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Immunology |
CRC Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, United Kingdom [A. L. N. C., A. B. R., W. A. T., S. P. L.]; Leukaemia Research Fund Virus Centre, Department of Veterinary Pathology, Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom [R. F. J.]; and Department of Cellular Pathology, Birmingham Heartlands Hospital, Birmingham B9 5SS, United Kingdom [J. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of HD3
cases in Western countries and a far higher proportion in some developing regions are associated with EBV infection of the malignant H-RS cells (1, 2, 3)
. The role of EBV in the etiology of HD remains unclear, but its oncogenic potential is apparent from its ability to transform human B cells in vitro into permanently growing LCLs and its association with a number of other human malignancies (4
, 5)
. Despite its oncogenic potential, EBV is widespread in the human population, where it persists as an asymptomatic infection of B cells. The evidence suggests that HLA class I-restricted CTLs play a key role in controlling EBV in healthy virus carriers (reviewed in Ref. 6 ). Given the importance of CTLs in the control of EBV infection, clinical studies have explored the possibility of infusing EBV-specific T-cell lines to treat or prevent the outgrowth of EBV-positive lymphomas that can occur in transplant recipients (7, 8, 9) . The results of these studies not only support the role of CD8+ CTLs in controlling EBV infection but also demonstrate the efficacy of adoptive T-cell therapy for treating EBV-positive lymphomas in immunosuppressed individuals. Therefore, there is now considerable interest in the possibility of extending this approach to treat other EBV-positive malignancies. A T-cell-based therapy for EBV-positive cases of HD may be of particular value for treating advanced stage or relapsed disease, in which conventional therapeutic strategies are rarely curative, and would also avoid the late complications seen with radiotherapy and chemotherapy, such as myocardial damage and the development of secondary tumors (reviewed Refs. 10 , 11 ).
EBV-specific CTL responses in healthy virus carriers have been studied extensively by cocultivating PBMCs in vitro with the autologous EBV-transformed LCL. Within an LCL, EBV establishes a predominantly latent infection with the expression of at least eight viral proteins, i.e., six nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C, and LP) and two LMPs (LMP1 and LMP2; Ref. 4 ). RNA transcripts from the BARF0 open reading frame in the BamHI A region of the viral genome have also been detected (12) . Studies on T-cell responses in healthy virus carriers have demonstrated a hierarchy of immunodominance among EBV latent antigens. Thus, for most donors, the T-cell response generated in vitro after LCL stimulation is dominated by cells specific for the EBNA3 family of proteins (EBNA3A, EBNA3B, and EBNA3C) with subdominant reactivities detectable to EBNA2, LP, LMP1, and/or LMP2 (13 , 14) . EBNA1 is protected from processing by the classical HLA class I route, because of the presence of an internal GAr region (15 , 16) . However, using targets expressing a GAr-deleted EBNA1 molecule, CTLs specific for this protein have been identified in several donors (17) . CTLs that recognize an HLA-A2-restricted epitope within BARF0 have also been described (18 , 19) .
In contrast to an LCL, EBV-infected H-RS cells display a more restricted pattern of viral latent gene expression. Thus, immunohistochemical studies have demonstrated the expression of EBNA1, LMP1, and LMP2 proteins in H-RS cells (20, 21, 23) , and transcriptional studies indicate that a BARF0 protein product may also be expressed (24) . However, the immunodominant EBNA3 family of proteins is not present. Nevertheless, several CTL target epitopes have now been defined in LMP1 and particularly in LMP2, many of which are restricted through common HLA alleles (e.g., HLA-A2; Refs. 25, 26, 27 ).
The expression of known CTL target antigens in H-RS cells offers the potential for a CTL-based therapy for HD. However, to develop an effective therapy we must first determine why the host’s immune response has failed to clear the tumor. One possibility is that H-RS cells cannot be recognized by CTLs because of a defect in the HLA class I antigen processing pathway. However, most EBV-positive Hodgkin’s tumors have high levels of expression of HLA class I molecules and of the transporters associated with antigen processing (TAPs 1 and 2; Refs. 28, 29, 30 ). Furthermore, HD-derived cell lines can process and present vector-introduced EBV antigens to specific HLA class I-restricted CTLs with resultant killing of the H-RS cell (28 , 31) .
