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IMMUNOBIOLOGY
Allogeneic T regulatory cell–mediated transplantation tolerance in adoptive
therapy depends on dominant peripheral suppression and central tolerance
Dennis Adeegbe,1 Robert B. Levy,1 and Thomas R. Malek1,2
1Department
of Microbiology and Immunology and 2Diabetes Research Institute, Miller School of Medicine, University of Miami, FL
T regulatory cells (Tregs) represent agents
to mediate tolerance to allografts so that
the use of immunosuppressive drugs is
avoided. In this regard, we previously
demonstrated that the adoptive transfer
of allogeneic Tregs into IL-2R␤ⴚ/ⴚ mice
prevented autoimmunity and led to allograft tolerance. Here, we investigated the
requirements and mechanisms that favor
this long-lasting tolerance. The most potent tolerance required exact matching of
all alloantigens between the adoptively
transferred allogeneic Tregs and allo-
geneic skin grafts, but tolerance to such
allografts that lacked expression of major
histocompatibility complex class I or II
molecules also occurred. Thus, Tregs are
not required to directly recognize major
histocompatibility complex class II alloantigens to suppress skin transplant rejection. Depletion of allogeneic Tregs substantially, but not completely, abrogated
this form of tolerance. However, thymocytes from allogeneic Treg adoptively
transferred IL-2R␤ⴚ/ⴚ mice did not reject
the corresponding allogeneic skin graft in
secondary Scid recipients. Consistent
with a requirement for a deletional mechanism in this IL-2R␤ⴚ/ⴚ model, a small
number of wild-type T cells readily abrogated the immune tolerant state. Collectively, these findings indicate that full
tolerance induction is largely dependent
on substantial Treg-mediated suppression and thymic deletion of alloreactive
T cells and may represent general conditions for Treg-mediated transplantation
tolerance. (Blood. 2010;115:1932-1940)
Introduction
CD4⫹CD25⫹Foxp3⫹ T regulatory (Treg) cells inhibit a wide array
of immune responses1-3 and effectively prevent the destruction of
host tissue in graft-versus-host disease (GVHD).4-8 Treg cells also
inhibit autoreactive and alloreactive T-cell responses in various
autoimmune and transplantation studies, respectively, thus promoting tolerance.9-15 The potent suppressive activity of Treg cells has
led to substantial interest in investigating their therapeutic use to
facilitate tissue/organ transplantation.16-18 In most of these studies,
the Treg cells infused to promote tolerance are syngeneic to the
recipient. With respect to transplantation tolerance, current data
support the notion that Treg cells use mainly the indirect antigen
recognition pathway in the control of alloreactive immune
responses.19-21
One key issue is whether adoptively transferred histoincompatible Treg cells induce transplantation tolerance without a requirement for additional immunosuppression. The use of major histocompatibility complex (MHC)–mismatched Treg cells may be required
in settings in which endogenous Treg cells exhibit intrinsic defects
that limit their efficacy to suppress auto- and/or alloimmunity and
increase the potential donor pool for adoptive Treg cell immunotherapy. In experimental allogeneic bone marrow transplantation of
mice, infusion of donor-derived allogeneic Treg cells at the time of
the bone marrow transplant effectively suppresses acute
GVHD.4,22,23 These preclinical findings provide the rationale for
the initial Treg cell trials, which administer donor-type allogeneic
Treg cells in patients undergoing human leukocyte antigen–
matched allogeneic bone marrow transplantation.24 Thus, there is
heightened interest in the basis by which allogeneic Treg cells
mediate transplantation tolerance.
In this regard, we previously reported that adoptive therapy that
uses allogeneic donor Treg cells in IL-2R␤–deficient mice not only
fully prevented their lethal systemic autoimmune disease but also
resulted in long-lasting transplantation tolerance when such mice
received allogeneic skin grafts that expressed the MHC type of the
donor Treg cells.25 However, little is known about the T-cell
receptor (TCR):MHC requirements governing Treg-cell recognition and suppression of graft rejection across MHC barriers in
adoptive transfer settings in which donor Treg cells are MHC
disparate from the recipient. Here we address that issue as well as
more broadly investigate that mechanisms by which allo-Treg cells
support skin graft tolerance by using Treg-cell transfers into
IL-2R␤–deficient mice as a model.
Methods
Mice
C57BL/6 (B6), BALB/c, C3H/HeJ, B6 ␤2microglobulin-deficient (B6
␤2m⫺/⫺; B6.129p2-B2mtm1Unc), BALB.B (C.B10-H2b/LiMcdj), B6bm3
(B6J-H2bm3/Egj), B6bm12 (B6(C)-H-2-AB1bm12/KhEgj), and B6 MHC
class II–deficient (B6 II⫺/⫺; B6.129S-H2dlAb1⫺Ea/J) mice were obtained
from The Jackson Laboratory. B6 mice congenic for CD45 and expressing
the CD45.1 allele (B6.SJL-Ptprc/BoAiTac), B6 Rag2/␥c double-deficient
mice (B6 ⫻ C57BL/10SgSnAi-[KO]␥c-[KO]Rag2), and BALB/cscid (C.B-17
scid/scid) mice were obtained from Taconic Farms. B6 and BALB/c
IL-2R␤⫺/⫺ mice have been previously described.12 These mice were
maintained in our animal colony by breeding homozygous IL-2R␤⫺/⫺ pairs
that were rendered autoimmune-free by neonatal adoptive transfer of
purified wild-type (WT) syngeneic CD4⫹ or CD4⫹CD25⫹ T cells. All
Submitted August 13, 2009; accepted December 8, 2009. Prepublished online
as Blood First Edition paper, December 29, 2009; DOI 10.1182/blood-2009-08238584.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2010 by The American Society of Hematology
1932
BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
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BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
LONG-TERM ALLOTOLERANCE AND DONOR Treg CELLS
1933
Table 1. Summary of autoimmune status, donor engraftment, and skin graft tolerance for IL-2R␤ⴚ/ⴚ mice adoptively transferred with Treg
cells
Donor Treg
strain
Skin graft (MST)
IL-2R␤ⴚ/ⴚ
recipient strain
Percent donor cell engraftment (ⴞ SEM)
Percent
CD4ⴙCD69ⴙ
(ⴞ SEM)
BALB/c
B6
C3H
n
B6
B6
9.1 ⫾ 0.7
10.1 ⫾ 4.5
31
⬎ 90
33
8
BALB/c
B6
8.2 ⫾ 1.9
14.2 ⫾ 4.7
⬎ 90
⬎ 90
29
8
BALB/c
BALB/c
8.5 ⫾ 0.9
12.5 ⫾ 3.8
⬎ 90
29
31
8
B6
BALB/c
7.6 ⫾ 1.4
13.6 ⫾ 3.4
⬎ 90
⬎ 90
30
9
BALB/c
8.0 ⫾ 1.6
15.1 ⫾ 4.0
⬎ 90
32
⬎ 90
4
C3H
B6 or BALB/c IL-2R␤⫺/⫺ mice were adoptively transferred at birth with Treg cells from the indicated strains of mice. The recipients were judged to be autoimmune-free. Host
CD4⫹ T cells that express ⬍ 20% CD69⫹ cells represent one indicator of lack of autoimmunity. These mice received BALB/c, B6, and C3H skin grafts 8 weeks after the Treg cell
transfers. Mice were monitored up to at least 90 days after skin transplantation. The percentage of donor cell engraftment was then determined as the percentage of CD4⫹
splenic T cells that were donor Treg cells, defined by expression of CD25 and/or Foxp3. The mean survival time (MST) of the skin grafts and number of recipient mice (n) are
indicated. Data are a composite of previously published data19 and additional skin graft recipients.
experiments were approved by the Institutional Animal Use Committee at
the University of Miami.
Antibodies and FACS analysis
Biotin-conjugated monoclonal antibodies (mAbs) to H-2Kb (AF6-88.5),
Cy-Chrome–conjugated mAbs to CD4 (H129.19), and Cy-Chrome–
streptavidin, phycoerythrin (PE)–conjugated mAbs to CD25 (PC61), CD4
(GK1.5), H-2Kb (AF6-88.5), H-2Kd (SF1-1.1), PerCP-conjugated mAbs to
CD4 (RM4-5), and allophycocyanin-streptavidin were all purchased from
BD Biosciences. Fluorescein isothiocyanate–anti-CD4 (GK1.5), fluorescein isothiocyanate–anti-CD45.1, biotin-conjugated mAbs to CD45.1
(A20.1), and CD25 (7D4) were prepared in our laboratory. PE–anti-Foxp3
(FJK16s) was obtained from eBiosciences and was used in intracellular
fluorescence-activated cell sorting (FACS) analysis by the use of a kit
according to the manufacturer’s instructions. FACS analysis was performed
as previously described26 with a Becton Dickinson LSR1 cytometer and
CellQuest software or a BD Biosciences LSRII cytometer and Diva
software. Typically 30 000 to 50 000 events were collected per sample.
Purification of T-cell subsets
Unfractionated or CD4⫹ T cells were isolated by the use of Thy 1.2 MACS
or anti-CD4 MicroBeads, respectively, by positive selection on MS MACS
separation columns (Miltenyi Biotec). CD4⫹CD25⫹ T cells were purified
from the spleen as previously described12 by initial depletion of CD8⫹
T cells and B cells, followed by positive selection of Treg cells targeting
CD25 with magnetic micro beads. Cell purity typically ranged from 90% to
95% as analyzed by FACS. Donor B6 (H-2b) cells were depleted by
negative selection after incubating spleen cell suspensions with biotinconjugated anti-H-2kb (AF6-88.5) and streptavidin MACS MicroBeads.
Donor BALB/c (H-2d) cells were depleted either by negative selection after
spleen cell suspensions were incubated with PE-conjugated H-2kd (SF11.1) mAb and anti-PE MACS MicroBeads or by cell sorting by the use of a
Becton Dickinson Vantage SE cell sorter. CD25⫹ cell depletion was
performed by incubating spleen cells with anti-CD25 (7D4) and complement.12 The source of antigen-presenting cells (APCs) was T cell–depleted
mitomycin C–treated spleen cells as previously described.12
In vivo mouse studies
For experimental studies, purified CD4⫹CD25⫹ Treg cells (2 ⫻ 105) were
adoptively transferred by intravenous injection into the superficial facial
vein of 1- to 2-day-old neonatal IL-2R␤⫺/⫺ mice. At 8 to 12 weeks later, the
adoptively treated mice were used for skin graft experiments and other in
vitro assays. In transplantation studies that used Rag2⫺/⫺/␥c⫺/⫺ or BALB/
cscid mice, cell inoculums were injected in the tail vein of recipient mice.
Skin grafting was performed as previously described.25 In brief, syngeneic,
test tolerogenic allogeneic, and third-party allogeneic full-thickness tail
skin from WT female donors were serially placed in tandem on separate
graft beds on the tail of individual anesthetized recipients. Protective glass
tubing was placed over the length of the tail bearing the grafts for 7 days.
Grafts were monitored every other day and scored as rejected when greater
than 75% or more of the original graft tissue had become necrotic as
assessed by visual examination. Grafts were scored from 0 to 4 as follows:
0, graft is healthy and is intact; 1, graft is inflamed, but no signs of necrosis;
2, graft is inflamed, necrosis of less than 50% of graft area; 3, necrosis of up
to 75% of graft area; and 4, complete loss of graft.
Statistical analysis
A paired 1-tailed Student t test was used to compare the mean survival time
(MST) between allogeneic third-party and test tolerogenic allogeneic skin
grafts and an unpaired 2-tailed Student t test was used to compare the mixed
lymphocyte response (MLR) responses.
Results
Allogeneic donor Treg cells mediate donor-specific skin graft
tolerance
IL-2R␤⫺/⫺ mice lack mature and functional CD4⫹CD25⫹Foxp3⫹
Treg cells and consequently develop severe lethal systemic autoimmune disease that in many ways resembles GVHD.12,27 This
absence of mature endogenous Treg cells provides a model to
follow a defined population of transferred donor Treg cells and
investigate their suppressive function and mechanisms of action in
vivo. We previously demonstrated that the adoptive transfer of
either syngeneic or allogeneic Treg cells into neonatal IL-2R␤⫺/⫺
mice fully prevented their lethal autoimmunity such that they now
lived a normal life span with a normal fraction of donor Treg
cells.12,25 Thus, the allo-Treg cells were not rejected by the
IL-2R␤⫺/⫺ recipients. Remarkably, autoimmune-free IL-2R␤⫺/⫺
mice that received allogeneic Treg cells consistently exhibited
long-term (defined typically as ⬎ 90 days) tolerance to allogeneic
skin grafts that expressed the same genetic background as the donor
Treg cells.25 The effectiveness of allogeneic Treg cells in this
setting has been found for all 3 strain combinations that have been
tested so far, that is, BALB/c (H2d) Treg cells into B6 (H2b)
IL-2R␤⫺/⫺ recipients, B6 Treg cells into BALB/c IL-2R␤⫺/⫺
recipients, and C3H (H2k) Treg cells into BALB/c IL-2R␤⫺/⫺
recipients. A summary of all recipients to date for these strain
combinations with respect to skin graft tolerance and donor Treg
cell persistence is shown in Table 1. For simplicity in this report,
the donor Treg cells strain into IL-2R␤⫺/⫺ recipient strain will be
designated, for example, as B6 Treg3BALB/c⫺/⫺.
Transplantation tolerance with alloantigen disparities between
donor Treg cells and skin graft
Past work25 also showed delayed (MST ⫽ 48 days) rejection when
grafting allogeneic skin from F1 mice as long as 1 haplotype was
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ADEEGBE et al
BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
Figure 1. Skin graft survival when the donor Treg cells and
allograft differ in only a single MHC class I or II alloantigen.
Eight- to 12-week-old BALB/c IL-2R␤⫺/⫺ mice that were adoptively treated with the indicated population of Treg cells at birth
(denoted as the donor Treg strain3IL-2R␤⫺/⫺ recipient strain)
received BALB/c, C3H, and B6bm3 (B6bm3) or B6bm12 (B6bm12) skin
grafts. (A) Tolerance to B6bm3 skin. (B) Tolerance to B6bm12 skin.
The number of mice/group, combined from 2 independent experiments, and the MST of the allogeneic skin grafts are listed within
each panel of the figure.
matched with the donor allogeneic Treg cells. This finding suggested that a dominant tolerance mechanism may be operative in
our system. Therefore, we more fully investigated the requirements
for alloantigen similarity between the donor Treg cells and the skin
graft. Accordingly, skin transplantation experiments were performed by the use of grafts that differed in only a single MHC
molecule or expressed only minor alloantigenic disparities relative
to the donor allogeneic Treg cells. Initially, the suppression of graft
rejection to B6bm3 or B6bm12 skin grafts was examined in
B6 Treg3BALB/c⫺/⫺ recipients. These skin grafts express MHC
class I or class II molecules, respectively, that differ by 3 amino
acids from conventional B6 MHC class I (H-2b) or class II
(I-Ab).28,29 For B6 Treg3BALB/c⫺/⫺ recipients, substantial but not
long-lasting tolerance was noted for B6bm3 (Figure 1A, right;
P ⬍ .001) or B6bm12 (Figure 1B right; P ⬍ .001) skin grafts with
MST of 64 to 69 days, whereas fully allogeneic C3H skin grafts
were rejected rapidly with MST of 28 to 29 days.
For B6bm3 Treg3B6⫺/⫺ (Figure 2A right) or B6 Treg3B6⫺/⫺
(Figure 2B center) recipients, B6bm12 (P ⬍ .001) or BALB.B
(P ⬍ .001) skin grafts, respectively, exhibited delayed rejection.
The eventual rejection of BALB.B skin grafts by B6 Treg3B6⫺/⫺
Figure 2. Skin graft survival when the donor
Treg cells and allograft differ in major or
minor alloantigens. Eight- to 12-week-old
B6 IL-2R␤⫺/⫺ mice that were adoptively treated
with the indicated population of Treg cells at
birth (denoted as the donor Treg strain3
IL-2R␤⫺/⫺ recipient strain) received BALB/c,
C3H, B6bm3, and B6bm12 (A) or BALB/c, BALB.B,
B6, and C3H (B) skin grafts. (A) Tolerance to
B6bm3 skin. (B) Tolerance to BALB.B skin. The
number of mice/group from a single experiment
and the MST of allogeneic skin grafts are listed
within each panel of the figure.
mice indicates that Treg selection and recognition of self-peptide/
MHC class II is not sufficient to mediate long-lasting tolerance.
However, long-lasting (MST ⫽ ⬎90 days) tolerance for B6bm3 and
BALB.B skin grafts was only observed for B6bm3 Treg3B6⫺/⫺
(Figure 2A right) or BALB.B Treg3B6⫺/⫺ (Figure 2B right)
recipients, respectively. As additional controls for these experiments, when the donor Treg cells were syngeneic to the recipients,
as in WT BALB/c mice (Figure 1A-B left), BALB/c Treg3
BALB/c⫺/⫺ recipients (Figure 1A-B center), WT B6 mice (Figure
2A-B left), and B6 Treg3B6⫺/⫺ recipients (Figure 2A-B center),
mismatched C3H, B6bm3, B6bm12, and BALB.B skin grafts were
rapidly rejected (MST ⫽ 17-30 days). We have always noted
that WT mice rejected allogeneic skin grafts with more rapid
kinetics than syngeneic Treg cells3IL-2R␤⫺/⫺ mice, and this
difference is most likely accounted for by the lack of IL-2
responsiveness by the rejecting effector T cells in the latter
mice. Collectively, these data indicate that tolerance is not
sustained and is eventually abrogated when there is any
disparity in major or minor alloantigens between the skin grafts
and the donor allogeneic Treg cells.
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BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
LONG-TERM ALLOTOLERANCE AND DONOR Treg CELLS
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Figure 3. Skin graft survival when the
allograft lacks surface expression of MHC
class I or II molecules. Eight- to 12-weekold BALB/c IL-2R␤⫺/⫺ mice that were adoptively treated with the indicated population of
Treg cells at birth (denoted as the donor
Treg strain3IL-2R␤⫺/⫺ recipient strain) received skin grafts from BALB/c, C3H, and
B6 MHC-deficient mice. (A) Tolerance to
B6 MHC class II–deficient (B6 II⫺/⫺) skin.
(B) Tolerance to MHC class I–deficient
(B6 ␤2m⫺/⫺) skin. The number of mice/group
from a single experiment and the MST of
allogeneic skin grafts are listed within each
panel of the figure.
Transplantation tolerance toward allografts that lack
expression of MHC class I or II molecules
The preceding data demonstrate that the most effective tolerance
occurs when the donor allogeneic Treg cells are identical with the
allo-skin grafts. Because Treg cells express MHC class II–
restricted TCRs, it was of interest to determine whether the skin
graft must express MHC class II molecules because this may
indicate that suppression is the result of a mechanism that depends
on direct recognition of MHC class II molecules associated with
the graft. To test this hypothesis, B6 Treg3BALB/c⫺/⫺ recipients
received skin grafts from BALB/c, C3H, and MHC class II–
deficient B6 II⫺/⫺ mice (Figure 3A right). These recipients retained
BALB/c and B6 II⫺/⫺ donor skin for greater than 90 days whereas they
readily rejected C3H skin (MST ⫽ 28 days). Control experiments
revealed that WT BALB/c mice (Figure 3A left) and BALB/c
Treg3BALB/c⫺/⫺ recipients (Figure 3A center) readily rejected C3H
and B6 II⫺/⫺ skin grafts. These data indicate that tolerance is independent of class II expression by the donor skin transplants.
Given the ubiquitous expression of MHC class I antigens on
cells of the transplanted tissue, we also tested the requirement for
graft-derived MHC class I antigens for allo-Treg cell–mediated
tolerance. Similar skin graft experiments were performed with the
use of B6 ␤2m⫺/⫺ donor skin, which lacks surface expression of
MHC class I molecules.30 This skin graft also was retained
long-term by B6 Treg3BALB/c⫺/⫺ recipient mice (Figure 3B
right), whereas it was readily rejected by WT BALB/c mice (Figure
3B left) and BALB/c Treg3BALB/c⫺/⫺ (Figure 3B center)
recipients. Thus, allogeneic Treg cell–mediated tolerance also
readily occurs in the absence of graft-associated MHC class I.
Taken together, these data suggest that allogeneic donor Treg cells
likely suppress rejection that is not critically dependent on direct
recognition of graft-derived MHC molecules.
The dependency of tolerance on donor allogeneic Treg cells
One key feature of peripheral tolerance by Treg cells is active
suppression of antigen-responsive cells.31-33 The delayed rejection
of the partially matched skin grafts with the donor Treg cells is
consistent with that notion. However, the more long-lasting tolerance in settings where skin grafts were completely matched to the
donor allogeneic Treg cells in this and past work25 raise the
possibility that additional mechanisms may also be in play. To
evaluate the role of donor Treg cells for skin graft tolerance when
the allogeneic Treg cells and the skin graft are fully matched, we
used a secondary transfer system where spleen cells from BALB/c
Treg3B6⫺/⫺ were transferred into B6 Rag2⫺/⫺/␥c⫺/⫺ mice and
2 days later these secondary recipients were grafted with skin from
BALB/c, B6, and C3H mice.
When unfractionated BALB/c Treg3B6⫺/⫺ spleen cells were
transferred into B6 ␥c⫺/⫺Rag⫺/⫺ mice, all of these secondary
recipients readily accepted syngeneic B6 and Treg cell-matched
BALB/c skin grafts for greater than 90 days, whereas they readily
rejected third-party C3H skin grafts (Figure 4A). The accepted
BALB/c donor skin showed no visual signs of inflammation. Donor
BALB/c Treg cells (3.1% ⫾ 0.4%) from the BALB/c Treg3B6⫺/⫺
spleen inoculum were readily detected 90 days later in the spleen of
the B6 Rag2⫺/⫺/␥c⫺/⫺ secondary recipients. In contrast, secondary
B6 Rag2⫺/⫺/␥c⫺/⫺ mice that received Treg cell–depleted BALB/c
Figure 4. Skin graft survival in the absence of donor Treg cells. Eight-week-old
B6 Rag2⫺/⫺/␥c⫺/⫺ mice (H-2b) received 7 ⫻ 106 unfractionated (A) or H-2d donor
cell–depleted (B) spleen cells from BALB/c3B6⫺/⫺ mice. Rag2⫺/⫺/␥c⫺/⫺ recipients
were then transplanted with BALB/c, C3H, and B6 (B6) skin 2 days after the cell
transfer. Mice were monitored up to 90 days after which they were sacrificed to
assess the presence of the donor Treg cells. MST of allogeneic skin grafts are listed
within each panel of the figure. Data represent 9 mice/group, combined from 2
independent experiments.
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ADEEGBE et al
BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
Figure 5. MLR by T cells or APCs from IL-2R␤ⴚ/ⴚ mice that were adoptively transferred with allogeneic Treg cells. (A) MLR responses by WT and recipient-derived
T cells to allogeneic APCs. The source of the responding T cells is listed below the x-axis. Unfractionated spleen cells (2 ⫻ 105) isolated from WT BALB/c and
BALB/c3BALB/c⫺/⫺ mice or spleen cells depleted of H-2b⫹ donor cells (2 ⫻ 105) isolated from B63BALB/c⫺/⫺ mice were cultured with BALB/c (left), B6 (center), or C3H (right)
APCs (2 ⫻ 105) for 96 hours. (B) MLR response of WT B6 T cells to APC derived from the spleen of indicated mice as listed below the x-axis. 3H-thymidine was added in the last
6 hours of culture. Data represent the mean ⫾ SEM of 2 to 3 independent experiments with the use of mice that were either tolerant skin graft recipients (B63BALB/c⫺/⫺) or
mice that rejected both donor skin (BALB/c⫹/⫹ and BALB/c3BALB/c⫺/⫺).
Treg3B6⫺/⫺ spleen cells rejected BALB/c skin grafts
(MST ⫽ 52 days), although this tempo of rejection was significantly slower (P ⫽ .002) than found for third-party C3H skin grafts
(MST ⫽ 34 days; Figure 4B). In this case very few donor Treg
cells (0.45% ⫾ 0.13%) were found in the spleens of the B6
Rag2⫺/⫺/␥c⫺/⫺ secondary recipients. A delay of 7 days in rejection
of BALB/c skin grafts was still noted for the 3 recipients within this
group with the lowest level (⬍0.2%) of residual Treg cells,
suggesting that this delay in rejection was Treg cell independent.
Nevertheless, the rejection of allogeneic BALB/c skin grafts after
Treg depletion establishes an important role for Treg cells for
tolerance to allogeneic skin graft.
T-cell anergy and defective APC function do not obviously
contribute to transplantation tolerance
Effective Treg cell–mediated dominant tolerance might be favorable to elicit other peripheral regulatory mechanisms, such as
anergy, ignorance, or induction of tolerogenic APCs to the alloantigens of the skin graft that together with Treg cells result in
long-lasting skin graft retention. To test this notion, MLRs were
performed with T cells and APCs from B6 Treg cell3BALB/c⫺/⫺
mice. When spleen cells from B6 Treg3BALB/c⫺/⫺ mice were
cultured with WT B6 or C3H APCs (Figure 5A), proliferative
responses were detected that were comparable with that when the
responding cells were obtained from BALB/c Treg3BALB/c⫺/⫺
mice. As expected, the MLRs from WT BALB/c mice to B6 or
C3H APC (Figure 5A) were 2- to 3-fold greater, and this difference
is attributable to the unresponsiveness of IL-2R␤⫺/⫺ T cells to IL-2
in vitro by T cells from the former groups of mice.34-36 These
findings indicate that recipient IL-2R␤⫺/⫺ T cells are present that
are capable of responding to the allo-skin grafts to which these
mice are tolerant. These data are consistent with the idea that
substantial T-cell alloantigen-specific anergy or ignorance cannot
account for the skin graft tolerance.
In an analogous manner, APCs from B6 Treg3BALB/c⫺/⫺
mice readily induced MLRs by WT B6 T cells that were very
similar to the responses induced by APCs from WT BALB/c or
BALB/c Treg3BALB/c⫺/⫺ mice (Figure 5B). In all 3 groups of
mice, the frequency of CD11cdimB220⫹Ly6C⫹ plasmacytoid dendritic cells, which have been implicated in some settings to
contribute to transplant tolerance,17 was similar (data not shown).
Taken together, these data suggest that a prominent tolerogenic
APC pool does not contribute to induction or maintenance of
donor-specific transplantation tolerance in B6 Treg3BALB/c
IL-2R␤⫺/⫺ mice.
Central tolerance mechanisms are induced in allogeneic
Treg-cell adoptively transferred IL-2R␤ⴚ/ⴚ mice
When IL-2R␤⫺/⫺ mice are adoptively transferred with Treg cells,
these cells undergo rapid homeostatic expansion to normal numbers and then are maintained at normal levels through extensive
proliferation and corresponding cell death.26 In the case of allogeneic Treg cells, this process might provide a substantial and
constant source of alloantigen for central tolerance. To test this
notion, we evaluated the capacity of a mixture CD4⫹ and CD8⫹
single-positive (SP) thymocytes derived from B6 Treg3
BALB/c⫺/⫺ mice to reject skin grafts upon transfer into secondary
lymphopenic BALB/cscid mice. Such secondary recipients containing thymocytes derived from B6 Treg3BALB/c⫺/⫺ mice readily
rejected C3H but not B6 skin grafts (Figure 6A right). These B6
grafts exhibited some signs of immune attack, as evident by the
somewhat-elevated rejection scores (Figure 6B), but this response
did not progress to complete loss of the graft 90 days after
thymocyte transfer. Spleen cells from these recipient BALB/cscid
mice essentially lacked Foxp3⫹ Treg cells (Figure 6C-D), indicating that this tolerance is not the result of residual donor B6 Treg
cells. As controls and in marked contrast, the transfer of SP
thymocytes from WT BALB/c (Figure 6A left) or BALB/c
Treg3BALB/c⫺/⫺ (Figure 6A center) mice into BALB/cscid secondary recipients resulted in complete rejection of B6 and C3H skin
grafts with high rejection scores (Figure 6B). The spleens of these
latter 2 groups of BALB/cscid mice contained Treg cells from the
thymocyte inoculums (Figure 6C-D), but these cells did not affect
rejection of the allogeneic skin grafts. The presence of Treg cells in
these 2 latter groups was expected because it was not possible to
excluded the syngeneic WT Treg cells during the sorting of the SP
thymocytes. Treg cells represent approximately 4% of CD4 SP
thymocytes in WT mice. WT donor syngeneic or allogeneic Treg
cells are found in the thymus of adoptively transferred IL-2R␤⫺/⫺
mice at a low frequency (0.1%-0.2%). These cells are available to
engraft and persist after thymocytes were transferred into BALB/cscid
recipients. This issue was avoided in the B6 Treg3BALB/c⫺/⫺
thymocyte group because H-2b⫹ cells were specifically excluded
during the cell sorting. Thus, the tolerance in secondary recipients
of thymocytes from B6 Treg3BALB/c⫺/⫺ mice is consistent with
the notion that the allogeneic Treg cells that are present primarily in
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BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
LONG-TERM ALLOTOLERANCE AND DONOR Treg CELLS
1937
Figure 6. Central tolerance in IL-2R␤ⴚ/ⴚ mice that
were adoptively transferred with allogeneic Treg
cells. Eight-week-old BALB/cscid mice (H-2d) received a
4:1 mixture of FACS-sorted SP CD4⫹ and CD8⫹ cells
(0.5 ⫻ 106 total) isolated from the thymus of WT BALB/c,
BALB/c3BALB/c⫺/⫺, or B63BALB/c⫺/⫺ mice, the latter
of which were further sorted to exclude any donor H-2b⫹
cells. BALB/cscid recipients were then transplanted with
BALB/c, C3H, and B6 donor skin 2 days later. Mice were
monitored up to 90 days, after which they were sacrificed
to assess the presence of Treg cells. (A). Skin graft
survival in BALB/cscid recipients that received the indicated donor thymocytes. The number of mice in each
group of recipients and the MST of allogeneic skin grafts
are listed within each the panel. (B) Rejection score of B6
skin grafts. (C) Percentage of CD4⫹Foxp3⫹ Treg cells in
the whole spleen of individual recipient mice. (D) Percentage of Treg cells within the splenic CD4⫹ T-cell subset.
Staining for MHC class Id and Ib confirmed that all CD4⫹
Foxp3⫹ T cells were of H-2d origin. Data are 4 to
5 mice/group from a single experiment.
the periphery of IL-2R␤–deficient mice led to substantial central
tolerance to the alloantigens expressed by the Treg cells.
T cells from naive mice readily abrogate transplantation
tolerance
The preceding experiments demonstrate that dominant Treg cellmediated suppressive and central thymic tolerance mechanisms
contribute to long-lasting skin graft acceptance when there is
complete genetic matching between the allogeneic Treg cells and
the allografts. To further investigate the resiliency of these mechanisms, we assessed whether such tolerance is maintained in the
presence of an exogenous source of T cells from naive WT or
autoimmune-free IL-2R␤⫺/⫺ mice. For these experiments, we
performed another set of secondary adoptive transfers where spleen
cells from BALB/c Treg3B6⫺/⫺ mice were transferred into B6
Rag2⫺/⫺/␥c⫺/⫺ recipients, which exhibit potent tolerance to BALB/c
skin graft (see Figure 4), but now defined numbers of T cells were
added to these spleen cell inoculums. In these transfers, all cells are
syngeneic to the recipient with the exception of the BALB/c
Treg cells present in the spleen cells from BALB/c Treg3B6⫺/⫺
mice. When the cotransferred T cells were derived from autoimmune-free B6 IL-2R␤⫺/⫺ mice (Figure 7A), tolerance to BALB/c
skin was maintained in the presence of 2 ⫻ 105 of cotransferred
IL-2R␤⫺/⫺ T cells, whereas delayed rejection occurred when
2 ⫻ 106 cells were cotransferred. In contrast, cotransferred WT
T cells resulted in efficient rejection of BALB/c skin grafts at
both doses of WT T cells (Figure 7B) that was largely comparable with the rejection of third-party C3H skin grafts (Figure 7B). We also noted (data not shown) rapid rejection
(MST ⫽ 24 days) of long-term tolerant C57BL/6 skin grafts
after the tolerant B6 Treg3BALB/c⫺/⫺ mice (n ⫽ 4) received
BALB/c T cells (2 ⫻ 106).
Figure 7. Ability of naive T cells to
break skin graft tolerance of IL-2R␤ⴚ/ⴚ
mice that were adoptively transferred
with allogeneic Treg cells. Eight-weekold Rag2⫺/⫺/␥c⫺/⫺ mice (H-2b) received
7 ⫻ 106 unfractionated spleen (SP) cells
isolated from BALB/c3B6⫺/⫺ mice along
with the indicated numbers of unfractionated (A-B) or CD25-depleted (C) naive
T cells isolated from IL-2R␤⫺/⫺ (A) or WT
CD45.1 B6 congenic (B-C) mice. The
naive T cells were from autoimmune-free
IL-2R␤⫺/⫺ mice due to the transfer of
syngeneic Treg cells at birth. Rag2⫺/⫺/
␥c⫺/⫺ recipients were then transplanted
with BALB/c, C3H, and B6 skin 2 days
later. The number of mice in each group
of recipients, combined from 2 independent experiments, and the MST of skin
grafts are listed within each the panel.
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1938
ADEEGBE et al
Upon further analysis, BALB/c Treg cells from the BALB/c
Treg3 B6⫺/⫺ spleen cells were only detected in the secondary
recipients that received 2 ⫻ 105 IL-2R␤⫺/⫺ T cells, which retained
the BALB/c skin grafts (Figure 7A top), whereas such Treg cells
were absent in all other B6 Rag2⫺/⫺/␥c⫺/⫺ secondary recipients
(data not shown). Reducing Treg cells in the WT T cells before
cotransfer by depletion of CD25⫹ T cells did not alter the time
course of rejection of the BALB/c skin grafts (Figure 7C) nor
promoted the persistence of the BALB/c Treg cells (data not
shown). These data suggest that T cells from naive mice eliminate
the allograft suppressive BALB/c Treg cells. Taken together, these
data provide direct evidence that the Treg cells from the tolerogenic
BALB/c Treg3B6⫺/⫺ spleen cells do not suppress WT T cells,
although the alloreactive response by new input IL-2R␤⫺/⫺ T cells
is more readily susceptible to suppression.
Discussion
The induction of effective tolerance to allogeneic skin in
IL-2R␤⫺/⫺ mice is relatively straightforward, that is, resulting from
singular transfer of small numbers of allogeneic Treg cells into
neonatal IL-2R␤⫺/⫺ mice. This finding and the application of donor
allogeneic Treg cells in clinical trials to suppress GVHD has led us
to further investigate the basis by which allogeneic Treg cells
mediate transplantation tolerance.24 Although tolerance induction
is efficient and highly reproducible, complex events ensue for
allograft acceptance. This study clarifies key requirements with
respect to alloantigen recognition and suppressive mechanisms by
which adoptively transferred allogeneic Treg cells mediated skin
graft tolerance in IL-2R␤–deficient mice.
We provide direct data for 2 mechanisms for this skin graft
tolerance. First, much of the tolerance between allogeneic Treg
cells and alloantigen-matched skin grafts is accounted for by donor
Treg cells. When secondary adoptive transfer experiments were
used, tolerance was largely abrogated upon depletion of the donor
Treg cells. Thus, there is a requirement for continued presence of
the donor Treg cells to mediate tolerance, consistent with active
suppression by these cells as shown by others.13,37,38 Nevertheless,
Treg-mediated suppression did not fully account for tolerance
because some delay in graft rejection remained upon depletion of
the donor Treg cells. Second, we found that central tolerance was
also induced to alloantigens associated with the donor Treg cells
because thymocytes from such mice did not efficiently reject skin
grafts that expressed these alloantigens when transferred into
secondary immunodeficient recipients. Such central tolerance
provides a mechanism to further reduce the number of potential
alloreactive T cells that require active suppression by Treg cells.
These 2 mechanisms work in tandem to establish a finely balanced
setting in which tolerance is maintained to the skin graft and the
allogeneic donor Treg cells. Consistent with this notion, the
addition of a few WT T cells to tolerogenic BALB/c Treg3B6⫺/⫺
spleen cells that were transferred to secondary Rag2⫺/⫺/␥c⫺/⫺
recipients readily broke transplantation tolerance to BALB/c skin
grafts and the donor Treg cells.
Our findings from in vitro MLR experiments suggest that
peripheral T-cell anergy or the development of tolerogenic APC
does not substantially account for allograft tolerance in IL-2R␤⫺/⫺
mice that are engrafted with allogeneic Treg cells, although some
subtle contribution by these inhibitory mechanisms cannot be
excluded. We believe it is noteworthy that MLR responses were
readily evident by recipient T cells toward B6 APCs when the
BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
responding cells were derived from B6 Treg3BALB/c⫺/⫺ mice.
This finding indicates that there is a substantial population of
T cells that directly recognize B6 or BALB/c MHC alloantigens by
these tolerant recipients and are suppressed by Treg cells. This
result strongly suggests that central tolerance is geared to primarily
eliminate alloreactive CD4⫹ and CD8⫹ T cells that recognize
indirectly presented donor-related antigens on host APCs. This
MLR experiment also rules out the remote possibility that our
highly purified donor Treg cells contained a rare progenitor cell that
support donor APC that directly mediated thymic-negative selection.
The Treg cells themselves provide a substantial source of
alloantigens that is available for indirect antigen-presentation/
donor antigen uptake by host APC. Adoptively transferred syngeneic or allogeneic Treg cells are maintained at normal numbers
life-long for autoimmune-free IL-2R␤⫺/⫺ recipients, whereas these
Treg cells undergo substantial homeostatic growth and death.39
Thus, the efficient long-term engraftment of Treg cells not only
results in a population of suppressive Treg cells but yield substantial chimerism to provide alloantigens to support long-term central
tolerance mechanisms. Such indirectly presented alloantigens may
mediate central tolerance though trafficking of host APC from the
periphery into the thymus. Support for this idea comes from recent
work that showed that approximately 50% of dendritic cells in the
adult thymus are derived from a circulating pool of dendritic cells
of peripheral origin.40,41 Furthermore, a few donor Treg cells are
found in the thymus of adoptively transferred IL-2R␤⫺/⫺ mice
(D.A. and T.R.M., unpublished data, August 2006). These cells
provide another source of donor alloantigens for transfer to resident
thymic APCs to mediate negative selection of T cells.
With respect to alloantigen recognition, our results demonstrated that there must be an exact genetic match between the
transferred donor Treg cells and the skin graft for the most
long-lasting allograft retention. As illustrated by the use of B6bm3,
B6bm12, and BALB.B skin grafts or donor Treg cells, any mismatch
in a single class I or class II molecule or in minor non-MHC
alloantigens between the donor Treg cells and skin graft ultimately
resulted in graft rejection. Nevertheless, in all these aforementioned settings, where there was substantial but not complete
matching between the donor Treg cells and the skin allografts, there
was a significant delay in rejection of these grafts in comparison
with third-party C3H allografts. This finding is consistent with an
important dominant suppressive mechanism by Treg cells in skin
graft tolerance. This “partial” tolerance may reflect suppressive
mechanisms by donor Treg cells that recognize the shared alloantigens of the allograft or represent a cross-reaction to the mismatched
alloantigens of the graft.
Treg cells need to be activated via the TCR to exert suppression.42 Thus, the observation that long-lasting allogeneic Treg cells
mediated skin graft tolerance by the use of skin from MHC class
II–deficient mice indicates that there is not an absolute requirement
for the Treg cells to become activated within the graft by
skin-derived APCs for the immune tolerance observed. Furthermore, tolerance to MHC class II–deficient skin cannot be the result
of failed direct class II recognition by alloreactive effector cells
because B6 class II⫺/⫺ skin was readily rejected by controls,
particularly BALB/c Treg3BALB/c⫺/⫺. Efficient tolerance to
allogeneic class II⫺/⫺ skin grafts further points to the importance of
indirect antigen presentation not only for central deletion of
potential graft rejecting cells but for activation of the suppressive
Treg cells. This finding, however, does not rule out that direct
recognition of donor class II by Treg cells may be operative in class
II–sufficient skin grafts, which may complement the activity of
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BLOOD, 11 MARCH 2010 䡠 VOLUME 115, NUMBER 10
LONG-TERM ALLOTOLERANCE AND DONOR Treg CELLS
those Treg cells that indirectly recognized donor antigens. Furthermore, tolerance to B6 class II⫺/⫺ allografts indicates that the
requirement for exact matching between the allogeneic Treg cells
and allo-skin grafts cannot be attributable to the Treg cells simply
“remembering” the MHC class II molecules on which they were
initially selected during their thymic development before adoptive
transfer. Thus, it is likely that indirect presentation of Treg
cell-derived allopeptides from donor Treg cells not only function
for inducing negative selection in the thymus but also to select/
activate at least a subset of the adoptively transferred engrafting
donor Treg cells.
In summary, our findings to date indicate that long-lasting
allograft tolerance by allogeneic Treg cells in IL-2R␤⫺/⫺ mice
depends on the following factors: (1) a substantial empty niche to
promote engraftment of the transferred Treg cells; (2) indirect
presentation of donor Treg cell–derived alloantigens that promote
some thymic negative selection of developing alloreactive T cells
and peripheral selection of suppressive Treg cells with TCRs
directed to such alloantigens; and (3) genetic identity between the
donor allogeneic Treg cells and the skin allografts. Besides these
points, there are several additional issues to consider. First, the
impaired T effector and perhaps memory responses associated with
the IL-2R␤–deficient genetic background34-36 dampen the allograft
rejection response, which may facilitate Treg cell-mediated tolerance. Second, all Treg cell-adoptive transfers, by necessity to
prevent autoimmunity, were performed by the use of neonatal
IL-2R␤⫺/⫺ recipients. The neonatal environment is particular
permissive to induce immune tolerance43-45 and represents a setting
related to early post–hematopoietic stem cell (HSC) transfers.
Finally, IL-2R␤–deficient mice lack natural killer (NK) and NK
T cells.34 The lack of these cells, especially NK cells, diminishes
the potential to eliminate transplanted allogeneic donor Treg cells
and rejection of MHC class-I negative allografts.46,47
These findings raise the intriguing possibility that clinical
scenarios that emulate the IL-2R␤–deficient model may enable
engraftment of allogeneic Treg cells and tolerance toward solid
tissues or HSC allografts. Identical genetic matching between
donor Treg cells and the transplanted tissues is readily accomplished simply by isolating Treg cells from the peripheral blood of
the same donor of the transplanted organ or tissue, such as
progenitor cell allografts. Meeting the other key factors may
require conditioning of the recipient to facilitate Treg cell engraftment. For example, reduction of recipient Treg cells after conditioning/chemotherapy before organ or HSC transfer, together with
intentional deletion of residual Treg cells, may provide a niche for
1939
the allogeneic Treg cell infusion. Application of less-vigorous
generalized immunosuppression, perhaps analogous to IL-2R␤
deficiency, to dampen rejection responses to allogeneic-Treg cells
and the allografts also may be beneficial. Inhibiting the IL-2R in
effector cells should not prevent immune responses against infectious agents because effective immunity occurs in the absence of
IL-2/IL-2R.35,48 Thus, immunosuppression in conjunction with
Treg cells may not need to be as robust as currently practiced. On
the basis of our data, once substantial stable allogeneic Treg cell
chimerism is achieved, graft-specific peripheral and central tolerance is vigorously promoted, which may permit suspension of
drug-mediated immunosuppression.
Several of the favorable conditions facilitating tolerance in the
IL-2R␤–deficient model also appear to be operative in suppression
of GVHD by allogeneic Treg cells. Not only does exact matching
of the allo-Treg cells to the bone marrow transplant occur, but the
lymphopenic setting during marrow reconstitution leads to an
environment that favors donor Treg engraftment and persistence to
readily suppress autoaggressive donor-derived T cells. Furthermore, the immunosuppressive drug rapamycin synergizes with
allo-Treg cells to enhance inhibition of alloresponses after HSC
transplantation.49,50 These similarities emphasize the value of
elaborating the mechanisms for the robust immune tolerance in
Treg cell transferred IL-2R␤⫺/⫺ mice as such understanding should
help better define the conditions required for successful adoptive
immunotherapy.
Acknowledgments
This work was supported by National Institutes of Health grant
R01AI055815.
Authorship
Contribution: D.A. designed and performed experiments, analyzed
data, and wrote the paper; R.B.L. analyzed the data and edited the
paper; and T.R.M. designed experiments, analyzed data, and wrote
the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Thomas Malek, Department of Microbiology and Immunology (R138), Miller School of Medicine, University of Miami, 1600 NW 10th Ave, Miami, FL 33136; e-mail:
[email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2010 115: 1932-1940
doi:10.1182/blood-2009-08-238584 originally published
online December 29, 2009
Allogeneic T regulatory cell−mediated transplantation tolerance in
adoptive therapy depends on dominant peripheral suppression and
central tolerance
Dennis Adeegbe, Robert B. Levy and Thomas R. Malek
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