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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3432-3438
Coexistence of Two Functioning T-Cell Repertoires in Healthy
Ex-Thalassemics Bearing a Persistent Mixed Chimerism Years After Bone
Marrow Transplantation
By
Manuela Battaglia,
Marco Andreani,
Marisa Manna,
Sonia Nesci,
Paola Tonucci,
Barbara Persini,
Gioacchino Robustelli della Cuna,
Arcangelo Nocera,
Jack Gorski,
Guido Lucarelli, and
Raffaele De Palma
From the Divisione di Ematologia e Centro Trapianto di Midollo
Osseo-Ospedale di Muraglia, Pesaro, Italy; the Lab di Medicina
Sperimentale, Fondazione "S. Maugeri," Pavia, Italy; the Sezione
di Immunologia, Ospedale S. Martino, Genova, Italy; The Blood Research
Institute, Milwaukee, WI; and the Dipartimento di Internistica Clinica
e Sperimentale, II Universita' di Napoli, Napoli, Italy.
 |
ABSTRACT |
Bone marrow transplantation (BMT) from an HLA-identical donor is an
established therapy to cure homozygous  -thalassemia. Approximately 10% of thalassemic patients developed a
persistent mixed chimerism (PMC) after BMT characterized by stable
coexistence of host and donor cells in all hematopoietic compartments.
Interestingly, in the erythrocytic lineage, close to normal levels of
hemoglobin can be observed in the absence of complete
donor engraftment. In the lymphocytic lineage, the striking feature is
the coexistence of immune cells. This implies a state of tolerance or
anergy, raising the issue of immunocompetence of the host. To
understand the state of the T cells in PMC, repertoire analysis and
functional studies were performed on cells from 3 ex-thalassemics.
Repertoire analysis showed a profound skewing. This was due to an
expansion of some T cells and not to a collapse of the repertoire,
because phytohemagglutinin stimulation showed the presence
of a complex repertoire. The immunocompetence of the chimeric immune
systems was further established by showing responses to
alloantigens and recall antigens in vitro. Both host and
donor lymphocytes were observed in the cultures. These data suggest
that the expanded T cells play a role in specific tolerance while
allowing a normal immune status in these patients.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BONE MARROW transplantation (BMT) from an
HLA-identical donor is a rational therapeutic modality for the cure of
-thalassemia major as well as for other
hemoglobinopathies.1-5 In the great majority of
transplanted thalassemic patients, a full donor engraftment is observed
and the donor's type pattern of globin chain synthesis is
established.1-6 In some cases, there is the presence of
mixed chimerism after BMT. This normally resolves into complete
engraftment or leads to graft loss. Mixed chimerism has also been
described after BMT for other diseases.7-9
We have previously reported that approximately 10% of thalassemic
patients develop a persistent mixed chimerism
(PMC).4,5,10,11 PMC is defined as the coexistence of donor
and recipient cells in the marrow of the host for a period longer than
24 months. Patients showing PMC have a functioning graft, normal levels
of hemoglobin (Hb), and are cured from
thalassemia.10 It still remains unclear how cells of
different origin may last without adverse immune response. If this
long-term coexistance is due to a tolerance/suppresion mechanism, it
remains unclear to what extent this would affect overall immune function.
To better understand the mechanisms underlying the establishment and
maintenance of the immune system in PMC, we analyzed the T-cell
repertoire of 3 well-characterized PMC patients by measuring the CDR3
length of TCR BV families.12-16 This showed a profound
skewed repertoire composed of a small number of specific T-cell
clonotypes. After in vitro stimulation by phytohemagglutinin (PHA), the normal gaussian distribution TCR CDR3 sizes
were reestablished. This shows that the skewing in the unmanipulated
peripheral blood mononuclear cell (PBMC) is associated
with an expansion of certain T cells but not with a collapse of the
whole repertoire.
The immune functions of these patients were tested by measuring the
response to recall antigens and third-party allogeneic cells in mixed
lymphocyte reaction (MLR). All 3 patients showed an active immune
response. Fluorescence in situ hybridization (FISH) or
restriction fragment length polymorphism of variable number tandem
repeats (RFLP-VNTR) analysis showed the presence of cells
of both host and donor origin after culture. These data suggest that
the skewed immune profile of these PMC patients plays a role in
maintaining a specific status of tolerance allowing the coexistence of
immune cells with different origin. However, this T-cell repertoire is
still functional, resulting in a state of normal immunocompetence.
| |
MATERIALS AND METHODS |
Patients.
Patients described in these studies were chosen among a cohort of more
than 800 patients affected by homozygous -thalassemia and were
subjected to allogenic BMT from an HLA-identical sibling. Clinical data
concerning these patients are summarized in
Table 1. Briefly, during periodical
follow-ups, we chose 3 patients who fullfilled the following criteria:
BMT performed at least 3 years before; persistence of at least 30%
RHCs; minimum Hb level of 12 g/dL; and absence of any
therapy. Two patients who developed rejection of the graft and 2 patients with full donor engraftment were included in the study as
controls. Three normal donors volunteered their blood to perform mixed
lymphocyte reactions. HLA typing on PBMC of normal donors was performed
using a standard lymphotoxicity assay. All of the individuals who
donated blood for this study were fully informed about the aims of the
study before giving their consent.
Cells and cultures.
PBMCs were obtained from whole blood by gradient density separation
using Ficoll according to a standard procedure, washed, and
viable-stored in liquid nitrogen until their use. Cell cultures to test
responses to PHA were prepared in flat-bottom 96-well multiplates
(Costar, Boston, MA). Cells were resuspended at 2 × 105 in 200 µL of complete medium (RPMI 1640, 10% heat-inactivated fetal calf serum [FCS], penicillin [100
U/mL], and streptomycin sulfate [100 µg/mL]) and then stimulated
with PHA at a final concentration of 5 µg/mL. After 3 days of culture
in an incubator at 37°C and 5% CO2, 0.5 µCi of
[3H]thymidine (Amersham-Pharmacia, Milano, Italy) was
added to each well; 16 hours later, the cells were harvested and
counted for thymidine incorporation following standard protocols. To
test the response to recall antigens, cells were resuspended as
described before and then stimulated with purified protein derivative
(PPD) or tetnus toxoid (TT) at a final concentration of 5 µg/mL. After 6 days of culture, 0.5 µCi of
[3H]thymidine was added to the culture and incorporated
radioactivity was measured as described above. For MLR cultures, PBMC
were irradiated (3,000 rad) using a 137Cs source and were
used to stimulate the PBMCs of PMC patients at a 1:2 ratio of
responders/stimulators. After 6 days of culture performed as described
above, a standard [3H]thymidine incorporation test was
performed. For T-cell repertoire analysis, cells were recovered after
cultures; we then prepared RNA and cDNA for spectratype analysis.
FISH analysis.
FISH was performed on cells recovered from cultures when the host and
donor were sex-mismatched. A biotinylated pY3.4 probe complementary to
a part of the Y chromosome was used as reported10,11; positive and negative controls were included in each test. After separation by means of Dynabeads, cells were analyzed on the membrane surface directly.11
RFLP-VNTR analysis.
This analysis was performed as described.10,11 Briefly, DNA
was extracted from cells recovered from the cultures after a Ficoll
separation, and the highly polymorphic VNTR genes were amplified. We
screened locus D1S80 and locus D17S30 as described. Conditions for
polymerase chain reaction (PCR) and gel electrophoresis have been
described as well. Southern blot analysis was performed with
hypervariable VNTR probes as reported.
RNA extraction and cDNA preparation.
RNA was prepared by a single-step procedure using Triazol (GIBCO, Grand
Island, NY) according to the manufacturer's
instructions. cDNA was prepared starting from 1 µg of total RNA as
described elsewhere using oligo(dT) as primer for the reverse
transcription reaction.12,13
PCR amplification and samples normalization for spectratype
analysis.
Amplification of cDNA was performed as previously
reported.12,13 Briefly, we used a fluoresceinated primer
with specificity for the -chain TCR constant region and 2 primers
bearing the specificity for the V region, as
published.14,15 The amplification conditions have been
described as well.12,13 To normalize the results, the
amounts of templates from different samples were titrated at different
dilution points of starting material by amplifying the TCR -chain
constant cDNA; the level of expression was then quantified by analyzing
the fluoresceinated signal.12 A coamplification, using 1 µL of a 1:1,000 dilution starting from 20 µmol/L solution of primer
pair specific for actin was performed in each PCR tube to check the
efficiency of the single PCR reaction.
Generation of spectratypes.
PCR product (0.5 µL) was diluted 1:1 with distilled water. The sample
was boiled and loaded on sequencing gel and then run in a
fluorescence-based DNA sequencer (Applied Biosystem 377 model; Applied
Biosystems, Foster City, CA) in the presence of
Rox-labeled size markers (Applied Biosystems).14 The data
were analyzed by means of Applied Byosistems Genescan software that
allows us to assign size and peak areas to the different PCR products.
The data for each TCR-V family were then visualized as chromatograms.
Single-strand conformational polymorphism (SSCP).
This analysis was performed according to standard procedures with minor
modifications.14 Briefly, PCR products of given TCR BV
families, having a predominant peak when analyzed by spectratyping, were serially diluted until only the predominant peak was visible by
Genescan analysis. PCR product (0.5 µL) was then further diluted in
0.1% sodium dodecyl sulfate (SDS)/10 mmol/L EDTA. Finally, 2 µL of
the obtained solution was mixed to loading buffer and run on a
nondenaturing gel (6% [wt/vol] acrylamide/10% [vol/vol] glycerol). The results were analyzed, as reported for spectratype analysis, by means of Genescan software and expressed as a gel file image.
| |
RESULTS |
Three patients were analyzed as part of this study. The patient data
are given in Table 1. All 3 were defined as showing PMC with at least
30% residual host cells at 3 or more years posttransplant. They have
normal lymphocyte counts with normal CD4/CD8 ratios and normal levels
of Hb. Leukocyte subpopulation distributions, determined by staining
with monoclonal antibodies and subsequent fluorescence-activated cell
sorting (FACS) analysis, were normal in all the patients (data not
shown). None of the 3 patients was receiving any therapeutic treatment,
and there were no reports of clinical complications during the periodic
posttransplant follow-ups.
TCR repertoire analysis of PMC patients.
To better understand the immunological phenomena underlying the
unexpected status found in these patients, we analyzed the T-cell
repertoire by means of TCR -chain CDR3 spectratyping. This technique
visualizes the T-cell repertoire for each TCR BV family as a series of
bands having a gaussian distribution.12-16 Any modification
in the normal gaussian profile and/or intensity of the bands represents
an alteration of the TCR repertoire. Because only 1 TCR BV gene is
productively rearranged for each T cell, the study of TCR-BV
repertoires allows us to follow the behavior of T-cell populations
under different conditions, both in vivo and in vitro.12-16
Repertoire analysis of the PBMCs from the 3 PMC patients showed severe
alterations. A representative part of these findings is shown in
Fig 1. The data for each of the TCR BV
families showed a normal gaussian profile in the donor PBMCs (top
panel) and a skewed repertoire in the recipient (bottom panel). Such a
skewed repertoire was observed in almost all of the TCR BV families
from all 3 patients, with the presence of predominant peaks and the
loss of the characteristic gaussian pattern. Such a profound skewing in
the T-cell repertoire is compatible with a collapse of the repertoire
and has been associated with severely compromised immune systems
posttransplant.13 In contrast, the 3 patients were healthy
and did not show any sign of a compromised immune system. Because there
was a disparity between the repertoire of these patients and their
immune status, we decided to investigate these patients further.
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| Fig 1.
Spectratype analysis of 1 representative donor and
recipient pair (UPN688). The donor data are from a pretransplant PBMC
sample. The recipient data are from PBMC obtained 7 years
posttransplant. The histograms show the TCR profile of 9 different BV
families (indicated on top of each pair of spectratypes). The donor or
recipient origin of each spectratype is identified on the right.
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T-cell repertoire analysis of PBMCs from patients who developed a
full donor engraftment or rejection.
As a control, we analyzed TCR on PBMCs derived from 2 patients with
full donor engraftment and 2 patients who rejected the transplant.
Patients with full donor engraftment showed TCR V spectratype
profiles comparable to those found in normal donors (data not shown).
Patients with rejection showed an hyperexpression of most of the TCR
V families' clonotypes (data not shown), reflecting a massive
activation of T cells. Similar results were previously reported by
several investigators.13,17,18
Evaluation of the complexity of the predominant clonotypes.
In spectratype analysis, each peak represents TCR having the same
BV-CDR3 length; therefore, each peak may consist of a variable number
of TCR sequences. To determine the complexity underlying of the
predominant peaks observed in the peripheral blood, we performed SSCP
on the DNA. This technique allows detection of single basepair
differences in 2 DNA strands of the same size. We selected the
prominent peaks present in different TCR BV families from each of the 3 patients for the SSCP analysis: BV 20 in UPN 855, BV2 in UPN 688, and
BV 5.3 in UPN1074. In all cases, the predominant spectratype peaks
could be resolved into 1 or 2 bands (Fig 2,
lanes 1 through 4, 7, and 8). Lanes 5 and 6 show the SSCP profile of
single spectratype peak known to be polyclonal. Rather than distinct
bands, a smear is observed. These results indicate that the repertoire
skewing is caused by a limited number of T-cell clonotypes. Although it
is possible that this may be the result of repertoire contraction, it
is more likely that this reflects an expansion of these cells in
response to some stimulus associated with the PMC state.
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| Fig 2.
SSCP analysis of predominant spectratype bands. Each pair
of lanes represents data derived from different samples. Lanes 1 and 2, BV2 from UPN688; lanes 3 and 4, BV20 from UPN855; and lanes
7 and 8, BV5.3 from UPN1074. Lanes 5 and 6 show results from the
analysis of a normal, polyclonal spectratype band. In each case, the
first lane represents PCR in which twice the amount of cDNA was used.
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PHA stimulation of the PBMC reestablishes a complex T-cell
repertoire.
As part of the investigation of the ability of the T cells from these
patients to mount an immune response to common stimuli, the
proliferative responses to the mitogen, PHA, was tested. It was
observed that the response to this mitogen induced the proliferation of
a large number of T cells. Spectratype analysis of the cultures showed
that, despite starting out with a heavily skewed repertoire, a normal
repertoire pattern was restored (Fig 3).
The starting spectratype pattern is shown at the top of each panel, and
the resulting spectratype after PHA stimulation is shown at the bottom. These results indicate that the T cells giving rise to the skewed starting repertoire do not have the same proliferative potential to PHA
as do the other T cells in the repertoire. These data also clearly
point out the existence of a complex repertoire in these individuals
that is masked in the PBMCs by the expansion of a select number of
T-cell clonotypes. Because a fixed amount of cDNA is used in these
analyses, the resulting patterns are always relative. It requires the
preferred expansion of the underlying repertoire by the PHA for it to
be visualized by the spectratype analysis.
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| Fig 3.
Spectratype analysis of in vitro culture with PHA. PBMCs
from UPN688 (A), UPN885 (B), and UPN1074 (C). For each patient, the TCR
profiles of 3 representative BV families (indicated on top of each pair
of spectratypes) are shown. The spectratype of the PBMC before culture
(pre) is shown at the top of each panel and that after the culture
(post) is shown on the bottom.
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Analysis of the in vitro response of T cells to antigens.
The immune status of the PMC individuals appears to be normal in that
they do not show any of the clinical signs associated with compromised
immune function. Although the results from the PHA stimulation showed
the existence of a complex repertoire in these individuals, it was
necessary to show that this repertoire was capable of standard
responses to recall antigens (PPD and TT) and unrelated third-party
alloantigens in culture. The results of these stimulation as assayed by
proliferation are shown in Fig 4. All of
the stimuli elicited a response from the T cells, and the proliferation
values were comparable to those obtained from cells of a normal donor.
The results for the stimulation with PHA are also included for
comparison.
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| Fig 4.
Proliferative analysis of the in vitro response to
different antigens. [3H]thymidine uptake, expressed in cpm, of PBMCs
of UPN 688, UPN855, and UPN1074 after in vitro culture with PPD, TT,
PHA, and an unrelated third-party alloantigen (see bar legend) is
shown. The same assay was made using a normal donor (ND) as a control.
One representative experiment of 3 performed is shown.
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In addition to the proliferation studies, T-cell repertoire analysis
was performed using spectratyping after stimulation with PPD or TT.
Several new peaks in a number of TCR-BV families were observed after
the cells were stimulated (data not shown). Others using this technique
have described similar results in response to nominal
antigens.12,14,16 It is interesting to point out that the
responding T cells did not correspond in CDR3 length to the skewed
cells present in the beginning of the culture. These experiments
indicate that standard immune responses can be observed in these
individuals and that these responses originate from the normal
underlying T-cell repertoire and not from the expanded T cells that
make up a large portion of the circulating lymphocytes.
Both donor and host cells are observed after in vitro responses to
different stimuli.
Because PMC is characterized by the presence of both host and donor T
cells,8 an important issue in understanding this system is
to determine if cells of both origins can contribute to the observed
immune response. Although it is normally the donor cells that are
responsible for responses in cases of complete engraftment, it is
possible that host cells may also contribute. This would show the
nature of the tolerance phenomenon. FISH or RFLP-VNTR analysis were
used to determine the origin of the cells after culture. FISH was used
for UPN688, because it depends on the presence of a sex-mismatch. We
show an example of the FISH analysis (Fig
5A) and of an RFLP-VNTR analysis (Fig 5B). The data are summarized in
Fig 5C, which plots the percentage of donor cells for each of the
cultures and each of the individual unstimulated PBMC. This shows that
the ratio of donor to host cells did not change appreciably during the
culture and that both were present after the culture period. This is
compatible with both donor and host cell responses in the cultures.
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| Fig 5.
Detection of donor and host cells after in vitro
responses to different stimuli. (A) A representative FISH analysis
performed on PBMCs of UPN688 after culture with PPD. The arrow
indicates the biotinylated pY3.4 probe complementary to a part of Y
chromosome, showing the presence of some recipient cells. (B) RFLP-VNTR
analysis performed on PBMCs of the donor (D) and the respective
recipient UPN855 (H) pretransplant, on PBMCs of the host
after culture with cells of a third party (A), and on the PBMCs of the
same transplanted recipient after in vitro culture with TT (T), PPD
(P), and PHA (Ph). The top arrow indicates the presence of recipient
cells and the bottom arrow indicated the presence of donor cells. (C)
Data summary in which the y-axis shows the percentage of donor cells
evaluated by FISH or RFLP-VNTR and the x-axis shows the different
samples analyzed before (PBMC) and after (PHA, PPD, TT, and Allo) in
vitro culture. Different color bars refer to the 3 different patients
(see legend).
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| |
DISCUSSION |
It is not a rare event to observe host hematopoietic cells after marrow
transplantation; this is referred to as mixed chimerism (MC). There is
evidence that, in some settings, MC is associated with an increased
risk of graft failure and/or disease recurrence. T-cell repertoires
with extensive gaps and contractions have been observed in cases of MC
and were associated with an impairment of immune response and recurrent
infections.17-21 These studies were mainly performed in
patients who underwent BMT for malignancies and before 1 year from the
transplant. In this report, we study T-cell responses to mitogens,
recall antigens, and T-cell repertoires in patients who, transplanted
years before, are cured from the disease and have developed a stable MC.
Our analysis showed a very skewed T-cell repertoire in PMC patients.
Such a finding is usually associated with a poor immune system
function,13,17,18,21 whereas these 3 patients had a normal
immune function, because they were not receiving any therapeutic
treatment and they did not suffer from recurrent infections. It should
also be pointed out that the skewing of the T-cell repertoire was
stable over time, because the repertoire analysis, which was performed
at different times after BMT, was found to be similar (Andreani et al,
unpublished data). Additional evidence for normal immune
system function is that the cells of PMC patients were able to mount a
normal immune response in culture to different stimuli, such as
mitogens, recall antigens, and alloantigens. We also determined that
both donor and recipient cells were contributing to the immune
response, as shown by RFLP-VNTR analysis and FISH.
A very striking observation came from the analysis of the T-cell
repertoire after culture with PHA. In these cultured cells, a gaussian
pattern of spectratype bands that is found in normal individuals was
reestablished. This result indicates the presence of a normal
repertoire in the peripheral blood of these patients that can be shown
by culturing with PHA. Thus, the skewing observed in the absence of PHA
results from the expansion of some T cells to the extent that they mask
the normal repertoire. The nature of this skewing was further analyzed
by SSCP analysis of the prominent peaks found by TCR repertoire
analysis in PMC individuals. This analysis demonstrated that those
peaks were oligoclonal or pauciclonal, supporting the idea that an
expansion of a few T-cell clonotypes was responsible for the
spectratype pattern found in the PMC patients.
Taken together, these data showed that preferential expansions of given
T-cell clonotypes were present in the peripheral blood of PMC patients
and suggest that these expansions could correlate with the establishing
of specific tolerance/anergy. A recent report has demonstrated that
mice spontaneously developing autoimmune disease may be cured by BMT
that establishes PMC. Unfortunately, there are no data concerning the
T-cell repertoire in this animal model.22 The existence in
mice of T cells that specifically inhibit a proliferation towards
specific antigens in presence of interleukin-10 has been
reported.23 It is possible that the cell populations found
expanded in our patients are similar to those present in the PMC
patients. They may expand specifically to maintain an equilibrium that
is needed for the coexistence of 2 different immune systems in the host
after BMT. Only in the presence of a specific trigger (eg, infections)
is this status altered and other T-cell populations start to
proliferate, as demonstrated by the experiment with PHA. More
experiments are needed to further clarify the origin of the expanded
cells found in the blood of these individuals and to assess if they are
from donor, recipient, or both or if they have specific phenotype
features. Moreover, the dynamics regulating the interactions between
host and donor repertoire after different stimuli need to be investigated.
The generalization of these findings to more cases of PMC is needed. It
should be pointed out that, in all 3 cases studied, the donor:host
engraftment was approximately equal. It will be interesting to
determine if the same phenomenon of repertoire skewing is observed in
patients with low host or donor engraftment. The mechanism of
maintianing more skewed chimerism may not be the same as that described here.
Our data support the idea that a condition of PMC is sustained by the
expansion of specific regulatory T cells able to induce a status of
tolerance or anergy, but the driving elements that select and expand
these cells as well as the functions of these cells remain to be
detailed. The understanding of the steps required for establishing the
PMC is of great importance and may contribute to improve greatly the
handling of allogeneic BMT. For example, the ability to generate PMC
could lead to a reduction of the total amount of drugs used for
conditioning regimens and a lowering of the doses of immunosuppressive
drugs administered before BMT, thus reducing the transplant-related
mortality. Furthermore, the analysis of the PMC phenomenon may shed new
light on the way the induction of tolerance takes place after
transplantation and, more generally, in the course of an immune response.
| |
ACKNOWLEDGMENT |
The authors thank Dr Emanuele Angelucci for several helpful suggestions
and comments and Dr Franco Locatelli for stimulating discussions.
| |
FOOTNOTES |
Submitted April 9, 1999; accepted July 7, 1999.
M.B. and M.A. contributed equally to this work.
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 Guido Lucarelli, MD, Divisione di
Ematologia e CentroTrapianto di Midollo Osseo, Ospedale di
Muraglia, Pesaro, Italy, Via Lombroso 19, 61100 Pesaro, Italia;
e-mail: g.lucarelli{at}wnt.it.
| |
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