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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 763-767
TRANSPLANTATION
Microchimerism in bone marrow-derived CD34+ cells of
patients after liver transplantation
Dirk Nierhoff,
Henrik Csaba Horvath,
Joannis Mytilineos,
Markus Golling,
Octavian Bud,
Ernst Klar,
Gerhard Opelz,
Maria Teresa Voso,
Anthony D. Ho,
Rainer Haas, and
Stefan Hohaus
From the Departments of Internal Medicine V and Surgery, Institute
of Transplantation Immunology, University of Heidelberg, Clinical
Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
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Abstract |
Lymphoid and dendritic cells of donor origin can be detected in the
recipient several years after a solid organ transplantation. This
phenomenon is termed microchimerism and could play a role in the
induction of tolerance. The fate of other hematopoietic cells
transferred by liver transplantation, in particular of stem and
progenitor cells, is unknown. For this reason, we studied peripheral
blood and bone marrow samples of 12 patients who had received a liver
transplant from an HLA-DR mismatched donor. Eight patients were
long-term survivors between 2.8 and 10.1 years after allografting.
CD34+ cells from bone marrow were highly enriched with
the use of a 2-step method, and a nested polymerase chain reaction was
applied to detect donor cells on the basis of allelic differences of
the HLA-DRB1 gene. Rigorous controls with DRB1 specificities equal to
the donor and host were included. In 5 of 8 long-term liver recipients,
donor-specific CD34+ cells could be detected in bone
marrow. Microchimerism in the CD34+ cell fraction did not
correlate to the chimeric status in peripheral blood. In conclusion,
our results demonstrate a frequent microchimerism among bone
marrow-derived CD34+ cells after liver transplantation.
The functional role of this phenomenon still needs to be defined.
(Blood. 2000;96:763-767)
© 2000 by The American Society of Hematology.
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Introduction |
The liver is a site of hematopoiesis in fetuses and,
under certain pathologic conditions, in adults. Evidence for the
presence of hematopoietic stem cells (HSCs) in the adult liver comes
from murine transplantation studies.1,2 Taniguchi et al
identified Sca1+ c-kit+ cells in the adult
mouse liver, which were capable of reconstituting bone marrow of
lethally irradiated recipient mice.1 In the same line,
lethally irradiated rats could be reliably rescued by orthotopic liver
transplantation.2 Recently, Crosbie et al detected and
characterized CD34+ HSCs in the adult human liver by flow
cytometry and tissue culture, showing multilineage hematopoietic colony
formation.3 CD34 expression was found on 0.81% to 2.35%
of isolated hepatic mononuclear cells by flow cytometry, and about 50%
of these cells expressed CD38 as a marker for lineage
commitment.3 These data suggest that the liver of the adult
may still be an important homing site for lymphohematopoietic cells.
The contribution of liver-derived HSCs to hematopoiesis is not known.
Liver transplantation can serve as a model to study the fate of
lymphohematopoietic cells derived from the liver.
Starzl et al demonstrated the persistence of donor cells in peripheral
blood, lymph nodes, skin, and bone marrow even years after liver
transplantation, a phenomenon that is called
microchimerism.4-6 Donor leukocytes are readily detectable
in the recipient's blood in the first days after transplantation but
usually decrease thereafter to a level undetectable by flow
cytometry.7 The transplantation of immunocompetent cells
along with the liver may result in graft versus host disease (GvHD), an
immunologic reaction against the recipient. Rare cases of GvHD after
liver transplantation have been reported in the literature, and
complete chimerism of peripheral blood in these patients has been
observed.8-10
The immunologic and clinical relevance of microchimerism still remains
a contentious issue. It has been postulated that microchimerism may
play a role in the induction of tolerance and hence result in a
reduction of rejection episodes and immunosuppressive
therapy.11,12
We hypothesized that human hematopoietic stem and progenitor cells are
transferred by liver transplantation and migrate to the recipient's
bone marrow. Therefore, we studied bone marrow-derived CD34+ cells and peripheral blood of patients who had
received a liver transplant 2.8 to 10.1 years ago to look for the
presence of donor cells using a nested polymerase chain reaction (PCR)
for major histocompatibility complex class II polymorphism.
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Patients and methods |
Patients
We enrolled 12 patients in our study after obtaining informed
consent from each. Eight patients had received a liver
transplant at least 2 years earlier. The median time after
transplantation was 5.1 years, with a range from 2.8 to 10.1 years. We
studied 4 patients before and 2 to 4 months following transplantation.
The patients' characteristics are detailed in Table
1. Clinical data, such as the number of
rejection episodes and immunosuppressive therapy at the time of the
study, are listed in Table 2. The patients
were treated in the Departments of Surgery and Internal Medicine of the
University of Heidelberg.
Cell preparation
Mononuclear cells (MNCs) from peripheral blood and bone
marrow were separated by Ficoll-Hypaque density gradient centrifugation (density 1.077g/cm3) (Ficoll-Hypaque, Pharmacia, Uppsala, Sweden). The
cells were washed twice with phosphate-buffered saline (PBS).
Aliquots from peripheral blood and bone marrow MNCs were incubated for
30 minutes at 4°C in the presence of an anti-CD45 fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody and an anti-CD34 (HPCA-2) phycoerythrin (PE)-conjugated monoclonal
antibody (all from Becton Dickinson, Heidelberg, Germany). The cells
were washed twice with PBS, and red blood cells were removed with the
use of a fluorescence-activated cell sorting (FACS) lysis solution (Becton Dickinson). A total of 10 000 cells were acquired.
Immunofluorescence analysis was performed with a 5-parameter FACScan
(Becton Dickinson) equipped with an argon-ion laser tuned at 488 nm and
15 mW. Emission from FITC and PE was measured with the use of filters
of 530 nm and 585 nm, respectively. The side scatter characteristics
(SSCs)-versus-CD45-fluorescence dot plot was used to
discriminate between the smallest hematopoietic cell population and
erythrocytes or debris. After Ficoll-Hypaque density centrifugation,
the fraction of CD45+ cells in peripheral blood and bone
marrow was greater than 95%. The CD34+ cells were analyzed
in a fluorescence-versus-SSC plot. Only cells with a
lymphoid or lymphomonocytoid appearance were counted as CD34+ cells, and their proportion was calculated in
relation to that of CD45+ cells. The percentage of
false-positive events determined by isotype-specific control was less
than 0.5% and was subtracted from the percentage of CD34+
cells.13
The CD34+ cell fraction from bone marrow samples was
enriched with the use of a 2-step method as previously
described.14 The first enrichment step consisted of an
immunomagnetic separation (Minimacs, Miltenyi-Biotec GmbH, Bergisch
Gladbach, Germany), according to the manufacturer's instructions. The
majority of the enriched CD34+ cells were incubated for 30 minutes at 4°C in the presence of an anti-CD34 (HPCA-2)
PE-conjugated monoclonal antibody (Becton Dickinson);
1 aliquot was incubated with an isotype-specific control. After the
CD34+ cells were washed with PBS, their purity was analyzed
in a fluorescence-versus-SSC plot (Figure
1). The percentage of false-positive events
determined by isotype-specific control was below 0.5% and was
subtracted from the percentage of CD34+ cells. The cell
fraction was further highly enriched by FACS with a FACSVantage (Becton
Dickinson). Only cells with a lymphoblastoid appearance were sorted
according to side and forward scatter characteristics. Final purity was
analyzed by flow cytometry (Figure 1). The high purity of this
procedure was demonstrated for patients with follicular lymphoma
showing no detection of contaminating cells positive for the
translocation t(14;18).14
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| Fig 1.
Bone marrow-derived CD34+ cells were
enriched with the use of a 2-step method.
Purity was analyzed after Minimacs (A, FSC/SSC; C,
CD34/SSC; E, isotype control IgG/SSC), obtaining a mean purity of
greater than 95%. Only cells with lymphoblastoid characteristics were
sorted. Final purity after FACS with a FACSVantage was greater than
99.5% (B, FSC/SSC; D, CD34/SSC).
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DNA preparation and PCR amplification
Total DNA was extracted from enriched CD34+
cells and from peripheral blood MNCs according to a standard protocol
based on cell lysis, proteinase K digestion, and chloroform
purification. The amount of DNA was measured by UV spectrophotometry.
The entire second exon of the HLA-DRB1 gene was amplified with the use
of commercially available primers to consensus regions (INNO-LiPA DRB
key, Innogenetics, Zwijndrecht, Belgium).15 The primers were added to a PCR mix containing KCl, Tris HCl, MgCl2,
gelatin, and deoxyribonucleoside trisphosphates
(dNTPs). PCR was carried out in a final volume of 50 µL, with 0.3 units recombinant Taq polymerase (Perkin
Elmer, Branchberg, NJ) and 0.1 µg template DNA.
The reactions were performed in a Perkin-Elmer 9600 thermocycler. The
amplification profile was 5 minutes' denaturation at 95°C followed
by 35 cycles of denaturation at 95°C for 30 seconds, primer
annealing at 58°C for 20 seconds, and primer extension at 72°C
for 30 seconds. At the end, an extension step of 10 minutes at 72°C
was added.15 PCR products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. After
successful amplification, the PCR product was diluted 1:10 in
H2O, and 1µL of the diluted PCR product was subjected to
a second round of amplification with sequence specific primers (SSPs) from the Collaborative Transplant Study.16 The cycling
conditions for this nested PCR assay were 30 cycles of denaturation at
94°C (20 seconds) and primer annealing/elongation at 69°C (50 seconds). Here also, 0.3 µL Taq polymerase was used per reaction.
Samples were electrophoresed on an ethidium bromide-stained 2%
agarose gel.
This procedure allowed the assignment of all broad HLA-DRB1
specificities as well as their serologic splits (HLA-DR 1-18). DNA
samples from healthy blood donors with DRB1 specificities identical to
those of the allograft donor and recipient were mixed and analyzed in
model reactions in order to evaluate allele combinations that could
result in either false-positive reactions or reduced amplification
efficiency. In 4 patients, samples of the donor and recipient before
transplantation were available to serve as rigorous baseline control.
Each patient's posttransplantation DNA was submitted to the complete
analysis repeatedly, and a chimeric state was defined if at least 2 out
of 3 assays showed donor-specific positive amplification signals.
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Results |
Microchimerism in peripheral blood
We used a nested PCR with SSPs (SSP-PCR) for exon 2 of the
HLA-DRB1 gene to detect donor cells in the allograft recipient (Figures
2 and 3). We
determined the sensitivity of this approach by spiking experiments to
be capable of detecting 1 donor cell among 1000 to 10 000 recipient
cells with high specificity (unpublished data, C. Horvath et al, August
1998). A chimeric status was defined when the results
of at least 2 out of 3 PCR reactions were positive. In the peripheral
blood, 5 out of 8 patients (63%) who had been followed for longer than
2 years after orthotopic liver transplantation were chimeras according
to PCR. We prospectively studied 4 patients and obtained DNA samples
before liver transplantation and 2 to 4 months following
transplantation. We could not assess 1 allograft recipient because of cross-reactivities of his HLA-type with the donor's type. Of the other 3 patients, 1 patient
clearly demonstrated donor-type HLA-DRB specificities in the PCR.
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| Fig 2.
Microchimerism in peripheral blood and
CD34+ cells in a patient (patient 1) 5.1 years after orthotopic liver transplantation as detected by nested
SSP-PCR for the HLA-DRB gene.
Donor-specific bands are indicated by an arrow. 14.* and 13.* indicate
different DRB subtypes. (A) Control DNA sample of a blood donor with
the same HLA-DR type (DR1 and DR2(15) as patient 1 before orthotopic
liver transplantation (patient 1's original DNA was no longer
available). DR15 and DR16 are both serological splits of DR2, and they
have a strong sequence homology. This causes a cross-reactivity in the
nested PCR-SSP assay. DR51 is the product of the DRB5 gene, which is in
linkage with DR15. (B) DNA mix containing the same sample as in panel A
in 1:1 dilution with a DNA sample of a control, who had the same
HLA type as patient 1's donor [(DR3(17)and DR6(13)]. The bands
with primers 14* and 18 are donor-specific bands
derived from sequence homology with DR13 and DR17,
respectively. The band at DR52 is the PCR product of the DRB3 gene,
which is in linkage with the donor's DR13. (C) DNA from peripheral
blood from patient 1 obtained 5.1 years after orthotopic liver
transplantation. Recipient-specific bands are 1.1, 15, (16), and 51, whereas donor-specific bands are 17, 13.*, and 52. No other bands are observed. (D) DNA from bone
marrow-derived CD34+ cells from patient 1. Bands specific
for the recipient are 1.1, 15, (16), and 51, whereas
donor-specific bands are 17, 13*, and 52. Further bands (7, 8, 12, and
53) may be associated with previous blood
transfusions.20
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| Fig 3.
Microchimerism in peripheral blood and
CD34+ cells in a patient (patient 9) surveyed before and
3 months after orthotopic liver transplantation as detected by nested
SSP-PCR for the HLA-DRB gene.
Donor-specific bands are indicated by an arrow. 14.* and 13.* indicate
different DRB subtypes. (A) DNA sample of patient 9 (DR215 and DR3) before transplantation,
used to determine the patient's chimeric state before orthotopic liver
transplantation. DR17 and DR18 are both serologic splits of DR3 and
they have a strong sequence homology. This causes a cross-reactivity in
the nested SSP-PCR assay. DR51 is the product of the DRB5 gene; DR52 is
the product of the DRB3 gene. These genes are in linkage with DR2 and
DR3, respectively. Unspecific additional bands are DR13.*. (B) DNA mix
containing the same sample as in panel A in 1:1 dilution with an
original DNA of patient 9's liver donor
(DR511). DR11 and DR12 are both serologic
splits of DR5, and they have a strong sequence homology. This may cause
a cross-reactivity in the nested PCR-SSP assay. (C) DNA from peripheral
blood from patient 9 at 3 months after orthotopic liver
transplantation. The recipient-specific band is 11, whereas the band in
103 is not specific for this donor/recipient combination. (D) DNA from
bone marrow-derived CD34+ cells. Bands specific for the
recipient are 11 and 12. No further bands can be detected.
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Microchimerism in bone marrow-derived CD34+ cells
CD34+ cells from bone marrow were enriched in a
2-step method consisting of an immunomagnetic procedure and FACS. In
the 8 long-term allograft survivors, the median proportion of bone
marrow-derived CD34+ cells before enrichment was
1.50% (range, 1.09% to 3.29%), whereas the median proportion of
CD34+ cells in the bone marrow of 4 patients studied at 2 to 4 months after transplantation ranged between 0.57% and 1.19%.
(median, 0.74%). After the first enrichment procedure, the use of the
Minimacs, the mean purity of the CD34+ cells obtained was
95.06% ± 2.20% (Figure 1). The purity of the CD34+
cells obtained after FACS was greater than 99.5% (median, 99.8%; range, 99.62% to 99.98%). In 5 of 8 (63%) long-term allograft recipients, donor-specific CD34+ cells could be detected in
the bone marrow (Figure 2, Table 2). No correlation was found between
chimerism in CD34+ cells and peripheral blood MNCs; 3 patients were positive in peripheral blood and HSCs, while 4 patients
were positive in either HSCs or peripheral blood. Only 1 patient was
negative in peripheral blood as well as in HSCs. In the prospectively
studied patients, CD34+ cells were chimeric in 1 patient,
who also demonstrated microchimerism in peripheral blood, whereas in 2 patients no cells of donor origin could be detected in blood or bone
marrow-derived CD34+ cells.
Clinical course
We included 8 long-term survivors after liver
transplantation in the study on the basis of a good graft function.
Nevertheless, most patients had experienced 2 or more rejection
episodes and required immunosuppressive therapy at the time of the
study. Only 1 patient with microchimerism in peripheral blood and in
the CD34+ cell fraction had not experienced any rejection
episode. In the long-term allograft recipient without microchimerism in
peripheral blood and CD34+ cells, cyclosporine had
previously been reduced to half of the dose as part of an earlier
study, but the patient developed a severe rejection episode and was
reset to full therapeutic dose.17 The cohort of patients
was too small to make any firm conclusions about microchimerism and
clinical outcome.
Patients studied before and at short term after liver transplantation
might be less biased for better graft function, and only 1 patient
demonstrated microchimerism in peripheral blood and
CD34+ cells.
| |
Discussion |
Cells of donor origin can be found in the recipient's peripheral
blood, skin, lymph nodes, and bone marrow even years after liver
transplantation. We could demonstrate microchimerism in the
CD34+ cell population of the recipient's bone marrow and
therefore conclude that hematopoietic stem and progenitor cells are
transferred with the liver transplant. Vascular endothelial cells also
express the CD34 antigen.18 These larger cells should have
been eliminated from the analysis by the process of isolation and
gating during FACS according to physical parameters. Nevertheless, we
cannot exclude that donor cells of endothelial origin could contribute to microchimerism in the CD34+ cell fraction.
We applied a nested PCR for the polymorphic allele of HLA-DRB1 to
detect cells of donor origin. Sensitivity and specificity depend on the
donor/recipient HLA-DRB1 type combinations and the degree of mismatch
between donor and recipient.19 We detected allogeneic
chimerism in the CD34+ cells in 4 of 5 patients with 2 mismatches and only in 1 of 3 patients with 1 HLA-DRB mismatch. Using a
similar nested PCR method, Spriewald et al reported
microchimerism in 87.5% of patients with 2 HLA-DRB mismatches, in
contrast to 50% in patients with 1 mismatch.19 Spiking
experiments with third-party DNA with the same HLA-DRB1 specificities
appear to be crucial to control for nonspecific amplifications. The use
of pretransplant DNA as control may further reduce the risk of misinterpretation.
In peripheral blood, we found microchimerism in 5 of 8 patients (63%),
which is in line with reports from other groups on the frequency of
long-term microchimerism after solid organ transplantation. Similarly,
we found cells of donor origin among the bone marrow-derived CD34+ cell fraction in 5 of 8 long-term liver recipients.
One can speculate that CD34+ cells egress from the liver
and engraft in the bone marrow during the early phase after liver transplantation. The permanent homing implies that the HSCs have escaped from the host-versus-graft reaction. On the other hand, the
presence of CD34+ cells in the recipient's bone marrow
could be due to a continuous production of HSCs in the liver. The
continuous production could sustain the permanent presence of
donor-derived HSCs in the recipient's bone marrow, although these
donor cells are still targets of the allograft reaction and are
perpetually being eliminated.
Our results give additional evidence for the presence of HSCs in the
adult human liver. Recently, Bodó et al described the development
of donor-derived acute promyelocytic leukemia in the recipient of a
liver transplant, indicating that donor myeloid cells had been
transferred with the graft 2 years earlier.20 A recent
study by Crosbie et al gave further evidence for the presence of HSCs
in the adult human liver.3 This group isolated CD34+ hepatic MNCs, which expressed CD45, CD38, and HLA-DR
and formed multilineage hematopoietic colonies after tissue culture.
The localization of HSCs in the liver is not clear. Endothelial or parenchymal cells might deliver the microenvironmental support, and an
association with endothelial cells might not be restricted to liver,
but also occur in other parenchymal organs. It will be interesting to
study microchimerism in HSCs after transplantation of solid organs
other than liver.
Microchimerism in peripheral blood might be the consequence of
proliferation and differentiation of HSCs engrafted in the recipient's
bone marrow. Other long-lived donor cells as memory T cells might
contribute to sustain microchimerism. This could explain our results in
2 patients in whom we could find microchimerism only in the peripheral
blood, not in the CD34+ cell fraction. On the other hand,
these results might reflect the limitations of the sensitivity of our method.
Apart from donor-specific bands, we could identify additional HLA-DRB1
specificities in 3 patients. Other mechanisms that lead to a state of
microchimerism have been described. For this reason, it appears
advantageous to use the polymorphic HLA-DRB region to discriminate
between donor and recipient cells rather than simply the Y chromosome,
which limits the analysis to the male-to-female transplant
situation.4 Additional chimeric bands could be due to blood
transfusions in the past.21 Bianchi et al showed that male
CD34+ cells could be detected in women up to 27 years after
delivery of a male child.22 Fetal lymphohematopoietic cells
that cross the placenta and enter the maternal circulation could play a
role in the induction of autoimmune diseases such as
scleroderma.23,24
The clinical and immunologic relevance of microchimerism and in
particular of HSCs in microchimerism is not defined. Starzl et al
developed a model in which tolerance is the consequence of a balance
between 2 immune systems.11 The presence of donor cells
indicates tolerance, but it could be the effect and not the cause of
tolerance. CD34+ cells themselves might function as veto
cells and suppress alloreactive T cells.25 Khan et al
demonstrated that allogeneic HSCs could differentiate into thymic
dendritic cells, which might induce central tolerance to an organ
transplant by negative selection.26 Our cohort of patients
was too small for a correlation between the presence of microchimerism
and clinical outcome. Nevertheless, most patients with or without
microchimerism had experienced 2 or more rejection episodes and
required immunosuppressive therapy at the time of the study.
The induction of tolerance may also depend on the quantity of chimeric
cells. Minute quantities of donor cells as in microchimerism are not
sufficient to induce a balance between 2 immune systems. Clinical
trials are underway in several institutions to determine whether donor
bone marrow cells can increase the proportion of chimeric cells and
enhance organ allograft survival.27,28
| |
Acknowledgments |
M. Weis, M. Pförsich, and E. Sollich for excellent technical
assistance and K. Hexel (Department of Tumor Immunology, German Cancer
Research Center, Heidelberg) and L. Volk for excellent technical
support with FACS-analysis and FACS-sorting.
| |
Footnotes |
Submitted October 22, 1999; accepted March 7, 2000.
Reprints: Stefan Hohaus, Istituto di Semeiotica Medica,
Divisione di Ematologia, Università Cattolica S. Cuore, Largo A. Gemelli, 1 00168 Rome, Italy.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction