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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1803-1813
Peripheral Blood T Cells Generated After Allogeneic Bone Marrow
Transplantation: Lower Levels of Bcl-2 Protein and Enhanced Sensitivity
to Spontaneous and CD95-Mediated Apoptosis In Vitro. Abrogation of the
Apoptotic Phenotype Coincides With the Recovery of Normal Naive/Primed
T-Cell Profiles
By
Nadia Chafika Hebib,
Olivier Déas,
Matthieu Rouleau,
Antoine Durrbach,
Bernard Charpentier,
Françoise Beaujean,
Jean-Paul Vernant, and
Anna Senik
From the UPR 420 of CNRS, Villejuif, the Service d'Hématologie
Clinique, and the Centre de Transfusion Sanguine, Hôpital Henri
Mondor, Créteil, France.
 |
ABSTRACT |
T-cell reconstitution after bone marrow transplant (BMT) is
characterized, for at least 1 year, by the expansion of populations of
T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were
obtained at various times after transplantation (from 45 to  730
days) and were tested for susceptibility to spontaneous apoptosis and
anti-Fas triggered apoptosis in vitro. Substantial proportions of
CD4+ and CD8+ cells generated during the
first year after transplantation, but not by day 730, exhibited in
these assays decreased mitochondrial membrane potential (  m) and
apoptotic DNA fragmentation. The apoptotic phenotype tended to
disappear late in the follow-up period, when substantial absolute
numbers of naive (CD45RA+/CD62-L+) T
cells had repopulated the peripheral blood compartment of the BMT
patients. The rate of spontaneous cell death in vitro was significantly
correlated with lower levels of ex vivo Bcl-2 protein, as assessed by
cytofluorometry and Western blot analysis. In contrast, the levels of
Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax
ratios. Cell death primarily concerned the expanded
CD8+/CD45R0+ subpopulation, although
CD45R0 subpopulations were also involved, albeit to a
lesser extent. These results show that the T-cell
regeneration/expansion occurring after BMT is accompanied by decreased
levels of Bcl-2 and susceptibility to apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IMMUNE RECONSTITUTION after allogeneic
bone marrow transplantation (BMT) is typified by the slow regeneration
of CD4+ T cells and the faster regeneration of
CD8+ T cells.1 There are also typically
functional abnormalities that persist for several years after the
quantitative recovery of T cells, whether the patient receives
autologous or allogeneic bone marrow.2 Most of the newly
generated CD4+ T cells of adult patients carry the
CD45RO+, CD29high, and CD11ahigh
activation/memory markers,3 and most CD8+ T
cells have the CD57+/CD28
phenotype,4 a characteristic of T cells that have undergone many rounds of replication.5 Total CD4+ counts
and the ability to produce naive
(CD45RA+/L-selectin+) T lymphocytes decrease
with increasing age, as shown for healthy BMT recipients examined 1 year after transplantation.3 Studies have been performed
with murine models involving the injection of small amounts of mature T
cells together with bone marrow cells into thymectomized and lethally
irradiated mice. In these models, T-cell regeneration occurs primarily
through the expansion of the injected T-cell population.6,7
Such studies suggest that in adult BMT patients (with aged and
irradiated thymus), the newly regenerated T-cell populations largely
result from the thymus-independent expansion of the population of
mature T cells contaminating the grafted bone marrow.6,8
The peripheral expansion of donor T cells may be driven by the
antigenic environment of the host, including histocompatibility
antigens9 and viral antigens,10,11 restricting
the diversity of the regenerated T-cell repertoire,9 and
occasionally leading to oligoclonality.12 It may also be driven by an antigen-independent, homeostatic process controlling the
size of the T-cell compartment13 or, for memory-phenotype CD8+ T cells, by interleukin-15 (IL-15) stimulation
alone.14
During the early phase of lymphocyte reconstitution after BMT, most
peripheral CD4+ and CD8+ T cells show enhanced
spontaneous apoptosis in vitro.15 The molecular mechanisms
underlying this phenomenon are unknown. In other clinical situations,
the amount of the anti-apoptotic bcl-2 gene product, known to
protect cells against a variety of apoptotic stimuli,16
critically regulates the survival of ex vivo T lymphocytes. Thus,
activated peripheral CD45RO+ T cells from patients
suffering from acute Epstein-Barr virus (EBV) and varicella zoster
virus (VZV) infections show enhanced spontaneous apoptosis in
short-term cultures; this is significantly correlated with low Bcl-2
levels.17 Peripheral T lymphocytes from human
immunodeficiency virus (HIV)-infected patients also undergo faster
spontaneous apoptosis in vitro than lymphocytes from normal
individuals, presumably reflecting the in vivo destruction of
lymphocytes in acquired immune deficiency syndrome
(AIDS).18,19 At least for the CD8+ T
lymphocytes of HIV-infected patients, apoptosis mostly affects activated (HLA-DR+) cells with decreased levels of Bcl-2
protein.20 This is also the case for peripheral blood
HLA-DR+ T cells from multiple myeloma
patients.21 An inverse relationship has been identified
between the amount of Bcl-2 protein and the expression of the
pro-apoptotic Fas/CD95 receptor in virus-infected cells20,21 and in normal peripheral T lymphocytes subjected to in vitro stimulation with mitogens, thereby rendered susceptible to
both accelerated spontaneous death and CD95-mediated
apoptosis.22,23
In this study, we examined, in a long follow-up period (from 45 days to
~730 days after transplantation), the rate of spontaneous and
anti-Fas-triggered apoptosis manifested in vitro by circulating CD4+ and CD8+ T cells from BMT patients. We
extended our analysis to the T-cell subsets defined by classical
markers of naive and memory T cells. We evaluated the amount of Bcl-2
in ex vivo T cells to explore its possible relationship with the rate
of spontaneous apoptosis in vitro.
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PATIENTS, MATERIALS, AND METHODS |
Patients and peripheral blood lymphocytes (PBL).
Twenty-six patients treated for hematological diseases (23 hematological malignancies, 2 sickle anemia, and 1 aplastic anemia) were included in this study. The patients had been subjected to a
conditioning regimen consisting of total body irradiation or high doses
of busulfan (or cyclophosphamide and total body irradiation for 1 patient). They had received a transplant with bone marrow from
HLA-identical sibling donors at the BMT unit of Hôpital Henri
Mondor. Prophylaxis of graft-versus-host disease (GVHD) consisted of an
association of cyclosporin-A and methotrexate. Six patients, suffering
from acute GVHD shortly after transplantation were given prednisone (2 mg/kg/d) for approximately 2 months. No symptomatic GVHD requiring
treatment was observed in any patient from day 180 after
transplantation onward. Twenty-one of the patients were 23 to 57 years
old, 2 were 17 years old, and the other 3 were 13 to 14 years old (mean
age, 36 years). Peripheral blood mononuclear cells (PBMC) from patients
who received transplant were sequentially obtained and frozen on days
45, 90, 180, 365, and eventually 730 to 740 after BMT. PBMC from the
corresponding bone marrow donors were used as control cells.
Monoclonal antibodies (MoAb) and reagents used in functional assays.
The agonistic anti-Fas MoAb CH-11 immunoglobin (IgM) was purchased
from DIACLONE (Besançon, France). The antagonistic anti-Fas MoAb
M3 (IgG1) and the control anti-Fas MoAb M33 (an IgG1 that is neither
agonistic or antagonistic) were kindly given by Dr D. Lynch (Immunex
Research and Development Corporation, Seattle, WA). The anti-CD3 MoAb
OKT3 (IgG2a), was obtained from the American Type Culture Collection
(Rockville, MD). Recombinant IL-2 was given by Roussel Uclaf
(Romainville, France), and IL-15 was kindly given by Dr B. Azzarone
(ICIG, Villejuif, France). The matrix metalloproteinase inhibitor
KB8301 was purchased from Becton Dickinson (Le Pont de Claix, France).
Phenotypic analysis.
CD45RO, CD45RA, HLA-DR, CD57, and CD95 expression on CD4+
and CD8+ T lymphocytes were analyzed by 3-color
immunofluorescence using Quantum RedTM conjugated anti-CD4 and anti-CD8
from Sigma (St Quentin Fallavier, France) emitting in FL3, in
conjunction with fluorescein isothiocyanate (FITC)- and phycoerythin
(PE)-labeled mouse MoAb. These were PE-conjugated anti-CD45RO from Dako
(Trappes, France), PE-conjugated anti-CD62L from Cymbus Biotechnology
(Biotest, Paris, France), and FITC-conjugated anti-CD45RA, anti-CD57,
anti-HLA-DR, and anti-CD95 from Immunotech (Marseille-Luminy, France).
For flow cytometric detection of membrane-bound Fas-L, PBMC were
incubated, immediately after thawing and thereafter, with 10 µmol/L
KB8301 in culture medium. After blocking Fc receptors with 20% human AB serum, the cells were incubated with Mike-1, a rat IgG2a directed at
human Fas-L from Alexis Corporation (obtained from Coger, Paris, France), or with control rat Lo-DNP-57, a rat anti-DNP IgG kindly provided by Dr D. Latinne (University of Louvain Medical School, Brussels, Belgium), then with PE-conjugated goat anti-rat IgG (Jackson
Laboratories, obtained from Becton Dickinson). After further incubation
with irrelevant polyclonal rat Ig, the cells were stained with
FITC-conjugated anti-CD4, anti-CD8, or anti-CD14 MoAb.
