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* Department of Immunology and
Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905
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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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-herpesvirus that infects mostly B cells and
is responsible for inducing their uncontrolled cell proliferation and
transformation (1). Under normal circumstances and in
healthy individuals, EBV infections are not life threatening and are
generally effectively controlled by the immune system through the
action of Ag-specific T lymphocytes (2). Both
CD8+ CTL and CD4+ helper T
lymphocytes (HTL)3 can
discriminate EBV-infected or EBV-transformed B cells and, as a
consequence, are able to inhibit their growth. EBV-specific T
lymphocytes recognize Ag as molecular complexes formed by viral peptide
epitopes with MHC molecules, which are expressed on the surface of the
infected/transformed B lymphocytes (2). Although lifetime immunity to EBV is apparently achieved in normal individuals, this virus is not completely eradicated and persists in a latent infection state (3, 4, 5), which is effectively controlled by the EBV-specific T lymphocytes (2). However, in immunosuppressed individuals such as transplant patients, primary EBV infection usually results in posttransplant lymphoproliferative disorders (PTLD) that often progress into B cell lymphomas (6, 7, 8). Current prophylactic and therapeutic approaches for PTLD and lymphomas are far from optimal. The reduction of immunosuppressive therapy facilitates the immune-mediated viral elimination, but with a high risk of organ rejection, and the use of antiviral agents is of unclear effectiveness (8). Results obtained using anti-CD20 mAb therapy appear promising, but have the disadvantage of long term B cell depletion and the occurrence of CD20-negative relapses (9, 10, 11). One of the most effective ways to prevent/treat PTLD and B cell lymphomas is via adoptive immunotherapy using EBV-specific T lymphocytes (12, 13), but this approach is labor intensive, costly, and not widely available. The risk of PTLD and lymphomas increases significantly in those patients who lack immunity to EBV before transplantation and undergoing immunosuppressive therapy (14). It is possible that this risk could be lowered if EBV-seronegative patients could be immunized to stimulate their T lymphocytes, but unfortunately such a vaccine is not yet available.
An attractive and relatively expedient approach to develop vaccines that are intended to elicit Ag-specific T cell responses is the use of synthetic peptides representing CTL and HTL epitopes. This strategy has been explored for various viral and malignant diseases (15, 16, 17, 18). A large number of T cell epitopes derived from EBV latent and lytic cycle Ags have been identified, and some of these are being considered as potential vaccine candidates (19, 20). Many of these efforts are focusing on using MHC class I-restricted peptide epitopes to induce CD8+ CTL responses to EBV, since these cells are considered to be the prime effector cells that will presumably annihilate the virus-infected and transformed cells. However, there is recent evidence that CD4+ T lymphocytes can also function as potent effectors for inhibiting EBV-induced B cell proliferation, which would be the initial step of PTLD (21).
One of the major potential drawbacks of T cell peptide epitope vaccines is the limitation of MHC restriction. Most CTL and HTL peptide epitopes will only be useful in the limited proportion of individuals who express the appropriate MHC allele. However, in the case of MHC class II epitopes, some peptides have been found to bind "promiscuously" to more than one MHC allele (22, 23, 24). In some cases these promiscuous epitopes can bind up to 10 frequently found MHC alleles, indicating that the majority of the population would recognize these epitopes (25).
In the present study we report the identification of a highly promiscuous MHC class II epitope from the EBNA2 Ag capable of stimulating CD4+ T cell responses. The EBNA2 epitope described herein can be presented to T cells in the context of HLA-DR1, -DR7, -DR16, -DR52, -DQ2, and -DQ7 alleles. Most significantly, the T cells induced by the EBNA2 peptide epitope were able to recognize EBV-transformed lymphoblastoid cell lines and were efficient in inhibiting early B cell proliferation induced by EBV infection. These results indicate that the promiscuous EBNA2 epitope is an excellent candidate for a vaccine to prevent and perhaps treat PTLD in transplant patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EBV-transformed lymphoblastoid cells (EBV-LCL) were generated from peripheral blood mononuclear cells of HLA-typed volunteers using culture supernatant from the EBV-producing B95-8 cell line (American Type Culture Collection, Manassas, VA). Mouse fibroblast cell lines (L cells), transfected and expressing individual human MHC class II molecules, were provided by R. W. Karr (Parke-Davis, Ann Arbor, MI).
Synthetic peptides and epitope prediction analysis
Potential MHC class II promiscuous helper T cell epitopes were predicted from the amino acid sequence of the EBNA2 Ag using the algorithm tables for three HLA-DR alleles (DRB1* 0101, DRB1*0401, and DRB1*0701) published by Southwood et al. (25). The algorithm calculates the average relative binding (ARB) value for each possible 9-mer core peptide sequence for the entire EBNA2 protein. The rationale for this approach is that the higher the ARB value of a peptide, the higher the probability that the peptide will bind to the corresponding HLA-DR allele. The selected peptides that displayed high ARB values were synthesized and purified as previously described (26). The purity (>95%) and identity of peptides were determined by HPLC and mass spectrometry.
In vitro induction of Ag-specific T cell lines using synthetic peptides
The methods used for generating tumor Ag-reactive HTL lines and clones using peptide-stimulated PBMC have been described in detail previously (26, 27). All blood donors used in the present study were seropositive for EBV. For donors M14 and M44, peptide-pulsed dendritic cells (DC) were used to stimulate purified CD4+ T cells. Briefly, DC were generated in tissue culture from adherent monocytes that were cultured for 7 days in the presence of GM-CSF and IL-4. A total of 1 x 104 peptide-pulsed DC (3 µg/ml for 2 h at room temperature) were irradiated (4200 rad) and cocultured with 3 x 104 autologous purified CD4+ T cells (using Ab-coated magnetic beads from Miltenyi Biotech, Auburn, CA) in each well of a 96-well, round-bottom culture plate. For the remainder of the blood donors (M1, M2, M9, M15, M20, M24, M25, M69, and M83), total unfractionated PBMC were stimulated with 3 µg/ml peptide in 48-well plates at 5 x 105 cells/well. Seven days later, all cultures were restimulated individually with peptide (3 µg/ml) and autologous irradiated PBMC, and 2 days later, human rIL-2 was added at a final concentration of 10 IU/ml. After two rounds of restimulation, the microcultures were tested for their proliferative responses to peptide as described below. Those wells showing a proliferative response to peptide of at least 2.5-fold over background were expanded in 24- or 48-well plates by weekly restimulation with peptides and irradiated autologous PBMC. Flow cytometric analysis after three rounds of peptide stimulation revealed that >95% of the cells were CD4+/CD8-. Culture medium for all procedures consisted of RPMI 1640 supplemented with 5% human male AB serum, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 µg/ml gentamicin. The institutional review board on human subjects (Mayo Foundation) approved this research, and informed consent for blood donation was obtained from all volunteers.
Analysis of Ag-specific response of T cells
T cells (3 x 104/well) were mixed
with irradiated APCs in the presence of various concentrations of Ag
(peptide) in 96-well culture plates. APC consisted of PBMC (1 x
105/well), HLA-DR-expressing L cells (3 x
104/well), or EBV-LCL (3 x
104/well). Cell proliferation assays were
incubated at 37°C in a 5% CO2 incubator for
72 h, and during the last 16 h, each well was pulsed with 0.5
µCi/well [3H]thymidine (Amersham Pharmacia
Biotech, Piscataway, NJ). In some cases culture supernatants were
collected before the addition of [3H]thymidine
for the determination of IFN- production using ELISA kits (BD
PharMingen, San Diego, CA). The radioactivity incorporated into DNA,
which correlates with cell proliferation, was measured in a liquid
scintillation counter after harvesting the cell cultures onto
glass-fiber filters. To identify the MHC restriction molecules involved
in Ag presentation, inhibition of the Ag-induced proliferative response
was determined by the addition of anti-HLA-DR mAb L243 (IgG2a,
prepared from hybridoma supernatants obtained from American Type
Culture Collection) or anti-HLA-DQ mAb SPVL3 (IgG2a; Beckman
Coulter, Fullerton, CA). Both Abs were used at a final concentration of
10 µg/ml throughout the 72-h assay. The specificity of these Abs and
their capacity to specifically inhibit Th responses have been
determined in our laboratory on numerous occasions. All assessments of
proliferative responses were conducted at least in triplicate, and
results correspond to the means. The stimulation index was calculated
by dividing the mean radioactivity (cpm) obtained in the presence of Ag
by the mean radioactivity (cpm) obtained in the absence of Ag but in
the presence of APC.
Cell-mediated cytotoxicity assays
Cytotoxic activity of CD4+ T cells was determined in a 51Cr release assay as previously described (28). Targets were prepared by incubating EBV-LCL (or T2 cells) with or without 10 µg/ml peptides at 37°C overnight. Target cells were labeled with 300 µCi [51Cr]sodium chromate (Amersham Pharmacia Biotech, Piscataway, NJ)/5 x 106 cells for 1.5 h at 37°C. T cells were mixed with 2 x 104 labeled targets at different E:T cell ratios in 96-well, round-bottom plates at a final volume of 0.2 ml. After 6- to 9-h incubation at 37°C, 30 µl supernatant was collected from each well, and the percentage of specific lysis was determined according to the formula: [(cpm of the test sample - cpm of spontaneous release)/(cpm of the maximal release - cpm of spontaneous release)] x 100. Results show the average specific lysis ± SE of triplicate determinations.
EBV-induced B lymphocyte proliferation assays
Infectious EBV stocks were prepared from culture supernatants of the EBV-producing B95-8 cell line. B95-8 cells were seeded at 2 x 105 cell/ml in complete RPMI 1640 medium and were incubated for 14 days at 37°C without medium exchange. The culture supernatant was adjusted to contain 10 g NaCl and 8% (w/v) polyethylene glycol (PEG-8000; Sigma, St. Louis, MO) per liter. The precipitate formed after overnight incubation at 4°C was collected by centrifugation at 7500 rpm in a Sorvall centrifuge with a GS3 rotor (Beckman Coulter). The pellet was resuspended in 1 ml complete medium for every 50 ml original EBV-containing supernatant, and was stored at -80°C until further use. The EBV infectivity assays were performed as recently described (21). Briefly, T cell-depleted PBMC were produced by negative selection using anti-CD3-coated magnetic beads (Miltenyi). The T cell-depleted PBMC (4 x 105 cells) were resuspended in 1 ml complete RPMI 1640 medium, and 20 µl concentrated EBV supernatant was added in the absence and the presence of various concentrations of T lymphocytes without the addition of cytokines. After 10–15 days in culture, the cultures were harvested, and the numbers of viable cells were determined using trypan blue exclusion. The percentage of CD23+ B cells in each culture condition was estimated by cytofluorometric analysis using an FITC-labeled specific mAb (BD PharMingen).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our goal was to identify promiscuous MHC class II
CD4+ T cell epitopes that could be used to
prevent/treat EBV-induced PTLD, which is a serious complication in
solid organ transplant patients. For our initial studies we selected
the EBNA2 latent viral Ag as a potential target for inducing T cell
responses against PTLD. Thus, we examined the amino acid sequence of
EBNA2 viral protein for the presence of peptide fragments containing
binding motifs for HLA-DR*0101, DR*0401, and
DR*0701 using the algorithm tables described by Southwood et
al. (25). Using the cut-off values of ARB necessary to
predict 75% of the HLA-DR binders, we found that of a possible 479
total peptides of a nine-amino acid length (EBNA2 has 487 residues), 35
peptides were predicted as potential binders for HLA-DR1, 23 peptides
for HLA-DR4, and only six peptides for HLA-DR7 (data not shown).
However, more relevant for the identification of promiscuous MHC
class II binders, we observed that only four peptide sequences from
EBNA2 exhibited ARB scores above the cut-off values for all three
alleles (Table I
). Interestingly, two of
these peptide sequences, EBNA2281and
EBNA2282, are almost identical with peptide
EBNA2280–290 (TVFYNIPPMPL), which was
reported to function as a T cell epitope restricted by
HLA-DQ2 and -DQ7 (29). In view of this finding, we
reasoned that peptide EBNA2280–290 would be the
prime candidate to be evaluated as a highly promiscuous epitope for
inducing CD4+ T cell responses in the context of
several additional HLA-class II molecules.
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Since peptide EBNA2280–290 was predicted to
function as a highly promiscuous MHC class II
CD4+ T cell epitope, this peptide was synthesized
and tested for its capacity to stimulate CD4+ T
cells isolated from 11 healthy, EBV-seropositive individuals. T cell
lines were prepared as described in Materials and Methods
using two different protocols. For two blood donors (M14 and M44)
purified CD4+ T cells were stimulated with
peptide-pulsed DC, and for the remaining nine donors peptide was added
to total unfractionated PBMC. All T cell cultures were restimulated
three or four times weekly with autologous irradiated PBMC and peptide,
after which the T cell responses to peptide were evaluated in a cell
proliferation assay. The majority of the individuals (7 of the 11) were
able to respond in vitro to peptide EBNA2280–290
in an Ag-specific manner (Fig. 1). The
four T cell lines that failed to exhibit peptide-induced proliferative
responses did so either because they had high background (high
proliferation in the absence of peptide) or because of lack of overall
proliferation.
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To define the HLA restriction alleles of the
EBNA2280–290 peptide-reactive T cell lines, cell
proliferation assays were performed using a panel of HLA-DR-transfected
mouse fibroblasts (L cells) and semiallogeneic human cells that were
used as APC. In addition, anti-HLA-DR or anti-HLA-DQ mAbs were
tested for their capacity to inhibit the peptide-induced proliferation
with autologous APC. The results from these experiments (Fig. 2) indicate that peptide
EBNA2280–290 could be presented to the
T cells in the context of HLA-DR1 (donor M1), HLA-DQ2
(M14), HLA-DR16 (M24), HLA-DR52 (M44 and M69), and HLA-DR7 (M83).
Cytofluorometric analysis revealed that the T cell lines expressed
the CD4 marker and did not express CD8 (data not shown), indicating
that these cells behaved as typical MHC class II-restricted T
lymphocytes. The T cell line from donor M9, which had previously
responded to peptide EBNA2280–290, could not be
analyzed for MHC restriction because it was not stable, and it ceased
to grow in tissue culture.
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One of the most critical attributes that peptide-induced
anti-EBV CD4+ T cells must exhibit is their
capacity to recognize the naturally processed viral Ag, which is
expressed by the EBV-infected or transformed B lymphocyte. Thus, it
became important to determine whether EBV-transformed lymphoblastoid
cell lines (EBV-LCL), which naturally process EBNA2, would be capable
of stimulating the peptide-reactive T cell lines. To carry out these
studies, EBV-LCL were generated from all blood donors to be used as APC
for each corresponding T cell line to be analyzed. In addition, T2
cells, which are negative for MHC class II, were used as a negative
control. The results from these experiments demonstrated that all the
peptide-reactive T cell lines were effective in recognizing Ag
presented directly by their respective EBV-LCL, as determined by their
secretion of IFN- (Fig. 3). On the
other hand, no apparent reactivity was observed toward T2 cells when
these were used as APC. These results indicate that the T cell epitope
represented by peptide EBNA2280–290 is processed
from the EBNA2 protein and can be expressed on the MHC class II
molecules of transformed EBV-LCL, enabling peptide-reactive
CD4+ T cells to efficiently recognize
them.
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There is ample evidence that CD4+ Th cells
are capable of exhibiting effector activity either in the form of
cytotoxicity or via the production of lymphokines against viral
infections. Moreover, recent studies showed that
CD4+ T cells are capable of suppressing the early
stages of B cell proliferation and transformation by EBV
(21), whereas CD8+ T cells appear to
play a more significant role in controlling EBV in the latent infection
state (2). In view of this, we explored whether
EBNA2280–290-reactive CD4+
T cells would be capable of exerting antiviral activity to control the
early events of EBV-induced B cell proliferation that ultimately lead
to malignant transformation. Various numbers of
EBNA2280–290-reactive CD4+
T cells derived from three separate donors (M1, M14, and M24) were
evaluated for their ability to inhibit EBV-mediated B cell
proliferation in a 10- to 15-day culture assay. For donor
M14, a T cell clone specific for an irrelevant non-EBV Ag (MAGE3) was
included as a negative control. In all cases the EBV-reactive T cells
were highly effective at inhibiting the proliferation of
CD23+ B lymphocytes, which was induced by EBV
infection (Fig. 4). In contrast, the
MAGE3-reactive T cell clone did not have a significant effect on the
proliferation of B cells (Fig. 4, middle panel, 
),
indicating that the inhibitory effect of the EBV-reactive T cells was
Ag specific.
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, left
panel, indicate that even in the presence of CsA (at 1 µg/ml),
which was added at the initiation of the experiments, the
EBNA2280–290-specific T cells were very
effective in inhibiting the EBV-induced proliferation of B lymphocytes.
On the other hand, the same concentration of CsA was capable of
suppressing the production of IFN- by PBMC that were stimulated with
antiCD3 Abs (Fig. 5, middle panel), indicating that this
compound was active at this dose. Similarly, the production of IFN-
by EBNA2280–290-specific T cells induced by Ag
presented by EBV-LCL was also inhibited by CsA (Fig. 5, right
panel). In another set of experiments performed under more
strenuous conditions, CsA was added to some of the cultures every
day (to a final 1 µg/ml) to take into account the possible effects
due to the decrease in the drug’s potency during the 10-day
experiment. Under these conditions, the
EBNA2280–290-specific T cells were still capable
of significantly inhibiting the EBV-induced B cell proliferation by
>70% (Fig. 6). Overall, these results
suggest that even in the presence of high doses of CsA,
EBNA2280–290-specific CD4+
T cells may be effective in preventing B lymphocyte proliferation
induced by EBV.
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The EBNA2280–290-reactive
CD4+ T cells may use various effector mechanisms,
such as cytotoxicity or the production IFN-, to inhibit EBV-mediated
B cell proliferation. However, the results presented in Figs. 5
and 6
suggest that for T cells to inhibit EBV infection and subsequent B cell
proliferation they could use a CsA-insensitive effector mechanism such
as cell-mediated cytotoxicity. The cytotoxic activity of the six
EBNA2280–290-specific T cell lines was evaluated
against various target cells. As shown in Fig. 7, the CD4+ T cell
lines from all six donors were capable of killing peptide-pulsed
autologous target cells. In all cases, with the exception of donor M1,
no cytotoxicity was detected against the MHC class II-negative T2
cells, indicating the requirement of TCR interaction with peptide MHC
complexes on the target cells. Moreover, in the case of donors M69 and
M44 (HLA-DR52-restricted) the T cells were also effective in killing
the autologous EBV-transformed LCL in the absence of exogenously added
peptide, indicating that the amount of naturally processed
EBNA2280–290 epitope in these cells was
sufficient to allow the cytolytic reaction to take place. Furthermore,
the EBNA2280–290-reactive T cell line from donor
M69 was not able to kill an EBV-LCL from an HLA-DR52-negative donor,
regardless of whether these cells were pulsed with synthetic peptide
(Fig. 7, upper left panel). These data indicate that lysis
by these cells is MHC restricted and requires the presence of the
specific MHC peptide complexes on the target cells. We noted that the
potency of the cytotoxic function of the M69 and M44 T cell lines was
different compared with that of the other cell lines. While 30%
specific lysis (50% of the maximal activity) was obtained at an E:T
cell ratio of 1:1 or less in the M69 and M44 T cell lines to obtain the
same level of cytotoxicity with the other T cell lines, an 
3-fold
greater E:T cell ratio was required (Fig. 7, dashed lines). These
results indicate that the M69 and M44 T cell lines were significantly
more potent in killing their target cells than the other T cell lines,
and this could help explain the inability of the latter cells in
killing the EBV-LCL in the absence of exogenously added peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Interestingly for one of the
candidate peptides, an almost exact sequence
(EBNA2280–290) had been previously described to
function as a T cell epitope restricted by the HLA-DQ2 and -DQ7 MHC
class II alleles (29). Thus, we reasoned that peptide
EBNA2280–290 could constitute a typical MHC
class II promiscuous epitope, capable of being presented to
CD4+ T cells by multiple HLA-DR and -DQ alleles.
Indeed, our data support this prediction, since we were able to elicit
T cell responses to peptide EBNA2280–290, which
were restricted by HLA-DR1, -DR7, -DR16, -DR52, and -DQ2 (Fig. 2). The
computer-based algorithm that we used predicted that this peptide
sequence would serve as an epitope restricted by DR1, DR4, and DR7.
However, we and others have observed that the many of the peptides
predicted by this algorithm can also bind to other MHC class II
alleles, such as DR9, DR13, DR15, DR51, DR52, DR53, and DQ6 (25, 26, 31). Although the predicted ARB of peptide
EBNA2280–290 for HLA-DR4 was very high (Table I), we have not yet observed T cell responses restricted by this
allele. Of all the responders, M9 expressed the DR4 allele, but
unfortunately the EBNA2280–290-reactive T cell
was not stable and could not be tested for its MHC restriction. Of the
four nonresponders (Fig. 1), M2 and M20 expressed DR4, but in neither
case we were able to establish an Ag-reactive T cell line. Additional
experiments will be required to determine whether HLA-DR4 is capable of
presenting peptide EBNA2280–290 to
CD4+ T lymphocytes.
Our results indicate that although peptide
EBNA2280–290 is promiscuous with regard to its
capacity to interact with various MHC class II molecules, the
presentation of the MHC/peptide complexes to the TCR itself is not
degenerate. For example, our data show that T cells recognizing peptide
EBNA2280–290 in the context of DR1 did not
become stimulated by APC expressing DR52 and DQ7 (M69 donor in Fig. 2, top left panel), which are alleles capable of presenting
this epitope to T cells restricted to their own alleles. Similarly,
DQ2-restricted T cells from M14 did not recognize peptide in the
context of DR7, DR52, or DQ7 (Fig. 2, top middle panel);
DR52-restricted T cells from M44 did not respond to the peptide
presented by DR1 (Fig. 2, bottom left panel); and
DR7-restricted T cells from M83 failed to recognize peptide presented
by DR16, DR52, and DQ7 (Fig. 2, bottom right panel). In
addition, in the cytotoxicity assays DR52-restricted T cells from M69
did not kill peptide-pulsed target cells that expressed DQ7 (Fig. 7, upper left panel). These results suggest that the TCR from
the various T cell lines that we isolated interact not only with
residues of peptide EBNA2280–290, but also with
polymorphic residues of the MHC class II molecules.
The lack of success in establishing
EBNA2280–290-reactive cell lines from the four
nonresponding individuals (Fig. 1) could be due to one of several
possibilities. One possible explanation could be the absence of an MHC
allele capable of binding this peptide to present it to the
CD4+ T cells. However, this was not the case in
the present study, since all nonresponding donors had at least one MHC
class II allele capable of presenting peptide
EBNA2280–290. Donor M2 expressed DQ7, donor M15
expressed DR1, M20 expressed DR7 and DQ2, and donor M25 expressed DR1
and DQ7 (data not shown). Another likely explanation for not being able
to isolate EBNA2280–290-reactive T cell lines
from these individuals could be the presence of a low precursor
frequency of T cells specific for this epitope in peripheral blood at
the time of sampling. With some of these donors (M20 and M25), we had
technical difficulties in isolating the peptide-reactive T cell lines,
because their cells proliferated significantly in the absence of Ag
(high background), making it impossible to select the
EBNA2280–290- specific T cell lines. Regardless
of the cause(s) for not responding to the
EBNA2280–290 epitope in some donors, it is clear
from our results and previous findings that a large segment of the
general population (50%) would be able to respond to this epitope,
since the probability of expressing at least one of the MHC class II
alleles capable of presenting this peptide is quite high.
Our selection of EBNA2 as a target Ag to induce T cell responses
to prevent PTLD appears appropriate, since
EBNA2280–290-reactive T cell lines were
shown to be effective at inhibiting the proliferation of B lymphocytes
induced by live EBV (Fig. 3). Although EBNA2 is considered to be one of
EBV’s latent cycle Ags, which is expressed on transformed EBV-LCL
(1, 4), this protein is also one of the first to be
produced after viral infection (32) and before
immortalization (33). Most importantly, EBNA2 functions as
a viral transcription factor that is essential for the initiation and
maintenance of the B lymphocyte growth and transformation states
(34, 35). Thus, T cell responses to EBNA2 would be
beneficial for controlling both the early infection of EBV (to prevent
PTLD) and the EBV-transformed B cells that may result in lymphomas. The
latter is supported by the data demonstrating that some of the
EBNA2280–290-reactive T cell lines were capable
of recognizing EBV-transformed LCL (Fig. 3), and in some cases the T
cells were also capable of killing the EBV-LCL (Fig. 7).
The effector mechanism(s) by which the
EBNA2280–290-specific CD4+
T cells are able to inhibit EBV-induced B cell proliferation (
Figs. 4–6) may be numerous. The production of lymphokines such as IFN- by
Ag-stimulated T cells could have an antiviral effect, inhibiting
infection and virus-induced B cell proliferation. However, this
mechanism would not be operative in the presence of some
immunosuppressive compounds such as CsA (Fig. 5, right
panel). Another more likely effector mechanism that
CD4+ Th cells might employ to inhibit
EBV-mediated B cell proliferation is via cytolysis of the
newly-infected cells. It has been reported that
CD4+ T cells can exhibit a high degree of
cytotoxicity via either the perforin or Fas/Fas ligand pathway
(36, 37, 38, 39, 40). Indeed, our results show that
EBNA2280–290-reactive CD4+
T cells displayed significant levels of Ag-specific cytotoxicity (Fig. 7). In the case of the DR52-restricted T cells from M69 and M44,
significant cytotoxicity was observed to the autologous EBV-LCL in the
absence of exogenous peptide, indicating the presence of optimal
amounts of naturally produced peptide/MHC complexes on these targets.
However, with other T cell lines the cytolytic activity against
autologous EBV-LCL was only observed with the addition of exogenous
peptide (Fig. 7). The cytotoxicity results contrast with the data
obtained in the IFN- release assay (Fig. 3), where all T cell lines
were capable of recognizing the naturally processed epitope presented
by autologous EBV-LCL. This discrepancy could be explained by
differences in the amount of time required to generate a signal in
these assays. In the cytotoxicity assays the T cells and targets were
allowed to interact with each other for 6–9 h, while in the cytokine
release assay this interaction lasted for 48 h. Thus, it is likely
that only those T cells with high avidity for their Ag will be able to
recognize autologous EBV-LCL in a short period of time (cytotoxicity
assay). The avidity of the T cells for their APC (or their targets) may
be influenced by numerous factors such as 1) the density of specific
MHC/peptide complexes on the APC, 2) the intrinsic affinity of the TCR
for the Ag, and 3) the level of expression of adhesion molecules and
their ligands, which help to stabilize cell to cell interactions. It is
possible that newly EBV-infected B cells could serve as better targets
for EBNA2280–290-reactive T cells than EBV-LCL,
which would help explain the strong, CsA-resistant
antilymphoproliferative effects observed (
Figs. 4–6). This possibility
would exist if the recently infected B cells were to express higher
levels of EBNA2280–290/MHC complexes (or
adhesion molecules) than the EBV-LCL.
In conclusion, our results indicate that the EBNA2280–290 epitope would be effective at eliciting potent anti-EBV CD4+ T cell responses capable of preventing PTLD in solid organ transplant candidates. Vaccination of patients with this epitope before the transplant and subsequent immunosuppressive therapy would allow the establishment of T cells capable of inhibiting B cell proliferation induced by EBV infection, which leads to PTLD. Alternatively, in vitro generated T cell lines reactive with the EBNA2280–290 epitope could be used for adoptive immunotherapy in patients with ongoing PTLD. Most importantly, our results suggest that once primed by Ag, the resulting effector EBNA2280–290-specific CD4+ T cells would be able to control PTLD even in the presence of some immunosuppressive drugs, such as CsA. Finally, because the EBNA2280–290 peptide is highly promiscuous toward various MHC class II alleles, it could be used as a vaccine in a large proportion of patients. A peptide vaccine clinical study in pretransplant patients using this epitope that is underway at our institution may help to answer some of these important questions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Esteban Celis, Department of Immunology, GU421A, Mayo Clinic, Rochester, MN 55905. E-mail address: celis.esteban{at}mayo.edu
3 Abbreviations used in this paper: HTL, helper T lymphocytes; ARB, average relative binding; CsA, cyclosporin A; DC, dendritic cells; LCL, lymphoblastoid cell line; PTLD, posttransplant lymphoproliferative disorder.
