The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 1643 -1654
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haigh, T. A.
Right arrow Articles by Taylor, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haigh, T. A.
Right arrow Articles by Taylor, G. S.

EBV Latent Membrane Proteins (LMPs) 1 and 2 as Immunotherapeutic Targets: LMP-Specific CD4+ Cytotoxic T Cell Recognition of EBV-Transformed B Cell Lines1

Tracey A. Haigh*, Xiaorong Lin{dagger}, Hui Jia*, Edwin P. Hui{dagger}, Anthony T. C. Chan{dagger}, Alan B. Rickinson2,* and Graham S. Taylor*

* Cancer Research United Kingdom Institute for Cancer Studies, University of Birmingham, Birmingham, United Kingdom; and {dagger} State Key Laboratory in Oncology in South China, Sir Y. K. Pao Centre for Cancer, Department of Clinical Oncology, Hong Kong Cancer Institute, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The EBV-latent membrane proteins (LMPs) 1 and 2 are among only three viral proteins expressed in EBV-associated Hodgkin’s lymphoma and nasopharyngeal carcinoma. Since these tumors are HLA class I and class II-positive, the LMPs could serve as both CD8+ and CD4+ T cell targets. In contrast to CD8 responses, very little is known about CD4 responses to LMPs. In this study, we describe CD4+ T cell clones defining four LMP1- and three LMP2-derived peptide epitopes and their restricting alleles. All clones produced Th1-like cytokines in response to peptide and most killed peptide-loaded target cells by perforin-mediated lysis. Although clones to different epitopes showed different functional avidities in peptide titration assays, avidity per se was a poor predictor of the ability to recognize naturally infected B lymphoblastoid cell lines (LCLs) expressing LMPs at physiologic levels. Some epitopes, particularly within LMP1, consistently mediated strong LCL recognition detectable in cytokine release, cytotoxicity, and outgrowth inhibition assays. Using cyclosporin A to selectively block cytokine release, we found that CD4+ T cell cytotoxicity is the key effector of LCL outgrowth control. We therefore infer that cytotoxic CD4+ T cells to a subset of LMP epitopes could have therapeutic potential against LMP-expressing tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virus-associated malignancies provide an important opportunity to develop immune T cell-based therapies targeting viral Ags expressed in tumor cells. The therapeutic potential of such an approach is best exemplified by an EBV-associated malignancy, posttransplant lymphoproliferative disease (PTLD),3 to which T cell-immunocompromised allograft recipients are particularly prone. PTLD lesions, particularly those arising early posttransplant, express the full spectrum of EBV-latent cycle proteins, the nuclear Ags EBNAs 1, 2, 3A, 3B, 3C, and –LP and the membrane proteins LMPs 1 and 2. In this respect, they closely resemble the lymphoblastoid cell lines (LCLs) generated when EBV transforms B cells into permanent growth in vitro. PTLD can be cured by adoptively transferring autologous or HLA-matched EBV-specific T cell preparations generated from PBMC by LCL stimulation in vitro (1, 2, 3). Such preparations tend to be dominated by CD8+ T cells specific for immunodominant EBV-latent cycle epitopes, most often drawn from the EBNA3A, 3B, 3C family (4, 5) and their clinical effectiveness is thought to derive principally from this virus-specific CD8+ T cell component.

The experience with PTLD has prompted interest in extending this approach to other EBV-positive malignancies such as Hodgkin’s lymphoma (HL), certain types of T/NK lymphoma, and nasopharyngeal carcinoma (NPC). However, these tumors lack the immunodominant EBNA3 proteins and the range of viral Ags available for CD8+ T cell targeting is limited to EBNA1, LMP1, and LMP2 (6). Of these, EBNA1 is now known to be an accessible CD8+ T cell target but EBNA1-specific CD8+ T cell memory is only detected in a small proportion of individuals with particular HLA class I alleles (7, 8). LMP1-specific responses are extremely rare, while LMP2 is more frequently immunogenic but almost always induces low-frequency subdominant responses (9, 10). Nevertheless, current trials of adoptive T cell therapy using LCL-stimulated preparations containing LMP2-specific CD8+ T cell reactivities have indicated possible clinical benefit against HL (11) and NPC (12). Further improvements to these protocols are clearly necessary and one attractive possibility would be to harness the power of the Ag-specific CD4+ as well as the CD8+ T cell response.

CD4+ T cells play an important role in generating and maintaining effective CD8+T cell immunity (13). More recently, however, attention has also focused on their potential role as direct effectors, capable of recognizing and killing target cells in situations where those targets express both MHC class II molecules and the relevant cognate Ag. This reflects the increasing number of examples in which endogenously expressed Ags are able to access the MHC class II presentation pathway within cells (14, 15, 16, 17, 18, 19, 20), an access once thought to be exclusive to exogenously acquired proteins. The fact that EBV-associated malignancies such as HL and NPC are HLA class II positive (6) has therefore prompted interest in the EBNA1, LMP1, and LMP2 proteins as targets of CD4+ T cells. Of these, EBNA1 appears to be a relatively strong immunogen for the CD4+ T cell response in EBV-infected individuals (21). However, although some in vitro-reactivated EBNA1-specific CD4+ T cell clones have been described which appear to recognize EBNA1-positive target B cells (17), the majority of such clones fail to recognize LCL target cells despite their endogenous expression of the EBNA1 protein (22, 23, 24). By contrast, very little is known about the LMPs as CD4+ T cell immunogens. This in fact reflects a general lack of information on multiple membrane-spanning proteins, of which LMPs are prime examples as CD4+ T cell targets. In this study, we describe the isolation of CD4+ T cell clones specific for a range of LMP1- and LMP2-derived epitopes, map their restricting alleles, and provide examples where such clones efficiently recognize latently infected targets and prevent their outgrowth through perforin-dependent cell killing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Donors, ELISPOT assays

PBMCs from healthy EBV-seropositive adult donors of Chinese origin were depleted of CD8+ T cells using CD8 Dynabeads (Dynal) and tested in ELISPOT assays of IFN {gamma} release (Mabtech), as previously described (21), using individual 20-mer peptides (overlapping by 15 aa) spanning the entire LMP1 and LMP2 sequences as seen in typical Chinese EBV strains (25, 26). Some donors were HLA class II typed by PCR using sequence specific primers (performed by the National Blood Service, Birmingham, U.K.).

In vitro reactivation and cloning protocols

CD8-depleted PBMCs were pulsed for 1 h with 5 µM of the appropriate peptide(s), then washed and cultured in RPMI 1640 medium supplemented with 5% human AB serum (Sigma-Aldrich). Cells were harvested on day 7 of culture and cloned by limiting dilution in IL-2-supplemented medium as described elsewhere (9). Microcultures showing peptide reactivity by IFN-{gamma} ELISA were expanded as previously described (23), now using FCS-supplemented medium. Microculture-derived T cell lines were analyzed for CD4/CD8 status by dual-staining cells with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 (Serotec) mAbs using standard protocols. T cell lines found to be exclusively CD4+ were assessed for clonality by PCR analysis of TCR Vβ chain usage using primers specific for each of the TCR Vβ chains.