Fluorochrome-labeled, isotype-matched Ig controls were from Dako. Flow
cytometry detection of Bcl-2 was performed with an anti-Bcl-2
purchased from Dako according to the manufacturer's instructions. CD57 antigen was detected with an anti-CD57 IgM (American
Type Culture Collection), followed by PE-conjugated goat
F(ab)'2 anti-mouse Ig (Tebu, Le
Perray-en-Yvelines, France). The cells were fixed with 1%
paraformaldehyde in phosphate-buffered saline (PBS) and analyzed by
flow cytometry using a FACScan (Becton Dickinson). Lymphocytes were
gated by a forward (FSC) versus right angle (90°) light scatter plot
(SSC). For each staining, data from at least 30,000 events in the
lymphocyte gate were collected and subsequently analyzed
using Cell Quest software (Becton Dickinson).
Culture conditions and induction of cell death.
Nonadherent PBMC (1 to 2 × 105) were seeded in the
wells of 96-well round-bottomed plates (Nunc, Roskide, Denmark) in 0.2 mL of RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. Spontaneous apoptosis was measured 18 hours later. The
death signal through CD95 was delivered by using the CH-11 anti-Fas
MoAb (1 µg/mL).
Flow cytometric analysis of cell death.
To evaluate mitochondrial  m whose decrease is an indicator of
apoptotic cell death,24 cells were stained for 15 minutes at 37° with 40 nmol/L of the potential sensitive fluorescent dye DiOC6 (3.3'-Diethyloxacarbocyanine), which emits in FL1
(Molecular Probes; Interchim, Montluçon, France). Apoptotic cells
with low m were identified on gated CD4+ and
CD8+ T cells. Electronic gates were also set on the
subpopulations of CD4+ or CD8+ T cells
coexpressing or lacking CD45RO.
Hypodiploid cell assessment.
5 × 105 cells were washed twice in PBS with 5.5 mmol/L
glucose and fixed overnight in ethanol (70% in water, at 4°C). Cells were then resuspended in 0.5 mL PBS containing 50 µg/mL propidium iodide, 100 U/mL RNAse A (Sigma) and incubated for 30 minutes at room
temperature under agitation. The DNA content of 104 cells
was monitored by cytofluorometry using a Coulter Epics profile II analyzer.
Immunoblot analysis.
5 × 105 cells were washed and solubilized in 20 mL of
lysis buffer. Cell lysates were then subjected to sodium dodecyl
sulfate polyacrilamide gel electrophoresis and electroblotted onto a
nitrocellulose membrane. Detection of Bcl-2 protein was performed with
an anti-Bcl-2 MoAb from Dako, and that of Bax with a rabbit antiserum
from Santa Cruz Biotechnology (obtained from Tebu). Blots
were stained with either anti-mouse or anti-rabbit horseradish
peroxidase-labeled secondary antibody (Amersham, Les Ulis, France).
They were then developed using an enhanced chemiluminescence detection
system (ECL kit, Amersham). Films were exposed for 1 to 5 minutes.
Densitometric scanning of the exposed films were performed using the
Bio-Rad gel DOC 1000 apparatus (Bio-Rad S.A., Ivry Sur Seine, France) and analyzed using the Molecular Analyst 2.1 software (Bio-Rad).
Statistical analysis.
Differences between groups were established by the Student's
t-test. Correlations were calculated by linear regression
analysis. A P value lower than .05 was regarded as
statistically significant.
| |
RESULTS |
Ex vivo peripheral T lymphocytes from BMT patients contain a large
proportion of primed/memory cells during the first year after
transplant but tend to recover substantial numbers of naive T cells in
the long term.
Phenotypic analyses of peripheral T lymphocytes from the 26 BMT
patients included in this study were sequentially performed after
transplantation, and the data were arbitrarily divided into 3 time
periods: (1) days 45 and 90 after transplantation (7 and 21 recipients,
respectively) plus 1 recipient examined on day 120; (2) days 180 and
360 (6 and 7 recipients respectively); and (3) days 730 to 740 (9 recipients). Absolute blood lymphocyte counts were similar in all 3 groups (mean ± SD: 2,047 ± 1,341, 2,267 ± 1,565, and
2,075 ± 732), consistent with the notion that the numbers of newly
generated lymphocytes of donor origin reach the lower limits of normal
levels within 2 to 3 months.25 More than 95% of T cells
were TCR- / +, irrespective of the time at which T
cells were assessed (not shown). As expected, the patients had higher
than normal numbers of CD8+ T cells and lower than normal
numbers of CD4+ T cells (Table
1), resulting in reverse CD4/CD8
proportions, particularly obvious for the first 2 groups of patients
(ie, days 45 to 365). In these 2 groups, most CD4+ T cells
(~80%) and a substantial proportion of CD8+ T cells
(~55%) expressed CD45RO, the low-molecular-weight isoform of the
leukocyte common antigen CD45, a marker of primed/memory T cells. (We
also included in this category cells coexpressing CD45RO and CD45RA.)
Increased expression of other activation markers such as CD57, and to a
lesser extent, HLA-DR, was also detected on these cells. All
lymphocytes appeared as small, nondividing cells by FSC analysis and
examination of their DNA profiles (see Fig 3B). This indicates that the
replication phase of these cells was almost completed. On day 730 or
greater, the levels of these activation markers had returned to normal
values, except for CD57, the level of which remained higher than normal
on CD8+ T cells.
T cells with a naive phenotype are most clearly distinguished by
coexpression of CD45RA and CD62L (the lymphocyte homing receptor L-selectin),26 so we used triple-color immunofluorescence
to assess changes in these markers after transplantation, as shown in
Fig 1 with the cells of unique patient
number (UPN) 324 (30 years old). Determinations performed with the
cells of 7 BMT recipients (mean age, 30.6 ± 14.2 years) showed that
the absolute numbers of CD4+ T cells with a naive
CD45RAhigh/CD62L+ phenotype were consistently
low on day 90 after transplantation (28 ± 21 cells/µL blood), but
tended to return to normal values toward the end of the follow-up
period, reaching 175 ± 168 cells/µL (v 350 ± 78 naive
CD4+ cells/µL in donors). The CD8+ T-cell
population contained lower than normal proportions of naive
CD45RAhigh/CD62L+ cells on day 90 (12% ± 7% v 38% ± 8% in donors); however, high absolute cell numbers were found in some cases. For example, UPN 564 (13 years old) had 231 naive CD8+ T cells/µL and UPN 282 (36 years old) had 322 naive CD8+ cells/µL. For the 5 other patients, a mean of 65 ± 40 naive CD8+ T cells/µL
was recorded (v 171 ± 39 cells/µL in donors). In the late
follow-up period, counts of naive CD8+ T cells generally
normalized at around 119 ± 97 cells/µL (n = 7). These data
suggest that post-BMT-generated naive T cells were present in the long
term, despite reduced thymic function, because of the patients' age.
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| Fig 1.
Recovery of naive peripheral
CD45RA+/CD62L+ T cells in long-term BMT
patients. Peripheral CD4+ and CD8+ T cells
from UPN 324 (30 years old) and his donor were examined by triple-color
immunofluorescence at various time points after BMT. Values in brackets
are numbers of CD45RA+/CD62L+ T cells per
microliter of blood.
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Cytofluorometric analysis of Fas/CD95 expression by T cells from BMT
patients.
The frequency of Fas-positive cells increased greatly among both
CD4+ and CD8+ T cells from all recipients
during the first year after transplantation (90% v ~45%
and ~50% in control CD4+ and CD8+ T cells,
P < .0001). However, on day 730 or greater after
transplantation, the percentages of Fas-positive cells had returned to
normal values (Fig 2A and B). Fas
expression on post-BMT T cells was of the same order of magnitude as
Fas expression on normal T cells (Fig 2C), in which it is generally
restricted to CD45RO+ memory T cells.27 Indeed,
most (70% to 90%) Fas-negative T cells were CD62L+,
whereas approximately only 25% Fas-positive T cells were
CD62L+ (not shown).
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| Fig 2.
Fas expression on CD4+ and
CD8+ peripheral T cells from BMT recipients and their
normal donors. (A and B) individual values obtained by cytofluorometric
analysis. Horizontal bars indicate the median values for each group.
(C) Representative histogram profiles generated from the T cells of UPN
324 showing the fluorescence obtained with an anti-Fas or irrelevant
isotype matched MoAb (vertical bars).
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Cultured peripheral T cells from BMT patients tend to undergo
enhanced spontaneous apoptosis in vitro and are susceptible to
anti-Fas-induced cell death during the first year after
transplantation.
Cells at various stages of apoptosis can be detected by
cytofluorometry, using the lipophilic dye, DiOC6 (3), which
accumulates in mitochondria as a function of m.24
Apoptotic cells also typically appear as hypoploid cells with decreased
DNA content.28 Freshly isolated T lymphocytes from BMT
patients had high levels of mitochondrial activity and their DNA was
not fragmented (Fig 3). However, following
an 18-hour incubation in culture medium alone, CD4+ and
CD8+ T cells obtained on day 90 after transplantation had
higher percentages of DiOC6 (3)low cells and
hypodiploid cells (Fig 3). Enhanced spontaneous apoptosis in vitro
affected T lymphocytes collected on days 45 to 90 and on days 180 to
365 after BMT (P < .0001 and P < .003,
respectively, in comparison with control T cells), but not those
collected on day 730 or thereafter (Fig 4).
The rate of spontaneous apoptosis on days 45 and 90 was higher for
CD8+ T cells than for CD4+ T cells
(51% ± 19% DiOC6 (3)low cells v
36% ± 16%, P < .0001). This difference remained
statistically significant for days 180 to 365 (P < .005).
In the same conditions, 14% ± 7% and 9% ± 8% apoptotic cells
were counted in donor CD8+ and CD4+ T cell
populations, respectively. The binding of an agonistic anti-Fas MoAb to
Fas receptors further increased the rate of cell death in post-BMT
lymphocytes. A mean of 68% ± 17% CD4+ T cells and 76% ± 15% CD8+ T cells disrupted mitochondrial potential on
days 45 to 90 (v 13% ± 9% and 20% ± 13% in the
homologous anti-Fas-treated donor T cells). The extent of
Fas-induced-specific apoptosis (ie, excess apoptosis over the
spontaneous background) slightly decreased for cells collected on days
180 to 365, and became negligible for cells collected on days 730 or
greater, consistent with the normalization of Fas expression and the
simultaneous decrease in the levels of other activation markers.
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| Fig 3.
Cytofluorometric analysis of accelerated spontaneous
apoptosis affecting post-BMT T cells in short-term cultures. PBL from
BMT patients and their donors were tested for apoptosis either directly
after harvesting (ex vivo) or after an 18-hour incubation in culture
medium. (A) A typical experiment performed with the cells of UPN 315 showing the percentages of cells with low m
(DiOC6(3)low). (B) Percentages of
hypodiploid cells detected in the same culture conditions by staining
ethanol-permeabilized lymphocytes from UPN 294 with propidium iodide
(linear scales are shown).
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| Fig 4.
Susceptibility to enhanced apoptosis in vitro and to
anti-Fas-triggered apoptosis at various time points after BMT.
Percentages of cells with low m were determined by
cytofluorometry in gated CD4+ and CD8+
T-cell subsets. Rates of apoptosis were much higher
(P > .0001) on days 45 to 90 and 180 to 365, but not on day
730 than those of control cells. The P values (asterisks) show
the levels of anti-Fas-triggered apoptosis relative to spontaneous
background apoptosis. Histograms are means ± SD for individual
determinations.
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In vitro Fas/Fas-L interactions are not involved in the high
incidence of spontaneous death occurring in unstimulated cultures of
post-BMT T lymphocytes.
At saturating concentration (10 µg/mL), the antagonistic anti-Fas
MoAb29 failed to reverse the accelerated spontaneous death occurring in vitro of T cells from BMT patients (Fig
5A). Similar results were obtained when the
assays were performed in the presence of 10 µmol/L K8301, a potent
inhibitor of matrix metalloproteinases, used to avoid the proteolytic
cleavage of membrane-bound Fas-L.30 In contrast, the M3
antibody almost completely inhibited anti-Fas-triggered apoptosis in
the same cells (control anti-Fas M33 antibody had no effect, Fig 5B),
and blocked activation-induced cell death (AICD) induced by CD3/TCR
crosslinking of activated control CD4+ T cells (Fig 5C). An
anti-Fas-L MoAb (Mik-1) specifically recognizing the extracellular
domain of Fas-L failed to detect significant Fas-L expression on the
surface of ex vivo T cells and monocytes from BMT patients. Moderate
but significant Fas-L expression was in contrast detected on the
surface of normal phytohemagglutinin-(PHA) IL-2-activated T blasts (Fig
5D). These data argue against the possibility of Fas/Fas-L interactions
being involved in the accelerated spontaneous death manifested in vitro
by the patients' T cells.
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| Fig 5.
In vitro Fas/Fas-L interactions are not involved in
accelerated spontaneous death of post-BMT T cells. (A) PBMC from 6 patients (on day 90 after BMT), were incubated for 18 hours in the
presence of 10 µg/mL of the blocking anti-Fas MoAb M3 or of the
negative control MoAb M33. Cell death was assessed according to trypan
blue uptake and morphological criteria. Histograms are means ± SD for
individual determinations. (B) Cytotoxic anti-Fas MoAb CH-11 was tested
in the same conditions using PBMC isolated on day 90 after BMT (3 patients). (C) Effect of MoAb M3 on AICD of in vitro preactivated
normal CD4+ T lymphocytes exposed for 8 to 14 hours to 10 µg/mL of plate-bound, anti-CD3 MoAb (OKT3). Percent cell loss is the
decrease in the absolute number of viable cells compared with that of
unstimulated control cells (means ± SD of 3 separate experiments).
(C) Flow cytometric analyses of anti-Fas-L (solid lines) and control
Lo-DNP-57 (dashed lines) MoAb reactivity with CD4+,
CD8+, and CD14+ cells from donor and
post-BMT T cells (from UPN 332) and with normal PHA-IL-2 blasts.
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Relationship between Bcl-2 protein levels and the rate of enhanced
spontaneous apoptosis displayed by CD4+ and
CD8+ T lymphocytes from BMT patients.
There is compelling evidence that Bcl-2 prevents the apoptotic death of
activated T lymphocytes deprived of growth factors and of quiescent T
cells that do not receive adequate stimuli.31 We therefore
investigated whether there was a correlation between Bcl-2 levels in ex
vivo T lymphocytes from BMT patients and their tendency toward
spontaneous apoptosis in vitro. Most PBL from patients who received a
transplant were stained with an anti-Bcl-2 MoAb. We therefore used
flow cytometry to estimate the amounts of intracellular Bcl-2 protein
(by measuring mean fluorescence intensity [MFI]) in individual ex
vivo CD4+ and CD8+ T cells. For example, UPN
342 had 39% CD4+ T cells and 63% CD8+ T cells
with decreased levels of Bcl-2 on day 90 after transplant, as assessed
by a shift in staining intensity relative to control T cells (Fig
6A). This was associated with a high rate
of cell death in culture (30% in CD4+ T cells and 60% in
CD8+ T cells). However, both Bcl-2 levels and spontaneous
cell death rates returned to normal values by day 840 after
transplantation. CD4+ and CD8+ T cells from UPN
342 and his bone marrow donor were also isolated by cell sorting. Their
levels of Bcl-2 were compared by Western blotting using an MoAb that
recognized the 26-kD Bcl-2 protein (Fig 6B) and were standardized using
the amount of actin detected in each sample (Fig 6C). This approach
confirmed the results obtained by flow cytometry. In contrast, the
level of Bax, another member of the Bcl-2 family, which promotes cell
death by antagonizing Bcl-2-mediated suppression of
apoptosis,32 remained constant (see the expected 22-kD band
in Fig 6B). Similar correlations between cytofluorometric and
immunoblot analyses of Bcl-2 levels were found for the T cells of 4 other patients (not shown).
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| Fig 6.
Ex vivo intracellular Bcl-2 and Bax protein expression by
T cells from UPN 342 at various times after BMT. (A) A follow-up study
of Bcl-2 levels in CD4+ and CD8+ T cells
from UPN 342 is shown. Percentages of cells with a Bcl-2 content lower
than that of donor cells are indicated. (B) Immunoblot analysis of
Bcl-2 and Bax protein expression in 5 × 105 T
FACS-purified CD4+ and CD8+ T cells from
UPN 342. Actin levels were assessed for the same samples. Lane 1, donor
T cells; lane 2, ex vivo T cells on day 90 after BMT; lane 3, ex vivo T
cells on day 840 after BMT. (C) Relative levels of Bcl-2 and Bax
proteins determined by scanning the exposed films with a densitometer
and expressed relative to the level of actin in the same samples.
Ratios were calculated for each experimental point as follows:
absorbance of Bcl-2 or Bax staining/absorbance of actin staining. The
ratio for sample 1 was set as 1 arbitrary unit. All the other ratios
were referred to this internal ratio.
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MFI values for Bcl-2 in individual T cells from bone marrow donors and
from BMT patients are shown in Fig 7A. On
days 45 to 90 and 180 to 365, but not on day 730 or greater after
transplantation, the MFIs of CD4+ T cells and
CD8+ T cells were significantly lower than those of donor
cells. The decrease in Bcl-2 levels was more pronounced in
CD8+ T cells than in CD4+ T cells. Linear
regression analysis (Fig 7B) showed that there was a significant
correlation between the proportions of patients' CD4+ and
CD8+ T cells undergoing enhanced spontaneous apoptosis in
culture and those showing ex vivo downregulation of Bcl-2. This
correlation was statistically more significant in CD8+ T
cells (P < .002, r = .5) than in CD4+
T cells (P < .04, r = .4), consistent with the
higher tendency of CD8+ T cells to undergo spontaneous
apoptosis in culture.
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| Fig 7.
Flow cytometric analysis of Bcl-2 levels in post-BMT
lymphocytes. Relationship with the rate of enhanced spontaneous
apoptosis in short-term culture. (A) MFI for intracellular Bcl-2
expression in ex vivo CD4+ and CD8+ T
lymphocytes. Horizontal bars are the arithmetic means of individual
data. (B) Inverse correlation between Bcl-2 expression in post-BMT T
cells and the rate of spontaneous apoptosis in vitro.
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Rates of spontaneous apoptosis in vitro and Bcl-2 levels in T- cell
subsets defined according to CD45RO isoform expression.
We used DiOC6(3) dye and triple-color immunofluorescence
to assess the rate of spontaneous apoptosis in short-term cultures of
CD4+ and CD8+ T-cell subpopulations with and
without CD45RO membrane expression. Typical cytofluorometric profiles,
obtained from UPN 440 (day 90 after transplantation) showed that both
the CD45RO+ and CD45RO T cells of this
recipient were susceptible to accelerated apoptosis in vitro (Fig
8A). Determinations were made for the cells
of 6 other BMT recipients (day 90) and their bone marrow donors (Fig 8B). They showed that CD8+/CD45RO+ T cells were
particularly susceptible to apoptosis, with approximately 50%
DiOC6low cells expressing the
CD8+/CD45RO+ phenotype and approximately 30%
DiOC6low cells belonging to the other
CD8+/CD45RO,
CD4+/CD45RO+, and
CD4+/CD45RO T-cell subsets. These
accelerated rates of apoptosis were associated with decreased levels of
Bcl-2, particularly evident in ex vivo CD8+/CD45RO+ T cells from BMT patients examined
on days 45 to 90 and days 180 to 365 (Fig
9). Normal
CD8+/CD45RO+ T cells also showed some tendency
to undergo enhanced spontaneous apoptosis in culture (~20%
DiOC6low cells v ~10% in other
normal T-cell subsets, see Fig 8B). This was also associated with lower
levels of Bcl-2 in normal CD8+/CD45RO+ T cells
than in their CD8+/CD45RO counterparts (Fig
9). Few differences were detected in the Bcl-2 levels of
CD4+/CD45RO+ T cells and
CD4+/CD45RO T cells from each group of
patients, consistent with the observation that the 2 types of cells had
similar rates of spontaneous apoptosis in vitro. All these data
strongly support the notion that the intracellular level of Bcl-2
dictates the extent of spontaneous in vitro apoptosis affecting newly
generated cells after BMT. That is, the lower the level of Bcl-2 in a
cell, the more susceptible it is to this type of death.
View larger version (36K):
[in this window]
[in a new window]
| Fig 8.
Susceptibility to enhanced spontaneous apoptosis in vitro
of T subpopulations according to CD45RO isoform expression. (A) A
typical triple-color immunofluorescence analysis performed with the
cells of UPN 440 and his bone marrow donor. The values in brackets are
the percentages of apoptotic cells
(DiOC6(3)low) within the
CD45RO+ and CD45RO T-cell subpopulations.
(B) Similar analyses performed with the cells of 6 other BMT
patients and their corresponding bone marrow donors. The histograms
show the means ± SD of individual determinations.
|
|
View larger version (40K):
[in this window]
[in a new window]
| Fig 9.
Bcl-2 expression in CD45RO+ and
CD45RO T-cell subpopulations from BMT patients and their
bone marrow donors. Bcl-2 levels (MFI values) were estimated by
triple-color immunofluorescence analysis. The P values at the
bottom of the figure refer to the MFI of donor CD45RO-negative T
cells.
|
|
| |
DISCUSSION |
Early after BMT,15 and for at least the first year after
transplantation (this study), a substantial proportion of newly generated peripheral CD4+ and CD8+ T
lymphocytes undergo enhanced spontaneous apoptosis in short-term culture. This suggests that these lymphocytes are in a fragile equilibrium between life and death in vivo. We found that the levels of
the bcl-2 gene product, which increases lymphoid
survival,33,34 were significantly lower than normal in
peripheral CD4+ and CD8+ T cells from many BMT
patients during the first year after transplantation, and that there
was a significant correlation between the extent of spontaneous
apoptosis in vitro and the decrease in Bcl-2 levels in these cells. In
contrast, the amounts of the pro-apoptotic protein Bax did not change,
suggesting that a decrease in the Bcl-2/Bax ratio contributed to the
tendency of post-BMT T lymphocytes to undergo spontaneous apoptosis.
Both the CD45RO+ and CD45RO
(CD45RA+) T-cell subsets circulating in BMT patients were
susceptible to spontaneous apoptosis and had reduced Bcl-2 levels.
These features were, however, much more pronounced in primed/memory
CD8+/CD45RO+ T cells, consistent with studies
of peripheral T cells from patients suffering from acute viral
infections such as those induced by EBV, VZV,17,35 or
HIV.18,19 It is unknown whether the few naive
CD45RA+/CD62-L+ T cells generated during the
first year after BMT were also subjected to spontaneous death in vitro.
Naive T cells slowly accumulate in vivo, so they presumably resist
apoptosis, whereas T cells with a primed/memory phenotype
(CD62L) are more likely to succumb, until homeostasis
of the immune T-cell system is reached. Further experiments are
required to investigate this possibility.
Our study shows that the expansion of the primed/memory T-cell
population, during the first year after BMT, was accompanied by the
upregulation of Fas expression and susceptibility to Fas-triggered apoptosis. In contrast, Fas-ligand expression was not upregulated on
the surface of post-BMT T cells, nor on the monocytes from the same
PBMC preparations. Accordingly, treatment with the antagonistic M3
anti-Fas MoAb failed to reverse the in vitro accelerated spontaneous death of post-BMT T cells, which suggests that Fas/Fas-ligand interactions are not involved. However, Fas+ post-BMT
lymphocytes may be in vivo the targets of Fas-L-expressing cells, so
the dysregulation of Fas expression may contribute, together with Bcl-2
dysregulation, to the presumed in vivo fragility of T lymphocytes after
BMT. A recent report showed that CD4+ T cells, generated
during peripheral expansion after intensive chemotherapy in adults, are
susceptible to AICD if stimulated with mitogenic lectins.36
We tested whether anti-CD3 MoAb induced AICD in the lymphocytes of BMT
patients (data not shown). They did not, probably because the cells
were in a quiescent state and were therefore insensitive to apoptotic
stimuli that otherwise operate on fully activated T cells.
After transplant, peripheral T cells with a low Bcl-2 content showed no
signs of apoptosis immediately after they were obtained. In particular,
their mitochondrial transmembrane potential (m), the dissipation
of which occurs very early in the apoptotic process,24 was
normal. This suggests that the cells were not yet committed to death
but that they were in a fragile equilibrium between life and death.
What then are their in vivo rescue signals? Constant T-cell receptor
(TCR)-major histocompatibility complex contacts ensure the survival of
mature CD4+ and CD8+ T cells in the
periphery.37,38 Interactions with fibroblasts are also
required to keep alive in vitro activated T cells with low Bcl-2 levels
that have reverted to a quiescent memory state.17 Recent
data indicate that type-1 interferons secreted by stromal cells are the
factors responsible for the rescue of such cells.39 Therefore, microenvironmental factors may counteract in vivo the tendency of post-BMT T lymphocytes (with low Bcl-2 content) toward spontaneous apoptosis. Cytokines may also be effective in this respect.
However, post-BMT lymphocytes could not be rescued from apoptosis by
cytokines such as IL-2 and IL-15 (data not shown), which are otherwise
efficient at attenuating the spontaneous death in vitro of T cells from
patients acutely infected with EBV and VZV,17 or
HIV.40,41
In the long term after BMT, the apoptotic phenotype tended to
disappear, and normal naive/memory T-cell profiles tended to emerge. In
the case of patients undergoing intensive chemotherapy for cancer, the
presence of a functional thymus makes possible the rapid regeneration
of naive (CD45RAhigh) CD4+ T cells from the
bone marrow progenitors, whereas in patients lacking thymus function
(>15 years old), the lymphocyte population consists mainly of T cells
with exclusively a primed/memory phenotype.42 These data
are consistent with the notion of thymic involution starting soon after
birth and continuing at a rate of approximately 3% per year until
middle age (and at 1% thereafter), resulting in a dramatic loss of
thymic function.43 However, remnants of thymic tissue
persist for decades and support residual T-cell differentiation in
adults.44 After BMT, these thymic remnants are profoundly
damaged, but thymic lymphopoiesis is eventually restored at a low rate
within small epithelial areas.45 This may account for the
accumulation of peripheral naive T cells in adult BMT patients.
However, it has been reported that short-term incubation in culture
medium of T-cell-depleted mouse marrow CD4
CD8 TcR / cells results in the
differentiation of CD4+ and CD8high
TCR-/+ cells similar to those of normal spleen T
cells, most with a CD62Lhigh CD44low naive
phenotype.46 These data suggest that there is a pathway of
T-cell lymphopoiesis in the bone marrow microenvironment, which may be
involved in T-cell reconstitution after BMT. In any case, the
accumulation of naive T cells in the long term and the normalization of
naive/primed T-cell profiles coincide with the restoration of normal
Bcl-2 levels, the disappearance of the apoptotic phenotype, and the
homeostatic equilibrium of the T-cell system.
| |
FOOTNOTES |
Submitted November 20, 1998; accepted April 23, 1999.
Supported by grants from the CNRS, the Etablissement Français des
Greffes, the Association pour la Recherche sur le Cancer, and the
Université de Paris Sud.
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 Anna Senik, PhD, Equipe d'Immunologie
Cellulaire et de Transplantation de L'UPR 420 du CNRS, 19 rue Guy
Moquet, 94801 Villejuif, France.
| |
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ABSTRACT
INTRODUCTION