Received for publication March 29, 2002. Accepted for publication June 4, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 H. Kobayashi, T. Nagato, M. Takahara, K. Sato, S. Kimura, N. Aoki, M. Azumi, M. Tateno, Y. Harabuchi, and E. Celis Induction of EBV-Latent Membrane Protein 1-Specific MHC Class II-Restricted T-Cell Responses against Natural Killer Lymphoma Cells Cancer Res., February 1, 2008; 68(3): 901 - 908. [Abstract] [Full Text] [PDF]
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T. A. Haigh, X. Lin, H. Jia, E. P. Hui, A. T. C. Chan, A. B. Rickinson, and G. S. Taylor EBV Latent Membrane Proteins (LMPs) 1 and 2 as Immunotherapeutic Targets: LMP-Specific CD4+ Cytotoxic T Cell Recognition of EBV-Transformed B Cell Lines J. Immunol., February 1, 2008; 180(3): 1643 - 1654. [Abstract] [Full Text] [PDF]
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 S. Bhaduri-McIntosh, M. J. Rotenberg, B. Gardner, M. Robert, and G. Miller Repertoire and frequency of immune cells reactive to Epstein-Barr virus-derived autologous lymphoblastoid cell lines Blood, February 1, 2008; 111(3): 1334 - 1343. [Abstract] [Full Text] [PDF]
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 B. P. O'Neill, S. Vernino, A. Dogan, and C. Giannini EBV-associated lymphoproliferative disorder of CNS associated with the use of mycophenolate mofetil Neuro-oncol, July 1, 2007; 9(3): 364 - 369. [Abstract] [Full Text] [PDF]
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H. Kobayashi, T. Nagato, K. Sato, N. Aoki, S. Kimura, M. Murakami, H. Iizuka, M. Azumi, H. Kakizaki, M. Tateno, et al. Recognition of Prostate and Melanoma Tumor Cells by Six-Transmembrane Epithelial Antigen of Prostate-Specific Helper T Lymphocytes in a Human Leukocyte Antigen Class II-Restricted Manner Cancer Res., June 1, 2007; 67(11): 5498 - 5504. [Abstract] [Full Text] [PDF]
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G. S. Taylor, H. M. Long, T. A. Haigh, M. Larsen, J. Brooks, and A. B. Rickinson A Role for Intercellular Antigen Transfer in the Recognition of EBV-Transformed B Cell Lines by EBV Nuclear Antigen-Specific CD4+ T Cells J. Immunol., September 15, 2006; 177(6): 3746 - 3756. [Abstract] [Full Text] [PDF]
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 H. Kobayashi, T. Ngato, K. Sato, N. Aoki, S. Kimura, Y. Tanaka, H. Aizawa, M. Tateno, and E. Celis In vitro Peptide Immunization of Target Tax Protein Human T-Cell Leukemia Virus Type 1-Specific CD4+ Helper T Lymphocytes. Clin. Cancer Res., June 15, 2006; 12(12): 3814 - 3822. [Abstract] [Full Text] [PDF]
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 K. N. Heller, C. Gurer, and C. Munz Virus-specific CD4+ T cells: ready for direct attack J. Exp. Med., April 17, 2006; 203(4): 805 - 808. [Abstract] [Full Text] [PDF]
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E. Landais, X. Saulquin, M. Bonneville, and E. Houssaint Long-Term MHC Class II Presentation of the EBV Lytic Protein BHRF1 by EBV Latently Infected B Cells following Capture of BHRF1 Antigen J. Immunol., December 15, 2005; 175(12): 7939 - 7946. [Abstract] [Full Text] [PDF]
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E. Piriou, K. van Dort, N. M. Nanlohy, M. H. J. van Oers, F. Miedema, and D. van Baarle Loss of EBNA1-specific memory CD4+ and CD8+ T cells in HIV-infected patients progressing to AIDS-related non-Hodgkin lymphoma Blood, November 1, 2005; 106(9): 3166 - 3174. [Abstract] [Full Text] [PDF]
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H. Kobayashi, T. Nagato, K. Oikawa, K. Sato, S. Kimura, N. Aoki, R. Omiya, M. Tateno, and E. Celis Recognition of Prostate and Breast Tumor Cells by Helper T Lymphocytes Specific for a Prostate and Breast Tumor-Associated Antigen, TARP Clin. Cancer Res., May 15, 2005; 11(10): 3869 - 3878. [Abstract] [Full Text] [PDF]
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, Proliferative responses where SI is >3 (considered significant);
). The production of IFN-
, Numbers of CD23+ B
cells obtained in the absence of T cells; 
),
peptide-pulsed autologous EBV-LCL (•), and MHC class II-negative T2
cells (
) and pulsed with peptide
(
).