Transplantation tolerance induced in humans at the fetal or the neonatal stage.

Abstract

Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens. Engraftment needs no conditioning regimen prior to transplantation in neonates with severe combined immunodeficiency disease or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.

Full text

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Hindawi Publishing Corporation
Journal of Transplantation
Volume 2011, Article ID 760319, 4 pages
doi:10.1155/2011/760319
Research Article
Transplantation ToleranceInducedinHumans at
theFetal orthe NeonatalStage
Jean-LouisTouraine1,2andKamel Sanhadji1,2
1Claude Bernard University and Hospices Civils de Lyon, France
2Transplant Unit, Pav. P., Hˆ opital Ed. Herriot, 69437 Lyon Cedex 3, France
Correspondence should be addressed to Jean-Louis Touraine, jean-louis.touraine@chu-lyon.fr
Received 2 March 2011; Accepted 6 May 2011
Academic Editor: Hargovind L. Trivedi
Copyright © 2011 J.-L. Touraine and K. Sanhadji. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Patients transplanted with HLA-mismatched stem cells from fetal livers develop transplantation tolerance to donor antigens.
Engraftmentneeds noconditioningregimenpriortotransplantationinneonates withseverecombined immunodeficiency disease
or in human fetal patients having not yet developed any immune maturity, especially T-cell differentiation. The chimeric patients
have donor-derived T lymphocytes which progressively demonstrate positive interactions with other host cells. They also can
be shown to be tolerant toward both host and donor antigens. The latter tolerance relies upon clonal deletion from the T-cell
repertoire, and it results from the contact between thymocytes of donor origin and dendritic cells or macrophages also deriving
from donor stem cells. The former tolerance does not imply clonal deletion of T-cells with host reactivity. Numerous T-cells
recognizing the allogeneic, host-type antigens are identified in these patients, but these cells are anergized, following interaction
with epithelial cells of the host thymus. Induction of transplantation tolerance at the fetal stage requires minimal engraftment
only; in the future it will be possible to further amplify the clinical benefit, using additional cell transplants after birth.
1.Introduction
Following the pioneering and most promising work of
Billingham, Brent, and Medawar in newborn mice [1],
many experimental and some clinical studies have focused
on means to induce transplantation tolerance. In human
patients, full tolerance has yet been developed in two
circumstances only: (a) when the transplant recipient is not
immunologically competent or mature and (b) when the
transplant involves full replacement of host lymphocytes
by donor lymphocytes. The latter condition is obtained
by myeloablation and immunosuppression, followed by
stem cell transplantation (SCT) [2]. The former mode of
tolerance induction is seen in patients with severe combined
immunodeficiency disease (SCID) treated with SCT [3] or in
fetal patients subjected to SCT prior to their immunological
maturation [4].
We review herein our results in infants and human
fetuses who were treated by fetal liver SCT and who devel-
oped full tolerance to both donor and host antigens [5–13].
2.Patients,Materials,and Methods
Nineteen patients with SCID, including 17 infants and 2
fetuses, received fetal liver SCT [4, 7]. Fetal livers were
obtained from dead human fetuses under conditions
approved by the French National Committee for Bioethics.
Donors were aged less than 14 weeks after fertilization. A
cell suspension was prepared, cell viability was checked, and
cells were administered by intravenous or intraperitoneal
injection. In the two fetal recipients with SCID, the cells were
injected into the umbilical vein in utero [4]. Fourteen of
the 19 patients had evidence of donor cell engraftment and
developed immunological reconstitution. All were subjected
to immunological investigations, especially on peripheral
blood lymphocytes (PBLs). The studies reported herein
mainly concern three children who were analyzed more
extensively over a long period of time (21–34 years).
Three nonimmunodeficient fetuses with various diseases
(thalassemia major, Niemann-Pick type A disease and
hemophilia A) were also analyzed for at least 2 years after the

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2 Journal of Transplantation
in utero SCT (which was performed by the intraperitoneal
route).
HLA typing was initially carried out on PBL, T-cell
clones, and EBV-transformed B-cell lines using a previously
described cytotoxicity assay [14] and was confirmed more
recently using molecular biological methods.
To analyze responses in mixed leukocyte cultures
(MLCs), PBL from the patients were cultured together with
a variety of irradiated stimulator cells. The proliferative
responseofTlymphocytestothesestimulatorcellswasdeter-
mined by the degree of tritiated thymidine incorporation
[12]. The experiments were performed in triplicate, and the
results are expressed as the mean ± standard deviation (SD).
3.Results
3.1. HLA Typing. Table 1 summarizes the HLA phenotypes
(classIandclassII)ofcellsofdonorandhostoriginthatwere
prepared from the three most extensively studied patients,
all of whom have had a stable chimerism for many years.
They had received fetal liver SCTs from several donors but
only the HLA phenotype of the permanently engrafted cells
is reported here.
Over the past 34 years, many investigations have been
carried out in these three patients. On all occasions, most B-
cells and antigen-presenting cells were found to be of host
origin while all T-cells were of donor origin. Accordingly,
T-cell clones prepared from the PBL of these patients only
exhibited the HLA determinants of the donor.
With the exception of patient 2, for whom the HLA-A2
antigen was shared by the donor and the host, all patients
had a complete mismatch between host-derived and donor-
derived cells. T lymphocytes and B lymphocytes or antigen-
presenting cells share no class II determinant and almost no
class I HLA antigen, since the former are derived from donor
stem cells and the latter from host cells.
3.2. Mixed Leukocyte Cultures. After immunological recon-
stitution had developed in the SCID patients treated post-
natally, PBLs were prepared and used as responders while
irradiated cells of various origins were employed as stimula-
tors [12]. The 2 patients reported in Table 2 demonstrate an
inability to mount a proliferative response to host stimulator
cells. In contrast, the T lymphocytes from these patients
proliferated readily when stimulated by allogeneic cells or by
cells from their parents (Table 2).
Among T-cell clones (e.g., tetanus-toxoid-specific T-cell
clones) prepared from patients’ PBL, virtually none recog-
nized donor HLA antigens, whereas 15 of 50 clones directly
recognizedHLAantigensofthehost[9]andmountedstrong
proliferative and cytotoxic responses to host-derived cells
[9,13].ThehighfrequencyofCD8+host-reactivecellsinthe
chimeric patients was comparable with that of alloreactive
cells, in contrast with the lack of cells that reacted with the
donors [12].
MLCs were performed in 6 additional patients, including
the 2 SCID patients treated at the fetal stage, in utero. The
results were comparable with those described above, with
a specific lack of proliferative response to host stimulator
Table 1: HLA phenotypes of host cells and of cells of donor origin
found in three chimeric patients.
PatientHost/donor cells
HLA
B
14–47
8–18
37–62
8–35
27–44
35–60
ADR
4–11
3–9
4-5
6-7
4–14
1–13
1
Host
Donor
Host
Donor
Host
Donor
3–33
1-2
2–31
2–30
2-3
26–29
2
3
Reproduced from [7].
cells. Again, such an absence of proliferation in MLC did not
indicate lack of recognition of host antigens, since some T-
cell clones could be shown to be host reactive.
3.3. Lymphokine Synthesis and Secretion. The production of
lymphokines by host-reactive T-cells from these patients
was characterized by high levels of gamma-interferon
and, following activation, granulocytic-monocyte-colony-
stimulating factor, interleukin-5, and interleukin-2 [12].
Interestingly, no interleukin-4 was produced, irrespective of
the mode of activation [12, 13]. Spontaneous secretion of
interleukin-10 by the patients’ cells was regularly found to
be increased [5, 6].
3.4. Results in Nonimmunodeficient Patients. We report here
3humanfetalpatientswhohavebeentreatedbyinuteroSCT
at the age of 12–14 weeks postfertilization. After birth, the
children were repeatedly studied.
In the thalassemic girl, engraftment was ascertained in
blood and bone marrow by the presence of (a) hemoglobin
A, (b) cells with the donor Y chromosome, and (c) cells
with the donor HLA phenotype. However, the number of
donor cells remained limited and tended to decrease with
age. At 4 years of age, 0.5–1% of bone marrow cells only
expressed simultaneously the CD34 marker and the HLA-
A32 phenotype of donor origin. This low chimerism on the
long-term was not sufficient to ensure significant clinical
benefit, but it suggested maintenance of tolerance to donor
antigens.
The two other patients had evidence of donor cell
survival, in the absence of immunosuppression at any time,
with the prolonged presence of cells with HLA markers of
the donor [4]. This engraftment was made possible by the
immune immaturity of recipients at 12–14 weeks of fetal
age. However the number of donor cells did not increase
with time. Actually, it became lower after the first 1 or 2
years. The hemophiliac did not generate any antifactor VIII
antibody, suggesting tolerance to this factor, possibly as the
resultoffactorVIIIproductionbydonor-derivedcellsandits
presentation to the immune system of the developing fetus.
4.Discussion
Because of immune incompetence, SCID patients on the one
hand and humans in the early stage of fetal development on

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Journal of Transplantation3
Table 2: Proliferative response of PBL from 2 patients and a normal donor to host, parental, and allogeneic cells.
Patient
Stimulator cells from
Mother
64.7 ± 3.5
12.1 ± 0.4
22.7 ± 0.6
Medium
0.6 ± 0.0
1.7 ± 0.3
2.1 ± 0.5
Host
1.4 ± 0.3
4.1 ± 0.1
38.6 ± 1.1
Father
40.0 ± 5.8
ND
Allogeneic
54.3 ± 1.2
24.6 ± 2.0
47.2 ± 3.7
Allogeneic
75.6 ± 5.6
22.4 ± 2.2
36.0 ± 2.9
1
2
Normal donor
(control)
The indicated data are cpm × 10−3[3H] TdR incorporation expressed as the mean ± SD.
Reproduced from [12].
ND
the other hand can benefit from engraftment of mismatched
stem cells. As a source of stem cells to treat our patients,
we have used fetal livers, taking advantage of the relative
competitive engraftment superiority of fetal liver cells over
adult bone marrow cells, especially in fetal recipients [15].
Despite the lack of HLA antigens shared by donor-
derived T lymphocytes and the other cells of the body,
efficient immune interactions develop in SCID patients
treatedpre-orpostnatally[3,4,7,16,17].Inparticular,there
appearstobenorestrictionoffunctionofhelperorcytotoxic
T-cells [3, 5, 7, 8, 11], and immune reconstitution of the host
progresses up to a full degree [7, 18].
Tolerance toward both host and donor is achieved in
these chimeric patients. The immune immaturity of the
host explains the lack of donor cell rejection that of the
donor explains the lack of graft-versus-host disease (GvHD)
induced by transplanted cells.
Following SCT in our SCID patients, donor-reactive (but
not host-reactive) cells have been shown to be deleted from
the T-cell repertoire. Clonal deletion is therefore responsible
for immunological tolerance to antigens of the donor and
this process of negative selection is likely to occur in the
host thymus, as a result of contact between thymocytes and
dendritic cells or macrophages of donor origin (Figure 1).
In contrast, host-reactive T-cells (also designed as allore-
active T-cells since the stem cells that generated T-cells are
allogeneic to other cells of the host body) remained present
and relatively numerous in these chimeric patients. That
no detrimental effect (GvHD or autoimmunity) occurs at
any stage of T-cell development suggests that donor-derived
T lymphocytes have been suppressed or anergized in the
host. This hypothesis is supported by the specific absence
of proliferative response to host stimulator cells in MLC.
Further evidence of clonal anergy or suppression has been
obtained in experiments involving transplantation of human
fetal liver and thymus of similar or different origins in SCID
mice [19]. Tolerance to host antigens appears to develop
in human T-cells present in these experimental animals as
the result of a clonal anergy that follows contact of human
thymocytes with human epithelial cells of the host thymus
fragment.
Transplantation tolerance is therefore induced by two
different mechanisms: tolerance to donor by clonal deletion
and tolerance to host by clonal anergy (Figure 1).
In nonimmunodeficient fetuses, tolerance was also
apparently induced but, in contrast with SCID patients,
the number of donor cells did not expand significantly
acquisition of immunological tolerance to
donor and to host
Thymus
(recipient)
Stem cell (donor)
T cell
-
Clonal deletion
(due to donor derived macrophages
and dendritic cells)
to donor
Tolerance
Clonal anergy
(due to host thymic epithelial cells)
to host
T-cellfrom donor stem cells:
differentiation
-
Figure 1: Differentiation of donor stem cells into mature T lym-
phocytes within the host thymus: acquisition of tolerance by thy-
mocytes in contact with other donor cells and with host thymic
epithelial cells.
over the years. Various hypotheses may account for this
limited development of donor cells: selective advantage of
host stem cells over donor stem cells, lack of “space” in
the hematopoietic niches, allogeneic reactions from the
progressively immunomature lymphocytes of the host, and
allogeneic reactions from the maternal T-cells that have been
shown to reduce engraftment after in utero SCT [20]. When
allogeneicandautologousSCTinfetalsheepwerecompared,
however,nosignificantdifferencewasfoundbetweenthetwo
groups[21].Lowengraftmentdidnotappeartoresultmostly
from major histocompatibility complex-driven allogeneic
reactionsbutratherfromdonor/hostcompetitionofanother
kind. Nevertheless, since donor-specific tolerance induction
requires relatively minimal engraftment [18], clinical appli-
cation may take advantage of such a tolerance produced at
thefetalagetoprocurefurthertreatmentwithallogeneiccells
of similar origin, in larger numbers and with appropriate
adjunctive treatment, later on in life.
Acknowledgments
The authors are grateful to Drs. M. G. Roncarolo, R.
Bacchetta, H. Plotnicky, H. Spits, H. Yssel, H. B´ etuel, J. E. de
Vries, and J. Banchereau for their contributions and advice
in these studies.

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4Journal of Transplantation
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