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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 390-400
Massive Activation-Induced Cell Death of Alloreactive T Cells With
Apoptosis of Bystander Postthymic T Cells Prevents Immune
Reconstitution in Mice With Graft-Versus-Host Disease
By
Sylvie Brochu,
Benjamin Rioux-Massé,
Jean Roy,
Denis-Claude Roy, and
Claude Perreault
From the Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital,
Montreal, Quebec, Canada.
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ABSTRACT |
After hematopoietic stem cell transplantation, the persistence and
expansion of grafted mature postthymic T cells allow both transfer of
donor immunologic memory and generation of a diverse T repertoire. This
thymic-independent process, which is particularly important in humans,
because most transplant recipients present severe thymus atrophy, is
impaired by graft-versus-host disease (GVHD). The goal of this study
was to decipher how GVHD influences the fate of grafted postthymic T
cells. Two major findings emerged. First, we found that, after a brisk
proliferation phase, alloreactive antihost T cells underwent a massive
activation-induced cell death (AICD). For both CD4+ and
CD8+ T cells, the Fas pathway was found to play a major
role in this AICD: alloreactive T cells upregulated Fas and FasL, and
AICD of antihost T cells was much decreased in the case of lpr
(Fas-deficient) donors. Second, whereas non-host-reactive donor T
cells neither upregulated Fas nor suffered apoptosis when transplanted
alone, they showed increased membrane Fas expression and apoptosis when coinjected with host-reactive T cells. We conclude that GVHD-associated AICD of antihost T cells coupled with bystander lysis of grafted non-host-reactive T cells abrogate immune reconstitution by
donor-derived postthymic T lymphocytes. Furthermore, we speculate that
massive lymphoid apoptosis observed in the acute phase of GVHD might be responsible for the occurrence of autoimmunity in the chronic phase of GVHD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN NORMAL INDIVIDUALS, the size of
peripheral T-cell compartments is maintained by thymic and extrathymic
pathways. First, thymic output is responsible for initial seeding of
secondary lymphoid organs in young subjects.1 Continuous
thymic output in later life contributes to the diversity of the T-cell
repertoire,2,3 although the level of thymic function is
known to decrease with age as the thymus undergoes progressive
involution.4 Second, most T lymphocytes produced daily in
adults derive from the proliferation of postthymic T cells that retain
an impressive proliferative potential.5-7 Finally,
extrathymic differentiation of hematopoietic progenitors has been
detected in selected organs.8-12 The possible contribution
of the latter pathway to T-cell homeostasis remains to be better
defined but is generally believed to play a minor role compared with
the first two other pathways.
It has been shown that, after transplantation of syngeneic
hematopoietic stem cells, both thymic and extrathymic pathways can
contribute to the reconstitution of peripheral T-cell compartments. In
this model, the relative contribution of each pathway depends on the
recipient's thymic function and the number of postthymic T cells in
the donor inoculum.13,14 In contrast, when the transplant is performed into an allogeneic recipient, graft-versus-host disease (GVHD) initiated by the graft postthymic T cells causes severe lymphoid
hypoplasia.15-17 Disappearance of host lymphoid cells at
the time of GVHD is easily accounted for by the generation of antihost
alloreactive T cells that eradicate recipient mature lymphocytes and
hematopoietic progenitors via both perforin and Fas-mediated
cytotoxicity.16,18,19 However, there is no satisfactory explanation for the failure of donor-derived T cells to reconstitute GVHD+ hosts. It has been suggested that, during the course
of GVHD, Fas/FasL interactions may contribute to failure of donor
T-cell reconstitution, because donor T-cell hypoplasia is less severe when the donor is FasL defective or when the recipient is treated with
anti-FasL monoclonal antibody (MoAb).16,20,21 However, interpretation of these interesting observations is limited by the fact
that Fas/FasL interactions costimulate T-cell activation and
proliferation in such a way that signaling through Fas in the early
stages of an immune response augments the generation of effector
cells.22,23 Indeed, after transplantation of FasL-defective cells, the generation of antihost effector cells has been shown to be
severely impaired.20 Thus, it is not clear whether
interference with Fas signaling decreases the severity of GVHD-related
T-cell hypoplasia because (1) hypoplasia involves Fas-dependent
apoptotic events or (2) because, similar to other molecules (eg, CD28,
CD40, etc),24,25 the costimulatory effect of Fas is
required for the expansion of alloreactive T cells.
The severity of GVHD-associated T-cell hypoplasia implies that GVHD
abrogates both thymic and extrathymic T-cell reconstitution. Mechanistically, each differentiation pathway should be approached separately. It has been shown that the thymus stroma is a direct target
of GVHD, and damage to the host thymus provides a rational explanation
for the failure of donor-derived progenitors to differentiate via the
classic central pathway.15,17,26 The problem in this case
is probably one of soil rather than seed. In contrast, the failure of
the graft mature postthymic T cells to expand and repopulate the host
peripheral compartments, as observed in athymic syngeneic recipients,
remains unexplained. This question is particularly relevant in the case
of human recipients. Indeed, because of age- and disease-related
factors such as chemotherapy, radiotherapy, or infections, most
patients have thymus hypoplasia.27-31 In addition, T-cell
reconstitution has been shown to depend mostly on proliferation of
grafted postthymic T cells, at least during the first year posttransplant.32-34 Consequently, the failure of grafted
postthymic T cells to expand in GVHD+ recipients can have a
dramatic impact on immune reconstitution and is probably responsible to
a large extent for the high frequency and severity of infections in
these patients. Moreover, impairment of donor T-cell expansion could
represent a major obstacle for the implementation of adoptive
immunotherapy strategies. The latter point is increasingly important,
because innovative approaches aimed at transferring activated/memory T
cells specific for pathogens such as cytomegalovirus (CMV)
and Epstein-Barr virus (EBV) hold great promise for the
treatment of infections in immunocompromised hosts.35-37
Thence, the goal of the present work was to determine the fate of
grafted postthymic T cells in GVHD+ recipients and to
understand why these mature donor T cells fail to repopulate the
host's peripheral T-cell compartments.
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MATERIALS AND METHODS |
Mice.
The following strains of mice were purchased from the Jackson
Laboratory (Bar Harbor, ME): A/J, C57BL/6J (B6),
B6.SJL-PtprcaPep3b/BoyJ (Ly5a)
(B6.SJL), B6.PL-Thy1a/Cy (Thy1.1) (B6.PL),
B6.MRL-Faslpr (lpr), and
B6Smn.C3H-Faslgld (gld). Mice with a B6
background have the H2b haplotype, whereas A/J mice are
H2a. All inbred mice are Ly5.2+, except for
B6.SJL mice, which are Ly5.1+. F1 hybrids from B6.SJL and
A/J mice (B6.SAF1), and from B6 and A/J mice (B6AF1) were bred at the
Guy-Bernier Research Center (Montreal, Quebec, Canada). Animals were
maintained in specific pathogen-free conditions according to the
standards of the Canadian Committee for Animal Protection. Lpr donors
were between 5 and 8 weeks old, whereas other mice used as cell donors
or recipients were between 5 and 16 weeks of age.
Induction of GVHD.
Spleen and lymph nodes (cervical, axillary, and inguinal) from donor
mice with a B6 background were pooled into single-cell suspensions in
which the number of Thy1+ T cells was assessed by flow
cytometry. GVHD was induced by intravenous injection of a spleen/lymph
node cell suspension containing 60 × 106 T cells into
unirradiated F1 hybrid recipients.
Production of non-host-reactive cells.
Bone marrow cells were obtained from the tibiae and femurs of B6.PL
donor mice and T-cell-depleted with specific anti-Thy-1 antiserum
(Cedarlane, Hornby, Ontario, Canada) as previously
described.13,38 Then, 107 bone marrow cells
were intravenously injected into irradiated (12 Gy total body
irradiation from a 60Co source at a dose rate of 128 cGy/min) B6AF1recipients on day 0. On day 60, spleen and lymph nodes of
these B6AF1 recipients were harvested and used as a source of
non-host-reactive cells.
MoAbs.
The following MoAbs were obtained from Pharmingen (San Diego, CA):
fluorescein isothiocyanate (FITC)-labeled anti-V panel, FITC- and
phycoerythrin (PE)-labeled anti-Thy1.1 (MRC OX-7) and anti-Thy1.2
(53-2.1 and 30-H12, respectively), FITC-labeled and biotin-conjugated
anti-Ly5.1 (A20) and anti-Ly5.2 (104), PE-labeled antibodies against
CD19 (1D3), CD44 (1M7) and Fas (Jo2), Cy-chrome-labeled anti-CD4
(RM4-5) and anti-CD8 (53-7.7), purified anti-FasL (MFL3), biotin-conjugated goat-antihamster IgG, PE-conjugated streptavidin, and
isotypic controls.
Flow cytometric analysis.
Cells were analyzed on a FACScalibur (Becton Dickinson, San Jose,
CA) using the CellQuest program (Becton Dickinson) or on a
FACScan (Becton Dickinson) using the Lysis II program (Becton Dickinson). Lymphocytes were gated by forward and side scatter, and
fluorescence data were collected for 10,000 cells. Studies of selected
T-cell populations were performed on 5,000 to 10,000 gated cells that
were CD4+ or CD8+ and expressed the Ly5/Thy1
phenotype of interest. Direct immunofluorescence staining was performed
as previously described.38 A sensitive three-step indirect
staining method was used to assess the low-level expression of cell
surface FasL. First, cells were stained with purifed anti-FasL Ab.
After two washes, cells were incubated with biotinylated second-step
reagent and then, after two additional washes, PE-conjugated
streptavidin was added together with other selected
fluorochrome-conjugated antibodies for three-color staining.
Measurement of FITC-labeled Annexin-V binding.
Apoptosis was analyzed by quantifying phosphatidylserine residues
exposed on the cell membrane. Spleen cells were first stained with
membrane-specific antibodies. After two washes with phosphate-buffered saline, 3 µL of recombinant FITC-labeled Annexin-V (Pharmingen) was
added to cells resuspended in 100 µL of binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L
CaCl2). After 15 minutes of incubation in the dark at room
temperature, 500 µL of binding buffer was added and the samples were
analyzed on a FACScan flow cytometer. In control samples, propidium
iodide (2 µg/mL) staining was performed to help set the limit used to discriminate Annexin-V-positive and -negative cells. In some
experiments, Annexin-V staining was performed after stimulation with
Concanavalin A (Con A). Briefly, cells diluted at 2 × 106/mL in RPMI 1640 supplemented with 10% fetal calf
serum, 100 U/mL penicillin G, 100 µg/mL streptomycin, 2 mmol/L
L-glutamine, 1 mmol/L sodium pyruvate, 5 × 10 5
mol 2-mercaptoethanol (2-ME), and 2 µg/mL of
Concanavalin were incubated at 37°C in a humidified atmosphere of
5% CO2. After 40 hours of culture, cells were washed twice
before antibodies and Annexin-V staining.
Statistical analysis.
Results for group means were compared using the Student's
t-test.
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RESULTS |
Experimental model.
GVHD was induced by injecting a mixture of C57BL/6 or B6.SJL
(H-2b) lymph node + spleen cells containing 6 × 107 T lymphocytes into unirradiated B6AF1 or B6.SAF1
recipients (H-2b/a). The origin of cells in chimeras was
determined according to their Ly5 phenotype: C57BL/6J, A/J, and B6AF1
mice are Ly5.1Ly5.2+, B6.SJL are
Ly5.1+Ly5.2, and B6.SAF1 mice are
Ly5.1+Ly5.2+. We elected to use unirradiated
recipients because irradiation per se can induce a number of effects
that could have confounded our analyses. Indeed, irradiation can (1)
increase Fas expression,39 (2) induce extrathymic T-cell
development,40 (3) impair the function of thymus stromal
cells,41 and (4) trigger the release of inflammatory
cytokines.42 By using unirradiated recipients, we ensured
that changes found in our chimeras were caused only by GVHD.
Expansion and disappearance of postthymic T cells during the course
of GVHD.
After injection of B6.SJL cells into B6AF1 hosts, a transient increase
of host lymphocytes was followed by total and irreversible disappearance of host B cells by day 12 (Fig 1A) and of host T cells between days
16 and 50 (Fig 1B). Between days 4 and 12, donor-derived T cells showed
a notable proliferation, involving mainly the CD8+ subset
(Fig 1B and D). This was followed by a conspicuous lymphoid hypoplasia
of longer duration for T cells than for B cells. On day 100, (donor-derived) B-cell numbers had regained normal levels, whereas the
numbers of (donor-derived) T cells were decreased by 70% compared with
age-matched controls. These observations are concordant with the
description of T-cell reconstitution reported by Hakim et
al43 in a similar model of GVHD.
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| Fig 1.
Expansion and disappearance of postthymic T cells during
the course of GVHD. A cell suspension containing 6 × 107
B6.SJL T cells was injected into unirradiated B6AF1 recipients. Shown
is a time course evaluation of the numbers of host- and donor-derived B
cells (A) and T cells (B) found in the spleen of GVHD+
recipients. CD44 phenotype of host (C) and donor (D) T cells found in
the spleen of GVHD+ recipients. Three-color staining was
performed using PE-labeled anti-CD19, anti-Thy1.2, or anti-CD44;
Cy-chrome-labeled anti-CD4 or anti-CD8; and FITC-labeled anti-Ly5.1 or
anti-Ly5.2. Results are presented as the mean ± SD in (A) and (B);
for the sake of clarity, only the mean is shown in (C) and (D). There
were three to four mice per group.
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T-cell proliferation can be observed after cognate interactions with
antigen-presenting cells or after exposure of bystander cells to high
concentrations of cytokines (such as interferon [IFN], interleukin-2
[IL-2], IL-6, and tumor necrosis factor [TNF]) produced by
antigen-specific T cells.44-46 Acquisition of an
activated/memory phenotype, characterized by upregulation of CD44, is
observed in antigen-specific T cells after cognate interactions, but
not in proliferating bystander T cells.44,47,48
Nevertheless, at least in the context of some viral infections, T cells
that are antigen-experienced, and thus have already upregulated CD44, may be more susceptible to bystander activation than naïve
(CD44lo) T cells.47 Analysis of the CD44
phenotype of expanded host- and donor-derived postthymic T-cell
populations showed divergent phenotypes. At its time of maximal
expansion (day 12), the population of donor-derived T cells was
composed mainly of CD44hi cells, with very few
CD44lo elements (Fig 1). In contrast, at its peak (day 8),
expansion of recipient T cells involved mainly CD44lo
cells, with a lesser increase in the number of CD44int
cells and a notable decrease in the amount of CD44hi
elements. These results suggest that the transient expansion of host T
cells was a bystander effect induced by the GVHD-associated cytokine
storm, whereas donor T-cell proliferation was induced by cognate
interactions with host antigens. Considering that we infused 6 × 107 donor T cells and that approximately 3% of T cells
respond to a given allo-H2 haplotype,49 the grafted
inoculum used in the preceding experiments contained  2 × 106 host-reactive T cells and approximately 5.8 × 107 non-host-reactive T cells. Taking into account that
only a portion of infused T cells home to the spleen, host-reactive T
cells (2 × 106) would have to expand more than
30-fold to account for the approximately 6 × 107
donor T cells that were found in the spleen of day-12 recipients (Fig
1). By comparison, the bystander expansion of host T cells was much
more modest, because the number of host T cells increased to a maximum
of twofold on day 8 (Fig 1).
Assessment of V usage by CD4+ and CD8+ cell
populations on day 12 showed that expansion of donor T cells involved
all V families tested. Indeed, the V profile of expanded donor
CD4+ T cells was similar to that of normal B6.SJL controls,
whereas the V usage by the expanded CD8+ subset showed
only minor differences when compared with controls (Fig 2). These observations show that the
cell expansion was of polyclonal origin and was not elicited by a
superantigen.50,51
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| Fig 2.
Donor-derived postthymic T cells express a diverse V
repertoire. A cell suspension containing 6 × 107 B6.SJL T
cells was injected into unirradiated B6AF1 recipients. Three-color
staining was performed using the following antibodies: FITC-labeled
anti-V, Cy-chrome-labeled anti-CD4 or anti-CD8, and biotin-coupled
anti-Ly5.1 plus Streptavidin-PE. Chimeras were studied on day +12.
Results are presented as the mean ± SD (3 mice per group). *P < .05 when compared with B6.SJL controls.
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Expansion of donor T cells essentially involves host-reactive cells
and is thus antigen-specific.
To evaluate more directly the importance of antigen-specific versus
bystander proliferation in donor T-cell expansion, we produced and
studied the fate of a donor (B6.PL) T-lymphocyte population selectively
depleted in host (H2a)-reactive cells. These
(Ly5.2+Thy1.1+) non-host-reactive T cells were
harvested from the spleen and lymph nodes of B6AF1 mice that had been
irradiated (12 Gy) and injected 60 days before with 107
T-cell-depleted B6.PL bone marrow cells. After differentiation of
B6.PL progenitors in the B6AF1 thymus, the lymphoid organs of these
chimeras are repopulated with B6.PL T cells from which anti-H2a reactive elements have been deleted. As
expected,13 the proliferative activity of these T cells was
normal when assessed after in vitro stimulation with mitogens (Con A
and phytohemagglutinin [PHA]) or third-party major
histocompatibility complex (MHC) antigens (H2d; data not shown).
A cell suspension containing 4 × 107
non-host-reactive T cells (of B6.PL origin) and 4 × 107 B6.SJL T cells (containing host-reactive T cells) was
injected into B6.AF1 hosts, and we compared the expansion of these two donor T-cell populations. The rationale was that, if donor T-cell proliferation was host antigen-specific, B6.PL-derived
non-host-reactive T cells would not proliferate and the donor T-cell
compartment would contain almost exclusively B6.SJL cells. On the
contrary, if donor T-cell proliferation were mainly due to bystander
activation, the ratio of B6.PL vs. B6.SJL cells would approach 1:1.
When assessed on days 8 and 12 posttransplant, the ratio of B6.PL
versus B6.SJL cells was 1:99 (Table 1). At
the time of maximal T-cell expansion, on day 12, the mean absolute
numbers of B6.PL-versus B6.SJL-derived T cells found in the spleen of
GVHD+ recipients were 61 × 106 and 0.5 × 106, respectively (data not shown). This indicates
that donor T-cell expansion involved host-reactive cells and that the
importance of bystander proliferation was negligible. Thus, the intense
donor T-cell proliferation was consecutive to cognate interactions with host antigens and not the result of paracrine stimulation of bystander (non-host-reactive) T cells.
Host-reactive cells show massive apoptosis involving Fas/FasL
interactions.
The observation that brisk expansion of host-reactive T cells is
followed by severe and prolonged lymphoid hypoplasia suggests that
activation of donor T cells leads to massive activation-induced cell
death (AICD). We therefore used Annexin-V staining to measure the
number of apoptotic B6.SJL donor T cells in the spleen of B6AF1 hosts.
The percentage of apoptotic T cells in fresh spleen cell suspensions
reached 22% for the CD4+ subset on day 16 and 32% for the
CD8+ subset on day 12 (Fig 3).
These numbers of apoptotic cells are significantly higher than those
observed in control B6.SJL T cells (3% to 4.5%). Because the in vivo
clearance of apoptotic cells is very rapid,52,53 our
results suggest that extensive amounts of donor T cells are most likely
eliminated by apoptosis during the course of acute GVHD.
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| Fig 3.
Host-reactive cells show massive apoptosis involving
Fas/FasL interactions. (A) Proportion of apoptotic
(Annexin-V-positive) T cells in F1 recipients injected with B6.SJL vs.
lpr cells. The mean numbers of apoptotic
CD4+/CD8+ T cells was 4.5%/3% in B6.SJL
controls and 5.8%/3.4% in lpr controls (n = 3; data not shown).
Results are expressed as the mean ± SD, n = 3. *P < .05, **P < .01, ***P < .005 relative to B6.SJL donors.
(B) Histograms showing results for a representative F1 recipient
studied on day 16 after injection of B6.SJL or lpr cells (shaded
diagram). The clear histogram represents a negative control (untreated
B6.SJL or lpr mouse).
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With this in mind, we asked whether Fas/FasL interactions played a
significant role in the apoptosis of donor T cells. We inferred that,
if apoptosis was Fas-dependent, it should not occur when the donor is
Fas- or FasL-deficient. In a first set of experiments, we determined
that only the study of Fas-deficient (lpr), but not of FasL-deficient
(gld) donors would be appropriate to address this question. Indeed,
after injection into B6.SAF1 hosts, T cells from gld donors showed no
measurable expansion and were rapidly eliminated
(Fig 4). This observation might be
explained by the fact that Fas/FasL interactions have a costimulatory
effect on T-cell activation and generation of effector
cells.20,22,23 Moreover, the failure of T cells from gld
donors to proliferate demonstrates that, at least in our model, the
absence of GVHD with gld donors may not be accepted as an evidence that
Fas-mediated apoptosis (as opposed to Fas-mediated costimulation) plays
a role in GVHD. In contrast to gld donors, cells of lpr donors expanded vigorously in recipients (Fig 4). Furthermore, from day 12 to 16, the
number of lpr-derived CD8+ T cells increased, in contrast
to CD8+ cells obtained from normal donors, which decreased.
More importantly, the number of apoptotic donor-derived
CD4+ and CD8+ T cells was strikingly decreased
in the case of lpr donors as compared with normal donors (Fig 3). These
observations suggest that Fas signaling plays a prominent role in the
AICD of host-reactive T cells. However, it should be noted that, in the
case of lpr donors, CD4+ T cells did not expand to the same
extent as CD8+ cells between days 12 and 16 (Fig 4).
Although the reason for this observation is unclear, it suggests that
there may be differences in the mechanisms controlling CD4+
versus CD8+ cells in this setting.
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| Fig 4.
Influence of Fas/FasL interactions on donor T-cell
expansion. Expansion of donor T cells obtained from B6.SJL, gld
(FasL-deficient), or lpr (Fas-deficient) mice after transplantation in
B6AF1 or B6.SAF1 recipients. Three-color staining was performed with
the following fluorochrome-conjugated antibodies: anti-Ly5.1 or
anti-Ly5.2, anti-CD4 or anti-CD8, and anti-Thy1.1 or anti-Thy1.2. On
day 16, the number of donor-derived CD8+ T cells was
greater with lpr donors than with B6.SJL donors (***P < .005).
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We then analyzed the kinetics of Fas and FasL expression on T-cell
subsets between days 8 and 16 in B6AF1 recipients of a B6.SJL graft. A
significant upregulation of Fas expression was found in
CD4+ and CD8+ host and donor T cells
(Fig 5). Fas upregulation was most
noticeable on donor CD4+ T cells and was of limited
duration, being observed on day 8 but not later. In addition, a
significant increase in the proportion of FasL+ cells was
found specifically among the donor CD8+ T-cell subset
(Fig 6). Donor CD4+ T cells
showed only a minimal increase in FasL expression that did not reach
significance. Upregulation of FasL on donor CD8+ T cells
was present at the three time points studied from days 8 to 16. These
results suggest that Fas-dependent AICD of donor T cells is initiated
specifically by FasL+ CD8+ host-reactive T
cells.
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| Fig 5.
Expression of Fas on donor and host T-cell subsets. A
cell suspension containing 6 × 107 B6.SJL T cells was
injected into unirradiated B6AF1 recipients. (A) Increased Fas
expression on day 8. Three-color staining was performed with the
following antibodies: FITC-labeled anti-Ly5.1 or anti-Ly5.2,
Cy-chrome-labeled anti-CD4 or anti-CD8, and PE-labeled anti-Fas
(shaded histogram) or its isotypic control (clear histogram). Results
from one representative experiment of three are shown. (B) Time course
evaluation of Fas expression. Mean ± SD, N = 3; nd, not done.
*P < .05, **P < .01, ***P < .005 relative
to control.
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| Fig 6.
Upregulation of FasL expression on CD8+ T
cells. A cell suspension containing 6 × 107 B6.SJL T
cells was injected into unirradiated B6AF1 recipients. (A) Increased
FasL expression on donor (Ly5.1+) CD8+ T
cells on day 8. Cells were first labeled with hamster anti-FasL
antibody, then with biotin-coupled goat antihamster antibody, and
lastly with PE-conjugated streptavidin together with FITC-labeled
anti-Ly5.1 and Cy-chrome-labeled anti-CD4 or anti-CD8 antibodies; only
the last two steps were performed in staining control. FasL staining is
represented by a shaded histogram and staining control as a clear
histogram. The value shown in each box represents the specific staining
of FasL+ cells. Results from one representative
experiment of three are shown. (B) Time course evaluation of FasL
expression. Mean ± SD, N = 3. **P < .01, ****P < .001 relative to control.
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The influence of host-reactive cells on the fate of
non-host-reactive cells.
Although AICD entails the disappearance of most, if not all,
host-reactive T cells, what is the fate of non-host-reactive donor T
cells? To evaluate this question, we compared the fate of
non-host-reactive T cells when injected either alone or with host-reactive T cells. After injection of 2 × 107
(B6.PL B6AF1) non-host-reactive T cells into B6AF1 hosts, 3.8 × 106 Ly5.2+Thy1.1+ T cells
were found in day-12 recipients (Fig 7A).
Strikingly, the recovery of non-host-reactive T cells was decreased by
approximately fivefold when 4 × 107 B6.SJL T cells
were coinjected (Fig 7A). As a negative control, we verified that
coinjection of syngeneic (B6.SAF1) T cells did not decrease the
recovery of non-host-reactive T cells. Thus, clearance of
non-host-reactive T cells was accelerated in the presence of
host-reactive T cells. We hypothesized that two mechanisms could be
responsible for the rapid demise of non-host-reactive T cells in the
presence of host-reactive T cells: (1) competition for space between
host-reactive and nonreactive T cells and (2) apoptosis of bystander
cells induced by host-reactive T cells. Three series of observations
showed characteristic features of apoptosis in non-host-reactive T
cells when coinjected with host-reactive T cells, but not when injected
alone: (1) upregulation of Fas expression (Fig 7B), (2) downregulation
of Thy1 (Fig 7C; decreased expression of several cell surface membrane
antigens such as Thy1 is one of the characteristic changes found on
apoptotic cells54), and (3) increased numbers of
Annexin-V-positive T cells (Fig 8). Increased numbers of Annexin-V-positive elements among
non-host-reactive T cells coinjected with host reactive T cells were
observed when analyses were performed on fresh recipients' splenocyte
suspensions, but became more impressive when analyses were performed on
mitogen-stimulated T cells (Fig 8). In a control group, we observed
that coinjection of syngeneic (B6.SAF1) T cells did not increase the
proportion of apoptotic elements among non-host-reactive cells (Fig
8B). These results lead us to contend that the failure of grafted
non-host-reactive postthymic T cells to reconstitute the immune system
of GVHD+ hosts is a consequence of bystander killing
mediated by host-reactive effector T cells.
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| Fig 7.
Accelerated clearance of non-host-reactive donor
(Ly5.2+Thy1.1+) T cells when coinjected
with host-reactive (Ly5.1+Thy1.2+) T cells.
(A) Numbers of CD4+ and CD8+
non-host-reactive T cells found in the spleen of B6AF1 recipients on
day 12. Host-reactive and non-host-reactive cells were prepared as
described for Fig 3A. Non-host-reactive T cells (20 × 106) were injected alone or with either 40 × 106 host-reactive T cells or 40 × 106
syngeneic (B6.SAF1) T cells. N = 3; *P < .05 when compared
with results in mice transplanted with non-host-reactive T cells alone
or non-host-reactive T cells + syngeneic T cells. No differences
were found between mice transplanted with non-host-reactive T cells
alone versus non-host-reactive T cells + syngeneic T cells. (B)
Non-host-reactive grafted CD4+ T cells overexpress Fas
when coinjected with host-reactive T cells (right panel), but not when
injected alone (left panel). Fas expression was assessed on day 8 as
described in Fig 5. Results from one representative experiment of three
are shown. (C) Downregulation of Thy1 expression was observed on
CD4+ and CD8+ non-host-reactive grafted T
cells when coinjected with host-reactive T cells (right panel), but not
when injected alone (left panel). Two- and three-color staining was
performed with the following fluorochrome-conjugated antibodies:
anti-Ly5.2 or anti-Thy1.1, anti-Fas, and anti-CD4 or anti-CD8. Results
from one representative experiment of three are shown.
|
|
View larger version (30K):
[in this window]
[in a new window]
| Fig 8.
Apoptosis of non-host-reactive T cells when coinjected
with host-reactive T cells. Non-host-reactive cells were injected
alone or with either host-reactive T cells or syngeneic T cells as
described in Fig 7. (A) The proportion of non-host-reactive cells
stained by Annexin-V was evaluated in fresh uncultured spleen cell
suspensions from day-8 B6AF1 hosts. Three-color staining was performed
with the following antibodies: PE-labeled anti-Ly5.2,
Cy-chrome-labeled anti-CD4 or anti-CD8, and FITC-labeled Annexin-V.
Results from one representative experiment of three are shown. (B)
Annexin-V staining was performed after culturing recipient spleen cells
during 40 hours in the presence of Con A. Results are expressed as the
mean ± SD (N = 3) in the lower panel, and results from one
representative experiment are depicted in the upper panel. **P < .01, ***P < .005 when compared with the results obtained
in mice transplanted with non-host-reactive T cells alone or
non-host-reactive T cells + syngeneic T cells. No differences were
found between mice transplanted with non-host-reactive T cells alone
versus non-host-reactive T cells + syngeneic T cells.
|
|
| |
DISCUSSION |
The fate of alloreactive antihost T cells.
After transplantation of T-cell-replete hematopoietic cell grafts,
host-reactive T cells are initially sequestered in the spleen, where
they proliferate before reentering the circulation and disseminating to
other organs.55,56 By examining host spleen cell
suspensions, we observed, in the early days posttransplant, a brisk
expansion of alloreactive T cells followed by massive in situ AICD.
Expanded populations of donor T cells comprised more CD8+
than CD4+ T cells and were quite diverse in terms of V
repertoire. Expansion selectively involved antihost T cells.
Non-host-reactive T cells did not expand when transplanted alone or
with host-reactive cells. The latter observation indicates that T-cell
proliferation triggered by host H2 antigens basically involves
antigen-specific and not bystander T cells. Our finding is consistent
with other recent studies on T-cell responses to viral
antigens.46,57-61
The very high numbers (up to 32%) of Annexin-V-labeled donor T cells
found in fresh spleen cell suspensions from GVHD+ mice
point to a massive GVHD-associated AICD. AICD is a physiologically important process that contributes to the termination of immune responses and can be mediated by numerous molecular pathways, including
Fas, TNF, TRAIL, galectin, and CTLA-4.62-67 Analyses of
T-cell responses to various antigens have shown that Fas usually plays
a dominant role in AICD of CD4+ T cells.68-70
The situation is more complex regarding AICD of CD8+ T
cells. In the latter case, the role of Fas in various systems ranges
from negligible to predominant.68,71-76 In models in which the Fas pathway seems to be of little significance, AICD is mediated predominantly by the TNF pathway.71,74
Our results demonstrate the upregulation of FasL on CD8+
host-reactive T cells, an increased expression of Fas on donor
CD4+ and CD8+ T cells, and a major decrease in
apoptotic donor CD4+ and CD8+ T cells in the
case of lpr donors. They provide strong evidence that the Fas pathway
plays a critical role in the AICD associated with GVHD. Furthermore,
although it appears that both CD4+ and CD8+ T
cells are targets of this AICD, effector function (FasL expression) seems to be performed only by host-reactive CD8+ T cells.
Our data are concordant with results from others showing that the level
of FasL upregulation detected on antigen reactive T cells after massive
in vivo expansion is inferior to that observed on T cells after
short-term in vitro culture with mitogens.18 This may well
be explained by the recent observation that upregulation of FasL is an
early and transient event that is maximal after one cell division but
decreases after two to three cell divisions.77 Thus, when
studies are performed on T-cell populations that have undergone massive
in vivo expansion (here  30-fold for host-reactive T cells, ie, 5
cell divisions), FasL expression may not be at its maximum level. The
molecular interactions responsible for the low levels of apoptosis
observed in T cells from lpr donors remain to be defined. This could
represent Fas-dependent apoptosis mediated by the low levels of Fas
proteins expressed by lpr cells.78 Alternatively, residual
levels of apoptosis detected with lpr donors could result from AICD
mediated by TNF or other pathways and/or from passive cell death caused
by deprivation of survival stimuli leading to decreased expression of
antiapoptotic proteins, mainly of the Bcl family.64
The fate of non-host-reactive T cells.
When injected alone, non-host-reactive T cells neither proliferated
nor upregulated expression Fas expression. However, coinjection of
host-reactive T cells entailed an increased expression of Fas on, in
addition to apoptosis and accelerated disappearance of, non-host-reactive T cells. Therefore, apoptosis of non-host-reactive T cells and their subsequent failure to repopulate host secondary lymphoid organs likely represent collateral damage induced by FasL-expressing CD8+ host-reactive T cells. Bystander lysis
of Fas+ T cells by FasL+ antigen-specific T
cells has been reported in the course of viral infections.79-83 However, this collateral damage is usually
of minimal significance because of its limited magnitude and because normal thymic output can replenish the peripheral T-cell pool. In
contrast, we propose that, in the context of GVHD, bystander lysis of
postthymic T cells can likely have far-reaching consequences. Indeed,
severe thymic hypoplasia is commonly found in recipients of allogeneic
hematopoietic stem cell transplants, particularly those with GVHD.
Thymic-independent T-cell reconstitution via expansion of grafted
postthymic T cells should be able to compensate for thymic
failure.13,14 However, by abrogating this salvage pathway,
bystander lysis of non-host-reactive donor T cells can dramatically
compromise immune reconstitution.
Two different signaling pathways can increase Fas expression on T
cells: TCR ligation and cytokines such as IFN- , IL-2, IL-7, and
TNF- .84-87 By definition, non-host-reactive T cells do
not express antihost specific T-cell receptors. Thus, upregulation of
Fas on these cells is presumably caused by secretion of IFN-, IL-2,
IL-7, and TNF- during the GVHD-associated cytokine
storm.18,42,88,89 Interestingly, it has recently been shown
in humans that acute GVHD, but not infection, is associated with
increased soluble Fas serum levels.90 In the latter case,
it remains to be determined whether increased serum Fas levels are
caused by upregulation of Fas on donor T cells.
Patients with GVHD are profoundly immunodeficient. Therefore, they
represent prime candidates for adoptive immunotherapy based on
injection of donor-derived T cells specific for pathogens such as CMV
or EBV.35-37 The results presented here suggest that, at least during the acute phase of GVHD, the brisk expansion of activated antihost CD8+ T cells can severely curtail the survival of
transferred postthymic T cells. It will therefore be important to
determine whether lysis of bystander postthymic T cells can be avoided
by delaying adoptive transfer after the cytokine storm has subsided or
after host-reactive T cells have gone through AICD.
Massive apoptosis: A link between GVHD and autoimmunity?
In normal individuals, apoptotic cells are readily cleared by the
monocyte-macrophage system and do not elicit immune
responses.52,53 However, under circumstances in which the
clearance of apoptotic cells is impaired, it has been proposed that
accumulation of high numbers of apoptotic cells could lead to
immunogenic presentation of intracellular self-antigens and thereby
initiate autoimmune responses.91,92 Direct evidence
supporting this concept has recently been presented. Indeed, it has
been shown that normal mice injected with large amounts of apoptotic
syngeneic thymocytes (107 cells intravenously weekly for a
total of 4 injections) develop a picture similar to that of systemic
lupus erythematosus, characterized by autoantibodies and IgG deposition
in the glomeruli.93 The investigators speculate that, when
confronted with massive amounts of apoptotic (lymphoid) cells, the
clearance capacity of macrophages could be overwhelmed and that
abnormal (because of its level and/or duration) autoantigen
presentation could pave the way to autoimmunity.93 Additionally, studies in mice and humans have shown that chronic GVHD
is more prevalent in individuals who have presented acute GVHD and
shares many clinical, histologic, and immunologic features with
autoimmune diseases such as systemic lupus erythematosus and Sjogren's
syndrome.94-96 How an alloimmune reaction (acute GVHD) can
initiate autoimmunity (chronic GVHD) remains elusive. Our observations
raise the possibility that massive apoptosis of donor T cells (via AICD
and bystander lysis) could represent the missing link between these two processes.
| |
FOOTNOTES |
Submitted November 17, 1998; accepted March 11, 1999.
Supported by the National Cancer Institute of Canada (C.P.). D.-C.R. is
a senior scholar of the Fonds de la Recherche en Santé du
Québec.
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 Claude Perreault, MD, Guy-Bernier Research
Center, Maisonneuve-Rosemont Hospital, 5415 de l'Assomption Blvd,
Montreal, Quebec, Canada H1T 2M4; e-mail: c.perreault{at}videotron.ca.
| |
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