|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3439-3447
Epstein-Barr Virus Induces Fas (CD95) in T Cells and Fas Ligand in B
Cells Leading to T-Cell Apoptosis
By
Jerome E. Tanner and
Caroline Alfieri
From the Department of Pediatrics, Children's Hospital of Eastern
Ontario, University of Ottawa Medical School, Ottawa, Ontario, Canada;
and the Laboratory of Molecular Virology, Ste-Justine Hospital Research
Center, Department of Microbiology and Immunology, University of
Montréal, Montréal, Québec, Canada.
 |
ABSTRACT |
Epstein-Barr virus (EBV) acute infectious mononucleosis (AIM) is
characterized by transient immunosuppression in vivo and increased
T-cell apoptosis after ex vivo culture of AIM peripheral blood
mononuclear cells. We undertook experiments to test whether EBV or
purified virion envelope glycoprotein gp350 could contribute to
Fas-mediated T-cell apoptosis. Our in vitro results indicate that EBV
increased Fas expression in CD4+ T cells and Fas ligand
(FasL) expression in B cells and macrophages. Purified gp350 was also
shown to significantly increase CD95 expression in CD4+ T
cells. When T-cell CD95 was cross-linked, EBV-stimulated T cells
underwent apoptosis. The induction of T-cell CD95 by EBV followed by
CD95 cross-linking with anti-CD95 monoclonal antibody resulted in a
loss in the number of T cells responding to the T-cell mitogens,
anti-CD3 antibody, and interleukin-2. These results indicate that, in
addition to serving as a principal ligand for the attachment of virus
to target cells, gp350 may also act as an immunomodulatory molecule
that promotes T-cell apoptosis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE INFECTIOUS mononucleosis
(AIM) is a self-limiting lymphoproliferative disease caused by the
Epstein-Barr virus (EBV).1 AIM is characterized by a 2-fold
or greater expansion in the number and percentage of CD8+ T
lymphocytes and a 3-fold decrease in the percentage of CD4+
T lymphocytes, a marked impairment of normal T-cell responses, ie,
anergy, as well as a high number of peripheral blood T cells predisposed to undergo apoptosis when cultured ex vivo.2-10
Recently, the Fas antigen, now designated as CD95, has been shown to be
an important mediator of T-cell apoptosis.11,12 After
cross-linking of surface CD95 by its natural ligand, Fas ligand (FasL),
or by an anti-Fas monoclonal antibody (MoAb), cells will undergo
apoptosis.13,14 In addition to EBV, CD95 expression has
been shown to be elevated in T cells after infection by a number of
viruses, including those that lead to transient or progressive immunosuppression.9,15-22
EBV displays a unique tropism for B cells that express the C3d
receptor, CD21, due to C3d homologous amino acid sequences in the
virion envelope glycoproteins, gp350 and gp220 (gp350).23 The EBV receptor, CD21, is found principally on B cells, but it is also
expressed, albeit at lower levels, in epithelial cells and T
cells.24, 25 In addition to CD21, T cells may also express
an additional 70-kD EBV binding protein.26
Because gp350 is capable of binding to and activating B lymphocytes in
vitro27 through a receptor that may also be expressed on T
cells and because AIM T cells are susceptible to apoptosis, we
undertook experiments to determine whether the EB virion, or purified
EBV glycoprotein gp350, could bind to T cells and induce CD95
expression. We also investigated whether CD95 expression predisposed T
cells to undergo apoptosis. The results from such experiments may
suggest a mechanism to explain why T cells from AIM patients undergo apoptosis.
| |
MATERIALS AND METHODS |
Lymphocyte donors.
Peripheral blood mononuclear cells (PBMCs) were obtained from patients
diagnosed at Sainte-Justine Hospital (Montreal, Quebec, Canada) with
EBV AIM, as determined by the presence of fever, pharyngitis,
lymphadenopathy, lymphocytosis with atypical lymphocytes, and
heterophile antibodies (determined by monospot testing; Monosticon Dri-Dot; Organon Teknika, Durham, NC),28 and from healthy
volunteer blood donors (kindly supplied by the Ottawa Red Cross
[Ottawa, Ontario, Canada] and the Children's Hospital of Eastern
Ontario [Ottawa, Ontario, Canada]). PBMCs were isolated from
heparinized whole blood by Ficoll-hypaque density gradient centrifugation.
Cells and culture.
PBMCs, peripheral blood T and B cells, the EBV-producing cell lines
B95-8 (CRL 1621; American Type Culture Collection
[ATCC], Manassas, VA) and Akata29 (gift from
Dr K. Takada, Nihon University School of Medicine, Tokyo, Japan) or the
EBV-negative B-cell line Louckes (gift from Dr E. Kieff, Harvard
Medical School, Boston, MA) were all maintained in Iscove's modified
Dulbecco's medium, supplemented with 10% heat-inactivated fetal calf
serum (FCS), 2 mmol/L glutamine, 50 U/mL penicillin, and 50 µg/mL
streptomycin (GIBCO BRL, Burlington, Ontario, Canada).
T- and B-cell purification.
Peripheral blood T cells were obtained from healthy blood donors after
separation on Ficoll-hypaque density gradients and rosetting with
2-aminoethylisothiouronium-treated sheep red blood cells.30,31 B cells were obtained after Ficoll-hypaque
density gradient centrifugation and CD19-dynabead affinity-purification (Dynal, Success Lake, NY). T-cell preparations were found to be 93% ± 2.3% (mean ± standard error of the mean [SEM])
CD3+ (T cells), 1.5% ± 1.5%
CD19+ (B cells), and 1.5% ± 0.25% CD14+
(monocytes/macrophages). B-cell preparations were found to be 98% ± 2.4% CD21+, 2.3% ± 2.6% CD3+, and
0.94% ± 0.032% CD14+ as determined by immunostaining
with phycoerythrin (PE)-coupled antihuman CD3 mouse MoAb (clone Leu-4;
Becton Dickinson, San Jose, CA), PE-coupled antihuman CD19
mouse MoAb (clone Leu 12; Becton Dickinson), PE-coupled antihuman CD21
mouse MoAb (clone HB-5; Becton Dickinson), and PE-coupled antihuman
CD14 mouse MoAb (clone Leu M p9; Becton Dickinson) and flow
cytometric analysis.
EBV and gp350 preparation.