Another possibility to explain the persistence of EBV-positive H-RS cells is that the host fails to mount a CTL response to those viral proteins present in the tumor. A few reports have attempted to study EBV-specific CTL responses in the blood of HD patients with EBV-positive disease, but these have largely analyzed polyclonal T-cell populations generated after stimulation of PBMCs with the autologous LCL (32 , 33) . Such polyclonal populations are usually dominated by reactivities to the EBNA3 antigens, and thus weaker responses to tumor-associated viral antigens, such as the LMPs, may have been masked. In one study, an LCL-reactivated polyclonal line from a single HD patient was cloned by limiting dilution, and a single LMP2-specific clone identified; however, the EBV status of this patient’s tumor was not reported (31) .
A third possibility is that LMP-specific CTLs fail to access the tumor site or fail to function in the tumor microenvironment. Currently, there is little information available on this issue, although one study reported that EBV-specific CTLs are suppressed in EBV-positive tumors while still present in the circulating lymphocyte pool of the same patient (32) . Furthermore, EBV-positive H-RS cells produce a number of immunosuppressive cytokines including IL-10 and transforming growth factor-ß (34 , 35) .
In the present study, we have addressed some of these issues by conducting a detailed analysis of the EBV-specific CTL response in HD patients: (a) EBV-specific CTLs were reactivated from peripheral blood using the autologous LCL, followed by limiting dilution cloning of responder cells to enable detection of subdominant responses. The same approach was also used to analyze EBV-specific responses in TILs from EBV-positive and -negative Hodgkin’s tumors; and (b) an Elispot assay was used to quantitate CTL responses to defined LMP1 and LMP2 epitopes in PBMCs and TILs from HD patients.
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MATERIALS AND METHODS |
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), having previously obtained informed consent. Blood was fractionated on Ficoll density gradients, and PBMCs were cryopreserved. Tumor biopsy material was collected, rinsed with RPMI 1640 to remove any traces of blood, and then disaggregated using a scalpel or Medimachine (Dako, Ltd.). Cell suspensions were centrifuged on Ficoll density gradients to purify mononuclear cells and stored frozen.
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Cell Lines.
LCLs were generated in vitro by transformation of B cells using the EBV isolate B95.8 (37)
and cultured in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin (growth medium). PHA-activated T-cell blasts were generated by stimulation of PBMCs with PHA (10 µg/ml; Murex Biotek, Chattillon, France).
Reactivation of EBV-specific CTLs from Blood and Tumor Biopsy Material.
PBMCs and TILs from HD patients were stimulated in vitro with the autologous LCL (irradiated at 4000 rads) at a responder:stimulator ratio of 40:1. Cells were cultured in T-cell medium (growth medium containing 1% pooled human AB serum; Sigma Chemical Co., Poole, United Kingdom). After 7 days, fresh medium and autologous LCL (irradiated) were added to the culture. On day 14, cells were cloned by limiting dilution to 0.3 and 3 cells/well (five 96-well plates for each cell dilution) and maintained in IL-2-conditioned medium by intermittent restimulation with irradiated autologous LCL, as described previously (26)
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Chromium Release Assays.
Clones derived from PBMCs and TILs were screened using a standard 4-h chromium release assay for EBV specificity. Clones were tested against a panel of target cells expressing individual EBV antigens from recombinant vaccinia vectors as described previously (26)
. The vaccinia constructs used in this study have all been described previously (13
, 38)
. Clones were screened for EBNA1 specificity using a truncated form of EBNA1 (termed E1
GA) lacking the GAr region. In most cases, the target cell used for this study was the autologous LCL that expresses all EBV latent proteins. As reported previously, many EBV-specific clones generated in vitro from healthy virus carriers can lyse an LCL coated with the cognate viral peptide or expressing the target EBV antigen from a vaccinia vector but mediate little or no lysis of the LCL alone, although the CTLs were initially reactivated using this EBV-positive cell line (39)
. Clones that mediated high background levels of lysis of the LCL alone on initial screening were retested after 7–14 days of culture, by which time killing of the LCL alone had reduced sufficiently to identify the target EBV antigen. For the purposes of this study, the definition of EBV target specificity required that the percentage of specific lysis of a target cell expressing one EBV antigen be at least double that observed with targets expressing the other EBV antigens, and that this value exceeded all others by at least 15% specific lysis. All antigen-specific responses were confirmed in at least two replicate assays.
In some cases, having identified the target viral protein, clones were tested for recognition of peptide epitopes defined previously in this molecule and that were appropriate for the HLA type of the donor (see Tables 1
and 2
). Where assays involved the use of synthetic peptides (peptide sensitization assays) 51CrO4-labeled targets were plated out in growth medium (100 µl/well) containing a known concentration of peptide. Cells were then incubated for 1 h before the addition of CTLs (100 µl/well). Recorded peptide concentrations refer to those in the final 200-µl volume. Peptides were synthesized using fluorenylmethoxycarbonyl chemistry by Dr. John Fox (Alta Bioscience, University of Birmingham, Birmingham, United Kingdom). They were dissolved in DMSO, and protein concentrations were measured using a modified Biuret assay. None of the peptides mediated target cell lysis in the absence of CTLs.
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monoclonal antibody 1-DIK (Mabtech, Stockholm, Sweden) for 3 h at room temperature. Plates were then washed six times with RPMI 1640 and blocked with RPMI 1640 + 10% FCS for 1 h. PBMCs or TILs were added in duplicate wells in 100-µl volumes at 105, 2 x 105, and 4 x 105 cells/well (cell input number) in the presence of 2 µg/ml peptide. Wells containing PHA (100 µg/ml) or an equivalent dilution of DMSO solvent were used as positive and negative controls, respectively. Plates were incubated overnight at 37°C with 5% CO2. Cells were discarded the next day, and plates were washed six times with 0.05% Tween 20 diluted in PBS. A biotinylated anti-IFN- monoclonal antibody 7-B6-1 (Mabtech) was added at 1 µg/ml and left for 3 h at room temperature. After 6 additional washes, a 1:1000 dilution of streptavidin-conjugated alkaline phosphatase was added for 2 h. Plates were washed again six times. Individual cytokine-producing cells were detected as dark spots after a 30-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium using an alkaline phosphatase-conjugate substrate kit (Bio-Rad, Richmond, CA). Spots were counted under a dissection microscope. The number of specific T-cell responders was calculated by taking the mean number of spots from duplicate wells and subtracting the mean number of spots in negative control wells with the same cell input number. Where more than one input cell number was used, mean corrected values were plotted on a graph, and a line of best fit was drawn through the points to obtain the frequency of specific precursors. Results are expressed as number of SFCs/106 PBMCs or TILs. |
RESULTS |
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, where clones demonstrate clear specificity for GAr-deleted EBNA1, EBNA3A, EBNA3C or LMP2; results from all donors are summarized in Table 3
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GA, LMP2, and EBNA2 were seen less frequently, whereas CTLs specific for LP, LMP1, or BARF0 were not detected in this patient cohort. A similar pattern of responses was seen in patients with EBV-negative and -positive HD. Donor HD1 was EBV-seronegative but was included to investigate whether LCL reactivation of PBMCs was capable of eliciting a primary EBV-specific response in vitro. Two hundred and forty clones were generated from the blood of this donor, but none was specific for any of the viral antigens tested.
Two individuals in each group showed relatively weak reactivity to LMP2, one of the antigens expressed in EBV-positive HD. In two cases (HD4 and HD14), responses were HLA-A2 restricted (data not shown), and the target epitopes were defined as LLW and CLG, respectively (Fig. 2)
. Responses to the GAr-deleted form of EBNA1 (E1GA) were seen in three individuals, and in one case (HD5) mapped to the HLA B35-restricted epitope HPV.
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, the response was clearly restricted through HLA B38, and although the donor carries the B38.01 subtype, clones recognized EBNA2 on a background of either B38.01 or B38.02 subtypes. Using peptide sensitization assays, the minimal target epitope was mapped to residues 14–23 (sequence YHLIVDTDSL; Fig. 3B
). This epitope sequence accords well with the predicted HLA B38.01 peptide binding motif (49)
, having a histidine residue at position 2 and leucine at the COOH terminus.
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. Representative results of TIL-derived EBNA3A-specific CTL clones from an EBV-positive tumor (HD18) are illustrated in Fig. 4A
. Of 30 such clones derived from this tumor biopsy, 18 were specific for the HLA B8-restricted epitope QAK (Fig. 4B)
. In one case (HD5), we identified CTLs specific for the B35-restricted EBNA1 epitope HPV, but LMP1 or LMP2-specific CTLs were not detected in any biopsy. No EBV-specific CTLs were isolated from the biopsy of patient HD2. This donor was EBV seronegative, which again suggests that our reactivation protocol had not generated primary responses in vitro.
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and 4
). In most cases, similar reactivities were seen at both sites, and where the target epitope was determined, this was also conserved. Only one of these patients (HD14) carried a detectable LMP2-specific response in the blood (5 clones of 235 tested). Such a response was not detected in the tumor biopsy from this patient; however, this may reflect the fact that considerably fewer clones were available for testing from the biopsy (n = 67).
Quantitation of Circulating LMP1/2-specific CTL Precursor Frequencies by Elispot Assay.
The above results indicate that CTL precursors specific for the tumor-associated viral antigens LMP1 and LMP2 are reactivated only infrequently from peripheral blood of HD patients after in vitro stimulation with the autologous LCL. However, this approach may underestimate the true number of precursors because it requires that antigen-specific T cells proliferate over at least 5 weeks of in vitro culture and that they mediate cytotoxic function. Therefore, to complement our studies using LCL reactivation, we analyzed EBV-specific T-cell responses in HD patients and healthy EBV carriers using the Elispot assay. This avoids the need for prolonged in vitro culture and provides more quantitative data on the frequency of T cells specific for a given epitope.
The Elispot assay was applied to blood samples from 11 HD patients and 12 healthy EBV carriers to measure precursor frequencies of T cells specific for known peptide epitopes within LMP1 and LMP2. The epitopes studied included two HLA-A2-restricted epitopes in LMP1 (YLL and YLQ) and four epitopes within LMP2 [CLG, LLW, and FLY (HLA-A2-restricted); TYG (HLA-A24-restricted)]. The immunodominant HLA-A2-restricted GLC epitope from the EBV lytic cycle protein BMLF1 was included as a positive control (50)
. Eight patients had EBV-negative tumors, whereas three had EBV-positive disease. Assays were performed in duplicate at three input cell numbers for all patients except HD12 (performed at 125,000 cells/well, in duplicate) and HD21 (performed at 170,000 cells/well, in duplicate). Results are shown in Table 5
.
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Quantitation of EBV-specific CTL Precursor Frequencies in TILs from HD Biopsies by Elispot Assay.
Cryopreserved TILs were available from six HD patients, two with EBV-negative tumors, and four with EBV-positive tumors. TILs were thawed, purified on a Ficoll density gradient, and used directly in Elispot assays. Epitopes used in this study were selected for each individual according to their HLA type and in some cases also on the reactivities detected previously by LCL stimulation (Table 4)
. Results are summarized in Table 6
and, where possible, are compared with data obtained from the patients’ blood samples.
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and also with data obtained by LCL reactivation of PBMCs and TILs (Tables 3
and 4
). It should be noted, however, that the frequency of responses in TILs was almost always significantly lower than that observed in PBMCs for this patient; indeed, the relatively strong responses to the LMP1 epitopes YLL and YLQ were almost undetectable in the TIL population. In contrast, Elispot results from donor HD9 (EBV-negative HD) showed an increased response to the LMP1 epitope YLL in TILs compared with PBMCs. HD9 also showed a strong response both in TILs and PBMCs to the EBNA3A epitope RPP, which confirmed results obtained by LCL reactivation (see Table 3
and 4
). TILs from four EBV-positive tumors were examined, and EBV-specific T-cell precursors were detected in all cases. The HLA types of these patients meant that in only one case (HD24) was it possible to screen for a known epitope in LMP2. T cells specific for the HLA-A24-restricted TYG epitope were detected at a frequency of 35 SFCs/106 TILs from this patient.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); minor responses were only detectable after cloning. Immunohistochemical studies on HD biopsies and in vitro analysis of H-RS cell lines suggest that H-RS cells are capable of presenting endogenously synthesized EBV proteins. Therefore, because of an increased antigenic load, it was conceivable that CTL responses to viral proteins expressed in the tumor might be increased in EBV-positive HD patients. However, the results summarized in Table 3
indicated that HD patients display a similar pattern of immunodominance to that observed in healthy EBV carriers (13
, 14
, 38)
. Thus, in the majority of cases, the dominant response was to one or other of the EBNA3 family of proteins. Responses to LMP2 were relatively weak, and responses to LMP1 and BARF0 were undetectable. Note that CTLs specific for the GAr-deleted form of EBNA1 (E1GA) were detected in blood samples from some HD patients, but these effectors are unlikely to target an EBV-positive H-RS cell because full-length EBNA1 protein expressed in the tumor will not be processed and presented to HLA class I-restricted T cells (15
, 16)
. Although numbers were small, there was no obvious difference between patients with EBV-negative and -positive HD in the frequency and pattern of CTL responses reactivated using this protocol.
Using the more sensitive Elispot assay, we measured circulating precursor frequencies to predefined A2- and A24-restricted CTL target epitopes in LMP1 and LMP2. Responses to at least one LMP-derived epitope were detectable in the blood of all EBV-seropositive HD patients; however, precursor frequencies were generally low when compared with the immunodominant EBV lytic cycle epitope GLC. These results were again comparable with those seen in healthy EBV carriers (Table 5)
. Previous reports have claimed that HD patients often possess a generalized defect in cell-mediated immunity, including an impaired response to T-cell mitogens and a decreased capacity of T cells to respond in a mixed lymphocyte response (51)
. However, using both LCL reactivation and Elispot assays, we observed no obvious suppression of the EBV-specific CTL response in HD patients when compared with healthy EBV carriers.
Having observed a weak LMP-specific CTL response in the blood of EBV-positive HD cases, it was important to determine whether such responses could also be detected at the tumor site. The only other study to examine virus-specific CTL responses at the tumor site of EBV-positive HD patients was reported by Frisan et al. (32)
and revealed evidence for local suppression of virus-specific CTLs. Thus, by culturing TILs in IL-2-conditioned medium, they were able to isolate EBV-specific polyclonal CTL lines from three of three EBV-negative HD biopsies but none of six EBV-positive tumors. Furthermore, by studying a single patient with EBV-positive disease, they were able to use the autologous LCL to reactivate an EBV-specific polyclonal CTL line from the blood but not from the tumor biopsy. However, again using the autologous LCL, we were able to reactivate EBV-specific CTL clones not only from the biopsy of EBV-negative tumors but also from three of three EBV-positive tumors (Table 4
and Fig. 4
). The difference between our findings and those of Frisan et al. (32)
may be explained by the fact that in our study tumor-derived EBV-specific responses were often weak and therefore may have gone undetected in a T-cell line without limiting dilution cloning. Our results therefore demonstrate that EBV-specific effectors are present in at least some EBV-positive HD tumors, and that given the appropriate stimulus, they can be reactivated and expanded in vitro. Nevertheless, it should be noted that none of the tumor-derived clones targeted EBV proteins known to be expressed in H-RS cells. In one case (HD14), the donor was known to possess a relatively weak LMP2-specific response in their blood (Table 3)
; yet no such response could be identified in their tumor. This may simply reflect the fact that fewer clones were isolated from the tumor than from the blood of this patient, but it demonstrates that LMP2-specific CTLs have not accumulated and/or expanded at the tumor site, despite the presence of their target antigen.
Using the Elispot assay, we were able to demonstrate that EBV-specific T cells are not only present in EBV-positive tumors but they are active directly ex vivo, releasing IFN- in response to antigenic stimulation (Table 6)
. Only one EBV-positive HD tumor (HD24) was available for study that carried an appropriate HLA type to examine responses to predefined LMP-derived epitopes. A clear response to an EBV lytic cycle epitope was detected in this tumor, but only a very weak response was detected to the LMP2-derived epitope TYG. In contrast, a clear LMP1-specific response was detected in TILs from one of two EBV-negative HD cases studied.
The failure of LMP-specific CTLs to accumulate and/or expand within an EBV-positive tumor may be explained if they are functionally impaired in vivo, e.g., by down-regulation of the T-cell receptor 
chain (52)
, and/or because of the action of immunosuppressive cytokines, such as IL-10 and transforming growth factor-ß (34
, 35)
. Furthermore, H-RS cells secrete the chemokine TARC, which may cause an influx of activated T cells with a Th2 phenotype that prevents the generation of an effective cell-mediated immune response (53)
. If there is some degree of inactivation of circulating CTLs in vivo, it is clearly possible, as demonstrated here, to overcome this by a period of in vitro culture. It remains to be seen, however, if patient-derived T cells activated in vitro and then returned to the donor can retain their function when they enter the tumor site.
The present study represents a first step in the detailed analysis of EBV-specific responses in the blood and tumor sites of HD patients and points the way to further studies involving larger numbers of patients. Nevertheless, our data suggest that EBV-positive H-RS cells may persist despite an existing EBV-specific CTL response in the blood and the tumor of HD patients because of the low frequency of CTL precursors that target EBV proteins expressed in H-RS cells. This may also explain why the HLA-A2 allele, through which many LMP-specific CTL responses are mediated, is not associated with increased protection from HD (54) . Furthermore, our findings have important clinical implications in that they suggest that boosting/eliciting the relevant component of the EBV-specific CTL response, either by immunization or adoptive transfer of T cells expanded in vitro, may prove an effective therapy for EBV-positive HD. A recent clinical study has attempted to treat three patients with EBV-positive HD by adoptive transfer of virus-specific polyclonal CTL lines (33) . After infusion of T cells, some patients showed an improvement of stage B symptoms with stabilization of disease and/or a decrease in EBV load. The lack of a complete response after T-cell infusions may partly be explained by the use of CTL lines reactivated in vitro using the autologous LCL. As mentioned above, one might expect only a minor component of the total EBV-specific response in such lines to target proteins expressed in H-RS cells. Therefore, the relevant EBV-specific CTL response may not have been boosted sufficiently. Reactivating PBMCs with the autologous LCL, followed by limiting dilution cloning, we succeeded in isolating functional LMP-specific CTLs from two of four EBV-positive HD patients. However, in many such patients, LMP-specific precursor frequencies may be too low to isolate potentially therapeutic T cells using this method. Strategies that selectively reactivate LMP-specific CTLs are likely to yield T-cell populations with improved therapeutic potential. One approach would be the use of autologous dendritic cells pulsed with LMP-derived peptide epitopes, a strategy that has been successful previously in reactivating LMP-specific responses from healthy EBV carriers (55) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a Medical Research Council (MRC) Clinical Training Fellowship Award (to A. L. N. C.) and a MRC Career Development Award (to S. P. L.). 
2 To whom requests for reprints should be addressed, at CRC Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 0121-414-2803; Fax: 0121-414-4486; E-mail: s.p.lee{at}bham.ac.uk
3 The abbreviations used are: HD, Hodgkin’s disease; H-RS, Hodgkin-Reed-Sternberg; LCL, lymphoblastoid cell line; PBMC, peripheral blood mononuclear cell; LMP, latent membrane protein; GAr, glycine-alanine repeat; IL, interleukin; TIL, tumor-infiltrating lymphocyte; Elispot, enzyme-linked immunospot; PHA, phytohemagglutinin; SFC, spot-forming cell; HLA, human leukocyte antigen.
Received 2/26/01. Accepted 6/ 7/01.
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B. F. Skinnider and T. W. Mak The role of cytokines in classical Hodgkin lymphoma Blood, May 29, 2002; 99(12): 4283 - 4297. [Abstract] [Full Text] [PDF]
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A. D. Hislop, N. E. Annels, N. H. Gudgeon, A. M. Leese, and A. B. Rickinson Epitope-specific Evolution of Human CD8+ T Cell Responses from Primary to Persistent Phases of Epstein-Barr Virus Infection J. Exp. Med., April 1, 2002; 195(7): 893 - 905. [Abstract] [Full Text] [PDF]
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