Target cells

B lymphoblasts were generated by CD40L/IL-4 stimulation as previously described (27).

EBV-transformed LCLs were generated by infection with the prototype CKL (Chinese) or B95.8 (Caucasian) EBV strains. Unless otherwise stated, all target cell recognition assays used CKL-transformed LCLs.

ELISA of IFN-{gamma} release and mAb blocking

A range of numbers of CD4+ T cells was incubated in U-bottom microtest plate wells with a standard number of autologous, HLA-matched or HLA-mismatched LCL cells that had been either prepulsed for 1 h with 5 µM peptide or exposed to an equivalent concentration of DMSO solvent as a control, and then washed. Supernatant medium was harvested after 18 h and assayed for IFN-{gamma} by ELISA (Endogen) following the manufacturer’s recommended protocol. In mAb-blocking assays, LCLs (either loaded with epitope peptide or exposed to DMSO alone as a control) were preincubated with mAbs specific for HLA-class I (W6/32 ATCC clone HB-95), HLA-DR (L243, ATCC clone HB-55), HLA-DQ (SPV-L3; Serotec) and HLA-DP (B7.21, Cancer Research U.K. Research Services) at 10 µg/ml for 1 h before T cell addition, and the assay was conducted as above with mAb in the assay medium.

Chromium release assays

Effector CD4+ T cells were added to target LCL cells (peptide-loaded or DMSO-exposed as above) at known E:T ratios in 5- or 18-h chromium release assays and results are expressed as percentage of specific lysis of the target line. In some experiments, T cells were pretreated for 2 h with 10 or 100 nM concanamycin A (Sigma-Aldrich) before addition to LCL targets in the presence of the same concentration of inhibitor. In other experiments, LCLs were pretreated for 2 h with the Fas-specific mAb ZB4 (2 µg/ml; Immunotech) before adding T cells in an equal volume of medium and conducting the assay as above. As a control for the ability of ZB4 to inhibit Fas-mediated lysis, in some cases ZB4-pretreated cells were then exposed to the agonistic anti-Fas mAb CH11 (0.5 µg/ml; Immunotech) or to an irrelevant control Ab instead of cells, and the assay was conducted as above.

Perforin/granzyme staining of T cells

T cells were first stained with anti-CD4 mAbs conjugated to appropriate fluorophores, then fixed in 1% paraformaldehyde for 10 min, permeabilized by incubation in 0.1% saponin in PBS for 30 min, and stained with perforin-PE or granzyme A-FITC Abs (BD Pharmingen). Cell staining was analyzed on a Coulter Epics Excel Flow Cytometer.

Outgrowth assays

Target cell lines (peptide loaded or DMSO exposed as above) were seeded into round-bottom microtest plate wells at doubling dilutions from 10,000 to 78 cells/well, with 6 replicate wells per dilution, with or without the addition of T cells (10,000 cells/well). All cultures were set up in standard LCL medium without cytokine supplements and were refed weekly by replacing half the medium in each well. Outgrowth was scored visually at 4 wk, with large pellets of actively growing CD19-positive cells indicating successful outgrowth of the seeded LCL cells. In contrast, only small quantities of cellular debris were present in wells where outgrowth had been inhibited by T cells. In some cases, the outgrowth assays were performed in parallel with or without the addition of cyclosporin A (CSA; Sandimmune; Novartis Pharmaceuticals) to standard culture medium at a final concentration of 0.25 µg/ml.

The range of cytokines produced by such cultures with and without CSA addition was determined using a multiplex bead assay detecting IFN-{gamma}, TNF-{alpha}, IL-2, IL-4, and IL-10 (Beadlyte-Upstate Biotechnology).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4+ T cell clones against defined LMP1 peptide epitopes

Based on screening CD8-depleted PBMCs from EBV-immune donors against a full panel of 74 LMP1 peptides in IFN-{gamma} ELISPOT assays, we identified a number of relatively frequently recognized epitope regions and therefore selected for detailed study four individuals with clear evidence of responses to these regions. The ELISPOT results from these four donors are shown in Fig. 1 (left-hand column) as numbers of spot-forming cells per 106 CD8-depleted PBMCs; reactivities were observed against LMP1 peptides 43/44 (recognized by donors 1 and 4), peptides 42/43 (recognized by donor 2), peptides 36/37 (recognized by donor 3), and peptides 3/4 (recognized by donors 2 and 4). ELISPOT responses to these four epitope regions were relatively common in the Chinese donors tested; 21% of healthy donors and 37% of NPC patients had reactivity to one or more of these epitopes (28). To characterize these responses in greater detail, we generated CD4+ T cell clones from each of the responses illustrated above by exposing CD8-depleted PBMCs for 1 h to the relevant peptide pair, then washing the cells and culturing for 7 days before limiting dilution cloning. For each epitope region, we selected two to three microcultures showing peptide reactivity by IFN-{gamma} release in a screening assay and evidence of monoclonality based on being exclusively CD4+ and monotypic by PCR analysis of TCR Vβ chain usage (data not shown).


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		FIGURE 1. Identification of CD4+ T cell epitopes in the LMP1 protein. Left panels, The results of ELISPOT assays of IFN-{gamma} release in which CD8+ T cell-depleted PBMCs from EBV-immune donors 1–4 were tested for reactivity to 74 peptides (20-mer overlapping by 15 aa) covering the primary sequence of LMP1. Results are shown as the number of spot-forming-cells per 106 CD8-depleted PBMCs for each peptide tested. Right panels, The results of peptide titration assays for one representative CD4+ T cell clone raised from the donor in question against one of the epitope peptide regions (arrowed) identified by ELISPOT assay. In each case, the clone was exposed to individual peptides from the epitope region itself and from neighboring peptides in the LMP1 sequence (peptide numbers shown in key box) using peptide concentrations in the range 10–5–10–10 M or to a DMSO control (neg). Responses were assayed by IFN-{gamma} ELISA and are expressed as a percentage of the maximal IFN-{gamma} release obtained with the optimal peptide.

 
These selected clones were then assayed against target cells pre-exposed to titrated concentrations of the two epitope region peptides and their neighboring 20-mer in the LMP1 sequence. Representative results for one clone per epitope region are shown in Fig. 1 (right-hand column). Clones reactive to epitope region 43/44 (c1.63 from donor 1 and, data not shown, c4.2 and 4.4 from donor 4) all recognized peptide 43 slightly better than peptide 44; we therefore identified peptide 43 (LMP1 aa 211–230) as the optimal epitope and named this epitope SSH, based on the first three amino acids of the epitope sequence. Likewise, we identified the optimal epitope for the peptide 42/43 region as peptide 42 (LMP1 206–225, designated QAT), for the peptide 36/37 region as peptide 37 (LMP1 181–200, designated LIW) and for the peptide 3/4 region as peptide 3 (LMP1 11–30, designated GPP). These titration assays also allowed us to ascertain the functional avidity of each clone, defined as that concentration of epitope peptide giving half maximal IFN-{gamma} release. Clones reactive to the same epitope, whether from the same or different donors, gave similar functional avidities, but there were significant interepitope differences in avidity values, ranging from 1000 nM for QAT-specific clones to 1 nM for LIW-specific clones.