EBV was isolated as filtered concentrated cell supernatant from B95-8
cell cultures in which virus was induced by starving cells for 14 days32 or as purified virus obtained from Akata cells after
stimulation with rabbit antihuman IgG and 2 cycles of dextran T-10
gradient centrifugation.29,33 Virus purity was confirmed by
electron microscopy. Mock-EBV samples were obtained using an identical
procedure with the EBV-negative B-cell line, Louckes, or from
unstimulated Akata cell culture supernatant also banded by dextran
gradient centrifugation.
The gp350 complex was generated by initially diluting purified
anti-gp350 MoAb 2L10 (gift from Dr Gary Pearson, Georgetown University,
Washington, DC) or 72A1 (HIB 167; ATCC and Hoffman et al34)
or an isotype-matched negative control antibody (antirespiratory syncytial virus nuclear protein MoAb IgG 858-3; Chemicon International, Los Angeles, CA) to 1 mg/mL in 100 µL of 0.1 mol/L sodium bicarbonate (pH 8.2), followed by incubation in a sterile flat-bottom 96-well enzyme-linked immunosorbent assay (ELISA) plate (Nunc, Canadian Life
Technologies, Burlington, Ontario, Canada). After 18 hours, antibody
was removed, plates were washed with phosphate-buffered saline (PBS),
and wells were blocked with culture medium. A total of 100 µL of
culture medium containing 2 µg of purified gp35035 was
added per well, and plates were incubated at 4°C for an additional 3 hours. Unbound gp350 was then removed by PBS washes. The purity of
gp350 was checked by gel electrophoresis and silver
staining.36 The anti-gp350 MoAb, 2L10, has previously been
shown not to block EBV or gp350 binding to the EBV receptor, whereas
72A1 has been shown to block EBV or gp350 binding to the CD21
molecule.35
Induction of T-cell CD95 expression and apoptosis.
T cells or PBMCs, which were initially incubated at 4°C for 2 hours
with 1/10 vol of concentrated virus stock preparation, 1/10 vol of
purified virus, or 1/10 vol of mock-virus preparation, were washed 3 times with cold medium and seeded at 5 × 105 cells/mL
in 2 mL complete medium in a 24-well flat-bottom plate. Parallel
cultures of cells, which were cultured in the presence of
phytohemagglutinin (PHA; 1:200 vol/vol) served as control for apoptosis
and CD95 expression. Cells were cultured for 24-hour intervals,
followed by collection and staining with either fluorescein isothiocyanate (FITC)- or PE-conjugated MoAb, FITC-conjugated Annexin V
(R&D Systems, Minneapolis, MN), or propidium iodide.
In experiments that involved the blocking of EBV binding to T cells for
subsequent inhibition of CD95 expression, concentrated virus was first
incubated for 1 hour on ice with purified anti-gp350 MoAb 72A1 (1 µg/mL) or an irrelevant monoclonal (antirespiratory syncytial virus
nuclear protein monoclonal 858-3; 1 µg/mL) before its addition to
purified T cells. The cell:virus mixture was then incubated for 2 hours
on ice to allow for virus binding, followed by several washes with cold
medium, and cultured for 24 hours.
In several experiments, T cells that had been stimulated with EBV or
with mock-virus preparations were also cultured in the presence or
absence of 5 µg/mL antihuman CD95 IgM MoAb (clone CH-11 antibody; MBL
International, Watertown, MA) for 12 or 24 hours before cell staining
for CD95, apoptosis analysis, or continued stimulation with antihuman
CD3 mouse monoclonal, OKT-3, and 50 U/mL interleukin-2 (IL-2; Cetus,
Emeryville, CA).37
Flow cytometric analysis.
Cells were stained with FITC-conjugated antihuman CD95 mouse MoAb
(UB-2; MBL International) and PE-coupled antihuman CD4 mouse MoAb
(clone SK3; Becton Dickinson), PE-coupled antihuman CD8 mouse MoAb
(clone SK1; Becton Dickinson), or PE-coupled antihuman CD56 mouse MoAb
(clone MY31; Becton Dickinson). In some experiments, cells were also
incubated with the above-mentioned PE-conjugated monoclonals or with
PE-coupled antihuman CD20 mouse MoAb (clone 2H7; Pharmingen Canada,
Mississauga, Ontario, Canada), PE-conjugated antihuman CD14 mouse MoAb
(clone m p9; Becton Dickinson), and rabbit antihuman FasL peptide
polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA).
Cell-bound antihuman FasL was detected with FITC-coupled goat
antirabbit Ig antibody (GIBCO BRL). Cells were analyzed for FITC and PE
dual immunofluorescence by flow cytometry. Appropriate FITC and PE
control antibodies (Becton Dickinson) were also included to monitor
nonspecific antibody binding.
Measurement of apoptosis by flow cytometry.
The proportion of cells undergoing apoptosis was determined by Annexin
V (R&D Systems) or propidium iodide staining.38 The distinct cell cycle region of apoptosis (Ao region) seen after propidium iodide staining has been shown previously to be that found
below the Go/G1 diploid peak.
Statistical analyses.
Statistical comparison of AIM and matched control populations used the
Mann-Whitney U test. Z values greater than 1.96 were considered
significant. All other statistical analyses used in this study used the
Student's t-test.
| |
RESULTS |
Expression of CD95 on lymphocytes from AIM patients.
Patients with AIM have been shown to exhibit high numbers of apoptotic
T cells after their culture in vitro.8 To determine whether
lymphocytes from AIM patients also exhibit elevated levels of the CD95
antigen, PBMCs obtained from AIM patients or matched healthy blood
donors were stained with FITC-labeled anti-CD95 MoAb and processed for
flow cytometry. We also determined the level of CD95 expression within
the CD4+ and CD8+ lymphocyte populations. As
shown in Fig 1A through F, PBMCs obtained from an AIM patient contained a greater number of CD95+
lymphocytes compared with those from a matched control blood donor
(75% v 6%). CD95 expression in the CD4+ and
CD8+ populations was 12% and 51% for this particular AIM
patient, versus 4% and 0.8% for a matched control donor,
respectively. Results from additional healthy blood donors and AIM
patients indicated that PBMCs from AIM patients contained 73.5% ± 6.4% (mean ± standard deviation [SD]) CD95+
lymphocytes, versus 26.1% ± 13.6% for healthy donor PBMCs (z = 3.15; Fig 1G). Analysis of CD95 expression in AIM CD4+ and
CD8+ populations showed that they also expressed a greater
number of CD95+ cells versus the control CD4+
and CD8+ cell populations. The CD4+ cells from
AIM patients were 12.6% ± 1.4% CD95+, versus 6.4% ± 3.8% CD95+ in the CD4+ control
population (z = 1.66; Fig 1G). The CD8+ cells from AIM
patients were 51.8% ± 6.3% CD95+ versus 3.8% ± 4.1% CD95+ for the CD8+ control population
(z = 3.09; Fig 1G). Although we did not address the nature of
the non-T-cell population in AIM PBMCs that expressed CD95, the
additional 9.1% CD95+ cells (73.5% CD95+
cells in PBMCs v 64.4% CD95+ cells in the
CD4+ and CD8+ lymphocyte population) may be EBV
or cytokine-stimulated B cells and phagocytic cells.39,40
View larger version (31K):
[in this window]
[in a new window]
| Fig 1.
Levels of CD95 expression on PBMCs and CD4+
and CD8+ peripheral blood T cells from AIM patients.
PBMCs from a representative healthy blood donor (A through C) or from
an AIM patient (D through F) were stained with FITC- and PE-conjugated
control IgG (A and D), FITC-conjugated anti-CD95 and PE-conjugated
anti-CD4 (B and E), or FITC-conjugated anti-CD95 and PE-conjugated
anti-CD8 (C and F). Results from gated cells (upper right inserts in
[A] and [D]) were plotted on a logarithmic scale. CD95 expression
in gated peripheral blood lymphocytes from 25 healthy blood donors
( ) or 7 AIM patients ( ) (G). Cells were analyzed by 2-color
immunofluorescence after staining with FITC-conjugated CD95 (x-axis)
and PE-conjugated anti-CD4 or anti-CD8 MoAbs (y-axis). Results are
expressed as the mean percentage positive ± SD. Statistically
significant values analyzed by the Mann-Whitney U test are designated
by an asterisk.
|
|
Induction of CD95 expression after exposure to EBV or EBV gp350.
Because AIM patients have a significant number of EBV-positive B cells,
which in turn have the potential to undergo virus replication and gp350
expression in the lymph node,41-45 we investigated whether
the EB virion was capable of inducing CD95 expression in peripheral
blood T cells. Initially, we determined the percentage of T cells that
bound to EBV, because T cells or T-cell lines have previously been
shown to express the EBV receptor.26,46 As shown in
Fig 2A, up to 34% of the peripheral blood
T cells were found to bind EBV, as determined by gp350
immunofluorescence. Peripheral blood B cells from the same donor showed
an expected higher positive percentage (83%) for gp350
immunofluorescence (Fig 2B). These results are in agreement with those
of Levy et al46 and Hedrick et al,26 who found
that primary peripheral blood T cells or T-cell lines expressed an EBV
receptor that bound EBV in up to 23% and 50% of the cells,
respectively.
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
Binding of EBV to T and B cells (A and B, respectively).
Affinity-purified T or B cells derived from a healthy donor's
peripheral blood (shown in upper right inserts) were incubated at 1 × 106 cells/200 µL on ice for 2 hours with 1/10 vol of
concentrated B95-8 virus or mock virus stock preparations. Cells were
washed with cold PBS and stained for bound gp350 using anti-gp350 MoAb,
2L10. Samples were developed with FITC-conjugated goat antimouse IgG.
Results were plotted on a logarithmic scale. Virus- and mock-infected
cells are indicated by solid or open histograms, respectively. The
percentage of fluorescent cells greater than that of the mock-infected
cells is indicated above the bar. T and B cells were 87% and 92%
viable, respectively, after affinity purification, as measured by
trypan blue exclusion.
|
|
To investigate whether CD95 is expressed on T cells after exposure to
EBV, as well as to study the kinetics of CD95 expression, we measured
CD95 immunofluorescence on T cells over a 48-hour period. As shown in
Fig 3A and B, increased CD95 expression was detected as early as 12 hours postinfection in both the
CD4+ and CD8+ T-cell populations. CD95 was seen
to peak at 24 hours, displaying 15% and 4% positive-cell
immunofluorescence on CD4+ and CD8+ cells,
respectively. CD95 continued to be expressed on the T cells during the
remainder of the 48-hour period, albeit at reduced levels (Fig 3A and
B). This was compared with mock-virus-treated CD4+ and
CD8+ T cells that displayed no more than 2% and 0.7%
CD95+ immunofluorescence, respectively, during the 48-hour
period (Fig 3A and B). The lower percentage of CD95-expressing cells
seen during the latter part of the 48-hour period in T cells that were exposed to EBV may be due to a loss of surface-bound virus through cellular internalization and proteolytic processing. Further evidence that EBV specifically induced CD95 expression in T cells can be seen in
Fig 3C. CD56+ NK cells that were exposed to EBV or
mock-virus treatment did not express significant levels of CD95 over
the entire 72-hour period (<1%; Fig 3C).
View larger version (11K):
[in this window]
[in a new window]
| Fig 3.
Time course of CD95 expression for CD4+,
CD8+, and CD56+ lymphocytes after EBV
infection. PBMCs were incubated on ice for 2 hours with 1/10 vol of
concentrated B95-8 virus () or mock virus ( ) preparations, washed
with PBS, and cultured at 5 × 105 cells/mL in 2 mL of
culture medium. Aliquots were taken at hourly intervals and stained by
2-color immunofluorescence using FITC-conjugated CD95 and PE-conjugated
anti-CD4 (A), anti-CD8 (B), or anti-CD56 (C) MoAbs. (D) PBMCs from 4 different blood donors were stained at 24 hours for CD95 (), for
CD95 and CD56 ( ), for CD95 and CD4 ( ), or for CD95 and CD8
(). Results are expressed as the mean percentage
positive ± SEM. CD95-fluorescence activity found in untreated cells
(medium only) was subtracted from both mock- and virus-treated samples
before plotting for each time point.
|
|
Results from in vitro testing of 4 additional blood donor T-cell
preparations for their CD95 expression after EBV stimulation indicated
that EBV induced 30% more T lymphocytes to express CD95 as compared
with mock treatment (P = .03; Fig 3D). Similarly, 16%
(P = .01) and 8% (P = .08) more CD4+ and
CD8+ cells, respectively, expressed CD95 after EBV
treatment, as compared with mock treatment (Fig 3D). CD56+
cells expressed 0.7% and 0.5% CD95 after either EBV or mock
stimulation, respectively (Fig 3D). The induction of CD95 expression in
our donor T-cell populations did not appear to be due simply to the presence of EBV-reactive memory T cells, because 1 of the blood donors
(PR) was EBV-seronegative and displayed 12% and 8% increases, respectively, in the number of CD4+ and CD8+ T
cells expressing CD95.
