an additional 2 d, followed by FACS analysis. Funds (to B.Z.).

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