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Cancer Research Campaign Institute for Cancer Studies, and
Medical Research Council Centre for Immune Regulation, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom
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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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It is still not known how important the processing/presentation of
exogenously acquired Ag might be in the induction of human
CD8+ CTL responses. The present paper seeks to
address this issue in the context of the CTL response to a natural
pathogen of humans, EBV. This is a 
-herpesvirus that in normal
circumstances is largely if not exclusively restricted to target cells
of the B lymphoid lineage and that can establish either latent
(nonproductive) or lytic (productive) infection in such cells
(11). Both forms of infection induce an array of
CD8+ CTL responses that are apparent during
primary EBV infection and persist in the memory of long-term virus
carriers (12, 13, 14, 15). The present work focuses on the latent
Ag-specific response because here the full range of target Ags is well
defined, namely, six nuclear Ags (EBV-encoded nuclear Ags (EBNAs) 1, 2,
3A, 3B, 3C, and -LP) and two latent membrane proteins (LMP 1 and 2), as
is their apparent heirarchy of immunodominance as
CD8+ T cell targets. Thus, when the
CD8+ T cell pool of virus carriers is
rechallenged in vitro with cells of the autologous EBV latent
Ag-expressing B lymphoblastoid cell line (LCL), the dominant responses
almost always map to epitopes derived from the EBNA3A, 3B, and 3C
family of proteins, sometimes accompanied by subdominant responses to
one of the other latent proteins but apparently never to EBNA1
(14, 15). Such findings led to the important discovery
that endogenously expressed EBNA1 could not be presented to the
CD8+ T cell repertoire because an internal
glycine-alanine repeat (GAr) domain protected the protein from a key
step in the MHC class I processing pathway, namely proteasomal
degradation to peptides (16, 17). This GAr-mediated
protection proved to be extremely robust, holding firm even when the Ag
was deliberately over-expressed in LCL cells, despite the fact that
such latently infected B cells have an otherwise highly efficient APC
phenotype (16, 18). Therefore, in subsequent work we were
surprised to find, albeit from a very small number of EBV-immune
donors, rare CD8+ T cell clones derived by in
vitro outgrowth that were specific for EBNA1 peptides
(18). We reasoned that these rare reactivities must have
arisen in vivo by uptake and processing of EBNA1 as an exogenous Ag,
perhaps via DCs. Here, we show that DCs can indeed process full-length
EBNA1 in this way and go on to demonstrate that EBNA1 epitope-specific
responses are much more abundant than hitherto imagined, being seen
both in the primary and memory phases of infection at magnitudes at
least the equal of responses to immunodominant epitopes from the
"conventionally processed" EBNA3 proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whole blood was obtained from healthy EBV-seropositive adults and from infectious mononucleosis (IM) patients undergoing primary EBV infection. IM patients identified on clinical grounds and by heterophile Ab positivity were sampled during the first 10 days of illness. PBMC were isolated by Lymphoprep (Nycomed Pharma, Olso, Norway) density gradient centrifugation and were either used fresh or cryopreserved until required. Aliquots of PBMC were used to establish EBV-transformed LCLs using the B95.8 virus strain and maintained in RPMI 1640 containing 2 mM glutamine, 10% v/v FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (growth medium). PHA-activated T blasts (PHA blasts) were prepared by culturing PBMCs in the presence of PHA (20 µg/ml) and thereafter expanded in standard culture medium supplemented with 1% pooled human serum and 30% v/v supernatant from the MLA-144 cell line. Cryopreserved IM PBMC were thawed into IL-2-supplemented medium before immediate use in flow cytometry, or for ex vivo assays used as effectors within 2 h. All donors were HLA class I typed using PCR-based DNA typing. HLA B35 subtyping was conducted by Dr. M. Bunce (The Oxford Transplant Center, Churchill Hospital, Oxford, U.K.).