To determine whether the major virion envelope glycoprotein, gp350,
alone was responsible for inducing CD95 in T cells, as opposed to
stimulation in conjunction with another virion surface protein, T cells
were incubated with EBV, with EBV plus the gp350 neutralizing antibody
(72A1),34 or with gp350 coated onto microplate wells. As
seen in Fig 4, gp350 was capable of
inducing CD95 expression in CD4+ T cells to a level
comparable with that of EBV. CD4+ and CD8+ T
cells were 14% and 3% CD95+, respectively, after
2L10-gp350 exposure, versus 1% and less than 1% CD95+,
respectively, in CD4+ and CD8+ T cells exposed
to 2L10 antibody alone (P = .024 for 2L10-gp350 exposure
v 2L10 antibody exposure in CD4+ T cells).
CD4+ and CD8+ T cells were 22% and 7%
CD95+, respectively, after EBV exposure, versus 3% and 5%
CD95+, respectively, in CD4+ and
CD8+ T cells exposed to mock-treatment (P = .03 for
EBV-treatment v mock-treatment in CD4+ T cells).
PHA induced CD95 levels in CD4+ and CD8+ T
cells to 34% and 39%, respectively, compared with mock treatment (P = .02 and P = .03 in CD4+ and
CD8+, PHA-treated T cells v mock-treated
CD4+ and CD8+ T cells, respectively). MoAbs
2L10, 72A1, and 858-3 coated onto the wells failed to induce
significant levels of CD95 in T cells (no more than 5% and 0.6% for
CD4+ and CD8+ T cells, respectively, as
compared with medium). Interestingly, T cells treated with 72A1 Ig
plate-bound gp350 did not increase their level of CD95 expression to
that of 2L10, suggesting that the T-cell binding epitope for gp350 may
be at the same site as that for the B-cell EBV binding epitope that is
found in proximity to the gp350 amino-terminus.35
View larger version (27K):
[in this window]
[in a new window]
| Fig 4.
Induction of CD95 in T cells by EBV and purified gp350.
(A) Affinity-purified T cells were incubated at 1 × 106 T
cells/200 µL for 2 hours on ice with 1/10 vol of mock virus, 1/10 vol
of purified Akata virus, or 1/10 vol of purified Akata virus that was
previously incubated on ice for 1 hour with either 1 µg/mL 72A1 or
858-3 antibody. Cells were washed 2 times with cold PBS and cultured
for 24 hours. Cells were stained with FITC-conjugated CD95 MoAb and
PE-conjugated antihuman CD4 MoAb () or antihuman CD8 MoAb ().
Purified T cells (2 × 105/200 µL) were cultured for 18 hours with either plate-bound anti-gp350/220 MoAbs 72A1, 2L10, control
MoAb 858-3, antibody-gp350 complexes, or PHA. Cells were stained with
FITC-conjugated anti-CD95 MoAb and PE-conjugated antihuman CD4 MoAb or
antihuman CD8 MoAb. CD95 expression in medium-treated
CD4+ and CD8+ T cells for the 3 separate
donors was subtracted during analysis and the mean CD95 percentage
positive cells ± SD was plotted. Statistically significant values
after analysis by the Student's t-test are designated by an
asterisk. (B) Purified Akata virus used in the EBV stimulation
experiments is shown (original magnification × 86,500). (C) The
purified gp350 used in the formation of the antibody-gp350 complexes
was resolved in a 6.5% gel by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and silver stained.
|
|
Induction of T-cell CD95-mediated apoptosis after EBV exposure.
Because T cells from AIM patients have been shown to undergo apoptosis
when cultured in vitro, we sought to determine whether in vitro
EBV-exposed T cells underwent apoptosis. Exposure of PBMCs to purified
virus or PHA and subsequent staining for CD4 or CD8 and Annexin V
showed that 24% of the CD4+ cells underwent EBV-specific
apoptosis compared with mock treatment (P = .03).
CD8+ cells only underwent 6% EBV-specific apoptosis
(Fig 5). The lack of significant
CD8+ cell apoptosis after EBV stimulation was not due to an
inability of these cells to undergo apoptosis, because PHA treatment
resulted in significant levels of CD8+ cell apoptosis
(56%; P = .0007; Fig 5). Interestingly, and in contrast,
purified T cells exposed to EBV did not undergo spontaneous apoptosis,
but were capable of undergoing apoptosis if subsequently treated with
the anti-CD95 mouse monoclonal, CH-11 (Fig
6). Comparison of mock-infected versus EBV-infected T cells showed that
apoptotic cells increased by only 4% in the EBV-infected population,
but increased by 7% and 37% in the anti-CD95 treated, mock- and
EBV-infected population, respectively (Fig 6). Because our T cells did
not undergo significant apoptosis unless treated with anti-CD95 MoAb, these results suggested that EBV stimulation alone failed to induce significant levels of FasL in T cells.
View larger version (15K):
[in this window]
[in a new window]
| Fig 5.
Level of CD4+ and CD8+ cell
apoptosis in PBMCs after EBV stimulation. PBMCs were stimulated with
purified Akata virus or mock virus and were then seeded at 5 × 105 cells/mL and cultured for 24 hours. Cells were
subsequently stained with FITC-coupled Annexin V and PE-labeled
anti-CD4 or PE-labeled anti-CD8. The mean percentage values of Annexin
V-positive CD4+ or CD8+ cells ± SEM
obtained from 4 separate donors are plotted.
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 6.
Induction of apoptosis in T cells by EBV. T cells were
mock-infected (A and B) or EBV-infected (C and D) in the absence (A and
C) or presence (B and D) of anti-CD95 IgM. The percentage of apoptotic
cells (Ao) is indicated.
|
|
Further testing of FasL levels in PBMC CD14+ monocytes,
CD56+ NK cells, CD20+ B cells, or
CD4+ and CD8+ T cells from EBV-stimulated PBMCs
indicated that EBV markedly increased the number of B cells expressing
FasL as compared with both the untreated B-cell population or with
those that were mock infected (2- and 6-fold increase in the percentage
of FasL-positive B cells; P = .0001 and P = .005, respectively; Fig 7). CD14+
monocytes also appeared to increase their FasL after EBV infection compared with mock-infected cells (2.6-fold, P = .009);
however, when they were compared with the untreated population, they
did not show a significant statistical difference (P = .44; Fig
7). CD56+ NK cells increased FasL expression upon in vitro
culture, regardless of the stimulant, making it unclear whether EBV
plays a specific role in increasing NK FasL (Fig 7). CD4+
and CD8+ T cells stimulated with EBV or PHA did not show a
statistically significant increase in their FasL expression when
compared with either the untreated CD4+ and
CD8+ population or mock-infected cells (Fig 7).
