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[Cancer Research 65, 1577-1586, February 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Functional Characterization of EBV-Encoded Nuclear Antigen 1–Specific CD4+ Helper and Regulatory T Cells Elicited by In vitro Peptide Stimulation

Kui Shin Voo1,2, Guangyong Peng1, Zhong Guo1, Tihui Fu1, Yanchun Li1, Lori Frappier3 and Rong-Fu Wang1,2

1 The Center for Cell and Gene Therapy and 2 Department of Immunology, Baylor College of Medicine, Houston, Texas; and 3 Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Rong-Fu Wang, The Center for Cell and Gene Therapy, Baylor College of Medicine, ALKEK Building, N1120, One Baylor Plaza, Houston, TX 77030. Phone: 713-798-1244; Fax: 713-798-1263; E-mail: rongfuw{at}bcm.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ helper and regulatory T (Treg) cells play important but opposing roles in regulating host immune responses against cancer and other diseases. However, very little is known about the antigen specificity of CD4+ Treg cells. Here we describe the generation of a panel of EBV-encoded nuclear antigen 1 (EBNA1)–specific CD4+ T-cell lines and clones that recognize naturally processed EBNA1-P607-619 and -P561-573 peptides in the context of HLA-DQ2 and HLA-DR11, -DR12, and -DR13 molecules, respectively. Phenotypic and functional analyses of these CD4+ T cells revealed that they represent EBNA1-specific CD4+ T helper as well as Treg cells. CD4+ Treg cells do not secrete interleukin (IL)-10 and transforming growth factor ß cytokines but express CD25, the glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR), and Forkhead Box P3 (Foxp3), and are capable of suppressing the proliferative responses of naïve CD4+ and CD8+ T cells to stimulation with mitogenic anti-CD3 antibody. The suppressive activity of these CD4+ Treg cells is mediated via cell-cell contact or in part by a cytokine-dependent manner. Importantly, these Treg cells suppress IL-2 secretion by CD4+ effector T cells specific for either EBNA1 or a melanoma antigen, suggesting that these CD4+ Treg cells induce immune suppression. These observations suggest that the success of peptide-based vaccines against EBV-associated cancer and other diseases may likely depend upon our ability to identify antigens/peptides that preferentially activate helper T cells and/or to design strategies to regulate the balance between CD4+ helper and Treg cells.

Key Words: Tumor Immunity • Vaccination • Regulatory T cells • T Lymphocytes • EBV Antigens


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ helper and regulatory T (Treg) cells recognize peptides presented by MHC class II molecules, but play distinct roles in regulating host immune responses (1, 2). CD4+ helper cells provide help for the priming and maintenance of CD8+ T cells, thus enhancing the overall immune response to viruses and cancer (3). Recent studies further showed that CD4+ T cells are required for the expansion of adoptively transferred memory CD8+ T cells in vivo (4, 5). Thus, the identification of MHC class II–restricted tumor antigens capable of stimulating CD4+ helper cell responses is a critical step in the development of effective cancer vaccines (6).

However, recent studies indicate that a small subset of CD4+ Treg cells profoundly suppress host immune responses and induce self-tolerance (1, 2, 7). These Treg cells are commonly divided into naturally occurring and antigen-induced Treg cells. The naturally occurring Treg cells develop during the normal process of T-cell maturation in the thymus, whereas antigen-induced Treg cells develop as a consequence of T-cell activation under certain conditions of suboptimal antigen exposure, costimulation, and/or cytokine milieu (8). Although the full range of these antigen-induced Treg cells are not fully understood, many subsets of such T cells have been characterized, which include TH1-like, TH2-like, TH3, and Tr1 cells that secrete predominantly IFN-{gamma}, interleukin (IL)-4, transforming growth factor (TGF)-ß and IL-10 cytokines, respectively (7, 9–12). CD25, glucocorticoid-induced tumor necrosis factor receptor (GITR) family molecule, and cytotoxic T-lymphocyte antigen 4 (CTLA4) are useful markers, although they are not specific for CD4+ Treg cells (2, 13). The transcriptional factor Forkhead Box P3 (Foxp3) may serve as a relatively specific marker for CD4+ Treg cells in mice and humans (14–17). Despite these markers for CD4+ Treg cells and cytokine profiles, it is still difficult to define CD4+ Treg cells solely based on expression of a certain phenotypic marker or cytokine. The definitive evidence for CD4+ Treg cells is primarily defined by the functional proliferation assay (10). Although the precise mechanisms of this suppression remain to be determined, CD4+ Treg cells inhibit immune-cell functions either directly through cell-cell contact or indirectly through the secretion of anti-inflammatory mediators, such as IL-10, TGF-ß, or IL-4 (1, 18). It has long been speculated that the generation and maintenance of CD4+ Treg cells require the presence of target antigen or tissues (19, 20). However, the antigen specificity of CD4+ Treg cells remains largely unknown (1, 9). Our recent work showed the presence of LAGE1-specific CD4+ Treg cells in a tumor-infiltrating lymphocyte (TIL) line of a melanoma patient (17), providing evidence that tumor-specific ligands may play a critical role in inducing tumor-specific immune tolerance through recruiting and activating CD4+ Treg cells at tumor sites.

EBV is a human {gamma} herpesvirus with a tropism for B cells and infects more than 95% of the adult population (21). Infected healthy individuals carry EBV in their memory B cells for the rest of their lives as an asymptomatic infection (22). EBV has been associated with several B and epithelial cell malignancies, including Burkitt's lymphoma, posttransplant lymphoproliferative disorder, Hodgkin disease, and nasopharyngeal carcinoma (23). One of the key proteins in EBV-mediated cell transformation is the viral oncoprotein EBV-encoded nuclear antigen 1 (EBNA1) that is essential for the maintenance of the viral episome in tumor cells (24) and is the only EBV latent antigen expressed in all EBV-associated tumors. Thus, EBNA1 serves as an important target antigen for immunotherapy. Although the role of EBNA1-specific CD8+ T cells remains to be established (25–27), nearly all healthy EBV carriers mount CD4+ T-cell responses against EBNA1 (28–30). Additional studies have identified several EBNA1-derived endogenous peptides recognized by CD4+ T cells in the context of HLA-DR1, -DR7, or -DP3 (31, 32). However, the functional properties of these EBNA1-specific CD4+ T cells were poorly defined.

In this study we describe the generation and characterization of new EBNA1-specific CD4+ T cells following in vitro stimulation of human peripheral blood mononuclear cells (PBMC) with EBNA1 peptides. Interestingly, some of these CD4+ T-cell clones were Treg cells based on their phenotypic and functional analyses. These Treg cells recognized the same epitopes as CD4+ helper cell clones. Because CD4+ helper and Treg cells play opposing roles in regulating host immune responses, the induction of CD4+ Treg and T helper cells by the same peptide may negatively regulate overall immune responses and thus may have important implications for the development of peptide-based vaccines against cancer and other diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Reagents. Melanoma cell lines, HEK293 cell lines, and EBV-transformed lymphoblastoid cell lines (LCL) were maintained in RPMI 1640 growth medium supplemented with 10% FCS. Buffy coats for PBMC isolation were obtained from the Gulf Coast Regional Blood Center (Houston, TX). Autologous LCL 10 and 111 were generated in our laboratory using the supernatant of the B95.8 marmoset cell line. Established LCLs were maintained in growth medium, and the expression of human B-cell marker was confirmed by staining with anti-CD19 monoclonal antibody (mAb, PharMingen, San Diego, CA). Ten EBNA1 peptides and antibodies used for T-cell recognition blocking assays were described previously (32). EBNA1 protein was expressed in SF-9 cells and purified to homogeneity as described (33).

Fluorescence-Activated Cell-Sorting Analysis. Phycoerythrin- or FITC-conjugated anti-CD4, CD8, and GITR antibodies were purchased from PharMingen (San Diego, CA). The expression of CD25 was determined after staining T cells with an anti-CD25 antibody (R&D Systems, Minneapolis, MN) followed by a secondary goat anti-mouse mAb conjugated to FITC.

Human Leukocyte Antigen Typing of Donor PBMCs. The human leukocyte antigen (HLA) serotypes and DNA genotypes of PBMCs from healthy human donors were determined by the NIH HLA Laboratory. The HLA genotype of PBMCs from donor 111 was HLA-DRB1*0301, 0701, DQB1*0201, 0202, DRB3*0101, and DRB4*0103; for donor 10 was HLA-DRB1*1, 12, DQB1*05, and DRB3.

ELISPOT and Cytokine Release Assays. We used the ELISPOT assay to detect antigen-specific T cells in fresh PBMCs as previously described (34). Human EBNA1-specific T-cell clones were identified by their ability to release IFN-{gamma} upon recognition of autologous LCL 10 or LCL 111 cells pulsed with EBNA1 peptides. Autologous LCL targets cells were incubated with 5 µmol/L peptides for 1.5 hours in RPMI medium containing 2% human serum at 37°C, followed by three washes with serum-free RPMI medium. Unless otherwise specified, 5 x 104 target cells were cocultured with 5 x 104 effector T cells for 16 hours in T-cell assay medium containing RPMI plus 2% human serum, glutamine, and 120 IU/mL IL-2. IL-2, IL-4, IL-10, and IFN-{gamma} cytokines released from T cells were measured by ELISA kits (Endogen, Woburn, MA). TGF-ß cytokine release from T cells was determined by ELISA kits purchased from R&D Systems and BD Biosciences (Palo Alto, CA).

Generation of Human CD4+ T-Cell Lines and Clones. We had previously shown that an algorithm-predicted HLA-DR4-binding EBNA1-P518-530 peptide was instead presented by HLA-DP3 molecules (32). Hence, we revised our strategy to identify EBNA1 T-cell epitopes presented by various MHC class II molecules. We first selected PBMCs from donors of unknown haplotypes with a low ELISPOT background. PBMCs were stimulated in vitro in lymphocyte culture medium containing RPMI 1640 supplemented with 2 mmol/L L-glutamine, 0.05 mmol/L ß-mercaptoethanol, and 10% human male AB serum (Valley Biochemicals, Winchester, VA) at 2 x 105 cells per well in a flat-bottomed 96-well plate in the presence of 5 µmol/L of EBNA1 peptides. On days 7 and 14, cells were restimulated with autologous irradiated (5,000 rad) PBMCs pulsed with the same peptide. On days 8 and 15, 300 IU/mL of IL-2 were added to cell cultures. On day 19, all wells showing marked T-cell growth were tested for peptide-specific reactivity using autologous LCL 111 cells or autologous PBMCs pulsed with EBNA1-P607-619 or -P561-573 peptides, respectively. Several T-cell lines were generated that showed peptide-specific T-cell reactivity and were further cloned by using the limiting dilution methods as described (35) using one T cell per well and 5 x 104 cells per well of irradiated allogeneic PBMCs as feeder cells.

Proliferation Assays. Proliferation assays were done as previously described (17) using 1 x 105 CD4+ or CD8+ T cells purified from human PBMCs and U-bottomed 96-well plates containing 1 x 105 CD3-depleted antigen-presenting cells, 0.1 µg /mL anti-CD3 mAb, and different numbers of CD4+ regulatory or helper T cells. Transwell experiments were done as previously described except that the anti-CD3 antibody used was 0.1 µg /mL (17).

Suppression Assay for IL-2 Release. Treg and helper T cells were cultured in RPMI 1640 growth medium containing 30 IU/mL IL-2 supplemented with 10% human serum. EBNA1-specific T-cell clones were precultured (1:1 ratio) with TIL1363-2D1- or C5-C9 EBNA1-P561-573-specific CD4+ T helper cells in the presence of HLA-matched target cells pulsed with EBNA1 peptides for 24 hours. IL-2 secretion in the culture supernatants was determined by ELISA after 18 hours incubation of the mixture of T cells (Treg and TIL1363-2D1 with 1363mel cells, or Treg and EBNA1-specific C5-C9 CD4+ T cells with EBNA1-P561-573-pulsed 1087mel). CD4–C5 T cells derived from human PBMCs and EBNA1-P518-526-specific M2-B1 CD8+ T cells were used as negative controls. Parallel experiments were also done using 2 µg/mL of anti-CD3 to activate Treg cells to evaluate inhibition of effector T cell IL-2 mRNA expression by Treg cells. Effector T cells were precultured with Treg cells for 36 hours. IL-2 mRNA expression was determined after 15 hours incubation of the mixture of Treg cells and effector T cells with 1363mel cells using IL-2 and glyceraldehyde-3-phosphate dehydrogenase primer sets and conditions as previously described (36).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of EBNA1-Specific CD4+ T-Cell Lines and Clones. To generate CD4+ T cells specific for EBNA1, we screened PBMCs from 10 donors with 10 EBNA1 peptides (Figure 1A). PBMCs from donors 10 and 111 were found to have the highest levels of precursors for EBNA1-P561-573 or EBNA1-P607-619 peptide-specific T cells (Fig. 1B) and were used for the generation of EBNA1-specific T-cell lines and clones by in vitro stimulation with EBNA1 peptides. After three cycles of stimulation with the appropriate EBNA1-peptides, wells showing marked T-cell growth were tested for recognition of autologous peptide-pulsed PBMCs or LCL cells. Five T-cell lines were generated (designated C5, A6, G6, B8, and D12) and found to have EBNA1 peptide specificity against the peptide-pulsed PBMCs or autologous LCLs (Fig. 1C and D).



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  		Figure 1. Generation of EBNA1-specific T cells. A, schematic presentation of EBNA1 showing the relative positions of the GA-rich domain and peptides used for in vitro stimulation. Top, published epitopes. Bottom, peptides used in this study. B, detection of PBMCs from donors 10 and 111 that responded to EBNA1 peptides with an ELISPOT assay. PBMCs were used at 2 x 105 cells per well and results are expressed as spot-forming cells (SFC) per million PBMCs. C and D, generation of EBNA1-P561-573 and P607-619-specific T-cell lines. EBNA1 peptides were pulsed on autologous PBMCs (C) or LCL 111 (D) and used for T-cell recognition. Results are the mean of IFN-{gamma} release from two replicate wells.

 
Because bulk T-cell lines C5 and G6 showed some background activity against EBNA1-P506-520 control peptide-pulsed autologous PBMCs, we did limiting dilution cloning of the EBNA1 reactive T-cell lines. Figure 2A shows T-cell line C5 yielded 10 T-cell clones capable of recognizing autologous LCL 10, whereas T-cell line D12 yielded 4 clones capable of responding to LCL 111 (Fig. 2B). The T-cell clones with the highest T-cell reactivity against autologous LCLs were chosen for further study. Fluorescence-activated cell-sorting (FACS) analysis of P3-C9 and D12-H12 T cells confirmed that both were CD4 positive (Fig. 2C and D). To determine the restriction elements of these T cells, we tested the T-cell recognition in the presence of antibodies against MHC class I and II as well as HLA-DP, -DQ, and -DR molecules. Figure 2E and F shows that the T-cell recognition of clones P3-C9 and D12-H12 (specific for EBNA1-P561-573 and EBNA1-P607-619, respectively) was specifically blocked by mAbs against MHC class II, HLA-DR (for P3-C9), or HLA-DQ (for D12-H12), but not by antibodies against HLA-DP or MHC class I molecules. These results suggest that human T-cell clones P3-C9 and D12-H12 recognized EBNA1-derived peptides presented by HLA-DR and -DQ molecules, respectively.



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  		Figure 2. Characterization of EBNA1-specific T cells. A, T-cell recognition of EBNA1-P561-573-specific T-cell clones against autologous LCL 10. T-cell clones alone secreted less than 50 pg/mL of IFN-{gamma}. B, T-cell recognition of EBNA1-P607-619-specific T-cell clones against autologous LCL 111. C and D, FACS analysis of P3-C9 and D12-H12 T cells for CD4 expression. Ab, antibody. E, HLA-DR restriction of P3-C9 T cells. F, T-cell recognition of D12-H12 T cells against 1 x 104 LCL 1359 (DQ2, 3) cells pulsed with the EBNA1-P607-619 peptide was inhibited by anti-HLA-DQ antibodies. Results are the mean of IFN-{gamma} release from duplicate experiments.

 
T-Cell Affinity and Recognition of Naturally Processed Peptides. To determine the minimum concentration of the EBNA1-P561-573 and EBNA1-P607-619 peptides required for T-cell recognition, we pulsed HLA-DR or -DQ-matched 1297mel (DR11, 12) and LCL 1088 (DQ2, 3) cells with the corresponding EBNA1 peptides and then tested for their ability to stimulate T cells. Figure 3A shows that eight of the EBNA1-P561-573-specific CD4+ T-cell clones recognized the EBNA1-P561-573 peptide at concentrations as low as 0.5 nmol/L, whereas the D12-H12 CD4+ T cells required a relatively high concentration (100 nmol/L) of the EBNA1-P607-619 peptide for activation (Fig. 3B).



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  		Figure 3. T-cell affinity and natural processing and presentation of EBNA1 to CD4+ T cells. A, titration experiment for EBNA1-P561-573-specific T-cell clones. Peptides were pulsed on 1297mel (DR 11, 12) for use as target cells. A control peptide, EBNA1-P506-520, was also used at various concentrations (data not shown). B, titration experiment for EBNA1-P607-619-specific D12-H12 T cells. Peptides were pulsed on HLA-DQ2 expressing LCL 1088 for use as target cells. EBNA1-P518-530 served as a control. C, EBNA1-P561-573-specific CD4+ T cells recognized 1297mel cells transfected with Ii-EBNA1-aa 475-600 cDNA using LipofectAMINE reagent as previously described (32). D, full-length EBNA1 is presented to D12-H12 CD4+ T cells. Ten micrograms per milliliter of pure EBNA1 protein or bovine serum albumin (BSA) control protein was loaded onto autologous PBMCs overnight. Protein-pulsed PBMCs were then washed and cocultured with T cells overnight for IFN-{gamma} release assay. All results are the mean of two duplicate values.

 
A common problem encountered in experiments focusing on peptide stimulation in vitro is that T cells may respond to the peptide used for stimulation but fail to recognize tumor cells (29, 37) . To test whether the CD4+ T cells generated in this study were capable of recognizing naturally processed peptides on target cells, we transfected Ii-EBNA1. cDNA into HLA-DR12-matched 1297mel cells, or pulsed EBNA1 protein onto autologous PBMCs, and then tested for T-cell recognition. We found that EBNA1-P561-573-specific CD4+ T cells could recognize HLA-matched 1297mel target cells transfected with the Ii-EBNA1 cDNA (Fig. 3C), and D12-H12 CD4+ T cells specifically recognized autologous PBMCs pulsed with the full-length EBNA1 protein, but not those pulsed with bovine serum albumin (Fig. 3D). These results suggest that both EBNA1-P561-573- and EBNA1-P607-619-specific CD4+ T cells recognized naturally processed EBNA1 peptides.

Promiscuous Presentation of EBNA1 Peptides to CD4+ T Cells. To determine the restriction elements for the CD4+ T cells, we pulsed the EBNA1-P561-573 peptide onto melanoma and HEK293 cell lines expressing various HLA-DR alleles and tested for T-cell recognition. Figure 4A shows that three representative C5-D4, P3-D5, and P3-C9 CD4+ T cells recognized EBNA1 P561-573 peptide–pulsed target cells expressing HLA-DR11, -DR12, or -DR13 molecules, but no T-cell activity was detected in target cells expressing HLA-DR1, -DR3, -DR4, or -DR7 molecules. The clonality of one of such clones, P3-D5, was verified by TCR-Vß usage (Fig. 5 F, P3-D5), suggesting that single T-cell clones are capable of recognizing the same peptide presented by different HLA-DR alleles. These T cells also did not recognize any of the target cells pulsed with EBNA1 P516-520 control peptide (data not shown). To exclude the possibility that the T-cell clones present the EBNA1 peptide to themselves for recognition, we cultured the T cells in the presence of 50 nmol/L of the EBNA1-P561-573 peptide and did not detect any T-cell activation (data not shown). To determine whether the T cells could recognize HLA-DR-matched LCLs, we found that these T cells could recognize LCL cells expressing HLA-DR11, -DR12, or -DR13 molecules, suggesting that the EBNA1-P561-573 peptide can be presented by multiple HLA-DR molecules to T cells (Fig. 4A). To determine if the EBNA1-P561-573 peptide-specific T cells exhibit different peptide specificity we did epitope-mapping analysis by making 8 additional peptides. Figure 4B shows three representative EBNA1-P561-573 peptide–specific T-cell clones recognized the minimum 11-mer peptide (FMVFLQTHIFA), whereas further trimming from either end abolished T-cell recognition. The 11-mer minimal peptide can also be presented by HEK293IMDR11, HEK293IMDR13, and 1087mel (DR 12) to these T cells (data not shown). Taken together, these results suggest that these three T-cell clones recognized the same EBNA1 peptide presented by HLA-DR11, -12, or -13 molecules. This is similar to a previous report showing that a single CD4+ Th1 cell clone, ESL4.34, was capable of recognizing the same VP16 393-405 epitope presented by HLA-DR4, -DR11, or -DR13 allele (38).



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  		Figure 4. Presentation of EBNA1 peptides to CD4+ T cells. A, EBNA1-P561-573 peptide is presented on multiple HLA-DR molecules to CD4+ T cells. EBNA1-P561-573 peptide at 1 µmol/L concentration was pulsed on antigen-presenting cells, including HEK293IMDRs and melanoma cell lines, and tested for CD4+ T cell recognition. All three T-cell clones did not recognize EBNA1-P506-520 control peptide (data not shown). LCLs expressing HLA-DR11, -DR12, and -DR13 molecules serve as stimulators. LCLs alone did not secrete IFN-{gamma} (data not shown). B, identification of minimal EBNA1 peptide for HLA-DR12 binding. Peptides were pulsed as in A on 1087mel target cells for T cell recognition. C, EBNA1-P607-619-specific CD4+ D12-H12 T cells recognized HLA-DQ2-expressing LCLs.

 


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  		Figure 5. Identification of EBNA1-specific CD4+ Treg cells. A and B, functional characterization of the suppressive activity of EBNA1-P561-573 and -P607-619 peptide–specific CD4+ T-cell clones. Suppressive activity of EBNA1-specific T cells is shown on the right Y axis. C, functional screening of previously described (32) EBNA1-P518-530 peptide–specific CD4+ T-cell clones for suppressive activity. Proliferation of naïve CD4+ T cells was assayed as described in Materials and Methods. These T-cell clones were stimulated with 0.1 µg/mL anti-CD3 mAb. The ratio of responding CD4+ T cells to T-cell clones was 1:1. D and E, suppressive activity of activated Treg cells. Responder CD4+ (D) or CD8+ (E) T cells were cocultured with increasing numbers of Treg cells in the presence of anti-CD3 antibody. CD4+ P2-H9 and CD4+ C5-C9 T cells serve as controls that enhanced the proliferative activity of responder CD4+ T cells. F, TCR-Vß typing of EBNA1-specific T cells. The primers for the constant region Vß chain served as positive control. G, suppressive activity of activated EBNA1-specific T-cell lines. Experiments were done as in D. Results are the mean of two duplicate values.

 
Because the HLA-DQ haplotype of PBMCs from donor 111 is HLA-DQ*0201 and 0202, and T-cell activity could be blocked by an anti-DQ antibody (Fig. 2F), we tested whether D12-H12 CD4+ T cells could recognize LCL cells expressing HLA-DQ2 molecules. Indeed, these CD4+ T cells recognized HLA-DQ2-matched LCL 111 and 1359 cells, but not HLA-mismatched LCLs (Fig. 4C), suggesting that the T cells recognized the EBNA1-P607-619 peptide presented only by HLA-DQ2 molecule.

Functional Analysis of EBNA1-Specific CD4+ T Cells. To determine whether the T-cell clones generated in this study were CD4+ helper or Treg cells, we examined the ability of EBNA1-specific CD4+ T cells to suppress proliferation of naïve CD4+ T cells following anti-CD3 antibody stimulation. Interestingly, we found that 8 of 14 T-cell clones specific for the EBNA1-P561-573 peptide exhibited strong suppressive effects on the anti-CD3-induced proliferation of naïve CD4+ T cells, whereas 2 other clones enhanced the proliferation (Fig. 5A). We also found that the D12-H12 T-cell clone exhibited strong suppressive activity, whereas D12-E11 did not (Fig. 5B). Next, we tested the functional properties of 8 other CD4+ T-cell clones generated in a previous study (32). Among the T-cell clones, only P3-B7 was capable of suppressing anti-CD3-induced proliferation of naïve CD4+ T cells (Fig. 5C). To further characterize these T cells, we selected several representative clones and did suppressive assays in different ratios of naïve CD4+ or CD8+ T cells to Treg cells. As shown in Fig. 5D and E, P3-D5, P2-G11, and P3-B7 T-cell clones possessed potent suppressive activity, whereas D12-H12 exhibited a weak activity, but in a dose-dependent manner. By contrast, neither P2-H9 nor C5-C9 T-cell clones had the ability to suppress the proliferative response of both naïve CD4+ and CD8+ T cells. We next sought to determine the clonality of the T-cell clones. We determined TCR-Vß usage of the T-cell clones by reverse transcription–PCR using 25 pairs of TCR-Vß-specific primers as previously described (39). Figure 5F shows that D12-H12 and P3-D5 Treg cells express TCR-Vß9 and TCR-Vß13 genes, respectively, suggesting that they are pure T-cell clones. In contrast, P3-B7 Treg cells express three TCR-Vß genes, whereas C5-C9 effector T cells express two TCR-Vß genes, indicating that they are a mixture of different T-cell clones. To determine if Treg cells existed in the T-cell lines, we did suppressive assays on the EBNA1-specific T-cell lines. Figure 5G shows that the EBNA1-P561-573-, EBNA1-P607-619-, and EBNA1-P518-530-specific T-cell lines suppressed the proliferation of naïve CD4+ T cells, suggesting that Treg cells are present in the polyclonal T-cell population.

Phenotypic Markers of EBNA1-Specific CD4+ Treg Cells. To determine whether the Treg cells from Fig. 5 express the surface markers typically found on CD4+ Treg cells, we did FACS analysis. We found that EBNA1-specific Treg cells expressed significant levels of CD25 and GITR surface markers (Fig. 6A), whereas the C5-C9 helper T-cells were negative for both markers. TIL102-C6 melanoma–derived Treg cell clone served as a positive control (17). To further examine whether our T-cell clones expressed the Foxp3 marker associated with Treg cells, we did reverse transcription–PCR and real-time PCR to evaluate the expression level of Foxp3. The majority of the Treg cell clones expressed a reasonable high level of Foxp3 and CD25 markers compared with CD4+ effector T cells (C5-C9, TIL 110, 1363-5B10, and TIL 1363; Fig. 6B and C). Melanoma-specific 1359-2G6 Treg cells express high levels of Foxp3, and CD25 marker (data not shown) served as a positive control. Collectively, these results suggest that EBNA1-specific Treg cells express the markers typically found on CD4+ Treg cells (1, 40).



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  		Figure 6. Phenotypic and Foxp3 expression analyses of CD4+ Treg cells. A, expression of CD25 and GITR on EBNA1-specific CD4+ Treg cells. Melanoma CD4+ TIL102-C6 Treg cells serve as positive control for CD25 and GITR surface markers. CD4+ C5-C9 T cells serve as a negative control. B and C, expression level of Foxp3 in EBNA1-specific CD4+ Treg cells. Foxp3 expression was determined by reverse transcription–PCR (B) with 5'-TTCTGTCAGTCCACTTCACCAAGC-3' and 5'-GTTGAGAGCTGGTGCATGAAATGTGG-3' primers, and real-time PCR (C) using methods as described previously (17). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) and hypoxanthine phosphoribosyltransferase served as internal controls. Data are representative of two independent experiments.

 
Treg Cells Mediated Suppression of IL-2 Secretion by T Helper Cells. We next sought to determine whether the suppressive effect of EBNA1-specific CD4+ Treg cell clones on CD4+ helper melanoma TIL1363-2D1 cells required ligand-specific activation. As shown in Fig. 7A, secretion of IL-2 by TIL1363-2D1 T cells was completely suppressed after coculturing with CD4+ P3-D5, P3-B7, and D12-H12 Treg cells activated by EBNA1 peptides, whereas Treg cell clones treated with the control peptide had no such effect. The suppressive activity of Treg cells could not be reversed by the addition of IL-2 (3,000 IU/mL) into the culture medium before or during suppressive assays (data not shown). Figure 7B shows a parallel study in which the IL-2 mRNA expression level of activated effector T cells was inhibited by anti-CD3 activated Treg cells. Anti-CD3 activated Treg cells and 1363mel that do not secrete IL-2 served as negative controls. These data strongly suggest that the inhibition of IL-2 secretion by EBNA1-specific CD4+ Treg cells requires ligand-specific activation. Otherwise, they could not exert inhibitory effects on other CD4+ effector cells. To determine whether these Treg cells could also inhibit IL-2 secretion from EBNA1-specific helper cells, we used EBNA1-P561-573-specific C5-C9 CD4+ helper cells as targets. EBNA1-P518-526-specific M2-B1 CD8+ T cells were used as negative control. Figure 7C shows similar potent specific suppression of IL-2 secretion from C5-C9 CD4+ helper cells after coculture with the Treg cells, whereas effector M2-B1 CD8+ T cells had no such effect. Taken together, these data suggest that EBNA1-specific Treg cells could inhibit IL-2 secretion by EBNA1-specific T helper cells, naïve CD4+ and CD8+ T cells.



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  		Figure 7. Mechanisms of EBNA1-specific CD4+ Treg cell-mediated immune suppression. A, EBNA1 peptide-specific activation of CD4+ Treg cells suppresses IL-2 production from CD4+ TIL1363-2D1 T-cell recognition of autologous 1363mel cells. Effector T cells were cocultured with CD4+ Treg cell clones activated by the corresponding peptides (P3-D5 and C5-C9: 1087mel/EBNA1-P561-573; D12-H12: LCL 1359/EBNA1-P607-619; P3-B7: HEK293ECIIDP3/EBNA1-P518-530). EBNA1-P506-520 peptide served as control peptide. B, parallel studies as in A with IL-2 mRNA expression as readout. NADPH, nicotinamide adenine dinucleotide phosphate. C, parallel studies as in A using EBNA1-P561-573-specific C5-C9 CD4+ T cells as effector T cells. EBNA1-P518-526-specific CD8+ T cells were used as control. D, soluble factor or cell-cell contact is required for Treg cell–mediated suppression. Equal numbers of CD4+ responding T cells were cultured in both wells, whereas CD4+ Treg T cells or CD4-C5 T cells were cultured in inner wells. CD4-C5 T cells were derived from human PBMCs and served as non-Treg cell control. Proliferation of naïve CD4+ T cells was assayed by adding [3H]thymidine during the last 12 to 16 hours of culture.

 
Suppressive Mechanisms of EBNA1-Specific Treg Cells. To elucidate the mechanisms by which EBNA1-specific CD4+ Treg cells suppressed proliferation of naïve CD4+ T cells, we did transwell experiments. Although Treg cell clones P3-B7 and D12-H12 potently suppressed naïve CD4+ T-cell proliferation in a coculture system, they failed to do so once the naïve CD4+ T cells were separated from the Treg cell clones (Fig. 7D), suggesting that a cell-cell contact is required for their suppressive function. However, P3-D5 Treg cells were still capable of inhibiting the responding CD4+ T cells in transwell experiments, suggesting that soluble factors may contribute to the inhibitory activity of this clone. Cytokine profile analysis revealed that all EBNA1-P561-573-specific T-cell clones, including P3-D5 and C5-C9 T cells, secreted granulocyte-macrophage colony-stimulating factor, IFN-{gamma}, and IL-4, but not IL-10 or TGF-ß, whereas EBNA1-P518-530-specific P3-B7 T cells and EBNA1-P607-619-specific D12-H12 T cells secreted only granulocyte-macrophage colony-stimulating factor and IFN-{gamma} (data not shown). Because IL-4 is secreted by Treg and effector T cells, it could not be the soluble inhibitory factor. Consistent with this observation, an antibody against IL-4 failed to block their suppressive activity (data not shown). These data strongly suggest that the suppressive activity of P3-B7 and D12-H12 Treg cells is mediated by cell-cell contact, whereas the suppressive activity of P3-D5 Treg cells is mediated at least in part by soluble inhibitory factors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we identified two new EBNA1-derived peptidescapable of activating CD4+ T cells in the context of multiple MHC class II molecules. EBNA1-P607-619-specific CD4+ T cells possessed an intermediate binding affinity for the MHC-peptide complexes, whereas CD4+ T cells specific for the EBNA1-P561-573 peptide had a very high affinity. Thus, the EBNA1-P561-573 peptide was a more potent stimulator for T-cell recognition and could be presented by HLA-DR11, -DR12, or -DR13 molecules, which are expressed in more than 35% of the general populations. The EBNA1-P607-619 peptide can be presented by a prevalent HLA-DQ2 molecule, which is expressed in over 20% of the general population. EBNA1-specific CD4+ T-cell clones were capable of recognizing HLA-matched EBV-infected target cells (Fig. 4A and C), suggesting that the EBNA1 protein is naturally processed and presented by LCLs to CD4+ T cells.

Of particular interest is the finding that EBNA1-specific CD4+ T-cell populations generated after peptide stimulation contain both CD4+ T helper and Treg cell clones. Although our initial goal of this study was to generate EBNA1-specific CD4+ helper cells, additional studies indicated that 40% of CD4+ T-cell clones specific for EBNA1-P561-573 peptide were Treg cells that express CD25, GITR, and Foxp3 markers and suppress the proliferative responses of naïve CD4+ T cells to anti-CD3 antibody stimulation, whereas only one of eight T-cell clones specific for the EBNA1-P518-530 peptide had Treg characteristics. After several expansions in vitro, these Treg cells maintained their antigen specificity and suppressive function. Our in vitro peptide stimulation protocol employs repeated stimulation with peptide-pulsed autologous PBMCs in cell culture medium without IL-10 or TGF-ß, whereas others induced CD4+ T cells by stimulating PBMCs with peptide-pulsed dendritic cells or EBV+ B cells or in the presence of IL-10, TGF-ß, or dexamethasone/VitD3 (1, 41), which promoted the generation of CD4+ Treg cells. The fact that our protocol elicited both CD4+ T helper and Treg cell clones suggests the presence of EBNA1-specific CD4+ Treg cells in patients' PBMCs or T-cell lines we generated. Because of the low frequency of overall EBNA1-specific CD4+ T cells in human PBMCs, it is difficult to detect EBNA1-specific CD4+ Treg cells. However, we found that the bulk T-cell lines were capable of suppressing the proliferation of naïve CD4+ T cells in response to anti-CD3 antibody (Fig. 5G), suggesting that CD4+ Treg cells are present in these EBNA1-reactive T-cell lines. Although the origin of EBNA1-specific CD4+ Treg cells is unknown, antigen doses, peptide binding affinity, and cytokine milieu are important in the induction of CD4+ Treg cells (1, 42). Recently, it has been reported that prolonged infusion of low doses of HA peptides in mice developed CD4+CD25+ Treg cells in vivo (43). The chronic and persistent stimulation of some epitopes from the EBNA1 antigen might favor the generation of CD4+ Treg cells, but additional studies are required to investigate this issue.

Although many markers and cytokine profiles of CD4+ Treg cells are useful, it is still difficult to define CD4+ Treg cells solely based on the expression of certain markers or cytokine secretion (10). Similar to our IFN-{gamma}-secreting Treg cells, a recent study reported the existence of IFN-{gamma}-secreting TH1-like Treg cells (11). The majority of our Treg cells expressed a reasonable high level of Foxp3 and CD25 markers compared with CD4+ effector T cells. However, there is no correlation between the expression level of Foxp3 and suppressor activity of Treg cells (15). In one recent study, Vieira et al. (44) reported that Foxp3 negative mouse IL-10-Treg cells are capable of suppressing the proliferation of naive T cells. Hence, the definitive evidence for CD4+ Treg cells is to determine their functional ability to suppress the proliferation or IL-2 production by naïve or effector T cells. The EBNA1-specific, IFN-{gamma}-secreting CD4+ Treg cells described in this study were capable of suppressing the proliferation of naïve T cells in response to anti-CD3 antibody and inhibiting IL-2 secretion from antigen-specific effector T cells. Thus, they are truly antigen-specific CD4+ Treg cells.

The CD4+ Treg cell clones generated in this study used two different mechanisms for their suppressive activity. Transwell experiments revealed that P3-D5 Treg cells suppressed the proliferation of naïve CD4+ T cells in a soluble factor–dependent manner. By contrast, the suppressive function of P3-B7 and D12-H12 Treg cell clones was cell-cell contact dependent, similar to CD4+CD25+ naturally occurring Treg cells (13, 40). Regardless of their differences in action modes, these CD4+ Treg cell clones functionally suppressed the proliferation of naïve CD4+ and CD8+ T cells and inhibited the IL-2 secretion of EBNA1-specific and melanoma-specific CD4+ helper T cells. Thus, functional analysis of antigen-specific CD4+ T cells is critically important to understand their roles in regulating immune responses against cancer.

The fact that both CD4+ helper and Treg cells recognizing the same EBNA1 peptide can be generated raises an important issue regarding peptide-based immunotherapy against EBV-associated malignancies. Although antigen-specific CD4+ helper T cells can be induced by peptide-based vaccines, the simultaneous expansion of Treg cells with the same peptide specificity may ultimately inhibit effector (CD4+ and CD8+) T-cell responses against tumors. This may at least in part explain why overall immune responses in patients who received peptide vaccination are weak and transient (45), suggesting that antigen-specific Treg cells may dampen the immune responses elicited by peptide vaccines. Thus, identification of peptide ligands that differentially stimulate T helper and Treg cells would significantly advance the mechanistic appreciation of T cell–mediated T-cell responses against cancer. Our data suggest that the EBNA1-P561-573 peptide preferentially induced CD4+ Treg cells, whereas the EBNA1-P518-530 peptide preferentially elicited CD4+ effector T cells. Therefore, additional studies are necessary to understand the mechanisms that determine the fate of T cells to be effector or Treg cells. The success of peptide-based immunotherapy for EBV-associated cancer will depend on our ability to shift the balance between regulatory and helper T cells in clinical settings (46).


   Acknowledgments
 
Grant support: Baylor College of Medicine fund and NIH grants CA94237, CA101795, and CA90327 (R-F. Wang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Cliona M. Rooney (Baylor College of Medicine, Houston, Texas) for providing EBV+ B cell lines and Lizhong Zhou for technical assistance.


   Footnotes
 
Note: K.S. Voo and G. Peng contributed equally to this work.

Received 7/16/04. Revised 10/21/04. Accepted 12/ 9/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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