Synthetic peptides/baculovirus-expressed protein
Peptides were synthesized by standard fluorenyl-methoxycarbonyl chemistry (Alta Bioscience, University of Birmingham, Birmingham, U.K.) and dissolved in DMSO, and their concentration was determined by biuret assay. Full-length EBNA1 protein (bEBNA1) and a C-terminal fragment of EBNA1 (i.e., minus the GAr domain; b330–641) were prepared using the baculovirus expression system as described previously (18, 19) and were a kind gift from Dr. Lori Frappier (University of Toronto, Toronto, Canada). Human papilloma virus protein E4 prepared using the baculovirus expression system was a kind gift from Dr. Sally Roberts (University of Birmingham).
Enzyme-linked immunospot (ELISPOT) assay for single-cell IFN-
release
Detection of peptide-specific T cells in PBMC was conducted
essentially as described (20). Ninety-six well
polyvinylidene difluoride-backed plates (Millipor, Bedford, MA) were
precoated with 15 µg/ml of an anti-IFN- mAb, 1-DIK (MABTECH,
Stockholm, Sweden). PBMC were added to duplicate or triplicate wells at
known cell numbers in the presence of single or pooled peptides at a
final concentration of 2 µM per peptide. The plates were incubated
overnight at 37°C in 5% CO2. The cells were
discarded the following day and a biotinylated anti-IFN- mAb,
7B6-1 (MABTECH), was added at 1 µg/ml and left for 2–4 h at room
temperature, followed by streptavidin-conjugated alkaline phosphatase
(MABTECH) for an additional 2 h. Individual cytokine-producing
cells were detected as dark spots after a 30-min reaction with
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium using an
alkaline phosphatase-conjugated substrate kit (Bio-Rad, Richmond, CA).
The spots were counted under a dissection microscope. In all
experiments, results from ELISPOT assays are expressed as
IFN--positive cells per 106 PBMC. Depletion of
CD8-positive cells before some ELISPOTs was conducted using Dynabeads
M450-CD8 (Dynal, Merseyside, U.K.) according to the manufacturer’s
protocol. Efficient depletion of CD8+ cells was
confirmed by subsequent staining with FITC-conjugated anti-CD8 mAb
and flow cytometry.
Generation of HLA class I-peptide tetrameric constructs
Soluble MHC-peptide tetrameric constructs were produced according to methods previously described (21). Briefly, recombinant HLA-B*3501 heavy chain and human ß2-microglobulin proteins were purified in Escherichia coli cells after transformation with the appropriate expression plasmids, and expression was induced with isopropyl-ß-D-thiogalactopyranoside. The B*3501 heavy chain and ß2-microglobulin were then folded with the B*3501-restricted peptides from either EBNA1 (HPVGEADYFEY) or EBNA3A (YPLHEQHGM). Folding, purification, biotinylation, and tetramer production were performed according to Altman et al. (22).
Flow cytometry
Staining with HLA-tetrameric complexes was conducted as follows. PBMC (5 x 105 cells) were incubated at 37°C for 20 min in PBS with 0.1% BSA and 0.1% sodium azide containing 0.5 mg/ml of PE-labeled tetrameric complex before adding saturating amounts of an anti-CD8 mAb conjugated to FITC (PharMingen, San Diego, CA) followed by a further incubation on ice for 30 min. Labeled PBMC were then washed twice with PBS and fixed with 2% paraformaldehyde before analysis on an EPICS XL flow cytometer (Coulter Pharmaceutical, Palo Alto, CA). Data from flow cytometry was analyzed using WINMDI software (The Scripps Research Institute, San Diego, CA).
In vitro CTL reactivations
Peptide-specific CTL polyclonal cultures were generated as follows. Briefly, 2 x 106 PBMC were incubated with 50 µM peptide in a volume of 100 µl RPMI 1640 for 1–2 h at 37°C. Cells were then washed and resuspended in 2 ml growth medium supplemented with 25 ng/ml recombinant human IL-7 (Sigma, St. Louis, MO). After 3 days, IL-2 was added to the medium to a final concentration of 10 U/ml. Thereafter, cultures were fed twice weekly with growth medium containing 25 ng/ml IL-7 and 10 U/ml IL-2. Polyclonal CTL cultures were tested in cytotoxicity assays from day 12 onward and restimulated weekly with irradiated autologous LCL pulsed with 10 mM specific peptide. In vitro reactivation of CTL using DCs fed with EBNA1 protein were conducted as follows. DCs were prepared from PBMC by resuspending mononuclear cells in growth medium at 5 x 106 cells/ml then seeded into six-well plates (Costar, Cambridge, MA) at 107 cells/well. After 2 h at 37°C, nonadherent cells were removed, and the adherent population was cultured in growth medium supplemented with 50 ng/ml GM-CSF and 1000 U/ml IL-4. The cultures were re-fed on days 2 and 4 by replacing half the medium with fresh medium, as above. On day 6 or 7 the cells were harvested by gentle pipetting action, resuspended in 500 µl AIM-V serum-free medium (Life Technologies, Rockville, MD) containing full-length EBNA1 protein (bEBNA1) at 5 µg/ml and incubated for 14 h at 37°C. Cells were then washed twice with RPMI 1640 (with no supplements) and resuspended in growth medium containing GM-CSF and IL-4 as above, but now supplemented with 25% v/v macrophage-conditioned medium as a maturation stimulus. Cells were cultured for a further 24 h before being seeded as stimulators at 105 cells/2-ml well in growth medium supplemented with IL-7 at 5 ng/ml. Responder PBMCs were added at 2 x 106 cells/well to give a responder:stimulator ratio of 20:1. Cultures were fed twice weekly and screened for EBNA1 specificity in cytotoxicity assays from day 12. The cultures were restimulated on days 14 and 21 with DCs pulsed with EBNA1 protein as above and fed with growth medium supplemented with IL-7 (5 ng/ml) and IL-2 (20 U/ml). Limiting dilution cloning of selected polyclonal cultures was conducted as described (13).
Cytotoxicity assays
Chromium release assays were conducted as follows. LCL target cells were infected with the previously described recombinant vaccinia viruses (14, 18) for 90 min at a multiplicity of infection (moi) of 10, followed by incubation for a further 15 h. Targets were then labeled with 75–100 µCi of 51CrO4 for 90 min, washed, and incubated with polyclonal CTL cultures or CTL clones at known E:T ratios in a standard 5- to 7-h chromium release assay. Alternatively, LCL or PHA blast targets were first labeled with 75–100 µCi of 51CrO4 for 90 min in 100 µl final volume, and either specific peptide (at a final concentration of 2 x 10-8 M) or DMSO solvent was added for the final 60 min. Targets were then washed and incubated with effectors for 5- to 7-h in a standard chromium release assay. For assays involving protein loading, target cells were incubated overnight at 37°C with protein at 5 µg/ml in AIM-V serum before being labeled and used in a standard chromium release assay. The details of such assays and of relevant controls were as described (18). For all assays, the percentage specific lysis was calculated as (release by CTL - spontaneous release) x 100/(total release in 1% SDS - spontaneous release).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We began this work by re-examining the EBV latent Ag-specific
memory CTL response in one of the very few EBV-immune individuals,
donor RT (HLA-A2, A24, B*2705, B*3501), from whom an EBNA1
epitope-specific CD8+ T cell clone had been
isolated in earlier work and shown to recognize a B*3501-restricted
epitope HPVGEADYFEY (designated HPV) from the EBNA1 primary
sequence (aa 407–417) (18). A series of in vitro
reactivation experiments, using the conventional protocol of autologous
LCL stimulation followed by limiting dilution cloning and screening on
peptide-loaded target cells, yielded only an occasional HPV-specific
clone, and then only from a minority of experiments. By contrast, every
reactivation experiment yielded multiple clones reactive to two
immunodominant epitopes derived from EBNA3 proteins, namely a
B*2705-restricted epitope RRIYDLIEL (designated RRI) from
EBNA3C (aa 258–266) and a B*3501-restricted epitope YPL
HEQHGM (designated YPL) from EBNA3A (aa 458–466). However, when we
used the ELISPOT assay of rapid peptide-induced IFN- release to
measure the true frequency of epitope-reactive
CD8+ T cells in the blood of this donor, a quite
different picture emerged. As shown in Fig. 1
A, HPV-specific reactivity
was detectable at 290 IFN--secreting cells/106
PBMCs, almost equal to that seen against the immunodominant RRI epitope
and almost 40% of that seen against the immunodominant YPL epitope.
Furthermore, HPV-specific memory clearly exceeded that detectable
against five other known EBV epitopes potentially relevant to this
donor, namely the A*0201-restricted epitopes GLC and CLG, from the
BMLF1 lytic Ag and the LMP2 latent Ag, respectively, and
A*2402-restricted epitopes TYG, RYS, and TYS from LMP2, EBNA3A, and
EBNA3B, respectively.
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B). In two further
donors, 114 (Fig. 1C) and RK (Fig. 1D), the
YPL/B*3501 response was the most abundant component of memory, but the
HPV response again reached values of 568 and 725
cells/106 PBMCs, respectively. Such frequencies
were equivalent to those seen against two other strong latent cycle
epitopes (AVF and IED) in donor 114 and against the strongest of three
B8-restricted reactivities, to the EBNA3A epitope FLR, in donor
RK.
Table I presents the overall results
quantitating IFN--responsive cells in the blood of a series of
HLA-typed donors when challenged by peptide stimulation either with the
EBNA1 HPV epitope or with the EBNA3A YPL epitope. All 15 donors with
the B*3501 allele and serological evidence of prior EBV infection had
detectable responses to both epitopes within a similarly high frequency
range, from 120 to 1317 cells/106 PBMCs (mean
592) for the HPV epitope and from 63 to 2482
cells/106 PBMCs (mean 745) for the YPL epitope,
with HPV-specific T cell memory outnumbering YPL-specific memory in
5/15 cases. Furthermore, these two combined reactivities were often the
largest detectable components of EBV-latent epitope-specific memory
(Fig. 1, and data not shown). Interestingly, none of three
EBV-seropositive donors with a B*3502 and/or B*3503 allele contained
detectable HPV-specific memory, and only one had a detectable
YPL-specific response. For each of these nonresponders to the HPV
peptide, we amplified the relevant EBNA1 sequence from the EBV strain
present in circulating B cells and confirmed that the HPVGEADYFEY
epitope sequence was indeed conserved (data not shown). This strongly
suggests that the HPV epitope is immunogenic in the context of B*3501,
but not other B35 subtypes. As representative control donors in these
assays, we included two individuals (AL and NB) who were EBV
seropositive but lacked a B35 allele and one individual (MO) who was
B*3501 positive but EBV seronegative. None of these controls showed any
response either to the HPV or the YPL epitope.
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The implication from this work was that EBNA1, though not
accessible to the MHC class I pathway when expressed endogenously in
latently infected cells, was nevertheless presented to
CD8+ T cells in vivo, perhaps following uptake as
an exogenous Ag by DCs. To address this possibility, we first generated
DCs from B*3501-positive donors by culturing adherent PBMCs for 6 days
in GM-CSF/IL-4-conditioned medium, then exposed them to a purified
preparation of full-length EBNA1 protein made from a baculovirus
vector. After a further 24 h maturation in macrophage-conditioned
medium, the cells were then used as in vitro stimulators of CTL
responses by cocultivation with autologous PBMCs. Fig. 2, A and B show the
results obtained from two donors, EMc and RB, respectively, both known
to possess HPV-specific memory (see Table I). In both cases, these
polyclonal CTL preparations, assayed on day 21 following two rounds of
stimulation with EBNA1-loaded DCs, showed significant HPV
peptide-specific reactivity when tested on peptide-loaded targets.
Furthermore, these effectors showed significant lysis of autologous LCL
cells expressing the GAr-deleted form of EBNA1 endogenously from a
vaccinia vector, above the background lysis observed for cells infected
with a control vaccinia, but did not recognize target cells with
vectored expression of the full-length protein.
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C using as targets B*3501-positive DCs with
and without preexposure to baculovirus-expressed EBNA1 protein followed
by overnight processing. Both the exogenously loaded full-length EBNA1
protein (bEBNA1) and a C-terminal fragment (b330–641) containing the
HPV epitope region clearly sensitized the target cells to lysis at
levels similar to those observed on targets preexposed to the epitope
peptide. As a control, there was no recognition of targets loaded with
a baculovirus-expressed human papilloma virus protein, E4. Additional
control experiments, as previously described (18), showed
that EBNA1 protein-loaded DCs only became susceptible to T cell
recognition after several hours of incubation at 37°C, confirming
that this specific lysis required intracellular processing of the EBNA1
protein and was not due to contaminating peptide fragments in the
protein preparation (data not shown). In contrast to exogenously
acquired protein, when full-length EBNA1 was expressed endogenously
from a vaccinia vector either in LCL cells or in DCs, there was no
significant recognition by these HPV epitope-specific clones, whereas
the same target cells expressing GAr-deleted EBNA1 clearly did present
the epitope (Fig. 2D, and data not shown).
Identification of new EBNA1 epitope-specific CD8+ T
cell responses by peptide-induced IFN- release
Given the example of the strong B*3501-restricted reactivity to
the HPV epitope, we speculated that there would be additional
EBNA1-specific CD8+ T cell responses yet to be
identified. Therefore, we used the ELISPOT assay of rapid
peptide-induced IFN- release to screen a group of other EBV-immune
donors with a panel of 84 peptides (15-mers overlapping by 10 aa)
spanning the entire unique sequence of the EBNA1 protein. To minimize
the size of the screening, peptides were used in pools of three, an
approach that was first validated by its ability to detect HPV-specific
responses in B*3501-positive donors using a peptide pool that included
the 15-mer EBNA1 aa 403–417 containing the HPV epitope (data not
shown). Three of 16 individuals screened in this way (NA, CD, and GL)
gave evidence of specific IFN- release mapping to different EBNA1
15-mer peptide pools. The results shown in Fig. 3 indicate clear responses above
background to peptide pool 5 from donor NA (HLA-A3, -A24, -B7, and
-B37; top panel), to adjacent peptide pools 21 and 22 from
donor CD (HLA-A1, -B7, and -B57; middle panel), and to
peptide pool 13 from donor GL (HLA-A26, -A30, -B38, and -B53;
bottom panel).
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response was mediated by CD4+ or
CD8+ T cells within the PBMC population. As shown
in Fig. 4A, the response in
donor NA mapped to two adjacent peptides within pool 5 (EBNA1 aa 66–80
and 71–85) and was clearly CD8+ T cell-mediated
because it was no longer observed in CD8+ T
cell-depleted responder populations. This response was then minimized
to the 9-mer epitope EBNA1 aa 72–80, whose sequence, RPQKRPSCI,
accords well to the HLA-B7 motif with a Pro at position 2 and an Ile at
the C terminus (20). With donor CD, the response to
adjacent pools 21 and 22 in fact mapped to adjacent peptides aa
524–538 (in pool 21) and aa 528–543 (in pool 22) and was again shown
to be CD8+ T cell-mediated by depletion assays
(Fig. 4B). This response was minimized to a 9-mer epitope
EBNA1 aa 528–536, whose sequence, IPQCRLTPL, again accords with the
HLA-B7 consensus motif (23). Interestingly, donor GL’s
response to peptide pool 13 mapped to the same 15-mer peptide, EBNA1 aa
403–417, that contained the B*3501-restricted epitope 407–417.
However, in this case the 9-mer HPVGEADYF, aa 407–415, was recognized
as efficiently as the 11-mer B*3501 epitope HPVGEADYFEY (Fig. 4C). This 9-mer epitope, with Pro at position 2 and Phe at
the C terminus, accords with the concensus motif for binding to the
HLA-B53 allele of donor GL (24). It is important to note
the magnitude of these newly identified memory responses, especially to
the EBNA1 aa 528–538 and 407–415 epitopes, where frequencies of
600–750 IFN--secreting cells/106 PBMCs are in
the same high range as observed for the B*3501-restricted EBNA1
epitope.
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A (upper panel)
shows a representative result obtained with polyclonal effectors from
donor NA harvested on day 21 poststimulation. These cells mediated
significant lysis of LCL targets, either coated with the RPQKRPSCI
peptide or expressing the GAr-deleted form of EBNA1 from a vaccinia
vector. Again, lysis of LCLs expressing full-length EBNA1 was not
significantly greater than background levels seen for control
vaccinia-infected or mock peptide-loaded targets. A similar pattern of
results was observed with polyclonal effectors from cultures of donor
CD cells 21 days poststimulation with IPQCRLTPL peptide. Though overall
levels of lysis were lower with this donor, the results were again
consistent with induction of an epitope-specific CTL response, which
also showed recognition of endogenously expressed EBNA1 only in its
GAr-deleted form (Fig. 5B, upper panel). Similar
results were also observed with polyclonal effectors from donor GL
stimulated with the HPVGEADYF peptide (Fig. 5C, upper
panel). In subsequent assays, these polyclonal effector
populations were tested on a range of allogeneic LCL targets of known
HLA type, either preexposed to the relevant epitope peptide or to DMSO
as a control. The data from donor NA (Fig. 5A, lower
panel) and donor CD (Fig. 5B, lower panel)
are in each case consistent with HLA-B7-restricted recognition, and the
data from donor GL (Fig. 5C, lower panel) are
consistent with HLA-B53-restricted recognition.
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In the final set of experiments, we sought to determine whether an
EBNA1-specific CD8+ T cell response was
detectable during primary EBV infection and whether its magnitude ever
reached that shown by primary responses to immunodominant epitopes
within the EBNA3 proteins. To this end, we constructed PE-labeled
HLA-peptide tetramers, in which the B*3501 molecule was complexed with
the EBNA1 HPVGEADYFEY and EBNA3A YPLHEQHGM peptides, and used these
to track T cell reactivities in B*3501-positive IM patients. We
analyzed six B*3501-positive IM patients in the acute phase of disease
by B*3501/HPV and B*3501/YPL tetramer staining. Fig. 6A presents results from four
of these patients, IM107, IM122, IM135, and IM136, who collectively
illustrate the full range of responses observed. Thus, staining with
the B*3501/YPL tetramer ranged from undetectable in IM122 and IM135 to
1.6% CD8+ cells in IM136. In the same assays,
cells specific for the EBNA1-derived HPV epitope were detectable in
every case at frequencies that lay between 0.1%
CD8+ cells in IM107 and 5.0%
CD8+ T cells in IM135. This latter value is the
largest expansion ever recorded for a CD8+ T cell
response against a latent cycle epitope.
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B. High levels of
epitope-specific lysis were observed at total PBMC:target ratios of
40:1 and 20:1 in the ex vivo effector populations from both IM135 and
IM122, where respectively 5.0% and 1.0% of the circulating
CD8+ T cells stained with the B*3501/HPV
tetramer. Lower levels of specific lysis were apparent in ex vivo
effectors from IM108, but even here it was clear that a significant
component of the primary CTL response was directed against an EBNA1
epitope. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß- herpesviruses, which affect
the global Ag-presenting function of the cell but which are at least
partially reversible by cytokines such as IFN-
(26, 27, 28), the protection mediated by the GAr domain is
limited to EBNA1 only, appears to operate in a range of cell
backgrounds, and cannot be overcome even when the protein is
over-expressed endogenously from recombinant viral vectors (16, 18, 29). Therefore, it seemed highly probable that any
detectable CD8+ T cell response to EBNA1 could
only arise through its processing as an exogenous Ag.
Having identified rare CTL clones specific for a B*3501-restricted
EBNA1 epitope in one individual’s response to autologous LCL
stimulation, we used the ELISPOT assay of rapid peptide-induced IFN-
release to show that this reactivity actually constituted a major
component of EBV-specific T cell memory, not just in that individual
but in all 15 B*3501-positive EBV-carrying donors tested. The frequency
of HPV epitope-specific cells was in the same range as that seen for
the immunodominant B*3501-restricted YPL epitope derived from EBNA3A,
as well as for other immunodominant latent Ags (Fig. 1, Table I). If
these memory cells had indeed been induced in vivo by the processing of
exogenously acquired EBNA1, then one would predict that they would be
selectively reactivated in vitro by stimulating with DCs that had been
exogenously loaded with EBNA1 protein. This was indeed the case for
each B*3501-positive donor tested (Fig. 2), and the effectors thus
produced were both epitope- and Ag-specific, recognizing target cells
expressing a GAr-deleted form of EBNA1 from a vaccinia vector almost as
well as targets preexposed to the synthetic epitope peptide, but not
recognizing targets expressing the full-length protein. Importantly,
however, these same effectors did kill DCs preexposed to full-length
EBNA1 as an exogenous protein, confirming the capacity of such cells to
process and present exogenously acquired Ag via the MHC class I
pathway. The mechanism of this processing remains to be determined, but
is clearly cell type specific. Thus, both DCs and, as we have
previously shown (18), to some extent LCL cells themselves
have the capacity to present exogenously loaded EBNA1 in this way. In
contrast, when we conducted similar experiments with an
HLA-B35-positive keratinocyte cell line, no such recognition of
exogenously loaded EBNA1 protein was observed (data not shown). This is
consistent with previous studies demonstrating the inability of
epithilial cells to process exogenously acquired Ag unless provided
with a phagocytic stimulus (30). We presume that, when
-irradiated LCL cells are used as an in vitro stimulus for PBMC
responses, there is some presentation of EBNA1 acquired from
dying cells, probably by professional APCs present as a minor
fraction of the PBMC population, thereby explaining how EBNA1-specific
CTLs can be reactivated (albeit relatively inefficiently) by this
protocol. In this same context, in vitro studies have recently shown
that DCs are capable of processing and presenting EBNA1 from dying LCLs
to stimulate IFN- release from an EBNA1-specific
CD4+ T cell clone (31), thus clearly
showing that EBNA1 protein can be acquired from a LCL in a
cross-priming situation.
Further work showed that strong responses induced by processing of
exogenous EBNA1 protein are not limited to the B*3501 allele. ELISPOT
screening of peptide-induced IFN- responses across donors with a
range of HLA types identified new reactivities to two B7-restricted
epitopes and to one B53-restricted epitope. Interestingly, the B53
epitope (EBNA1 aa 407–415, HPVGEADYF) was actually a 9-mer that lay
within the 11-mer B*3501 epitope (EBNA1 aa 407–417, HPVGEADYFEY). The
HLA-B53 and -B35 molecules are very closely related alleles that belong
to the B5 cross-reactive group, but differ structurally at the F pocket
of the peptide binding groove, which interacts with the peptide C
terminus (32, 33). Although B35 has a strong preference
for Tyr residues at this position, the B53 allele can accommodate
either Leu, Ile, Val, Phe, or Tyr (24, 32, 33), explaining
why the shorter peptide with a Phe at position 9 can function as a B53
epitope.
As with the B*3501 allele, the analysis of B7- and B53-restricted responses emphasized: 1) that EBNA1 epitope reactivities can be an abundant component of EBV-latent Ag-specific memory when analyzed by direct ELISPOT assay (with frequencies up to 800 reactive cells/106 PBMC) and, more importantly, 2) that these memory cells can be efficiently reactivated in vitro to effector populations that recognize targets endogenously expressing the Ag in its GAr-deleted but not full-length form. In that context, the B7-restricted EBNA1 72–80 sequence represents the first example of an EBNA1 epitope derived from sequences N-terminal of the GAr domain and confirms that GAr-mediated protection extends upstream as well as downstream of the repeat (16). The fact that we have now detected strong EBNA1-specific responses through several different HLA class I alleles (Ref. 18 , and this study) emphasizes the generality of this exogenous processing pathway. Indeed, EBNA1 becomes directly comparable to the immunodominant EBNA3 Ags, not just in terms of the frequency of memory T cells to the most immunogenic EBNA1 vs EBNA3 epitopes, but also in terms of the range of HLA alleles that present such epitopes. For instance, strong responses to EBNA3B, a protein with more than twice the unique sequence content of EBNA1, have so far only been detected in the context of two alleles, HLA-A11 and -B*2702 (11). Using these same criteria, EBNA1 can certainly be considered a much stronger immunogen for CD8+ T cell reponses than latent cycle Ags such as EBNA2 or LMP1, which are equivalent to EBNA1 in unique sequence content and which when endogenously expressed can be processed via the MHC class I pathway (34, 35).
These observations strongly suggested that a T cell response generated by processing of exogenous Ag can provide a major component of virus-specific CTL memory. It was particularly interesting, then, to determine whether this could also be true for a primary response. In the EBV system, previous work has shown that the marked immunodominance of EBNA3-derived epitopes on particular HLA class I alleles is apparent not just in long-term memory but also during primary infection, as seen in IM patients (13). However, we speculated that the induction of the EBNA1 response might be delayed because this requires prior Ag release and, arguably, such release may depend upon an effective CTL response to latently infected cells having already been mounted. This would provide an interesting parallel to the humoral response to primary infection, in which Abs to EBNA1, but not to the other EBNAs, develop unusually late (36, 37). In fact, we consistently observed that the primary CTL response to the B*3501-restricted EBNA1/HPV epitope was at least as strong as that seen in the same individuals to the EBNA3A/YPL epitope and comparable to other immundominant latent epitopes. Indeed, the value of 5% CD8+ T cells being HPV specific in donor 135 represents the most dramatic expansion of any latent epitope-specific response yet recorded in an IM patient (21). On the evidence to date, we infer that the EBNA1-specific CTL response is not unduly delayed during primary infection, but can actually make a significant contribution to the large expansion of CD8+ T cell numbers characteristic of IM.
The source of the released EBNA1 required to prime the above response remains to be determined, but may well be from B cells that initiate but do not complete the virus-induced transformation process. This could be a significant source of all the latent proteins because it is clear from in vitro studies that only a minority of experimentally infected B cells survive to grow as colonies even though many initiate expression of the EBNAs and LMPs (38, 39). Another possibility is that latent Ags could be released from cells that have switched from latency to lytic cycle in vivo. Because there is no obvious distinction between CTL responses to EBNA1 and to the immunodominant EBNA3 proteins, either in magnitude or in kinetics, this raises the possibility that processing of exogenous Ag is in fact how all latent Ag-specific reactivities are induced. Such a scenario is attractive in that it also might help to explain the paradoxical finding that some EBNA3-derived epitopes that are immunodominant in vivo are nevertheless represented very poorly on the surface of LCL cells (40). If this were the case, then immunodominance may be more strongly influenced by the way latent proteins are handled exogenously by DCs than endogenously by latently infected B cells.
Therefore, the unique ability of EBNA1 to evade processing as an endogenously expressed Ag has allowed us to observe what is perhaps a general phenomenon: the significant contribution the "cross-priming" pathway can make to the induction of virus-specific CTL responses in humans, even in circumstances where the virus-infected cells themselves have efficient Ag-presenting function and are easily accessible to the CD8+ T cell repertoire. It is important to note that the EBNA1-specific T cells that are induced in such numbers in vivo are nevertheless unable to recognize their latently infected targets. We would argue that, in the specific context of the EBNA1 Ag, a priming pathway that normally helps induce highly efficient CTL responses to viral infection has been rendered biologically ineffective.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Neil Blake, Cancer Research Campaign Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.
3 Abbreviations used in this paper: DC, dendritic cell; EBV-encoded nuclear Ag; LMP, latent membrane protein; IM, infectious mononucleosis; LCL, lymphoblastoid cell line; ELISPOT, enzyme-linked immunospot assay; GAr, glycine-alanine repeat; PHA blasts, PHA-activated T blasts.
Received for publication July 20, 2000. Accepted for publication September 15, 2000.
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vß5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity 4:203.[Medline]
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GA (
), coinfected with vE1 and vT7 (
) or
with the control vTK (•), or preexposed to the EBNA1 407–417 epitope
peptide at 2 x 10-8 M (
) or to DMSO alone (
).
C, Selected CTL clones (EMc c2 and c14) generated from
limiting dilution cloning of the polyclonal culture in A
were tested on B*3501-positive DC targets preincubated with either
full-length EBNA1 protein (bEBNA1), a C-terminal fragment (b330–641),
or control human papilloma virus protein E4, all expressed from
baculovirus vectors. Target cells were exposed to protein at 5 µg/ml
for 12 h at 37°C before being used in a standard chromium
release assay. Note that controls to ensure that protein preparations
were free of epitope-containing peptide fragments capable of directly
loading onto surface B*3501 molecules were conducted as described
(
). D,
Selected CTL clones (EMc c114 and c125) were further tested on
autologous LCL targets treated as in A at E:T ratios of
10:1 (
).
The background observed when no peptide was used is shown. nt, Not
tested.
). All
results are expressed as the percentage of specific lysis in 5-h
chromium release assays.