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Induction of FasL in PBMCs after EBV or PHA stimulation.
PBMCs from healthy blood donors were incubated at 1 × 106
cells/200 µL for 2 hours on ice with 1/10 vol of mock virus, purified
Akata virus, or a final culture dilution of 1:500 (vol/vol) PHA. Cells
were washed 2 times with cold PBS and cultured for 24 hours at 5 × 105 cells/mL. Cells were incubated with PE-conjugated CD4,
CD8, CD20, CD16, or CD14 and rabbit anti-FasL, followed by incubation
with FITC-conjugated goat antirabbit IgG. Cells were analyzed by FACS
analysis and the results are expressed as the mean percentage positive
cells ± SEM for 5 separate blood donors.
|
|
When tested for the ability of EBV-treated, CD95-cross-linked T cells
to respond to anti-CD3 and IL-2, as measured by
[3H]-thymidine incorporation, EBV-stimulated,
CD95-cross-linked T cells were greatly impaired. As shown in
Table 1, T cells exposed to EBV and
anti-CD95 mouse MoAb lost more than 83% (average mock IL-2 v
average EBV IL-2, P = .03), 68% (average mock anti-CD3 v average EBV anti-CD3, P = .3), and 77% (average mock
anti-CD3 + IL-2 v average EBV anti-CD3 + IL-2, P = .04)
of their proliferative response to IL-2, anti-CD3, and anti-CD3 plus
IL-2, respectively.
To rule out the possibility that the loss of T-cell proliferation after
EBV stimulation and anti-CD95 antibody treatment was due to apoptosis
and a consequent decrease in T-cell number, PBMCs were first stimulated
for 24 hours with EBV. Thereafter, T cells or PBMCs were challenged
with the T-cell mitogens, anti-CD3, and IL-2. As shown in
Table 2, T cells stimulated with mitogen in the presence of B cells and other PBMC subpopulations were suppressed by 42% (P = .15), whereas T cells challenged in the absence of other lymphocyte populations were not suppressed (Table 2). These results, along with those seen in Figs 5 and 7, would suggest that B
cells or macrophages expressing FasL promote T-cell apoptosis, thus
lowering the number of available T cells responsive to mitogen. However, in the absence of FasL, T cells stimulated by EBV did not
undergo apoptosis (Fig 6) and maintained their responsiveness to
mitogen (Table 2).
| |
DISCUSSION |
Various mechanisms have been proposed for the transient
immunosuppression or anergy that accompanies primary EBV infection. These include the generation of suppressor T cells47 and
the expression of virally encoded immunosuppressive
cytokines.48,49 The finding that the T-cell apoptosis seen
in acquired immunodeficiency syndrome (AIDS) patients might be mimicked
in vitro by exposure of T cells to HIV gp120 for subsequent
activation-induced cell death and anergy37 led us to test
whether a similar mechanism might occur during EBV infection, thus
providing an explanation for AIM-induced T-cell apoptosis and anergy.
Our results using EBV particles or the major viral envelope
glycoprotein gp350 demonstrated that induction by EBV of CD95 in T
cells for their subsequent death after CD95-induced apoptosis can lead
to a loss in the number of T cells responsive to mitogen. It is
unlikely that EBV latent antigens expressed in immortalized B cells
significantly contributed to our in vitro observations of increased
CD95 expression in T cells, because the increased CD95 expression was
seen at 12 hours postinfection, a time when B cells are not yet
immortalized (Fig 3).50 Furthermore, we documented that
CD4+ T cells demonstrated an increase in CD95 expression
after exposure to purified gp350 (Fig 4).
CD95, along with the tumor necrosis factor (TNF) receptors, are
currently grouped together as the cell death receptors, which can
initiate apoptosis.12 Recent evidence indicates that their intracellular signaling pathways share both common and distinct sets of
proteins and signals.51 Our results strongly suggest that
CD95 is a principal mediator in EBV-induced T-cell apoptosis in vitro,
because T cells did not undergo apoptosis unless treated with an
anti-CD95 antibody. Our results also indicate that T cells stimulated
with EBV did not express significant levels of FasL (Fig 7) and did not
exhibit significant apoptosis (<4% as compared with mock infected;
Fig 6A and C). However, we did see a significant increase in the number
of B cells and macrophages expressing FasL after EBV stimulation (Fig
7). These results are comparable to those seen for HIV gp120, in which
HIV or gp120 stimulation resulted in increased CD95 expression in
purified T cells, but led to only low levels of apoptosis unless
subsequently exposed to anti-CD95 IgM or activated
monocytes.52,53 This relative absence of spontaneous apoptosis in HIV gp120-primed T cells was thought to be due to a lack
of significant T-cell FasL expression.53
Our in vitro experiments indicated that the CD4+ T-cell
population expressed higher levels of CD95 when exposed to virus or gp350 (Figs 3 and 4). We also observed a greater percentage of CD4+ cells undergoing apoptosis as compared with
CD8+ cells in 24-hour EBV-stimulated PBMCs (24% v
6% as compared with mock treatment), but an equal number of
CD4+ and CD8+ cells undergoing apoptosis in
24-hour PHA-stimulated PBMCs (43% v 45%; Fig 5), implying
that EBV-stimulated CD4+ cells may be more susceptible to
FasL killing. This is in contrast to our in vivo observation that
peripheral blood CD8+ T cells expressed higher levels of
CD95 (Fig 1). One possible explanation for this difference is that
CD8+ T cells from AIM patients, in addition to expressing
higher levels of CD95, may also express different ratios of Bcl-2 gene
products as compared with CD4+ T cells, thus making the
CD8+ T cells resistant to apoptosis. Several previously
reported in vitro models have demonstrated that CD4+ and
CD8+ T cells, both of which expressed significant levels of
CD95 upon stimulation, showed different ratios of Bax and
Bcl-XL expression and differential apoptosis
sensitivity.54,55 If CD95+CD8+
lymphocytes in AIM PBMCs express higher ratios of Bcl-2:Bax versus Bcl-XL:Bax due to EBV antigen recognition, then they may be
more resistant to FasL and hence reach higher levels in peripheral blood.10,55
An alternative explanation for our observed increase in CD95 expression
in peripheral blood CD8+ cells from AIM patients may be
that these lymphocytes are EBV antigen-activated CD8+ cells
in transition. Several laboratories have found that the increase in
CD8+ cells seen during AIM is due to an increase in the
number of TCR- clonotypes that recognize either the EBV lytic
antigens BZLF1 and BRLF1 or the latent antigen EBNA3.56-58
It is believed that these clonal populations may serve as temporary
cytotoxic T cells for the control of primary EBV
infection.56 Later, during the convalescent phase of IM,
these cytotoxic T cells disappear. Our observed increase in the number
of CD95+ CD8+ cells in AIM blood samples may
represent CD8+ T cells in transition. During early AIM,
these EBV-stimulated T cells would be CD95+ but FasL
resistant due to increased levels of Bcl-2 and Bcl-XL. Later, during the convalescent phase of AIM, these CD95+ T
cells would increasingly become FasL-sensitive and apoptotic, due to
insufficient EBV stimulation. Thus, expression of CD95 in AIM
CD8+ cells would serve as a check against their continued
cytotoxic actions by promoting FasL-mediated anergy and apoptosis.