All clones were then analyzed for their HLA class II-restricting alleles: first, by testing a panel of partially HLA-matched cells as peptide presenters and, second, by assaying mAbs against HLA-DP, DQ, or DR Ags (and against HLA class I as a negative control) for their ability to block the recognition of peptide-loaded autologous cells. Representative data from such assays are shown in Fig. 2 for each of the epitope-specific responses. This showed that SSH-specific clones (in this case c4.2) were specifically blocked by the anti-HLA DQ mAb and that epitope presentation required a HLA-DQB1*0601-matched presenting cell; both donors who responded to the SSH epitope (donors 1 and 4) were indeed DQB1*0601 positive. The corresponding experiments with other epitope-specific clones identified the QAT epitope as DQB1*02 restricted and the LIW epitope as DRB1*16 restricted. Interestingly, the GPP epitope was found to be presented by two different DR alleles, DRB1*07 in the case of donor 2 and DRB1*09 in the case of donor 4 (Fig. 2 and data not shown).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 2. HLA restriction of LMP1-specific CD4+ T cell clones. Representative clones specific for the SSH, QAT, LIW, and GPP epitopes within LMP1 were assayed for recognition of peptide-loaded LCL targets and the results are expressed as levels of IFN-{gamma} release. Upper panels, Assays using the autologous LCL, a partially HLA-matched LCL, and two HLA-mismatched LCLs; the HLA-DR and HLA-DQ types of these targets are shown; and the relevant restricting allele is shown in bold. Note that these assays used a wider panel of HLA-typed allogeneic targets than shown, but all results were consistent with the identification of the bolded restricting allele. Lower panels, Assays using the peptide-loaded autologous LCL target conducted either in standard medium (no Ab) or in the presence of blocking mAbs to HLA class I, HLA-DP, HLA-DQ, or HLA-DR molecules.

 
CD4+ T cell clones against defined LMP2 epitope peptides

We then used the same approach to establish and characterize a panel of CD4+ T cell clones against LMP2 epitopes. Fig. 3 (left) shows the original ELISPOT screen data from three donors who responded to distinct epitope regions in LMP2, namely, peptides 86/87 (donor 5), peptides 40/41 (donor 6), and peptides 39/40 (donor 7). Again, 10% of healthy Chinese donors and 17% of NPC patients had ELISPOT reactivity to one or more of these epitopes (28). For each epitope region, two epitope-specific CD4+ T cell clones were selected as above for detailed study. Fig. 3 (right) shows representative results from peptide titration assays using the two epitope region peptides identified in the ELISPOT assay and neighboring peptides in the LMP2 sequence. Fig. 4 shows the corresponding restriction data from assays conducted with partially HLA-matched peptide-presenting cells and with blocking mAbs.


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 3. Identification of CD4+ T cell epitopes in the LMP2 protein. Left panels, The results of ELISPOT assays of IFN-{gamma} release in which CD8+ T cell-depleted PBMCs from EBV-immune donors 5–7 were tested for reactivity to 98 peptides (20-mer overlapping by 15 aa) covering the primary sequence of LMP2. Results are shown as in Fig. 1. Right panels, The relevant peptide titration assays, results being presented as described in Fig. 1 legend.

 

Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 4. HLA restriction of LMP2-specific CD4+ T cell clones. Representative clones specific for the TYG, ICL, and VTF epitopes within LMP2 were assayed for restriction as in Fig. 2. Upper panels, Recognition of autologous HLA-matched and HLA-mismatched target LCLs (all pre-exposed to peptide; HLA DR and DQ types shown). Lower panels, Recognition of the autologous LCL in the presence of blocking mAbs to HLA class I, HLA-DP, HLA-DQ, or HLA-DR molecules.

 
Thus, the peptide 86/87 response mapped to epitope peptide 86 (LMP2 419–438, designated TYG) in titration assays and to the HLA DQB1*0601 allele in restriction assays. The peptide 40/41 response mapped to peptide 41 (LMP2 194–213, designated ICL) and was HLA DRB1*1001 restricted, while the 39–40 response mapped to peptide 40 (LMP2 189–208, designated VTF) and was HLA DRB1*0901 restricted. In contrast to the earlier study of LMP1-specific clones, in this study we found greater differences in avidity between clones to the same epitopes. Thus, the TYG-specific clone 5.162 had a peptide avidity of 80 nM (Fig. 3, right), whereas a second such clone from this same donor (c5.48; data not shown) had a much lower avidity of 1000 nM. Both ICL-specific clones showed avidities in the 30 nM region while the VTF-specific clones had avidities of 300-1000 nM (Fig. 3 and data not shown).

The specificity, restriction, and functional avidity data from all LMP1- and LMP2-specific clones analyzed are summarized in Table I. Note that all of the above experiments used peptides based on the LMP1 and LMP2 sequences of a typical Chinese EBV strain, CKL, since all of the donors were of Chinese origin. There is slight sequence divergence at the latent gene loci between Chinese EBV strains and the prototype Caucasian strain B95.8 and this leads to between 1 and 3 aa changes in the 20-mer peptide sequences being studied here. For reference, we also conducted peptide titration assays on the corresponding B95.8 sequence peptides and the results are shown in Table II. For two of the LMP1 epitopes (SSH and QAT) and for all three LMP2 epitopes (TYG, ICL, and VTF), there was only a single amino acid change and in each case this had little effect on CD4+ T cell recognition. By contrast, the other two LMP1 epitopes (LIW and GPP) had two or three residues altered and this was reflected in much poorer recognition of the B95.8 sequence peptide. Note that subsequent LCL recognition experiments were conducted on CKL-transformed LCLs except for some assays using effectors against the LMP2 ICL and VTF epitopes where there was equivalent recognition of the CKL and B95.8 epitope peptides.


View this table:
[in this window]
[in a new window]

  		Table I. Summary of LMP1 and LMP2 epitope-specific CD4+ T cell clones

 

View this table:
[in this window]
[in a new window]

  		Table II. Effect of EBV sequence polymorphism upon CD4+ T cell recognitiona

 
LCL recognition by CD4+ T cell clones

We next used assays first developed in work with EBNA-specific clones (23) to ask whether the above LMP1- and LMP2- specific CD4+ T cell clones were capable of recognizing unmanipulated (i.e., nonpeptide loaded) autologous LCL cells. For each clone, graded numbers of CD4+ T cells were added to a standard number of LCL cells from the autologous donor, from an appropriately HLA-matched donor, and from two HLA-mismatched donors. Each LCL was either left untreated or pre-exposed to the epitope peptide (at a dose known to produce an optimal response in the autologous presenting cell setting), and washed well before use; CD4+ T cell recognition was assayed by IFN-{gamma} release after overnight coculture. Representative data are shown in Fig. 5 for all of the epitope reactivities analyzed. Recognition of peptide-loaded targets is denoted by gray bars, recognition of unmanipulated targets by black bars.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 5. CD4+ T cell recognition of peptide-loaded and unmanipulated LCL targets. Using clones specific for the SSH, QAT, LIW, and GPP epitopes in LMP1 (A) and for the TYG, ICL, and VTF epitopes in LMP2 (B), graded numbers of T cells (100, 500, and 1000) were cocultured for 18 h with 5 x 104 HLA class II-matched or mismatched LCL targets that had been either pre-exposed to an optimal dose of epitope peptide (Figure 5) or left unmanipulated ({blacksquare}), and recognition was tested by ELISA for IFN-{gamma} release. Note the differences in scale on the IFN-{gamma} release axis between assays on peptide-loaded vs unmanipulated LCL targets. Shown below for each clone are the results from separate mAb-blocking assays conducted on the unmanipulated HLA-matched LCL target either in standard medium (no Ab) or in the presence of blocking mAbs to HLA class I, HLA-DP, HLA-DQ, or HLA-DR molecules. n.t., Not tested. All of the clones were tested in three independent experiments and one representative result is shown for one clone specific for each epitope.

 
Among the LMP1-specific clones (Fig. 5A), although all four reactivities responded well to HLA-matched LCL targets preloaded with peptide, clones specific for the SSH and QAT epitopes also showed strong recognition of unmanipulated target cells. This recognition appeared to be specific since it was restricted to HLA-matched targets, increased proportionately with greater T cell input, and could be blocked by the anti-HLA DQ mAb, just as would be predicted for these DQB1*0601-restricted and DQB1*02-restricted epitopes. Note that these same CD4+ T cells showed no detectable recognition of autologous or HLA-matched B lymphoblasts activated by CD40L/IL-4 stimulation rather than by EBV (data not shown), again consistent with their virus specificity. As illustrated in Fig. 5A, levels of unmanipulated LCL recognition could be as high as 60% of that seen in the same assay against peptide-loaded targets. We found that, for all three SSH- and for both QAT-specific clones, autologous LCL recognition varied between experiments but was always in the range of 15–60% of the maximal response against the peptide-loaded LCL. By contrast, clones specific for the other two LMP1-derived epitopes, LIW and GPP, showed much lower recognition of unmanipulated LCL targets, ~1% of the value seen with the corresponding peptide-loaded LCL. At these levels, significant IFN-{gamma} release could only be detected in cocultures with the highest CD4+ T cell input. However, this IFN-{gamma} response was reproducible between experiments and was never observed in cocultures with HLA-mismatched LCLs.

Data from individual LMP2-specific clones are illustrated in Fig. 5B. A clone specific for the DQB1*0601-restricted TYG epitope (c5.162) showed clear recognition of the unmanipulated HLA-matched LCL which increased with input T cell number and was specifically blocked by the anti-DQ mAb. However, the levels of recognition were relatively low, representing ~3% of the peptide-loaded LCL value. The other TYG specific but less avid clone, c5.48, failed to recognize unmanipulated LCL targets (data not shown). Both clones specific for the DRB1*1001-restricted ICL epitope recognized the unmanipulated HLA-matched LCL at levels varying from 9% of the peptide-loaded value for one of the clones (c6.142; Fig. 5B) to 2% for the other clone (c6.2; data not shown). Of the two clones specific for the DRB1*0901-restricted VTF epitope, c7.46 showed unmanipulated LCL recognition at ~5% of the peptide-loaded control value, whereas the less avid clone 7.95 was only capable of recognizing the peptide-loaded LCL. Data for all LMP1- and LMP2-specific clones analyzed are again summarized in Table I.

Cytotoxic capacity of CD4+ T cell clones

To examine their possible cytotoxic function, all clones were tested in 18-h cytotoxicity assays against unmanipulated and epitope peptide-loaded target LCLs, again both HLA matched and HLA mismatched with the relevant CD4 effectors (Fig. 6). Interestingly, all of the LMP1-specific clones mediated significant lysis of HLA-matched peptide-loaded LCL targets. Furthermore, the SSH- and QAT-specific CD4+ clones (i.e., those cells giving the highest recognition in IFN-{gamma} release assay) also showed significant killing of unmanipulated LCL targets at E:T ratios as low as 2.5:1 (Fig. 6A and data not shown). By contrast, none of the LMP2-specific clones gave detectable lysis of the unmanipulated LCL, although five of the six such clones clearly killed peptide-loaded HLA-matched targets effectively. The sixth clone, VTF-specific c7.95, failed to kill the relevant peptide-loaded LCL even though these cells clearly recognized the same targets by IFN-{gamma} release (cf VTF c7.95 data, Figs. 5B and 6B). This clone never gave detectable killing even when the E:T ratios were increased to 30:1.


Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 6. Cytotoxic function of CD4+ T cell clones. Results of cytotoxicity assays using clones specific for the LMP1 epitopes (A) and LMP2 epitopes (B) as in Fig. 5. Assays were conducted on HLA class II-matched and -mismatched LCL targets where the target cells were either pre-exposed to an optimal dose of epitope peptide (LCL + peptide) or left untreated (LCL). Unless otherwise indicated, assays were performed at E:T ratios of 5:1 ({blacksquare}) or 2.5:1 (Figure 6). Note that VTF c7.95 is unique in lacking cytotoxic function even at E:T ratios of 30:1. Representative results from three independent experiments are shown.

 
Because target cell lysis by the above CD4+ T cells was less obvious in 5-h assays (the usual time scale for CD8+ T cell cytotoxicity assays), we sought to determine whether target cells were being killed by perforin/granzyme release (as used by CD8+ T cells) or by the slower pathway involving Fas ligation sometimes observed for CD4+ T cells (17, 29). Interestingly, intracellular staining (Fig. 7A) showed that all of the cytotoxic clones were clearly perforin and granzyme A positive, whereas the one noncytotoxic clone, VTF c7.95, was distinct in lacking detectable perforin expression. We then performed cytotoxicity assays using peptide-loaded targets in the presence of either concanamycin A (30), to selectively block perforin/granzyme-dependent killing or the anti-Fas mAb ZB4 (31), to selectively block Fas-dependent killing (Fig. 7B). The results clearly show that peptide-loaded target cell lysis is significantly inhibited by concanamycin A but not by the mAb ZB4, whereas in a parallel assay this anti-Fas mAb did inhibit lysis of the same target cells induced by the agonistic anti-Fas mAb CH11 (31).


Figure 7
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 7. Mechanism of cytotoxicity. A, Flow cytometric analyses of permeabilized CD4+ T cells stained with mAbs specific for perforin and for granzyme A. Data are shown from clones specific for three LMP1 epitopes (SSH, QAT, and LIW) and for two LMP2 epitopes (ICL and VTF). Note that the noncytotoxic clone VTF c7.95 is unique in lacking perforin expression. B, Cytotoxicity assays using the above CD4+ T cell clones and matched LCL targets (pretreated with epitope peptide) where the assays were conducted over 18 h in standard medium (control) in the presence of 10–100 nM concanamycin A (ConA) or in the presence of the anti-Fas-blocking mAb ZB4 (+ZB4). Results are expressed as in Fig. 6 at E:T ratios of 5:1 ({blacksquare}) and 2.5:1 (Figure 7) unless otherwise indicated on the figure. In the same assays, the LCL targets were exposed to the agonistic anti-Fas mAb CH11 in standard medium (control) or in the presence of the anti-Fas-blocking mAb ZB4 as above (+ZB4) and specific lysis is shown as hatched bars. Representative results from three independent experiments are shown.

 
Control of LCL outgrowth by CD4+ T cell clones

All clones were then examined for their ability to prevent target cell outgrowth in 4-wk cocultures. In each case, two HLA-matched and two HLA-mismatched LCLs, with and without epitope peptide loading, were seeded into microtest plate wells across a range of cell inputs (104–78 cells/well). Outgrowth efficiency, defined as the minimum LCL input required to achieve successful outgrowth in 50% replicate wells, was then determined in the presence and absence of 104 added CD4+ T cells.

Representative results from such experiments are shown in Fig. 8. Outgrowth efficiency for LCLs cultured in the absence of T cells is shown by dotted lines and for LCLs in the presence of T cells by symbols (Fig. 8, • and {blacksquare}, unmanipulated LCL; {circ} and {square}, peptide-loaded LCL). Almost all clones tested inhibited the outgrowth of peptide-loaded LCLs of the correct HLA type without affecting the corresponding HLA-mismatched targets. Importantly, clones specific for the LMP1 epitopes SSH and QAT, that had shown efficient recognition of the unmanipulated LCL in IFN-{gamma} release and cytotoxicity assays, also significantly inhibited their outgrowth. Clones with lower levels of LCL recognition in those earlier assays, for example, clones to the LMP2 epitopes TYG and ICL, were correspondingly less active in outgrowth inhibition, but nevertheless still mediated a small effect apparent in HLA-matched but not HLA-mismatched LCL targets. By contrast, clones with only trace levels of unmanipulated LCL recognition in the earlier IFN-{gamma} assays, for example, clones against the LMP1 epitopes LIW and GPP, had no effect on outgrowth inhibition (Fig. 8 and data not shown). Particularly interesting was the result from the clone VTF c7.95, which produced IFN-{gamma} in response to the peptide-loaded HLA-matched LCL but failed to kill these cells; this was completely devoid of inhibitory activity in outgrowth assays.


Figure 8
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 8. Inhibition of LCL outgrowth. Clones specific for LMP1 epitopes SSH, QAT, and LIW (A) and LMP2 epitopes TYG, ICL, and VTF (B) were added at 10,000 cells/well to wells seeded with graded numbers (78–10,000) of LCL targets. These included HLA class II-matched LCLs, either pre-exposed to epitope peptide ({square}) of left unmanipulated ({blacksquare}) and HLA-mismatched LCLs, again peptide-loaded ({circ}) or unmanipulated (•). Representative results from one of two HLA-matched and one of two HLA-mismatched target LCLs are shown in each case. Results are expressed as the mean number of LCL cells necessary to achieve 50% LCL outgrowth after 4 wk. In each case, the dotted line gives the results for the LCL targets incubated without T cell addition. Note that the noncytotoxic clone VTF c7.95 is unique in showing no growth inhibition even of peptide-loaded targets. Representative results from three independent experiments are shown.

 
This last observation prompted a final series of experiments which sought to determine the relative importance of cytokine release and cytotoxicity as effector mechanisms contributing to LCL growth control. In this study, we took advantage of the fact that following TCR ligation, perforin/granzyme release from CTLs can be distinguished from cytokine secretion (32) in that only the latter requires NFAT-dependent gene transcription (33). We therefore conducted preliminary assays in which the CD4+ T cell clones were exposed to peptide-loaded HLA-matched LCL targets in the presence or absence of CSA, a potent inhibitor of TCR signaling via the NFAT pathway (33). As shown by representative results from one such clone (Fig. 9A), target cell lysis was virtually unaffected by the presence of CSA, whereas IFN-{gamma} production was almost completely blocked. This result was reproducible with many different clones. Furthermore, as shown in Fig. 9B, CSA also inhibited the production of other cytokines such as TNF-{alpha} and IL-2, which the LMP-specific clones were found to make in response to Ag stimulation; all cytokines were reduced to ~10% of control levels or less. The LCL outgrowth assays were therefore repeated, now seeding matched and mismatched LCLs at a range of dilutions with or without the addition of CD4+ T cells and in the presence or absence of CSA. Typical results for six different CD4+ T cell clones are shown in Fig. 9C, in this case using peptide-pulsed LCLs as targets to maximize the T cell-mediated effect. As before, all of these CD4+ T cell clones specifically inhibited outgrowth of the relevant HLA-matched peptide-loaded LCL but not of its HLA-mismatched counterpart. In each case, there was marked growth inhibition despite the presence of CSA in the culture medium. Measuring cytokines in supernatant medium harvested from the two sets of cultures at 24 h confirmed that CSA had reduced cytokine production by ≥90%; indeed, outgrowth inhibition frequently occurred in wells in which cytokine production had been abolished below detectable levels (data not shown).


Figure 9
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 9. Mechanism of LCL outgrowth control. A, Representative results of combined chromium release (cytotoxicity) and IFN-{gamma} release assays in which CD4+ T cell clones of defined specificity (in this case TYG c5.162) were cocultured with HLA-matched LCL targets (previously peptide loaded) in the presence or absence of CSA at 0.25 µg/ml. Supernatants were harvested after 5 or 18 h and the same samples were assayed for chromium release from target cells and for IFN-{gamma} production from T cells. Results are expressed as percentage of specific lysis based on chromium release (upper panel) and on IFN-{gamma} production (lower panel) at T cell:target ratios of 5:1 ({blacksquare}) and 2.5:1 (Figure 9). B, Representative results of cytokine release assays measuring the levels of IL-2, TNF-{alpha}, and IFN-{gamma} release from 10,000 T cells cocultured for 18 h with an equal number of peptide-loaded HLA-matched target cells. Assays were conducted in the presence or absence of CSA as above. Results are shown as pg/ml for each cytokine from clones specific for the LMP1 epitopes SSH and LIW and for the LMP2 epitope TYG. Note that no clone ever produced detectable levels of IL-4 or IL-10 assayed in parallel (data not shown). C, Representative results from three independent LCL outgrowth assays conducted on peptide-loaded LCL targets, HLA-matched and -mismatched as in Fig. 8. These assays were conducted in standard medium or in the continual presence of 0.25 µg/ml CSA (+CSA) as above. Results are shown for clones against the LMP1 epitopes SSH, QAT, and LIW and the LMP2 epitopes TYG and ICL and are expressed as in Fig. 8.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The LMPs are part of a very limited range of EBV-latent cycle proteins expressed in such malignancies as HL and NPC. Although at least one of these proteins, LMP2, can serve as a CD8+ T cell target (9, 27, 34) it is debatable whether restoring LMP-specific CD8+ T cell responses in HL and NPC patients will on its own be sufficient to achieve a long-lasting therapeutic response. Including relevant CD4+ T cell reactivities could offer additional therapeutic benefit, either through their helper function or possibly as direct effectors. Before the present study, little was known about the LMPs as CD4+ T cell targets apart from some preliminary data from ELISPOT screening assays (21) and the presence of a LMP2-specific CD4+ T cell component within polyclonal effector populations stimulated in vitro either with the autologous LCLs (35) or with LMP2-transfected autologous dendritic cells (36). As summarized in Table I, in this study, we describe CD4+ T cell clones reactive to seven previously unidentified epitopes, four within LMP1 and three within LMP2, each restricted through a defined HLA class II allele. All of the clones displayed Th1-like patterns of cytokine production in response to Ag stimulation, secreting IFN-{gamma}, TNF-{alpha}, and IL-2 in the absence of typical Th2-like cytokines such as IL-4 and IL-10. From peptide titration assays, the majority of clones analyzed showed functional avidities within the 1–100 nM range previously observed in our laboratory for CD4+ T cell clones against defined epitopes within EBNA1, EBNA2, EBNA3A, or EBNA3C (23). Again, as in that earlier work, clones to the same epitope in many cases showed similar avidities but there were marked differences between clones to different epitopes; e.g., two overlapping epitopes in the LMP1 sequence, SSH and QAT, elicited CD4+ T cell clones with markedly different avidities of 40 and 1000 nM.

With this as background, we went on to look in detail at the capacity of all of the clones to recognize unmanipulated LCL cells (i.e., HLA class II-positive targets expressing LMP1 and LMP2 at physiologic levels) as an indicator of their likely therapeutic value in targeting LMP-positive tumors. These assays showed that, for all seven epitopes studied, one or more of the epitope-specific clones was capable of LCL recognition but at levels which again varied substantially between epitopes. For example, recognition of the unmanipulated LCL by LMP1-specific clones ranged from 1% of that seen against peptide-loaded targets for clones against the LIW and GPP epitopes to between 15 and 60% for clones against the SSH and QAT epitopes. Note that these latter values exceeded the strongest LCL reactivity reported for any EBV-latent Ag-specific CD4+ T cell response, namely, against the well-characterized PRS epitope in EBNA2 (23, 37, 38).

An important conclusion from these experiments is that the ability to recognize unmanipulated LCL targets does not correlate with functional avidity of the response. Thus, clones specific for the LIW epitope in LMP1 were 1000-fold more avid in peptide titration assays than clones specific for the QAT epitope, also in LMP1, yet the latter showed much stronger LCL recognition. We infer that, just as for CD8+ T cell recognition (39), the cell surface level of CD4 epitope display is likely to be a key factor influencing the potential of any one particular epitope as a therapeutic target. Thus, in LCLs with the relevant HLA type, there must be substantially greater levels of the QAT-DQ2 complex than the LIW-DR16 complex on the cell surface. Such differences in epitope display can clearly override the effects of response avidity and, as a result, one cannot assume that epitopes inducing the highest avidity responses will necessarily be the most effective therapeutic targets. Within the context of any one epitope, however, clones with the highest avidity in peptide titration assays are likely to be the most useful therapeutically. For example, the more avid clones against LMP2 epitopes TYG and VTF gave detectable LCL recognition, whereas the less avid clones did not.

A second important conclusion from these experiments is that, although the different epitope responses vary greatly in their capacity for LCL recognition, this is not obviously reflected in the size of these responses in vivo as detected by ELISPOT screening. For example, responses to two DQ6-restricted epitopes, SSH and TYG, differ radically in LCL recognition, yet these responses are often of equivalent size (see Figs. 1 and 3). Larger scale ELISPOT screening of additional donors has confirmed that no one LMP1 or LMP2 epitope stands out as inducing either consistently high or consistently low responses (28). In light of these findings, it is clear that a numerically dominant response is not necessarily going to be the most valuable therapeutically. We infer that this lack of relationship between an LMP epitope’s immunogenicity and its level of display on naturally infected target cells reflects the fact that LMP-specific CD4+ T cell responses are unlikely to be primed by direct contact with EBV-infected B cells in vivo. More likely, these responses will be cross-primed by dendritic cells that have acquired the LMP proteins as exogenous Ags shed from infected cells. The implication, yet to be explored, is that these multiple membrane-spanning proteins are handled differently by the MHC class II processing pathway when exogenously acquired by APCs than when endogenously expressed in B cells.

Moving on to the functional analysis of the LMP-specific CD4+ T cell clones, we found that most were capable not only of secreting cytokines in response to peptide-loaded targets but also of killing these cells in cytotoxicity assays. As seen with cytotoxic CD4+ T cells to EBNA-derived epitopes (17, 23), killing was often better detected in overnight (18 h) than in shorter-term chromium release assays. The mechanism of killing by EBV-specific CD4+ clones remains unresolved in the literature with one report suggesting a Fas-dependent pathway (17), another a perforin-dependent pathway (36), and another a pathway that was perforin-independent but involved exocytosis of granulysin (40). Our study of LMP1- and LMP2-specific CD4 responses strongly suggests target cell lysis by a perforin- and granzyme-dependent mechanism. Thus, we noted that all 14 cytotoxic clones expressed both perforin and granzyme A, whereas the one noncytotoxic clone expressed granzyme A but was perforin negative; it is known that perforin is required for granzyme effector proteins to mediate target cell killing (41). Furthermore, concanamycin A, an inhibitor of the vacuole acidification required for perforin-dependent killing (30), blocked target cell lysis by these CD4 clones, whereas the antagonistic anti-Fas mAb ZB4 had no blocking effect. CD4+ T cell killing via the perforin pathway has now been reported in a number of human cell studies in vitro (42, 43, 44, 45, 46). Given the limited sensitivity of direct ex vivo cytotoxicity assays, we have been unable to determine the cytotoxic function of LMP-specific CD4+ T cells before in vitro expansion. However, it is interesting to note that perforin-positive CD4+ T cells have also been detected in vivo both in healthy individuals (47) and in disease states (48), strongly suggesting that the cytotoxic capacity of the presently described clones is not purely an in vitro artifact.

Arguably, the best in vitro correlate of a T cell clone’s therapeutic potential is the ability to prevent the outgrowth of target cells in long-term coculture assays. In this study, we show that those clones with the strongest reactivity against unmanipulated LCL targets in IFN-{gamma} and cytotoxicity assays are indeed by far the most efficient inhibitors of LCL outgrowth. This raised the question whether cytokine release or cell killing was the more important effector mechanism of growth control. In this regard, the only clone incapable of blocking the outgrowth of peptide-loaded LCL targets was the one clone that lacked cytotoxic ability. Furthermore, CSA was able to block cytokine release by our CD4+ T cell clones without affecting target cell lysis, and the presence of CSA in cocultures had little or no impact on outgrowth inhibition. Although we cannot rule out an as-yet-unidentified cytokine whose production by CD4+ T cells is CSA independent, all our data point to cytotoxicity as the key property required by CD4+ T cells to act as direct effectors of growth control.

The present work, which represents the first description of CD4+ T cell clones against defined epitopes in LMP1 and LMP2, is important in two respects. First, it provides tools with which to examine the route(s) whereby such highly hydrophobic membrane proteins access the HLA class II pathway for presentation to CD4+ T cells. Second, it proves that there are components of CD4 memory that are specific for the LMPs, proteins expressed in a wide range of EBV-associated malignancies, and have the ability to directly recognize LMP-expressing target cells. Since LMP-specific CD4+ T cells are present in the blood of a sizeable proportion of patients with NPC, exploiting the therapeutic potential of these reactivities is an important priority for future work.


   Acknowledgments
 
We thank A. Milner for advice on Fas blocking Abs, J. Curnow for help with the multiplex cytokine assay, and N. Gudgeon and D. Sauce for advice and assistance with flow cytometry (all from the University of Birmingham, Birmingham, U.K.). We are also grateful to E. Houssaint and E. Landais (University of Nantes, Nantes, France) for advice on intracellular staining for perforin and granzyme.


   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 grants from Cancer Research U.K. and the Hong Kong Cancer Fund. Back

2 Address correspondence and reprint requests to Dr. Alan B. Rickinson, Cancer Research United Kingdom Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, U.K. E-mail address: a.b.rickinson{at}bham.ac.uk Back

3 Abbreviations used in this paper: PTLD, posttransplant lymphoproliferative disease; EBNA, EBV-encoded nuclear Ag; LCL, lymphoblastoid cell line; HL, Hodgkin’s lymphoma; NPC, nasopharyngeal carcinoma; CSA, cyclosporin A. Back

Received for publication July 11, 2007. Accepted for publication November 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Haque, T., G. M. Wilkie, C. Taylor, P. L. Amlot, P. Murad, A. Iley, D. Dombagoda, K. M. Britton, A. J. Swerdlow, D. H. Crawford. 2002. Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet 360: 436-442. [Medline]
  2. Rooney, C. M., C. A. Smith, C. Y. Ng, S. K. Loftin, J. W. Sixbey, Y. Gan, D. K. Srivastava, L. C. Bowman, R. A. Krance, M. K. Brenner, H. E. Heslop. 1998. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92: 1549-1555. [Abstract/Free Full Text]
  3. Khanna, R., S. Bell, M. Sherritt, A. Galbraith, S. R. Burrows, L. Rafter, B. Clarke, R. Slaughter, M. C. Falk, J. Douglass, et al 1999. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc. Natl. Acad. Sci. USA 96: 10391-10396. [Abstract/Free Full Text]
  4. Khanna, R., S. R. Burrows, M. G. Kurilla, C. A. Jacob, I. S. Misko, T. B. Sculley, E. Kieff, D. J. Moss. 1992. Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development. J. Exp. Med. 176: 169-176. [Abstract/Free Full Text]
  5. Murray, R. J., M. G. Kurilla, J. M. Brooks, W. A. Thomas, M. Rowe, E. Kieff, A. B. Rickinson. 1992. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J. Exp. Med. 176: 157-168. [Abstract/Free Full Text]
  6. Young, L. S., A. B. Rickinson. 2004. Epstein-Barr virus: 40 years on. Nat. Rev. Cancer. 4: 757-768. [Medline]
  7. Blake, N., S. Lee, I. Redchenko, W. Thomas, N. Steven, A. Leese, P. Steigerwald-Mullen, M. G. Kurilla, L. Frappier, A. Rickinson. 1997. Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 7: 791-802. [Medline]
  8. Blake, N., T. Haigh, G. Shaka’a, D. Croom-Carter, A. Rickinson. 2000. The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1. J. Immunol. 165: 7078-7087. [Abstract/Free Full Text]
  9. Lee, S. P., R. J. Tierney, W. A. Thomas, J. M. Brooks, A. B. Rickinson. 1997. Conserved CTL epitopes within EBV latent membrane protein 2: a potential target for CTL-based tumor therapy. J. Immunol. 158: 3325-3334. [Abstract]
  10. Woodberry, T., T. J. Suscovich, L. M. Henry, J. K. Davis, N. Frahm, B. D. Walker, D. T. Scadden, F. Wang, C. Brander. 2005. Differential targeting and shifts in the immunodominance of Epstein-Barr virus–specific CD8 and CD4 T cell responses during acute and persistent infection. J. Infect. Dis. 192: 1513-1524. [Medline]
  11. Bollard, C. M., L. Aguilar, K. C. Straathof, B. Gahn, M. H. Huls, A. Rousseau, J. Sixbey, M. V. Gresik, G. Carrum, M. Hudson, et al 2004. Cytotoxic T lymphocyte therapy for Epstein-Barr virus+ Hodgkin’s disease. J. Exp. Med. 200: 1623-1633. [Abstract/Free Full Text]
  12. Straathof, K. C., C. M. Bollard, U. Popat, M. H. Huls, T. Lopez, M. C. Morriss, M. V. Gresik, A. P. Gee, H. V. Russell, M. K. Brenner, et al 2005. Treatment of nasopharyngeal carcinoma with Epstein-Barr virus-specific T lymphocytes. Blood 105: 1898-1904. [Abstract/Free Full Text]
  13. Rocha, B., C. Tanchot. 2004. Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses. Curr. Opin. Immunol. 16: 259-263. [Medline]
  14. Lechler, R., G. Aichinger, L. Lightstone. 1996. The endogenous pathway of MHC class II antigen presentation. Immunol. Rev. 151: 51-79. [Medline]
  15. Lich, J. D., J. F. Elliott, J. S. Blum. 2000. Cytoplasmic processing is a prerequisite for presentation of an endogenous antigen by major histocompatibility complex class II proteins. J. Exp. Med. 191: 1513-1524. [Abstract/Free Full Text]
  16. Nimmerjahn, F., S. Milosevic, U. Behrends, E. M. Jaffee, D. M. Pardoll, G. W. Bornkamm, J. Mautner. 2003. Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy. Eur. J. Immunol. 33: 1250-1259. [Medline]
  17. Paludan, C., K. Bickham, S. Nikiforow, M. L. Tsang, K. Goodman, W. A. Hanekom, J. F. Fonteneau, S. Stevanovic, C. Munz. 2002. Epstein-Barr nuclear antigen 1-specific CD4+ Th1 cells kill Burkitt’s lymphoma cells. J. Immunol. 169: 1593-1603. [Abstract/Free Full Text]
  18. Hegde, N. R., C. Dunn, D. M. Lewinsohn, M. A. Jarvis, J. A. Nelson, D. C. Johnson. 2005. Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL. J. Exp. Med. 202: 1109-1119. [Abstract/Free Full Text]
  19. Wang, R. F., X. Wang, A. C. Atwood, S. L. Topalian, S. A. Rosenberg. 1999. Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen. Science 284: 1351-1354. [Abstract/Free Full Text]
  20. Weiss, S., B. Bogen. 1991. MHC class II-restricted presentation of intracellular antigen. Cell 64: 767-776. [Medline]
  21. Leen, A., P. Meij, I. Redchenko, J. Middeldorp, E. Bloemena, A. Rickinson, N. Blake. 2001. Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4+ T-helper 1 responses. J. Virol. 75: 8649-8659. [Abstract/Free Full Text]
  22. Khanna, R., S. R. Burrows, P. M. Steigerwald-Mullen, S. A. Thomson, M. G. Kurilla, D. J. Moss. 1995. Isolation of cytotoxic T lymphocytes from healthy seropositive individuals specific for peptide epitopes from Epstein-Barr virus nuclear antigen 1: implications for viral persistence and tumor surveillance. Virology 214: 633-637. [Medline]
  23. Long, H. M., T. A. Haigh, N. H. Gudgeon, A. M. Leen, C. W. Tsang, J. Brooks, E. Landais, E. Houssaint, S. P. Lee, A. B. Rickinson, G. S. Taylor. 2005. CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines. J. Virol. 79: 4896-4907. [Abstract/Free Full Text]
  24. Mautner, J., D. Pich, F. Nimmerjahn, S. Milosevic, D. Adhikary, H. Christoph, K. Witter, G. W. Bornkamm, W. Hammerschmidt, U. Behrends. 2004. Epstein-Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells. Eur. J. Immunol. 34: 2500-2509. [Medline]
  25. Cheung, S. T., S. F. Leung, K. W. Lo, K. W. Chiu, J. S. Tam, T. F. Fok, P. J. Johnson, J. C. Lee, D. P. Huang. 1998. Specific latent membrane protein 1 gene sequences in type 1 and type 2 Epstein-Barr virus from nasopharyngeal carcinoma in Hong Kong. Int. J. Cancer 76: 399-406. [Medline]
  26. Taylor, G. S., T. A. Haigh, N. H. Gudgeon, R. J. Phelps, S. P. Lee, N. M. Steven, A. B. Rickinson. 2004. Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma. J. Virol. 78: 768-778. [Abstract/Free Full Text]
  27. Lautscham, G., S. Mayrhofer, G. Taylor, T. Haigh, A. Leese, A. Rickinson, N. Blake. 2001. Processing of a multiple membrane spanning Epstein-Barr virus protein for CD8+ T cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway. J. Exp. Med. 194: 1053-1068. [Abstract/Free Full Text]
  28. Lin, X., N. H. Gudgeon, E. P. Hui, H. Jia, X. Qun, G. S. Taylor, M. C. N. M. Barnardo, C. K. Lin, A. B. Rickinson, and A. T. C. Chan. 2007. CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients. Cancer Immunol. Immunother. In press.
  29. Wilson, A. D., I. Redchenko, N. A. Williams, A. J. Morgan. 1998. CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis. Int. Immunol. 10: 1149-1157. [Abstract/Free Full Text]
  30. Kataoka, T., N. Shinohara, H. Takayama, K. Takaku, S. Kondo, S. Yonehara, K. Nagai. 1996. Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 156: 3678-3686. [Abstract]
  31. Milner, A. E., D. H. Palmer, E. A. Hodgkin, A. G. Eliopoulos, P. G. Knox, C. J. Poole, D. J. Kerr, L. S. Young. 2002. Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells. Cell Death Differ. 9: 287-300. [Medline]
  32. Slifka, M. K., F. Rodriguez, J. L. Whitton. 1999. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature 401: 76-79. [Medline]
  33. Rao, A., C. Luo, P. G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15: 707-747. [Medline]
  34. Hislop, A. D., G. S. Taylor, D. Sauce, A. B. Rickinson. 2007. Cellular responses to viral infection in humans: lessons from Epstein-Barr virus. Annu. Rev. Immunol. 25: 587-617. [Medline]
  35. Straathof, K. C., A. M. Leen, E. L. Buza, G. Taylor, M. H. Huls, H. E. Heslop, C. M. Rooney, C. M. Bollard. 2005. Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma. J. Immunol. 175: 4137-4147. [Abstract/Free Full Text]
  36. Su, Z., M. V. Peluso, S. H. Raffegerst, D. J. Schendel, M. A. Roskrow. 2002. Antigen presenting cells transfected with LMP2a RNA induce CD4+ LMP2a-specific cytotoxic T lymphocytes which kill via a Fas-independent mechanism. Leuk. Lymphoma. 43: 1651-1662. [Medline]
  37. Khanna, R., S. R. Burrows, S. A. Thomson, D. J. Moss, P. Cresswell, L. M. Poulsen, L. Cooper. 1997. Class I processing-defective Burkitt’s lymphoma cells are recognized efficiently by CD4+ EBV-specific CTLs. J. Immunol. 158: 3619-3625. [Abstract]
  38. Omiya, R., C. Buteau, H. Kobayashi, C. V. Paya, E. Celis. 2002. Inhibition of EBV-induced lymphoproliferation by CD4+ T cells specific for an MHC class II promiscuous epitope. J. Immunol. 169: 2172-2179. [Abstract/Free Full Text]
  39. Levitsky, V., Q. J. Zhang, J. Levitskaya, M. G. Masucci. 1996. The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4. J. Exp. Med. 183: 915-926. [Abstract/Free Full Text]
  40. Sun, Q., R. L. Burton, K. G. Lucas. 2002. Cytokine production and cytolytic mechanism of CD4+ cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus-specific T-cell cultures. Blood 99: 3302-3309. [Abstract/Free Full Text]
  41. Pinkoski, M. J., M. Hobman, J. A. Heibein, K. Tomaselli, F. Li, P. Seth, C. J. Froelich, R. C. Bleackley. 1998. Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis. Blood 92: 1044-1054. [Abstract/Free Full Text]
  42. Khanolkar, A., H. Yagita, M. J. Cannon. 2001. Preferential utilization of the perforin/granzyme pathway for lysis of Epstein-Barr virus-transformed lymphoblastoid cells by virus-specific CD4+ T cells. Virology 287: 79-88. [Medline]
  43. Miskovsky, E. P., A. Y. Liu, W. Pavlat, R. Viveen, P. E. Stanhope, D. Finzi, W. M. Fox, III, R. H. Hruban, E. R. Podack, R. F. Siliciano. 1994. Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines. J. Immunol. 153: 2787-2799. [Abstract]
  44. Vergelli, M., B. Hemmer, P. A. Muraro, L. Tranquill, W. E. Biddison, A. Sarin, H. F. McFarland, R. Martin. 1997. Human autoreactive CD4+ T cell clones use perforin- or Fas/Fas ligand-mediated pathways for target cell lysis. J. Immunol. 158: 2756-2761. [Abstract]
  45. Yasukawa, M., H. Ohminami, J. Arai, Y. Kasahara, Y. Ishida, S. Fujita. 2000. Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans. Blood 95: 2352-2355. [Abstract/Free Full Text]
  46. Yasukawa, M., H. Ohminami, Y. Yakushijin, J. Arai, A. Hasegawa, Y. Ishida, S. Fujita. 1999. Fas-independent cytotoxicity mediated by human CD4+ CTL directed against herpes simplex virus-infected cells. J. Immunol. 162: 6100-6106. [Abstract/Free Full Text]
  47. Appay, V., J. J. Zaunders, L. Papagno, J. Sutton, A. Jaramillo, A. Waters, P. Easterbrook, P. Grey, D. Smith, A. J. McMichael, et al 2002. Characterization of CD4+ CTLs ex vivo. J. Immunol. 168: 5954-5958. [Abstract/Free Full Text]
  48. Namekawa, T., U. G. Wagner, J. J. Goronzy, C. M. Weyand. 1998. Functional subsets of CD4 T cells in rheumatoid synovitis. Arthritis Rheum. 41: 2108-2116. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Haigh, T. A.
Right arrow Articles by Taylor, G. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haigh, T. A.
Right arrow Articles by Taylor, G. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS