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The Journal of Immunology, 2005, 175: 4137-4147.
Copyright © 2005 by The American Association of Immunologists

Characterization of Latent Membrane Protein 2 Specificity in CTL Lines from Patients with EBV-Positive Nasopharyngeal Carcinoma and Lymphoma1

Karin C. Straathof*, Ann M. Leen*, Elizabeth L. Buza*, Graham Taylor||, M. Helen Huls*, Helen E. Heslop*,{dagger},{ddagger}, Cliona M. Rooney*,{dagger},§,¶ and Catherine M. Bollard2,*,{dagger},{ddagger},¶

* Center for Cell and Gene Therapy, {dagger} Department of Pediatrics, {ddagger} Department of Medicine, § Department of Molecular Virology and Microbiology, and Department of Immunology, Baylor College of Medicine, Methodist Hospital and Texas Children’s Hospital, Houston, TX 77030; and || Cancer Research United Kingdom Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Viral proteins expressed by EBV-associated tumors provide target Ags for immunotherapy. Adoptive T cell therapy has proven effective for posttransplant EBV-associated lymphoma in which all EBV latent Ags are expressed (type III latency). Application of immunotherapeutic strategies to tumors such as nasopharyngeal carcinoma and Hodgkin’s lymphoma that have a restricted pattern of EBV Ag expression (type II latency) is under investigation. Potential EBV Ag targets for T cell therapy expressed by these tumors include latent membrane proteins (LMP) 1 and 2. A broad panel of epitopes must be identified from these target Ags to optimize vaccination strategies and facilitate monitoring of tumor-specific T cell populations after immunotherapeutic interventions. To date, LMP2 epitopes have been identified for only a limited number of HLA alleles. Using a peptide library spanning the entire LMP2 sequence, 25 CTL lines from patients with EBV-positive malignancies expressing type II latency were screened for the presence of LMP2-specific T cell populations. In 21 of 25 lines, T cell responses against one to five LMP2 epitopes were identified. These included responses to previously described epitopes as well as to newly identified HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-, B*3501-, B53-, and HLA-DR-restricted epitopes. Seven of the nine newly identified epitopes were antigenically conserved among virus isolates from nasopharyngeal carcinoma tumors. These new LMP2 epitopes broaden the diversity of HLA alleles with available epitopes, and, in particular, those epitopes conserved between EBV strains provide valuable tools for immunotherapy and immune monitoring.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virtually all undifferentiated nasopharyngeal carcinoma (NPC),3 up to 40% of HL, and 20–100% of non-Hodgkin lymphomas (NHL), depending on subtype and localization, are associated with EBV (1, 2, 3, 4). Regardless of the role of EBV in the pathogenesis of these malignancies, the viral Ags expressed by tumor cells provide target Ags for immunotherapeutic strategies. Adoptive transfer of EBV-specific T cells has proven an effective strategy to prevent and treat EBV-associated lymphomas that arise in immunocompromised patients (5, 6). These lymphomas, however, are highly immunogenic, as they express all latent EBV proteins, including EBV-encoded nuclear Ags (EBNAs) 1, 2, 3A, 3B, 3C, and LP, and latent membrane proteins (LMP) 1 and 2 (type III latency). In contrast, NPC, Hodgkin’s lymphoma (HL), and NHL tumors do not express the immunodominant EBNA3 Ags and have an EBV Ag expression pattern restricted to EBNA1, LMP1, and LMP2 (type II latency).

EBNA1, essential for maintaining the latent genome in dividing cells, is present in all EBV-positive malignancies. However, although Ag processing and presentation have been reported for EBNA1 proteins truncated during translation (7, 8, 9), an internal glycine-alanine repeat prevents processing of the full-length protein, and thereby inhibits its presentation to CD8+ T cells (10, 11, 12). LMP1 and LMP2 proteins are present in the majority of EBV-associated NPC and HL tumors, and although subdominant Ags, they provide targets for immunotherapeutic approaches (3, 13, 14, 15).

LMP2- and, to a lesser extent, LMP1-specific T cells are present in the peripheral blood of patients with EBV-positive HL and NPC (16, 17). This implies that these malignancies are able to develop despite the presence of circulating tumor-specific T cells. Secretion of immunosuppressive chemokines and cytokines and the presence of regulatory T cells at the tumor site may contribute to this escape from immune surveillance (18, 19, 20). Immunotherapeutic strategies that enhance the LMP-specific immune response may overcome this immunosuppressive environment. LMP-specific T cells can be actively boosted in vivo by vaccination with LMP peptides (21, 22). Alternatively, LMP-specific T cells can be removed from the immunosuppressive environment and expanded ex vivo. EBV-transformed B cell lines (LCL) provide an excellent source of APC for this purpose and are readily generated from patients with EBV-positive tumors (23, 24, 25). However, a major concern was whether LMP2-specific T cells could be reactivated and expanded using LCLs, in which all other latency proteins, including the immunodominant EBNA3 Ags, are expressed (17, 26).

To further develop and implement such immunotherapeutic strategies, detailed characterization of LMP-specific T cell immunity is required. To date, LMP2 epitopes presented in the context of HLA-A2, A11, A23, A24, A25, B27, B60, and B63 have been described (27, 28, 29, 30, 31, 32, 33). Expansion of this panel of LMP2 epitopes is desirable for multiple reasons. First, this will enable peptide- or epitope-based vaccination strategies for all patients regardless of their HLA type. Second, vaccination with multiple rather than a single epitope is desirable to prevent tumor escape as a result of mutation or strain-specific epitope variation (34). Third, a broad panel of LMP2 epitopes provides useful reagents, such as tetramers and peptides, that can be used for detailed characterization of ex vivo expanded LMP2-specific T cells for adoptive transfer as well as for monitoring of LMP2-directed immune responses following therapeutic intervention.

We were successful in generating EBV-specific CTL lines from 25 patients with type II tumors using LCL as APC (25, 35). In this study, we report the detailed epitope specificities of LMP2-directed immune responses in these patient CTL lines using a peptide library that spans the entire LMP2 protein. Using this strategy, we identified nine new LMP2-derived HLA class I- and class II-restricted epitopes, and we demonstrate the utility of these LMP2 eptiopes as reagents for immunological monitoring.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and EBV status of the tumors

The Baylor College of Medicine Institutional Review Board and the Food and Drug Administration approved the generation of EBV-specific CTL lines for use in clinical studies in patients with EBV-positive NPC, HL, and NHL (25, 35). In all patients, tumor samples had been established as EBV positive, using immunohistochemistry for LMP-1 and/or in situ hybridization for the small nonpolyadenylated viral RNA EBER1 (23).

Patient CTL lines

EBV-specific CTL lines were reactivated and expanded from PBMC using autologous LCL as APC, as described previously (36). Briefly, after informed consent, peripheral blood (40–60 ml) was collected from patients with EBV-positive NPC, HL, and NHL. First, 5 x 106 PBMC were incubated with concentrated supernatants from the EBV producer cell line B95-8, and cultured in RPMI 1640 (HyClone) supplemented with 10% FBS (HyClone) and 200 mM glutamine (Invitrogen Life Technologies), in the presence of 1 µg/ml cyclosporin A (Novartis Pharmaceuticals) to establish an LCL. Subsequently, PBMC (2 x 106 per well of a 24-well plate) were stimulated with LCL irradiated (40 Gy) at a responder:stimulator ratio of 40:1 in 50% RPMI 1640/50% Clicks medium (Irvine Scientific) supplemented with 10% FBS and 200 mM glutamine. After 9–12 days, viable cells were restimulated with irradiated LCL at a responder:stimulator ratio of 4:1, and subsequently further expanded by weekly stimulations with LCL in the presence of human rIL-2 (Proleukin; Chiron) (40–100 U/ml).

LMP2 peptides

A peptide library consisting of 122 15-mer peptides with 11-aa overlap covering the complete sequence of LMP2a (B95-8 strain; Swiss prot access P13285) was purchased from D. Stoll (Natural and Medical Sciences Institute, University of Tuebingen, Tuebingen, Germany). Lyophilized peptides were reconstituted at 20 mg/ml in DMSO. As described previously, these peptides were pooled in a total of 23 pools in such a manner that each 15-mer peptide was represented in 2 pools, according to the grid shown in Fig. 1B (37). Single 15-mer peptides were aliquoted at 8 mg/ml. To determine the minimal recognized LMP2 epitope sequence, additional peptides, varying in length from 9 to 14 aa, were obtained from Genemed Synthesis and reconstituted at 10 mg/ml in DMSO. Aliquots of peptides were stored at –80°C.



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  		FIGURE 1. Identification of LMP2 epitopes using an LMP2 peptide library. A, LCL-reactivated CTLs (1 x 105/well) from a patient with NPC (HLA-A*0206, A24, B51, B61) were stimulated with an LMP2 peptide library pooled into 23 pools. Responses were measured in an 18-h IFN-{gamma} ELISPOT assay. Shown are mean and SD of duplicate wells. The black horizontal line shows the threshold level used to determine significance (5x number of SFC/1 x 105 unstimulated CTL). B, All peptides were divided into 23 pools in such a way that each peptide is present in 2 pools. This method allows determining those single peptides that most likely induced responses to the peptide pools. Thus, responses to pools 1 and 17, 1 and 18, 12 and 17, and 12 and 18 may be induced by single peptides 49, 61, 60, and 72, respectively. C, Testing of these individual pentadecamers identifies the sequence of peptides 60 and 61, most likely the overlapping 11 aa, as the CTL epitope. D, Testing of the three potential nonamers within this sequence at indicated concentrations identifies the minimum recognized epitope.

 
ELISPOT assay

Ninety-six-well filtration plates (MultiScreen, MAHAS4510; Millipore) were coated overnight with 10 µg/ml anti-IFN-{gamma} Ab (catcher mAB91-DIK; Mabtech). CTL were plated at 1 x 105 cells/well and stimulated with LMP2 peptide pools (1 µg/ml each peptide) or individual peptides (5–5000 ng/ml, as indicated). Irradiated (40 Gy) autologous LCL were used as positive control. After 18–24 h, the plates were washed and incubated with the secondary biotin-conjugated anti-IFN-{gamma} mAb (detector mAb (7-B6-1 biotin); Mabtech). After incubation with avidin:biotinylated HRP complex (Vectastain Elite ABC Kit (standard), PK6100; Vector Laboratories), plates were developed with AEC substrate (Sigma-Aldrich). Plates were sent for evaluation to Zellnet Consulting. Results are shown as spot-forming cells (SFC) per 1 x 105 CTL. For the screening with LMP2 peptide pools, all assays were performed once in duplicate. Before using this method as screening for patient CTL lines, the reproducibility of this method was first confirmed using CTL lines from two healthy donors (data not shown). Those responses that exceeded 5x background level of nonstimulated CTL and were at least 5 SFC/1 x 105 CTL were regarded as significant. For 2 of 25 CTL lines screened, this threshold level was lowered to 1x background level. The relevance of the identified LMP2-specific responses was subsequently confirmed in ELISPOT assays using single LMP2 peptides, again using a threshold level of 5x background and >5 SFC/1 x 105. Responses to the identified LMP2 epitopes were consistently detected in the CTL lines studied; however, the strength of these responses varied between assays. The average response to each epitope within the same CTL line is reported in Table I.


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  		Table I. LMP2-specific T cell populations in patient CTL linesa

 
Determining HLA restriction of identified epitopes

To determine the HLA restriction of the novel LMP2 class I peptides, CTL with specificity for the LMP2 peptide were plated at 1 x 105/well in an IFN-{gamma} ELISPOT assay with partially HLA-matched PHA-activated lymphoblasts (40 Gy irradiated) used as APCs either alone or pulsed with peptide (10 µg/ml for 30 min at 37°C). All immunogenic peptides were analyzed for the presence of anchor sites for HLA alleles expressed by the patient using prediction databases from K. Parker (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html_112601) and H.-G. Rammensee (Heidelberg, Germany) (www.syfpeithi.de) and the HLA Factsbook (38). For seven of nine epitopes, the HLA restriction was subsequently confirmed by staining with the HLA tetramer derived from the newly identifed epitope. To confirm HLA class II restriction of identified epitopes, T cells were stained with CD4 and CD8 mAbs (BD Biosciences), sorted on a MoFlow Cytometer, and subsequently used in an IFN-{gamma} ELISPOT assay. HLA-DR restriction was confirmed by incubating the T cells for 30 min at 37°C with HLA-DR-blocking Ab (1 µg/well) before addition of the peptide.

Tetramer staining

Tetramers were prepared by the National Institute of Allergy and Infectious Diseases tetramer core facility, or by the Baylor College of Medicine tetramer core facility. CTL or PBMC (5–10 x 105) were incubated at room temperature for 30 min in PBS/1% FCS containing the PE-labeled tetrameric complex. Samples were costained with anti-CD8 FITC and anti-CD3 PerCP. Appropriate isotype controls were included. Stained cells were fixed in PBS containing 0.5% paraformaldehyde. For each sample, a minimum of 100,000 cells was analyzed using a FACSCalibur with the CellQuest software (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LMP2-specific T cells within patient CTL lines

Using LCL as APC, EBV-specific T cells were reactivated and expanded from the peripheral blood of 25 patients with EBV type II latency tumors: 13 patients with NPC, 10 patients with HL, and 2 patients with NHL. The presence of LMP2-specific and thus tumor-specific T cells within these patient CTL lines was assessed using a peptide library representing the entire LMP2a sequence (B95-8 strain). In 21 of 25 patient CTL lines, LMP2-specific T cells were detectable in an IFN-{gamma} ELISPOT assay after overnight incubation with each of the peptide pools. This result demonstrates that T cells specific for a subdominant EBV-Ag can regularly be reactivated using LCL even in patients. An example of one CTL line is shown in Fig. 1A: T cells that produced above background levels of IFN-{gamma} were detectable after stimulation with pools 1, 12, 17, and 18. In two patient CTL lines, the spontaneous IFN-{gamma} secretion of nonstimulated T cells resulted in a signal to noise ratio that was too high to detect LMP2-specific T cell responses. In two other patient CTL lines, none of the peptide pools induced IFN-{gamma} secretion of the T cells, although incubation with LCL resulted in a measurable response.

Determining minimal recognized LMP2 sequence

Following the initial screening with each of the LMP2 peptide pools, the minimal recognized T cell epitopes were identified. Based on the LMP2 peptide pools that induced IFN-{gamma} secretion, individual pentadecamers that were present in two of the peptide pools that tested positive were selected, e.g., pentadecamers 49, 60, 61, and 72 for the patient CTL line shown as example (Fig. 1B). Stimulation of the T cells with these single pentadecamers showed that the amino acid sequence of LMP2 that contained the recognized epitope was present in pentadecamers 60 and 61, most likely in the overlapping 11-aa sequence of these two adjacent peptides (Fig. 1C). In total, 35 LMP2-specific T cell responses were detected, 24 of which were targeted toward LMP2 sequences representing previously described LMP2 epitopes, including FLYALALLL, SSCSSCPLSKI, CLGGLLTMV, IEDPPFNSL, LLWTLVVLL, PYLFWLAAI, TYGPVFMSL, GLGTLGAAI, and RRRWRRLTV (27, 29, 31, 32, 33) (Table I). In 11 CTL lines, the recognized LMP2 sequence did not contain a known LMP2 epitope. The minimal recognized epitope was then identified by testing of the potential nonamers within the overlapping sequence of two adjacent pentadecamers to which responses were detected. For example, within the RLTVCGGIMFL sequence, TVCGGIMFL was shown to represent the minimum recognized epitope, whereas RLTVCGGIM and LTVCGGIMF were not recognized by the CTL (Fig. 1D). Using this strategy, seven nonamers representing new LMP2 epitopes were identified (Table I, epitopes in bold). Two of the epitopes identified in this study (LPVIVAPYL and FTASVSTVV) represent the minimum epitope within regions of LMP2 earlier reported as CD8+ T cells recognition sites in PBMC of healthy donors (33).

For one epitope, RRLTVCGGIMF (aa 240–250), the minimal recognized sequence consisted of 11 aa rather than 9 aa, analogous to a previously described LMP2 epitope SSCSSCPLSKI (aa 340–350). Interestingly, this epitope is located within a region that contains four overlapping CD8 epitopes, VLVMLVLLILAYRRRWRRLTVCGGIMFL, VLVMLVLLILAYRRRWRRLTVCGGIMFL, VLVMLVLLILAYRRRWRRLTVCGGIMFL, and VLVMLVLLILAYRRRWRRLTVCGGIMFL, and one CD4 epitope, VLVMLVLLILAYRRRRWRRLTVCGGIMFL (39). Similarly, ILLARLFLY is located in an epitope hotspot: SSCSSCPLSKILLARLFLYALALLL, SSCSSCPLSKILLARLFLYALALLL, and SSCSSCPLSKILLARLFLYALALLL.

HLA restriction of identified CD8 epitopes

To determine the HLA restriction of the identified LMP2 epitopes, we took advantage of described peptide-binding motifs (see Materials and Methods). The HLA type of the patient used as an example is as follows: A*0206, A24, B51, B61 (Fig. 2A). The identified epitope TVCGGIMFL contains a valine at position 2 and a leucine at position 9, which are anchor residues predicted to bind to HLA-A*0206. We subsequently confirmed this HLA restriction by using partially HLA-matched PHA-activated lymphoblasts pulsed with this LMP2 peptide as APC in an ELISPOT assay (Fig. 2A). T cells secreted IFN-{gamma} upon stimulation with all peptide-pulsed APC matched for the HLA-A2 allele, but not after stimulation with peptide-pulsed APC matched for HLA-A24. As no HLA-A*0206-matched APC were available, A*0201-typed APC were used in this experiment. Comparing IFN-{gamma} secretion upon stimulation with autologous (A*0206) and A*0201-typed APC pulsed with different concentrations of the TVCGGIMFL peptide demonstrates that, although less efficient, this epitope can also be presented in the context of HLA-A*0201 (Fig. 2B). HLA-A2 restriction was further confirmed by the identification of a T cell population staining positive with HLA-A*0201-TVCGGIMFL tetramer (Fig. 2C). Using this same strategy, the other newly identified LMP2 epitopes were found to be HLA-A*0204 or A*0217, HLA-A29, HLA-A68, HLA-B*1402, HLA-B27, HLA-B*3501, and HLA-B53 restricted (Table I).



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  		FIGURE 2. LMP2 epitope TVCGGIMFL is HLA-A*201/06 restricted. A, To determine the HLA restriction of the TVCGGIMFL peptide, T cells (1 x 105/well) with specificity for the TVCGGIMFL peptide were stimulated with PHA-activated lymphoblasts from donors with the indicated HLA types with and without peptide (1 x 105/well). Shown are the mean and SD of the response to peptide-loaded PHA blast after subtraction of the response to unloaded PHA blasts from the same donor. B, To determine whether this epitope was HLA-A2 subtype specific, HLA-A*0201- and HLA-A*0206-positive PHA lymphoblasts were pulsed with indicated amounts of TVCGGIMFL peptide and used as APC in an IFN-{gamma} ELISPOT with CTL as effectors. C, The polyclonal EBV-specific CTL line in which this epitope had been identified was stained with an HLA-A*0201 TVCGGIMFL tetramer. Indicated is the percentage of tetramer-positive cells within the CD8+ population.

 
Identification of a CD4 epitope

Although the LMP2 peptide library used in this study was designed to identify HLA class I-restricted epitopes with a length of 9–11 aa, an HLA class II-restricted epitope was identified in one of the patient CTL lines. T cells present in this CTL line recognized LMP2 sequence DYQPLGTQDQSLYLG (aa 73–87), but none of the shorter (9–14 aa) peptides derived from this pentadecamer (data not shown). Therefore, the pentadecamer appeared to represent the minimum recognized epitope. As the binding groove of MHC class II molecules can accommodate peptides with a length of up to 20 aa, we reasoned that this LMP2 peptide may be recognized in the context of HLA class II. Indeed, separation of CD4+ and CD8+ T cells within this polyclonal CTL line demonstrated that this peptide induces a CD4-mediated T cell response, whereas no CD8+ T cells were activated (Fig. 3A). The recognized pentadecamer contains anchor residues that are predicted for binding to HLA-DR4 (DYQPLGTQDQSLYLG), one of the HLA class II alleles of this patient (HLA-DR4/16, DQ5/7, DP not done). Complete abrogation of peptide recognition in the presence of an HLA-DR-blocking Ab confirmed this predicted HLA-DR restriction (Fig. 3B). Other class II epitopes may have been missed in this study because of the design of the peptide library, which for detection of class II epitopes should optimally be composed of overlapping 20-mers (39).



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  		FIGURE 3. Identification of an HLA-DR-restricted LMP2 epitope. A, The polyclonal CTL line in which a T cell population specific for LMP2 peptide DYQPLGTQDQSLYLG was identified was sorted for CD4+ and CD8+ T cells. Subsequently, recognition of this epitope by these separated CD4+ and CD8+ T cells was determined in an IFN-{gamma} ELISPOT assay. B, As this epitope contained anchor sites compatible with HLA-DR4, the HLA restriction of these epitopes was further characterized by blocking the HLA-DR molecules on the APCs before loading with the peptide. EBV-specific CTL from this patient were either nonstimulated or stimulated with APC loaded with peptide with and without prior HLA-DR blocking.

 
LMP2 epitopes partially conserved in NPC tumors

The LMP2 peptide library used in this study is based on the prototype EBV type I strain B95-8. However, different EBV strains may be present in the tumor, depending on the geographical origin of the patient (40). For these newly identified LMP2 epitopes to be useful for immunotherapy, their sequence must be conserved between the B95-8 strain and the EBV strain present in the tumor. We compared the amino acid sequence of the newly identified LMP2 epitopes with described LMP2 sequences in EBV isolates from NPC cell lines and biopsy samples (41). Six of the nine epitopes were fully conserved, whereas in three epitopes one or two mutations were present (Table II). Analysis of the immunogenicity of these variant epitopes with those derived from the B95-8 sequence shows that recognition of the CD8-restricted epitopes LPVIVAPYL and MGSLEMVPM is disrupted when indicated amino acids are altered (Fig. 4). The altered amino acids most likely compromise HLA binding (e.g., proline->leucine mutation at an anchor site for B53 binding of the LPVIVAPYL epitope) or TCR recognition of these epitopes. T cell responses to variants of the CD4-restricted epitope DYQPLGTQDQSLYLG, although reduced in number, appear to be preserved possibly because MHC class II-restricted epitopes are often promiscuous in their binding to HLA molecules (Fig. 4C). Using those LMP2 epitopes that are conserved between viral isolates for immunotherapeutic strategies is preferred so as to allow for their application in large patient groups worldwide.


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  		Table II. Comparison of LMP2 epitope sequence in B95-8 and virus isolates from NPC tumor (cell lines)a

 


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  		FIGURE 4. CTL recognition of LMP2 epitopes with altered amino acid sequences. CTL (1 x 105/well) were stimulated with the B95-8-derived LMP2 epitopes LPVIVAPYL (A), MGSLEMVPM (B), and DYQPLGTQDQSLYLG (C), as well as altered versions as identified in non-B95-8 EBV strains (see Table II), and responses were measured in an IFN-{gamma} ELISPOT assay. Average and SD of CTL stimulated with 500 ng/ml peptide are shown.

 
Assessing the breadth of the LMP2 response

EBV-specific CTL reactivated and expanded using LCL as APC contain both CD4+ (mean 7.4%, range 0.1–50.0%) and CD8+ (mean 83.8%, range 39.4–98.8%) T cells that can potentially recognize multiple LMP2-derived epitopes. Screening the patient CTL lines with the LMP2 peptide pools allows for assessment of the breadth of the LMP2-directed specificity. In 12 CTL lines, detectable LMP2 reactivity was directed against a single epitope, whereas in 9 CTL lines T cell responses against 2–5 LMP2 epitopes were present (Table III). In two CTL lines that were known to contain a FLYALALLL-specific T cell population as determined by tetramer staining, no IFN-{gamma}-secreting cells were detected upon stimulation with peptide pools that contained a pentadecamer representing the FLYALALLL sequence (data not shown). This observation suggests that screening with peptide pools may underestimate the true breadth of the LMP2 response in some cases. An example of a CTL line containing broad LMP2 specificity is shown in Fig. 5. Initial screening with the LMP2 peptide pools indicated recognition of multiple LMP2 sequences (Fig. 5A). Subsequently, these responses were mapped to 4 LMP2 epitopes: the earlier described HLA-A2-restricted FLYALALLL and LLWTLVVL, the HLA-B27-restricted RRRWRRLTV epitopes, and the newly identified HLA-A29-restricted ILLARLFLY epitope (Fig. 5B). The presence of T cells recognizing multiple epitopes is desirable, as this reduces the risk of immune escape by the tumor, and of strain sequence variations.


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  		Table III. Patient characteristics and responses to CTL therapya

 


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  		FIGURE 5. Breadth of the LMP2-specific T cell responses in patient CTL line. A, EBV-specific CTL from a patient with NHL (HLA type A2, A29, B13, B27) (1 x 105/well) were stimulated with the indicated LMP2 peptide pools. The black horizontal line shows the threshold level used to determine significance (5x number of SFC/1 x 105 unstimulated CTL). B, Responses to these peptide pools could subsequently be mapped to four different HLA-A2 (FLYALALLL and LLWTLVVL)-, A29 (ILLARLFLY)-, and B27 (RRRWRRLTV)-restricted LMP2 epitopes, demonstrating a broad LMP2 specificity in this CTL line.

 
Monitoring of LMP2-specific T cell populations postadoptive T cell therapy

The ability to monitor the frequency of LMP2-specific T cell populations in the peripheral blood or infiltrating at the tumor site is crucial to determine the efficacy of immunotherapeutic interventions. In our ongoing Food and Drug Administration-approved clinical studies, patients with relapsed EBV-positive NPC, HD, and NHL are being treated with autologous EBV-specific CTL. LMP2-specific T cell populations are identified in the infusion product by screening with the LMP2 peptide library and subsequent staining with tetramers derived from the identified epitopes. For example, 11.0% of the CD8+ T cells within the CTL line from a patient with EBV-positive Hodgkin’s disease were specific for one of the newly described LMP2 epitopes, MGSLEMVPM, which was found to be HLA-B*3501 restricted. Following infusion of this EBV-specific CTL line, the LMP2-specific T cell population was monitored in peripheral blood using tetramer analysis (Fig. 6). Preinfusion, 1.98% of CD8+ T cells in the peripheral blood were specific for this LMP2 epitope. The frequency of MGSLEMVPM-specific T cells increased to 5.37% 6 wk post-CTL infusion. These results indicate that the infused CTL (4 x 107/m2) proliferate in vivo and persist for at least 6 wk postinfusion and demonstrate the value of monitoring tools derived from LMP2 epitopes.



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  		FIGURE 6. Monitoring of an LMP2-specific T cell population in PBMC post-CTL infusion. A, Using an HLA-B35 LMP2aa 1–9:MGSLEMVPM tetramer, an LMP2-specific T cell population was detected in the CTL line from a patient with HL. B, Using the same HLA-B35 tetramer, the number of T cells specific for this epitope was determined in PBMC before and (C) 6 wk after the infusion of this CTL line to monitor the persistence and expansion of the infused CTL in vivo.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Detailed characterization of LMP2-directed T cell specificity will greatly enhance the potential application of immunotherapeutic strategies and our ability to evaluate their effect as treatment of EBV latency type II malignancies. Stimulation of patient EBV-specific cytotoxic T cell lines with an LMP2 peptide library using IFN-{gamma} secretion as readout proves to be a fast and sensitive method to evaluate the strength and breadth of the LMP2-specific immune response. As this technique is effective regardless of the patient’s HLA type, it can be applied to all patients. LMP2-specific T cell responses were detectable in 84% of LCL-reactivated CTL lines. This result may be viewed as surprising, as these patients have developed EBV-positive malignancies in the presence of a competent immune system. However, EBV+ve tumors expressing type II latency use multiple strategies to evade the immune response. For example, HL cells secrete the immunosuppressive cytokine, TG{beta}, and recruit regulatory T cells, which together have devastating effects on CTL proliferation and function (42). The apparent lack of efficacy of endogenous tumor-specific CTL circulating in the patient peripheral blood provides a rationale for ex vivo expansion of the tumor-specific CTL in isolation from tumor-derived immune suppressive factors.

The LMP2-specific T cell responses were mapped either to previously described epitopes or to nine newly identified HLA class I- and class II-restricted epitopes. The latter were shown to be HLA-A*0204/17, A*0206, A29, A68, B*1402, B27, B*3501, B53, and DR (most likely DR4) restricted, mostly alleles for which no LMP2 epitopes have previously been identified. Interestingly, the new class II epitope identified, which partially overlaps with a previously reported CD8+ recognition site 33, and the class I epitope MGSLEMVPM identified in this study are the only LMP2 epitopes located in the cytoplasmic region of LMP2, whereas all other LMP2 epitopes are located in the transmembrane region (Fig. 7).



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  		FIGURE 7. Location of epitopes with the LMP2 molecule. The location of newly identified (in black) and previously described (in gray) CD4 and CD8– epitopes within the LMP2 molecule is shown. Note that the amino acid sequences of multiple epitopes, including the newly identified LPVIVAPYL, FTASVSTVV, RRLTVCGGIM, TVCGGIMFL, and ILLARLFLY epitopes, partially overlap with the sequences of other LMP2 epitopes.

 
Although NPC, HL, and NHL occur worldwide, NPC is endemic in southern China (43), whereas EBV-positive HL is more common among Hispanics (44). As HLA-A*0206 is a common HLA allele in the Asian population and HLA-A29, A68, and B*3501 are common HLA alleles among the Hispanic populations (45), these are valuable additions to the panel of LMP2 epitopes particularly when conserved among geographically separated virus isolates.

Initial methods to identify LMP2 epitopes relied on the generation of CTL clones expanded from LCL-reactivated T cell lines from healthy donors, a relatively time-consuming process (27, 29, 30). More recently, an epitope-screening strategy based on a peptide library developed by Kern et al. (37) was used to analyze EBNA1-, LMP1-, and LMP2-specific immune responses in the peripheral blood of healthy donors (33, 39). This method with IFN-{gamma} release as measured in an ELISPOT assay as readout significantly simplified the epitope identification, and was therefore our method of choice. LCL-reactivated T cell lines increased the frequency of the LMP2-specific T cells compared with that in PBMC. Nevertheless, LMP2 specificity could rarely be detected in cytotoxicity assays because of the low frequency of the LMP2-specific component. However, LMP2 tetramer-reactive cells that were isolated and expanded demonstrated LMP2-specific cytotoxic effector function (data not shown). In this study, we have shown that, using LCL as APC, LMP2-specific T cells can be reactivated and expanded for the vast majority of patients with type II latency malignancies irrespective of the patient’s age, sex, type of cancer, and disease stage. LMP2-specific T cell populations that represent <0.1% of CD3+ T cells could be detected using the ELISPOT assay, suggesting underdetection of LMP2 specificity in previous studies.

One potential problem with the screening method used is that not all epitopes may be detected using the 15-mer peptide pools. In two CTL lines that contained a FLYALALLL-specific T cell population detectable by tetramer staining, no IFN-{gamma}-secreting cells were detected upon stimulation with peptide pools that contained a pentadecamer containing the FLYALALLL sequence. However, upon stimulation with the minimum 9-mer peptide, IFN-{gamma} secretion was induced. The pentadecamers used in this study are C and/or N terminus-extended versions of potential CD8 epitopes. Whereas peptide trimming by aminopeptidases is sufficient for MHC class I presentation of N terminus-extended epitopes, proteosomes are required for presentation of C terminus-extended epitopes (46, 47, 48). The FLYALALLL epitope differs from the other epitopes described, in that it relies on the immunoproteasome for its processing from whole Ag (29). Although T cells appear to be capable of peptide trimming to a certain extent, it is not clear whether they can express the immunoproteasome after IFN-{gamma} induction (49, 50), and thus may be unable to complete C-terminal trimming of the FLYALALLL epitope. Therefore, the absence of professional APC in our screening assay may explain why FLYALALLL-specific T cell responses were not detected in all cases. In addition, when APC are exposed to peptide mixtures, competition for binding to HLA molecules may lead to underdetection of LMP2 specificity.

If T cells specific for LMP2 epitopes are to have antitumor effects, epitopes originating from the Caucasian-derived B95-8 variant of LMP2 (51) must be conserved in the tumor strain of EBV. Comparison of the LMP2 epitope sequences in B95-8 with Asian and Mediterranean EBV isolates from NPC tumors showed that 1 or 2 aa were altered in 3 of the newly identified epitopes. Similarly, previous analysis showed amino acid alterations in 6 of 11 described/predicted LMP2 epitopes (including LPVIVAPYL characterized in this work) in isolates from NPC and HL tumors (30, 52). We and others (30) have shown that for 5 of these epitopes, the alterations did not disrupt CTL recognition. Mutations in 2 epitopes (LPVIVAPYL and MGSLEMVPM) were shown to compromise their interaction with CTL, and for 1 epitope (RRRWRRLTV) CTL recognition is predicted to be decreased (52). Overall, the majority of, but not all, LMP2 epitopes appear to be antigenically conserved among different isolates. These results imply that one should ideally use the LMP2 protein as expressed in the tumor as source of Ag for immunotherapeutic strategies. However, as this is not feasible in the manufacturing of a clinical grade therapeutic product, multiple LMP2 epitopes, including isolate-specific variant epitopes, should be used to activate tumor-specific T cells.

The ability of tumor cells to delete certain Ags or epitopes to escape from the immune response, as described both for the melanoma Ag MART1 and an immunodominant HLA-A11-restricted EBV EBNA3 epitope, further stresses the importance of targeting multiple tumor epitopes preferentially from multiple tumor Ags (34, 53). Although broad LMP2 specificity was found in a number of the LCL-reactivated CTL lines studied, in a significant number of CTL lines the LMP2 response was targeted toward a single epitope and 4 CTL lines lacked detectable numbers of LMP2-specific T cells. LMP1-specific responses were only detected in 1 of 25 CTL lines (data not shown). This is most likely a result of the preferential activation of immunodominant EBNA3 and lytic EBV-Ag-specific T cells using LCL as APC to establish these CTL lines. To improve tumor-Ag reactivity, reactivation and expansion methods using APC overexpressing LMP1 and LMP2 have been developed. Using this approach, the frequency of LMP-specific T cells and the number of epitopes that these are targeted toward can be increased (54, 55). Similarly, for vaccination approaches, vectors encoding whole protein or, to avoid possible oncogenicity of the Ag, multiple LMP1 and LMP2 epitopes (polytope approach) are being developed instead of single peptides, to boost LMP-specific T cells with a broad specificity (21). Incorporation of the LMP2 epitopes identified in this study into current polytopes will enhance the number of tumor Ag-derived epitopes targeted and allow for application of this strategy to an even broader patient group.

Valuable tools for immune monitoring following immunotherapeutic interventions can be derived from LMP2 epitopes. These include tetramers and peptides for stimulation of T cells in quantitative and functional assays to detect cytokine secretion, and in this study we have demonstrated how the in vivo expansion and persistence of LMP2-specific T cells can be monitored in the peripheral blood using LMP2 tetramers. Such immune studies that provide insight into functional changes in tumor immunity are crucial to evaluate efficacy and further optimize immunotherapeutic strategies. These newly identified LMP2 epitopes will contribute to a detailed characterization of the LMP2-directed T cell immunity required to achieve this goal.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grant PO1 CA94237, a Specialized Center of Research Award from the Leukemia Lymphoma Society, the Methodist Foundation (to K.C.S. and A.M.L.), the General Clinical Research Center at Baylor College of Medicine (RR00188), a Doris Duke Distinguished Clinical Scientist Award (to H.E.H.), and a Young Investigator Award from the Lymphoma Research Foundation and a Gillson Longenbaugh Foundation Award (to C.M.B.). Back

2 Address correspondence and reprint requests to Dr. Catherine M. Bollard, Center for Cell and Gene Therapy, 1102 Bates Street, Suite 750.07, Houston, TX 77030. E-mail address: cmbollar{at}txccc.org Back

3 Abbreviations used in this paper: NPC, nasopharyngeal carcinoma; EBNA, EBV-encoded nuclear Ag; HL, Hodgkin’s lymphoma; LCL, EBV-transformed B cell line; LMP, latent membrane protein; NHL, non-HL; SFC, spot-forming cell. Back

Received for publication December 6, 2004. Accepted for publication July 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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