Ultimately, the loss of these CD8+ cells by FasL would aid
in the reestablishment of immune cell homeostasis.59
Confirmation of our postulates concerning CD95 expression in AIM T
cells must await further analysis of both the changes in functionality
of CD95+ T cells and their expression of various
antiapoptotic genes over the course of AIM infection.
In conclusion, our results demonstrate that in vitro EBV can increase
the expression of CD95 in CD4+ T cells as well as FasL
expression in B cells and macrophages. Together, CD95 and FasL can lead
to increased CD4+ T-cell apoptosis. We also found that
gp350 can promote CD4+ T-cell apoptosis by inducing CD95
expression. Thus, gp350, in addition to serving as a ligand for virus
attachment and entry into B cells, may also function as an important
immunomodulator by inducing T-cell apoptosis. Such a role for gp350 may
be advantageous in promoting the establishment of EBV in B cells by
eliminating acute EBV-reactive T cells.
| |
ACKNOWLEDGMENT |
The authors thank Dr Elliott Kieff for his helpful criticisms and
suggestions, Dr Gerald Ahronheim for furnishing the AIM patient blood
samples, and all of the volunteer blood donors who furnished blood
samples for this study.
| |
FOOTNOTES |
Submitted May 26, 1998; accepted July 7, 1999.
Supported by the Canadian Foundation for AIDS Research (J.E.T.) and the
J. A. DeSéve Foundation (C.A.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jerome E. Tanner, PhD, Molecular Genetics
Research Laboratory, Room R306, Children's Hospital of Eastern Ontario
Research Center, 401 Smyth Rd, Ottawa, Ontario, Canada K1H 8L1.
| |
REFERENCES |
1.
Henle W, Henle G:
Epstein-Barr virus and infectious mononucleosis.
N Engl J Med
288:263, 1973
2.
Tosato G, Taga K, Angiolillo AL, Sadari C:
Epstein-Barr virus as an agent of haematological disease.
Clin Haematol
8:165, 1995
3.
Khanna R, Burrows SR, Moss DJ:
Immune regulation in Epstein-Barr virus-associated diseases.
Microbiol Rev
59:387, 1995[Abstract/Free Full Text]
4.
De Waele M, Thielmans C, Van Camp BKG:
Characterization of immunoregulatory T cells in EBV-induced infectious mononucleosis by monoclonal antibodies.
N Engl J Med
304:460, 1981[Medline]
[Order article via Infotrieve]
5.
Yoneyama A, Nakahara K, Higashihara M, Kurokawa K:
Increased levels of soluble CD8 and CD4 in patients with infectious mononucleosis.
Br J Haematol
89:47, 1995[Medline]
[Order article via Infotrieve]
6.
Biglino A, Sinicco A, Forno B, Pollono AM, Sciandra M, Martini C, Pich P, Gioannini P:
Serum cytokine profiles in acute primary HIV-1 infection and in infectious mononucleosis.
Clin Immunol Immunopathol
78:61, 1996[Medline]
[Order article via Infotrieve]
7.
Perez-Blas M, Regueiro JR, Ruiz-Contreras JR, Arnaiz-Villena A:
T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.
Clin Exp Immunol
89:83, 1992[Medline]
[Order article via Infotrieve]
8.
Moss DJ, Bishop CJ, Burrows SR, Ryan JM:
T lymphocytes in infectious mononucleosis. I. T cell death in vitro.
Clin Exp Immunol
60:61, 1985[Medline]
[Order article via Infotrieve]
9.
Uehara T, Miyawaki K, Ohata Y, Tamaru T, Yokoi S, Nakamura S, Taniguchi N:
Apoptotic cell death of primed CD45RO+ T lymphocytes in Epstein-Barr virus induced infectious mononucleosis.
Blood
80:452, 1992[Abstract/Free Full Text]
10.
Tamaru Y, Miyawaki T, Iwai K, Tsuji T, Nibu R, Yachie A, Koizumi S, Taniguchi N:
Presence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death.
Blood
82:521, 1993[Abstract/Free Full Text]
11.
Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, Li-Weber M, Richards S, Dhein J, Trauth BC, Posting H, Krammer PH:
Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily.
J Biol Chem
267:10709, 1992[Abstract/Free Full Text]
12.
Cleveland JL, Ihle JN:
Contenders in Fas/TNF death signaling.
Cell
81:479, 1995[Medline]
[Order article via Infotrieve]
13.
Ju S-T, Panka DJ, Cui H, Ettinger R, El-Khatib M, Sherr DH, Stanger BZ, Marshak-Rothstein A:
Fas(CD95)/Fas-L interactions required for programmed cell death after T cell activation.
Nature
373:444, 1995[Medline]
[Order article via Infotrieve]
14.
Kobayashi N, Hamamoto Y, Yamamoto N, Ishii A, Yonehara M, Yonehara S:
Anti-fas monoclonal antibody is cytocidal to human immunodeficiency virus-infected cells without augmenting viral replication.
Proc Natl Acad Sci USA
87:9620, 1990[Abstract/Free Full Text]
15.
Razvi ES, Welsh RM:
Apoptosis in viral infections.
Adv Virus Res
45:1, 1995[Medline]
[Order article via Infotrieve]
16.
Sieg S, Huang Y, Kaplan D:
Viral regulation of CD95 expression and apoptosis in T lymphocytes.
J Immunol
159:1192, 1997[Abstract]
17.
Ito M, Watanabe M, Ihara T, Kamiya H, Sakuiai M:
Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella-zoster virus antigen.
Cell Immunol
160:173, 1995[Medline]
[Order article via Infotrieve]
18.
Inoue Y, Yasukawa M, Fujita S:
Induction of T cell apoptosis by human herpesvirus 6.
J Virol
71:3751, 1997[Abstract]
19.
Esolen LM, Park SW, Hardwick JM, Griffin DE:
Apoptosis as a cause of death in measles virus-infected cells.
J Virol
69:3955, 1995[Abstract]
20.
Yoshida H, Sumichika H, Hamano S, He X, Minamishima Y, Kimura G, Nomoto K:
Induction of apoptosis of T cells by infected mice with murine cytomegalovirus.
J Virol
69:4768, 1995
21.
Hayashi EM, Iio S, Hijioka T, Kasahara A, Fusamoto H, Kamada T:
Role of Fas ligand in apoptosis induced by hepatitis C virus infection.
Biochem Biophys Res Commun
204:468, 1994[Medline]
[Order article via Infotrieve]
22.
Oyaizu N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, Pahwa S:
Cross-linking CD4 molecules upregulates FAS antigen expression in lymphocytes by inducing interferon- and tumor necrosis factor- secretion.
Blood
84:2622, 1994[Abstract/Free Full Text]
23.
Tanner J, Weis J, Fearon D, Whang Y, Kieff E:
Epstein-Barr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping and endocytosis.
Cell
50:203, 1987[Medline]
[Order article via Infotrieve]
24.
Tsoukas CD, Lambris JD:
Expression of CR2/EBV receptors on human thymocytes detected by monoclonals.
Eur J Immunol
18:1299, 1987
25.
Birkenbach M, Tong X, Bradbury LE, Tedder T, Kieff E:
Characterization of an Epstein-Barr virus receptor on human epithelial cells.
J Exp Med
176:405, 1992
26.
Hedrick JA, Lao Z, Lipps SG, Wang Y, Todd SC, Lambris JD, Tsoukas CD:
Characterization of a 70-kDa, EBV gp350/220 binding protein on HSB-2 T cells.
J Immunol
153:4418, 1994[Abstract]
27.
Tanner JE, Alfieri C, Diaz-Mitoma F:
Induction of interleukin-6 (IL-6) following stimulation of human B cell CD21 by the Epstein-Barr virus glycoproteins gp350 and gp220.
J Virol
70:570, 1996[Abstract]
28.
Bailey RE:
Diagnosis and treatment of infectious mononucleosis.
Am Fam Physician
49:879, 1994[Medline]
[Order article via Infotrieve]
29.
Takada K:
Crosslinking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines.
Int J Cancer
33:27, 1984[Medline]
[Order article via Infotrieve]
30.
Saxon A, Fieldhaus J, Robins RA:
Single step separation of human T and B cells using AET treated srbc rosettes.
J Immunol Methods
12:285, 1976[Medline]
[Order article via Infotrieve]
31.
Foung SKH, Coutre S, Engleman G:
Separation of human T and non-T lymphocytes from peripheral blood. In Engleman EG, Foung SKH, Larrick J, Raubitschek A (eds): Human Hybridomas and Monoclonal Antibodies. New York, NY, Plenum, 1985, p 437
32.
Gordon J, Walker L, Guy G, Brown G, Rowe M, Rickinson A:
Control of human B-lymphocyte replication. II. Transforming Epstein-Barr virus exploits three distinct viral signals to undermine three separate control points in B cell growth.
Immunology
58:591, 1986[Medline]
[Order article via Infotrieve]
33.
Dolyniuk M, Pritchett R, Kieff E:
Proteins of Epstein-Barr virus. I Analysis of the polypeptides of purified enveloped Epstein-Barr virus.
J Virol
17:289, 1976
34.
Hoffman GJ, Sondra GL, Hayward SD:
Monoclonal against a 250,000-dalton glycoprotein of Epstein-Barr virus identifies a membrane antigen and a neutralizing antigen.
Proc Natl Acad Sci USA
77:2979, 1980[Abstract/Free Full Text]
35.
Tanner J, Whang Y, Sample J, Sears A, Kieff E:
Soluble gp350/220 and deletion mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes.
J Virol
62:4452, 1988[Abstract/Free Full Text]
36.
Morrissey SH:
Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity.
Anal Biochem
117:307, 1981[Medline]
[Order article via Infotrieve]
37.
Groux H, Topier G, Monte D, Mouton Y, Capron A, Ameisen JC:
Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.
J Exp Med
175:331, 1992[Abstract/Free Full Text]
38.
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi CA:
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.
J Immunol Methods
139:271, 1991[Medline]
[Order article via Infotrieve]
39.
Durandy A, LeDeist F, Emile JF, Debatin K, Fisher A:
Sensitivity of Epstein-Barr virus-induced B cell tumor apoptosis mediated by anti-CD95/Apo-1/fas antibody.
Eur J Immunol
27:538, 1997[Medline]
[Order article via Infotrieve]
40.
Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff SJ:
Differential expression of fas (CD95) and fas ligand on normal human phagocytes: Implications for regulation of apoptosis in neutrophils.
J Exp Med
184:429, 1996[Abstract/Free Full Text]
41.
Anagnostopoulos I, Hummel H, Kreschel C, Stein H:
Morphology, immunophenotype, and distribution of latently and/or productively Epstein-Barr virus-infected cells in acute infectious mononucleosis: Implications for the inter individual infection route of Epstein-Barr virus.
Blood
85:744, 1995[Abstract/Free Full Text]
42.
Prang NS, Hornef MW, Jager M, Wagner HJ, Wolf H, Schwarzmann FM:
Lytic replication of Epstein-Barr virus in peripheral blood: Analysis of viral gene expression in B lymphocytes during infectious mononucleosis and in the normal carrier state.
Blood
89:1665, 1997[Abstract/Free Full Text]
43.
Niedobitek G, Agathanggelou A, Herbst H, Whitehead L, Wright DH, Young LS:
Epstein-Barr virus (EBV) infection in infectious mononucleosis: Virus latency, replication and phenotype of EBV-infected cells.
J Pathol
182:151, 1997[Medline]
[Order article via Infotrieve]
44.
Karajannis MA, Hummel M, Anagnostopoulos I, Stein H:
Strict lymphotropism of Epstein-Barr virus during acute infectious mononucleossis in nonimmunocompromised individuals.
Blood
89:2856, 1997[Abstract/Free Full Text]
45.
Thorley-Lawson DA, Myashita EM, Khan G:
Epstein-Barr virus and the B cell: That's all it takes.
Trends Microbiol
4:204, 1996[Medline]
[Order article via Infotrieve]
46.
Levy E, Ambrus J, Kahl L, Molina H, Tung K, Holers VM:
T lymphocyte expression of complement receptor 2 (CR2/CD21): A role in adhesive cell-cell interactions and dysregulation in a patient with systemic lupus erythematosis (SLE).
Clin Exp Immunol
90:235, 1992[Medline]
[Order article via Infotrieve]
47.
Haynes BF, Schooley RT, Payling-Wright CR, Grouse JE, Dolin R, Fauci AS:
Emergence of suppressor cells of immunoglobulin synthesis during acute Epstein-Barr virus-induced infectious mononucleosis.
J Immunol
123:2095, 1979[Abstract/Free Full Text]
48.
Moore KW, Viera P, Fiorentino DF, Trounstine ML, Khan TA, Mossmann TR:
Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRF-1.
Science
248:1230, 1990[Abstract/Free Full Text]
49.
de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE:
Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes.
J Exp Med
174:1209, 1991[Abstract/Free Full Text]
50.
Alfieri C, Birkenbach M, Kieff E:
Early events in Epstein-Barr virus infection of human B lymphocytes.
Virology
18:595, 1994
51.
Baker SJ, Reddy ED:
Transducers of life and death: TNF receptor specificity and associated TRAF proteins.
Oncogene
12:1, 1996[Medline]
[Order article via Infotrieve]
52.
Wu M, Dayley JF, Rasmussen RA, Schlossman SF:
Monocytes are required to promote peripheral blood T cells to undergo apoptosis.
Proc Natl Acad Sci USA
92:1525, 1995[Abstract/Free Full Text]
53.
Oyaizu N, Adachi Y, Hashimoto F, McCloskey TW, Hosaka N, Kayagaki N, Yagita H, Pahwa S:
Monocytes express Fas ligand upon CD4 cross-linking and induce CD4+ T cell apoptosis.
J Immunol
158:2456, 1997[Abstract]
54.
Boudet F, Lecoeur H, Gougeon ML:
Apoptosis associated with ex vivo down-regulation of bcl-2 and upregulation of fas in potential cytotoxic CD8+ T lymphocytes during HIV infection.
J Immunol
156:2282, 1996[Abstract]
55.
Boussiotis V, Lee BJ, Freeman GJ, Gribben JG, Nadler LM:
Induction of T cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes susceptibility to fas- and bax-mediated programmed cell death.
J Immunol
159:3156, 1997[Abstract]
56.
Callan MFC, Steven N, Krausa P, Wilson JDK, Moss PAH, Gillespie GM, Bell JI, Rickinson AB, McMichael AJ:
Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.
Nat Med
2:906, 1996[Medline]
[Order article via Infotrieve]
57.
Steven NM, Annels NE, Kumar A, Leese AM, Kurilla MG, Rickinson AB:
Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.
J Exp Med
185:1605, 1997[Abstract/Free Full Text]
58.
Silins SL, Cross SM, Elliott SL, Pye SJ, Burrows SR, Burrows JM, Moss DJ, Agaet VP, Miski IS:
Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.
J Exp Med
184:1815, 1996[Abstract/Free Full Text]
59.
Lynch DH, Ramsdell R, Anderson MR:
Fas and FasL in the homeostatic regulation of immune responses.
Immunol Today
16:569, 1995[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Bonardelle, K. Benihoud, N. Kiger, and P. Bobe
B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice
J. Leukoc. Biol.,
November 1, 2005;
78(5):
1052 - 1059.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kotsiopriftis, J. E. Tanner, and C. Alfieri
Heat Shock Protein 90 Expression in Epstein-Barr Virus-Infected B Cells Promotes {gamma}{delta} T-Cell Proliferation In Vitro
J. Virol.,
June 1, 2005;
79(11):
7255 - 7261.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Pages, J. Galon, G. Karaschuk, D. Dudziak, M. Camus, V. Lazar, S. Camilleri-Broet, C. Lagorce-Pages, S. Lebel-Binay, G. Laux, et al.
Epstein-Barr virus nuclear antigen 2 induces interleukin-18 receptor expression in B cells
Blood,
February 15, 2005;
105(4):
1632 - 1639.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Uda, T. Mima, N. Yamaguchi, Y. Katada, M. Fukuda, N. Fujii, K. Nakamura, and O. Saiki
Expansion of a CD28-Intermediate Subset among CD8 T Cells in Patients with Infectious Mononucleosis
J. Virol.,
June 5, 2002;
76(13):
6602 - 6608.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Zuniga, C. C. Motran, C. L. Montes, H. Yagita, and A. Gruppi
Trypanosoma cruzi Infection Selectively Renders Parasite-Specific IgG+ B Lymphocytes Susceptible to Fas/Fas Ligand-Mediated Fratricide
J. Immunol.,
April 15, 2002;
168(8):
3965 - 3973.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Lundy, S. P. Lerman, and D. L. Boros
Soluble Egg Antigen-Stimulated T Helper Lymphocyte Apoptosis and Evidence for Cell Death Mediated by FasL+ T and B Cells during Murine Schistosoma mansoni Infection
Infect. Immun.,
January 1, 2001;
69(1):
271 - 280.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Cinatl Jr., R. Blaheta, M. Bittoova, M. Scholz, S. Margraf, J.-U. Vogel, J. Cinatl, and H. W. Doerr
Decreased Neutrophil Adhesion to Human Cytomegalovirus-Infected Retinal Pigment Epithelial Cells Is Mediated by Virus-Induced Up-Regulation of Fas Ligand Independent of Neutrophil Apoptosis
J. Immunol.,
October 15, 2000;
165(8):
4405 - 4413.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION