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Developmental Exposure to Noninherited
Maternal Antigens Induces CD4 + T
Regulatory Cells: Relevance to Mechanism of
Heart Allograft Tolerance
Melanie L. Molitor-Dart, Joachim Andrassy, Jean Kwun, H.
Ayhan Kayaoglu, Drew A. Roenneburg, Lynn D. Haynes,
Jose R. Torrealba, Joseph L. Bobadilla, Hans W. Sollinger,
Stuart J. Knechtle and William J. Burlingham
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:6749-6761; ;
doi: 10.4049/jimmunol.179.10.6749
http://www.jimmunol.org/content/179/10/6749
The Journal of Immunology
Developmental Exposure to Noninherited Maternal Antigens
Induces CD4ⴙ T Regulatory Cells: Relevance to Mechanism
of Heart Allograft Tolerance1
Melanie L. Molitor-Dart,* Joachim Andrassy,† Jean Kwun,‡ H. Ayhan Kayaoglu,‡
Drew A. Roenneburg,‡ Lynn D. Haynes,‡ Jose R. Torrealba,*‡ Joseph L. Bobadilla,‡
Hans W. Sollinger,‡ Stuart J. Knechtle,‡ and William J. Burlingham2‡
S
olid organ and hemopoietic stem cell (HSC)3 transplantation have become common procedures for organ failure
and other end-stage diseases. The major limitation, rejection of the allograft by the host’s immune system, can be reduced
but not eliminated by various immunosuppressive drugs. Unfortunately, chronic rejection still occurs and maintenance immunosuppression leaves the patient susceptible to infectious complications
and a significantly increased risk of cancer development (1–3).
Therefore, it is important to search for alternative strategies such as
tolerance-based reduction or elimination of immunosuppressive
drugs in transplant recipients. One approach would be to take ad-
*Department of Pathology and Laboratory Medicine, Cellular and Molecular Pathology, University of Wisconsin, Madison, WI 53706; †Surgery Department Klinikum
Grosshadern, Ludwig-Maximilians University, Munich, Germany; and ‡Department
of Surgery, University of Wisconsin, Madison, WI 53792
Received for publication May 3, 2007. Accepted for publication September 4, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant RO1 AI066219 and
a grant from the Division of Transplantation, Department of Surgery, UW-Madison.
2
Address correspondence and reprint requests to Dr. William J. Burlingham, University of Wisconsin, G4/702 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792. E-mail address: [email protected]
3
Abbreviations used in this paper: HSC, hemopoietic stem cell; NIMA, noninherited
maternal Ag; NIPA, noninherited paternal Ag; GVHD, graft-vs-host disease; TR, T
regulatory; BFA, brefeldin A; LN, lymph node; DTH, delayed-type hypersensitivity;
lf, limits of flocculation; TT/DT, tetanus and diphtheria toxoid; TE, T effector; DST,
donor-specific transfusion; ILN, inguinal LN; GIC, graft-infiltrating cell; LAP, latency associated peptide.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
vantage of the patient’s history of exposure to heterozygous maternal cells and Ags during ontogeny. In the search for natural
mechanisms of allotolerance, Owen et al. (4) discovered more than
50 years ago that Rh-negative mothers of Rh⫹ babies had a significantly reduced likelihood of forming anti-Rh Abs if their own
mothers had been Rh⫹. Claas et al. (5) repeated this observation in
the HLA system while analyzing anti-HLA Abs in multiply transfused, highly sensitized patients awaiting renal transplantation,
when they discovered that such individuals frequently failed to
make Abs against the noninherited HLA of their mother. No such
immune privilege was afforded to HLA Ags not inherited from the
father. A possible explanation for this B cell hyporesponsiveness
to noninherited maternal Ags (NIMA) is that during early fetal
and neonatal life, maternal lymphocytes and/or soluble Ags enter the offspring’s circulation, which could induce immune tolerance in the baby. Several studies have demonstrated the passage of cells and Ags across the fetomaternal interface in both
directions, along with transfer of maternal cells and Ags orally
through nursing (6 – 8).
Compelling evidence of a lifelong influence of exposure to
NIMA on the immune system has been found in studies of living
related transplant recipients. A collaborative retrospective study
found superior long-term allograft survival in recipients of HLA
one haplotype-mismatched renal allografts donated by a sibling
that shared the same inherited paternal haplotype but was mismatched for the NIMA HLA haplotype. These grafts experienced
more early acute rejection episodes, but fared significantly better at
10 years compared with recipients of grafts that shared the maternal haplotype and were mismatched for noninherited paternal Ags
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We hypothesize that developmental exposure to noninherited maternal Ags (NIMA) results in alloantigen-specific natural and
adaptive T regulatory (TR) cells. We compared offspring exposed to maternal H-2d (NIMAd) with nonexposed controls. In vitro
assays did not reveal any differences in T cell responses pretransplant. Adoptive transfer assays revealed lower lymphoproliferation and greater cell surface TGF-␤ expression on CD4ⴙ T cells of NIMAd-exposed vs control splenocytes. NIMAd-exposed
splenocytes exhibited bystander suppression of tetanus-specific delayed-type hypersensitivity responses, which was reversed with
Abs to TGF-␤ and IL-10. Allospecific T effector cells were induced in all mice upon i.v. challenge with B6D2F1 splenocytes or a
DBA/2 heart transplant, but were controlled in NIMAd-exposed mice by TR cells to varying degrees. Some (40%) NIMAd-exposed
mice accepted a DBA/2 allograft while others (60%) rejected in delayed fashion. Rejector and acceptor NIMAd-exposed mice had
reduced T effector responses and increased Foxp3ⴙ TR cells (CD4ⴙCD25ⴙFoxp3ⴙ TR) in spleen and lymph nodes compared with
controls. The key features distinguishing NIMAd-exposed acceptors from all other mice were: 1) higher frequency of IL-10- and
TGF-␤-producing cells primarily in the CD4ⴙCD25ⴙ T cell subset within lymph nodes and allografts, 2) a suppressed delayedtype hypersensitivity response to B6D2F1 Ags, and 3) allografts enriched in LAPⴙ, Foxp3ⴙ, and CD4ⴙ T cells, with few CD8ⴙ T
cells. We conclude that the beneficial NIMA effect is due to induction of NIMA-specific TR cells during ontogeny. Their persistence
in the adult, and the ability of the host to mobilize them to the graft, may determine whether NIMA-specific tolerance is
achieved. The Journal of Immunology, 2007, 179: 6749 – 6761.
CD4⫹ TR CELLS TO NIMA
6750
FIGURE 1. NIMA breeding scheme and experimentation diagram. The F1 backcross breeding scheme used
to obtain NIMAd-exposed and NIPAd-nonexposed control offspring which receive challenge with either a DST
of B6D2F1 splenocytes or a DBA/2 heart allograft. NIMAd-exposed mice were considered tolerant if they had
a measurable heartbeat past 30 days and are termed NIMAd acceptors.
manner while preserving the graft-vs-leukemia effects and better
immune reconstitution (14).
The mechanisms underlying the phenomenon of NIMA-induced
tolerance remain unclear. In this study, we examine the role of
CD4⫹CD25⫹ T regulatory (TR) cells in the maternal effect promoting heart allograft tolerance.
Materials and Methods
Source of mice and typing
C57BL.6 (H-2b/b), DBA/2 (H-2d/d), and (C57BL.6 ⫻ DBA/2)F1 (B6D2F1;
H-2b/d) mice were obtained from Harlan Sprague Dawley. The care and
breeding of animals was in accordance with institutional guidelines. Offspring of F1 backcross pairs (B6D2F1 female ⫻ B6 male for NIMAdexposed; B6 female ⫻ B6D2F1 male for NIMAd control) were weaned
after 21 days and typed for H-2 locus-encoded Ags. Typing was performed
by flow cytometry on a FACSCalibur (BD Biosciences) using Abs specific
for H-2Kd (BD Biosciences). Homozygous H-2b male NIMAd-exposed and
NIPAd control offspring ages 5–10 wk were used for all experiments.
ELISPOT assay
Polyvinylidene difluoride membrane ELISPOT plates (Whatman) were
coated with primary Ab and incubated overnight at 4°C. Plates were
blocked for 2 h with 1% BSA in PBS (1% PBSA), washed in HL-1 serumfree medium (Cambrex BioScience) supplemented with penicillin/streptavidin and L-glutamine, and responder plus irradiated stimulator cells were
added in a 1:1 ratio. Plates were incubated at 37°C with 5% CO2. Twentyfour (for IFN-␥) or 48 (for IL-2 and IL-10) hours later, plates were washed
five times in PBS with 0.05% Tween 20 then five times in PBS. Secondary
Abs were diluted in 1% PBSA and added to plates and incubated at 4°C
overnight. Plates were washed and spot development was performed using
ELISPOT Blue Development Module (R&D Systems) according to the
manufacturer’s instructions. Spots were analyzed using an AID ELISpot
plate reader (AutoImmun Diagnostika). For IL-10 ELISPOTs, cells were
first incubated in a 96-well tissue-culture plate for 24 h before transferring
nonadherent cells to IL-10 ELISPOT plates to reduce background. The
following Abs were obtained from BD Biosciences: IFN-␥-coating Ab
used at 4 ␮g/ml; IFN-␥-biotinylated Ab used at 3 ␮g/ml; IL-2-coating Ab
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(NIPA). Indeed, 10-year graft survival of a NIMA HLA-mismatched graft was similar to that of HLA-identical sibling renal
transplants at the nine participating transplant centers (9). In follow-up studies of human bone marrow and HSC transplantation,
van Rood et al. (10) and Ichinohe et al. (11) found a powerful
tolerogenic effect of NIMA exposure suppressing graft-vs-host disease (GVHD) between adult living related donors. NIPA-mismatched transplants had a significantly higher incidence of GVHD.
Most recently, Japanese transplant centers have successfully used
NIMA-mismatched sibling and maternal donors in HSC transplantation introducing the parameter of mutual fetomaternal microchimerism in donor and recipients in the selection of donors (12).
We have described in a mouse heart transplant model a form of
maternally induced organ allograft tolerance (13) that closely parallels the human clinical findings in living related kidney transplantation (9). In this model, B6 male (H-2b/b) mice were crossed
with a (B6 ⫻ DBA/2)F1 (H-2b/d) female, resulting in 50% H-2b/b
homozygous offspring, all of which have been intimately exposed
to the NIMAd Ags in utero and orally via nursing. To control for
non-MHC genes that reassort in the F1 backcross, the parental
haplotypes were switched (B6 female ⫻ B6D2F1 male) resulting
in H-2b/b offspring with similar heterogeneity in non-MHC background genes that did not have the neonatal exposure to the H-2d
haplotype. Because the H-2d haplotype in this case encodes the
NIPA, such animals are referred to as NIPAd controls. Following
a fully allogeneic DBA/2 (H-2d/d) heterotopic heart transplant,
57% of NIMAd-exposed mice experienced allograft acceptance
(graft survival ⬎180 days) without any drug or conditioning treatment, whereas the NIPAd controls uniformly rejected around day
11 posttransplant (13). Using this same F1 backcross model, Matsuoka et al. (14) found that a bone marrow transplant from NIMAd-exposed donors into maternal-type B6D2F1 recipients reduced the morbidity and mortality of GVHD in an Ag-specific
The Journal of Immunology
6751
used at 3 ␮g/ml; and IL-2-biotinylated Ab used at 2 ␮g/ml. Abs for the
IL-10 ELISPOT assay were purchased as a pair from R&D Systems and
used according to the manufacturer’s instructions.
In vitro MLR assay
Splenocytes were harvested from normal F1 backcross NIMAd-exposed
and NIPAd control mice, labeled with CFSE, and incubated in a 1:1 ratio
with irradiated B6 or B6D2F1 splenocytes for 4 days in vitro in complete
RPMI 1640 plus 5% FCS (HyClone). Six hours before cell collection,
brefeldin A (Golgi Stop; BD Biosciences) was added to the cell cultures
and cells were analyzed for phenotype, cytokine production, and proliferation by CFSE dilution by flow cytometry. Proliferation analysis was performed using the computer program Modfit (Verity Software House).
In vivo MLR assay
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Splenocytes were harvested from normal F1 backcross NIMAd-exposed
and NIPAd control mice, labeled with CFSE, and 50 ⫻ 106 cells were
injected into a B6D2F1 recipient via tail vein injection and allowed to
incubate for 3 days. Six hours before cell harvest, recipient mice were
injected with 250 ␮g of brefeldin A (BFA; Sigma-Aldrich). Splenocytes
and inguinal lymph node (ILN) cells were harvested from the B6D2F1
recipients and H-2Kd-negative CFSE⫹ donor cells were analyzed for cell
phenotype, cytokine production, and proliferation by CFSE dilution by
flow cytometry. Proliferation analysis was performed using the computer
program Modfit.
Ag preparation for delayed-type hypersensitivity (DTH)
Ag was prepared from B6D2F1 splenocytes, which were harvested,
washed three times in PBS, and adjusted to a concentration of 120 million
cells/ml in PBS with 10 ␮g/ml PMSF (Sigma-Aldrich). The cells were
sonicated using a VR50 sonicator fitted with a 2-mm probe (Sonics). The
disrupted cells were centrifuged for 20 min at 14,000 ⫻ g at 4°C to remove
debris. The protein content of the supernatant was determined using a
microBCA Protein Assay kit (Pierce). A total of 20 ␮g of protein was used
for each injection in the DTH assays and referred to as BDF1 Ag.
Adoptive transfer DTH assays
In all adoptive transfer DTH assays, 10 ⫻ 106 splenocytes were injected
into footpads of naive B6 recipients along with coinjection inoculum. DTH
reactivity was measured as the change in footpad thickness 24 h postinjection over the preinjection reading using a dial-thickness gauge and
swelling is expressed in 10⫺4 inches. Coinjection inoculums include: PBS,
20 ␮g of BDF1 Ag, 0.25 limits of flocculation (lf) tetanus and diphtheria
toxoid (TT/DT) vaccine (Aventis Pasteur), 10 ␮g of anti-TGF-␤ mAb (BD
Biosciences), 10 ␮g of anti-IL-10 mAb (R&D Systems), and 10 ␮g of rat
IgG isotype (BD Biosciences).
Tetanus immunization to generate T effector (TE) memory T cell responses. F1 backcross NIMAd-exposed and NIPAd control mice were immunized s.c. in the inguinal pouch with 1 lf of TT/DT pediatric vaccine 2
wk before harvesting splenocytes.
Donor-specific transfusion (DST) to immunize for DTH assay. F1 backcross NIMAd-exposed and NIPAd controls were injected i.v. with 50 ⫻ 106
B6D2F1 splenocytes 2 wk before splenocyte harvest and standard adoptive
transfer or direct challenge DTH assay. Direct challenge DTH assay was
performed by injected 10 ⫻ 106 B6D2F1 splenocytes or PBS into NIMAdexposed or NIPAd control footpads.
Posttransplant DTH assays. Splenocytes were harvested from mice 4–6
wk posttransplant and injected with BDF1 Ag with or without anticytokine mAbs. The net swelling value obtained with cells plus PBS injections were subtracted from the test values, giving a corrected value, or
net swelling response. For the adoptive transfer DTH assays in which
CD4⫹CD25⫹ TR cells were depleted before injection, TR cells were separated out using the Miltenyi Mouse CD4⫹CD25⫹ Regulatory T Cell Isolation kit according to manufacturer’s directions using the AutoMacs Separator (Miltenyi Biotec). Flow cytometric analysis was used to confirm
purity of sorted cells and found them to by 92% pure.
Heart transplantation
Heterotopic vascularized heart transplantation was conducted using the intra-abdominal microsurgical technique described by Corry et al. (15). The
grafts were monitored by daily palpation and graded from 4⫹ (strongest
beat) to 0 (no beat). Graft rejection was determined by complete cessation
of heart beat (grade 0) and was confirmed by laparotomy.
FIGURE 2. In vitro cytokine production and proliferation analysis of F1
backcross offspring reveals no difference between NIMAd exposed and controls. NIMAd-exposed and control responder splenocytes were harvested and
mixed with irradiated B6 or B6D2F1 splenocytes in a 1:1 ratio in either IFN-␥
(A) or IL-2 ELISPOT or a traditional MLR for proliferation and phenotypic
analysis (B). A, IFN-␥ and IL-2 ELISPOT results shown as mean ⫾ SD of
spots per million cells with n ⫽ 5–9 mice in each group for each cytokine
analyzed. B, Representative staining of proliferation from in vitro MLR assay
as analyzed by flow cytometry. Responder cells were analyzed by first gating
on the H-2Kd-negative cells, then on CD4⫹ and CD8⫹T cells. C, Representative proliferation analysis by CFSE dilution using a proliferation analysis
program, which calculates the percent proliferated and number per generation.
Each daughter generation is represented by a shade of gray in each curve. The
white line profile is the CFSE-staining profile the program uses to best fit the
daughter generations under the curve to calculate the percent proliferation.
Flow cytometry
For posttransplant analysis, cell suspensions were prepared from spleen
and inguinal LN (ILN) from mice 4 – 6 wk posttransplant. Donor heart
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CD4⫹ TR CELLS TO NIMA
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FIGURE 3. In vivo MLR reveals differences between NIMAd exposed and controls. Splenocytes from NIMAd-exposed and control mice were labeled
with CFSE and injected i.v. into B6D2F1 recipients. Three days later, recipient mice are injected with BFA 6 h before harvest of spleen and ILN and
analyzed for responder cell proliferation and phenotype. A, Representative staining and analysis of NIMAd-exposed and control CD4⫹ responder cell
proliferation recovered from the ILN of B6D2F1 recipients. Analysis begins by gating on H-2Kd-negative donor responder cells, then gating on the CD4⫹
responder cell population for proliferation analysis using proliferation analysis software. B, Percent proliferation of NIMAd-exposed and control CD4⫹ and
CD8⫹ T cells recovered from the B6D2F1 recipient ILNs and spleen. C, Representative staining of cell surface TGF-␤, CD25, and Foxp3 on CD4⫹ cells
from NIMAd-exposed and control cells recovered from B6D2F1 recipient ILNs. D, Percent of donor responder CD4⫹TGF-␤⫹ and CD8⫹TGF-␤⫹ cells
recovered from the ILN and spleen of B6D2F1 recipients according to their proliferation status as indicated by the gating in the upper right dot plot in C
(CFSE bright ⫽ nonproliferated and CFSE dim ⫽ proliferated); n ⫽ 7 control, n ⫽ 8 NIMAd exposed. Results shown as mean ⫾ SD. ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.005; ⴱⴱⴱ, p ⬍ 0.0005 vs controls.
graft-infiltrating cells (GIC) were harvested at 4 – 6 wk posttransplant for
NIMAd-acceptor mice, and at the time of rejection for NIMAd-rejector and
NIPAd control mice. Mice that were analyzed for intracellular IL-10 pro-
duction posttransplant were injected with 250 ␮g of BFA (Sigma-Aldrich)
6 h before tissue harvest. For preparation of donor heart GICs, heart tissues
were teased apart using forceps and incubated in Liberase CL enzyme
The Journal of Immunology
blend (Roche) at a concentration of 0.4 mg/ml in complete RPMI 1640 for
1 h. The tissue was then gently pressed through a 0.45-␮m cell strainer
(Falcon; BD Biosciences) using a syringe plunger. RBC were lysed with
ACK buffer and remaining lymphocytes are washed in PBS and
resuspended in FACS wash buffer. All cells were kept on ice throughout
the staining procedure. A total of 106 cells were incubated with primary
biotinylated Abs against cell surface markers for 30 min and washed before
addition of secondary fluorochrome-labeled Abs against cell surface markers. Cells stained for intracellular cytokines and Foxp3 were fixed and
permeabilized using the Foxp3 staining kit according to instructions (eBioscience). Cells were analyzed using a FACSCalibur.
Abs used in all flow analyses include: H-2Kd-PE, H-2Kd biotinylated,
streptavidin-allophycocyanin, streptavidin-PerCP, CD4-allophycocyanin,
CD4-PerCP, CD8-allophycocyanin, CD8-PerCP, CD25-allophycocyanin,
IL-10-allophycocyanin, TGF-␤-PE (clone TB21; IQ Products); TGF-␤
(clone TB21; BioSource International) was FITC labeled using the FITC
EZ-Label Reagent and Accessory Pack (Pierce), the Foxp3-PE labeled
staining kit (eBioscience), mouse IgG1-PE, mouse IgG1-FITC, and rat
IgG2b-PE. All Abs were purchased from BD Biosciences unless otherwise
specified.
Histology
Statistics
All flow cytometry results were analyzed using the Student t test. Where a
significant difference in variance was found, a corrected Student t test
(Welch’s correction) was used. All other experiments were analyzed using
the nonparametric rank sums Mann-Whitney U test. Graft survival statistics were performed using a log-rank test.
Results
Effect of exposure to NIMA in F1 backcross offspring on
alloreactive T cell responses
As depicted in Fig. 1, we used the F1 ⫻ P backcross breeding
scheme originally described by Zhang and Miller (16) to generate
H-2b/b mice that were exposed to, but did not inherit, maternal
H-2d Ags from a B6D2F1 mother. These mice were weaned at 3
wk of age, allowing for maximum oral exposure to NIMAd
through nursing, and are referred to as NIMAd exposed. Control
breedings were performed by mating female B6 with male
B6D2F1 mice resulting in H-2b/b F1 backcross offspring that will
not have been exposed to maternal H-2d Ags, and are the NIPAd
nonexposed control mice, simply referred to as controls. Adult
mice were analyzed for effects of developmental exposure to H-2d
alloantigens using various assays without any conditioning treatments. Some F1 backcross offspring were challenged by a DST of
B6D2F1 splenocytes and then analyzed 1–2 wk later by DTH or
flow cytometry assays. Other mice were challenged with trans-
FIGURE 4. NIMAd-exposed mice exhibit TGF-␤- and IL-10-dependent
bystander suppression of a third-party recall DTH response in the presence
of NIMA. Left panel, Splenocytes from NIMAd-exposed (n ⫽ 10) and
control (n ⫽ 8) mice immunized with TT/DT were coinjected into B6recipient footpads with PBS, TT/DT, BDF1 Ag, or TT/DT ⫹ BDF1 Ag to
measure the bystander suppression of a TT/DT DTH TE response. ⴱ, p ⬍
0.05 vs control. Right panel, To reverse bystander suppression, splenocytes
from TT/DT-immunized NIMAd-exposed mice (n ⫽ 5) were coinjected
with BDF1 Ag ⫹ TT/DT ⫹ Abs directed against TGF-␤ or IL-10. ⴱ, p ⬍
0.05 vs isotype control.
plantation of a fully allogeneic (H-2d/d) DBA/2 heart and analyzed
posttransplant (Fig. 1).
We first compared the frequency of cytokine-producing alloreactive T cells in vitro using ELISPOT analysis. As shown in Fig.
2A, there were no differences between NIMAd-exposed and control mice in the production of IFN-␥ or IL-2 when stimulated with
irradiated B6D2F1 or B6 splenocytes. These results stand in contrast to our previous study which appeared to show a decreased
cytokine response in NIMAd-exposed mice (13); however, in those
experiments, B6 spleen cells were used as controls rather than
spleen cells from NIPAd F1 backcross control mice. In all experiments in this article, H-2b/b homozygous F1 backcross mice from
the NIPA breeding (Fig. 1) are used as controls.
Next, the proliferative response to NIMA stimulation in vitro
was compared between NIMAd-exposed and control splenocytes
by MLR. Fig. 2, B and C, show representative CFSE dilution assay
data of CD4⫹ and CD8⫹ responder cells. CD4⫹ or CD8⫹ cells in
control cultures with medium alone did not proliferate (top panels
in Fig. 2B), whereas a substantial portion of CD4⫹ and CD8⫹ of
cells proliferated in response to irradiated B6D2F1 cells. A proliferation analysis program that takes into account not only the
percent of cells that proliferated, but also the number of daughter
cells in each generation (Fig. 2C) was used to calculate the percent
of proliferated cells. Overall (n ⫽ 6 experiments), there were no in
vitro differences between splenocytes from NIMAd exposed and
controls either in CD4⫹ or CD8⫹ T cell proliferative responses to
B6D2F1 cells, or in expression of CD25, intracellular IL-10,
Foxp3, and cell surface TGF-␤ (data not shown).
Presence of surface TGF-␤⫹ CD4⫹ TR cells is revealed by in
vivo MLR
We next assessed lymphoproliferation in vivo by i.v. transfer of
CFSE-labeled responder splenocytes from NIMAd-exposed and
control mice into B6D2F1 recipients (“in vivo MLR”). Responder cells were recovered from spleen and ILN 3 days later
and analyzed for proliferation and phenotype ex vivo. In contrast to the in vitro MLR data, CFSE-labeled control responder
cells proliferated considerably more in B6D2F1 hosts than the
NIMAd-exposed responder cells (Fig. 3A). This difference was
evident both from a dot plot and by a proliferation analysis
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Donor heart grafts were collected at the time of rejection or 4 – 6 wk posttransplant for NIMAd-acceptor mice and subsequently frozen in OCT compound (Tissue-Tek). Frozen sections were fixed for 5 min with 4% paraformaldehyde/PBS (CD4 and CD8) or acetone (latency associated peptide
(LAP) and Foxp3). Foxp3 sections were permeabilized with 1% Triton/
PBS. All sections were then blocked with 10% BSA/TBS, followed by 5%
nonfat milk. Additional blocking for endogenous biotin was required for
LAP, using Biocare Medical’s avidin/biotin kit. Primary Abs were incubated overnight at 4°C and endogenous peroxidase was quenched using 3%
hydrogen peroxide/TBS. The CD4 and CD8 markers were detected using
a donkey anti-rat IgG-HRP-conjugated secondary (Jackson ImmunoResearch Laboratories). Biotinylated LAP was detected by streptavidin-HRP
(Biocare Medical). A rat-on-mouse HRP polymer kit (BioCare Medical)
was used for detection of Foxp3. Staining was visualized with DAB kit 2
(DakoCytomation) and the slides were counterstained with Harris hematoxylin. Abs used in histology include: CD4 and CD8 (BD Biosciences),
LAP biotinylated (R&D Systems), and Foxp3 (eBioscience).
Histopathology interpretation was performed using an Olympus microscope BX45 with incorporated digital camera MicroFire S99809 (Olympus
America) and data were analyzed with MicroSuite Five software (Soft
Imaging System). The number of positive cells per specific immunohistochemical marker was obtained in each mouse heart by counting the number
of positive cells per high power field (⫻400, surface 120,000 ␮M2) in 10
consecutive fields by a trained pathologist blinded to the experiment.
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CD4⫹ TR CELLS TO NIMA
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FIGURE 5. NIMAd-exposed mice
are capable of regulating a DTH response to maternal Ag after treatment
with a B6D2F1 DST. NIMAd-exposed and control mice were given a
DST of B6D2F1 splenocytes i.v. A,
Footpad swelling was measured preand postinjection with direct DTH
challenge of 10 ⫻ 106 B6D2F1
splenocytes into the footpads of normal (no DST) or DST-challenged NIMAd-exposed and control mice. Results given as mean ⫾ SD over the net
swelling PBS background. B, Left,
Representative flow analysis of
PBMC 7 days post-DST in which
CD4⫹ T cells are gated on (top panels) and then analyzed for expression
of CD25 and surface TGF-␤. Right,
PBMC were harvested from normal
or post-DST-treated mice and analyzed by flow cytometry for the expression of cell surface TGF-␤ and
CD25⫹ on CD4⫹ T cells; n ⫽ 3 for
each test group in A and B. Results
shown as mean ⫾ SD. ⴱ, p ⬍ 0.05 vs
control. C, Splenocytes were harvested from normal NIMAd exposed
or 2 wk post-DST challenge and coinjected with PBS or BDF1 Ag into the
footpad of a naive B6 mouse. To reveal a response to BDF1 Ag, splenocytes were coinjected with BDF1
Ag ⫹ Abs directed against TGF-␤
and IL-10; n ⫽ 5 for each test group.
Results shown are the mean ⫾ SD of
DTH swelling responses, measured in
units of 10⫺4 inches net footpad
swelling. ⴱ, p ⬍ 0.05 vs control.
program. In the example of cells recovered from spleen in Fig.
3A, CD4⫹ T cells proliferated best, while non-CD4⫹ cells (including CD8⫹ cells) divided, but fewer times. CD8⫹ responder
cell proliferation was analyzed separately using the same
gating strategies with Abs directed against CD8 instead of CD4.
As summarized in Fig. 3B, both CD4⫹ and CD8⫹ control responder cells recovered from the spleen of the B6D2F1
host had proliferated significantly more than the NIMAdexposed responder cells ( p ⫽ 0.02 for CD4⫹, p ⫽ 0.004 for
CD8⫹). This finding is consistent with the lower recovery of
Table I. Heart graft prolongation in NIMAd-exposed mice
Mouse Group
n
Graft Survival (Days)
Mean Graft Survival
Comparison
p Value
NIPAd rejector
NIMAd rejector
NIMAd acceptorb
21
21
14
5, 6, 7 ⫻ 2, 8 ⫻ 5,a 9 ⫻ 5,a 10 ⫻ 3, 11 ⫻ 3, 24
7, 8 ⫻ 3, 9, 10 ⫻ 3, 11, 12 13,a 14, 15 ⫻ 4, 16, 17,a 20 ⫻ 2, 27
28, 31, 32 ⫻ 5,a 33, 35, 42,a 100 ⫻ 4
9⫾4
13 ⫾ 5
⬎28 days
2 vs 1
3 vs 1 or 2
0.004
⬍0.001
a
b
Mice used in histological analysis.
Graft survival time points listed for the NIMAd acceptor are days on which animals were sacrificed and organs harvested for experimentation.
The Journal of Immunology
6755
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FIGURE 6. NIMAd-acceptor mice regulate to their maternal Ags
through CD4⫹CD25⫹ TR cells posttransplant. Splenocytes were harvested
from control, NIMAd-acceptor, and NIMAd-rejector mice 4 – 6 wk posttransplant and were (A) coinjected into B6-recipient footpads with PBS,
BDF1 Ag, BDF1 Ag ⫹ anti-TGF-␤ Ab, or BDF1 Ag ⫹ anti-IL-10 Ab. B,
Splenocytes were coinjected into B6-recipient footpads again with PBS or
BDF1 Ag. Next, CD4⫹CD25⫹ TR cells were sorted out and the
CD4⫹CD25⫹ TR-depleted cells were also injected with PBS or BDF1 Ag.
Lastly, the CD4⫹CD25⫹ TR cells were added back to the depleted splenocytes and injected with BDF1 Ag. Results are shown as the 24-h net swelling response subtracting the PBS background net swelling; n ⫽ 5 for each
test group. ⴱ, p ⫽ ⬍.05; ⴱⴱ, p ⫽ ⬍.005 vs control.
NI-MAd-exposed responder cells, which made up 2.3 ⫾ 1.3%
of the total splenocytes as compared with 5.26 ⫾ 2.49% of the
total splenocyte population for the control responder cells ( p ⫽
0.043). A similar trend for both CD4⫹ and CD8⫹ T cells was
seen in responder cells recovered from the ILNs of B6D2F1
hosts, although the difference in the percent of proliferation was
significant only for CD8⫹ T cells ( p ⫽ 0.02) (Fig. 3B).
NIMAd-exposed CD4⫹ T cells recovered from ILN showed differences from controls in expression of cell surface TGF-␤. As
shown in Fig. 3C, it was the CFSE bright cells, or cells that did not
proliferate, that expressed the most cell surface TGF-␤. This was
not the case with the control CD4⫹ cells recovered from B6D2F1
LN, few of which expressed cell surface TGF-␤, whether they had
proliferated or not (Fig. 3C). Differences between CD25 and
Foxp3 expression were minimal, regardless of proliferation status,
in the same LN-homing CD4⫹ T cell population (Fig. 3C), and no
differences were found between NIMAd-exposed and control
CD4⫹ responder cells when analyzed for expression of CTLA-4
and glucocorticoid-induced TNFR (data not shown). When the
data from all experiments were combined, the only significant difference between NIMAd-exposed responder cells and control cells
FIGURE 7. NIMAd-acceptor mice have significantly more TR cells posttransplant in spleen, ILNs, and in the graft itself. Splenocytes and ILN cells
were harvested 4 – 6 wk posttransplant from control (n ⫽ 9), NIMAd-acceptor
(n ⫽ 8a), and NIMAd-rejector (n ⫽ 16) mice. GIC were harvested at the time
of rejection for controls (n ⫽ 6) and NIMAd-rejector (n ⫽ 5) mice, and 4 – 6
wk posttransplant for NIMAd-acceptor (n ⫽ 6) mice. All cells were stained for
CD4, CD25, cell surface TGF-␤, and Foxp3. A, Representative dot plots of cell
surface TGF-␤ vs Foxp3 staining on CD4⫹ cells of splenocytes, ILN, and GIC
harvested from a NIMAd-acceptor mouse 5 wk posttransplant. B, Percent of
CD4⫹CD25⫹ TR cells expressing Foxp3, cell surface TGF-␤, or both Foxp3
and cell surface TGF-␤ in the spleen, ILNs, and GIC. Results shown as
mean ⫾ SD. ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.005, ⴱⴱⴱ, p ⬍ 0.001 vs control. n ⫽ 8 for
single-cell surface TGF-␤ or Foxp3 staining of CD4⫹CD25⫹ T cells, n ⫽ 4 for
dual staining (Foxp3 and TGF-␤ together).
recovered from recipient LN was in cell surface TGF-␤⫹ expression on both nonproliferated and proliferated CD4⫹ T cell fractions. The highest percentages of cell surface TGF-␤⫹ cells were
6756
CD4⫹ TR CELLS TO NIMA
in the nonproliferated population ( p ⬍ 0.005) (Fig. 3D). Recovered CD8⫹ T cells in the LN, and both CD4⫹ and CD8⫹ T cells
recovered from spleen tended to have higher percentages of surface TGF-␤⫹ cells in the nonproliferated fraction than controls,
however, these differences did not reach statistical significance
(Fig. 3D).
Bystander suppression of a recall DTH response in the presence
of noninherited maternal BDF1 Ags
Bystander suppression is the phenomenon associated with Ag-specific TR cells in a tolerant host (17–21). NIMAd-exposed and control mice were immunized with TT/DT. Splenocytes harvested 2
wk later were adoptively transferred into the footpads of naive B6
recipients along with TT/DT, resulting in swelling responses 3- to
5-fold higher than coinjection with PBS along (Fig. 4, left panel).
No DTH response was seen when splenocytes were injected with
a sonicate of B6D2F1 cells (BDF1 Ag) (Fig. 4, left panel). When
TT/DT-sensitized splenocytes from NIMAd-exposed mice were
injected with both TT/DT and BDF1 Ag, the DTH response was
suppressed, suggesting a dominant-negative effect mediated by a
TR cell response to maternal alloantigens (Fig. 4, left panel). Bystander suppression was not seen with the control splenocytes,
which retained a high TT/DT response in the presence of coinjected BDF1 Ag ( p ⫽ 0.009 vs NIMAd exposed) (Fig. 4, left
panel). Addition of either anti-TGF-␤ or anti-IL-10 Abs reversed
the bystander suppression of the TT/DT response to reveal a DTH
swelling similar to that seen when NIMAd-exposed splenocytes
were coinjected with TT/DT alone (Fig. 4, right panel), and significantly greater than the response to TT/DT-BDF1 Ag mixture in
the presence of isotype control Ab ( p ⫽ 0.015 vs anti-IL-10 or
anti-TGF-␤).
B6D2F1 i.v. challenge accentuates differences between
NIMAd-exposed and control mice
To determine whether semiallogeneic DST has a differential impact in NIMAd-exposed vs control adult F1 backcross mice,
B6D2F1 splenocytes were injected i.v. Neither NIMAd-exposed
nor controls mice made a DTH response to direct footpad challenge with viable B6D2F1 splenocytes pre-DST (Fig. 5A, left
panel). Both made a swelling response after DST challenge (Fig.
5A, right panel). Controls produced a more sustained response,
both on days 7 and 14 after DST challenge compared with NIMAdexposed mice, and this difference was significant 14 days postDST treatment ( p ⫽ 0.02) (Fig. 5A, right panel). Furthermore,
analysis of the PBMC 1 wk after DST treatment revealed that the
NIMAd-exposed mice had significantly more circulating CD4⫹ T
cells expressing CD25 and surface TGF-␤ compared either to controls or to pre-DST NIMAd-exposed mice (Fig. 5B).
The expression of TGF-␤ on CD4⫹CD25⫹ T cells in the peripheral blood led us to test whether the low response to B6D2F1
spleen cell challenge in the direct DTH assay seen in the DSTtreated NIMAd-exposed mice was due to regulation by TGF-␤producing cells. Although no differences were found in the expression of TGF-␤ between NIMAd-exposed and control cells from the
spleen and ILN at any time tested post-DST challenge as analyzed
by flow cytometry (data not shown), DST-primed control splenocytes coinjected with BDF1 Ag into the footpad of a B6 adoptive
host caused a significantly stronger swelling response than DSTprimed splenocytes from NIMAd-exposed mice ( p ⫽ 0.008 NIMAd exposed vs control) (Fig. 5C). The weak DTH response postDST in the NIMAd-exposed mice was not the result of an absence
of alloreactive indirect pathway TE cells: addition of Abs to TGF-␤
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FIGURE 8. NIMAd-acceptor mice harbor
TR cells in the graft. Representative immunohistochemistry on hearts from a control
rejected heart (right column), NIMAd rejector (middle column), and a NIMAd acceptor
(left column) harvested on day 40 posttransplant. Rejected hearts were harvested at the
time of rejection. Hearts were stained for
LAP expression (top row), Foxp3 (second
row), CD4 (third row), and CD8 (bottom
row).
The Journal of Immunology
6757
Table II. Histological analysis posttransplant
Cells/High-Powered Field
Mouse Group
d
NIPA control
NIMAd rejector
NIMAd acceptor
CD4
CD8
Foxp3
LAP
4 ⫾ 0.4
3⫾1
12 ⫾ 2
15 ⫾ 1
13 ⫾ 1
10 ⫾ 1
0.2 ⫾ 0.2
2 ⫾ 0.5
9⫾2
0.4 ⫾ 0.3
6⫾2
22 ⫾ 3
or IL-10, but not isotype control IgG, revealed a positive swelling
response to BDF1 Ags that was equivalent to the control DTH
swelling response after DST (Fig. 5C), and was significantly
higher than that elicited in response to the same Ag-Ab mixtures
before DST challenge ( p ⬍ 0.05, Fig. 5C). These data indicate that
NIMAd-exposed mice pre-DST do contain adaptive TR cells, detected via bystander suppression of a DTH recall response (Fig. 4),
but have few NIMAd-specific TE cells until after DST challenge.
To analyze the effects of re-exposure to NIMA via transplant, NIMAd-exposed and control mice were given a fully allogeneic
DBA/2 heart graft. Allograft recipients did not receive any DSTconditioning treatment or drug therapy. Our previous study (13)
indicated that if a NIMAd-exposed recipient was to reject their
allograft, they usually did so before the 30-day time point. For the
posttransplant analyses performed in these experiments, a beating
graft at day 30 was considered to be an accepted graft. Approximately 40% of NIMAd-exposed mice accepted their allografts
(⬎30-day graft survival) (Table I); these mice are referred to as
NIMAd-acceptor mice. Control mice uniformly rejected their cardiac allografts at a median survival time of 9 ⫾ 4 days. NIMAdexposed mice that rejected did so in a delayed fashion with a mean
survival time of 13 ⫾ 5 days ( p ⫽ 0.004 compared with controls)
(Table I). These mice are referred to as NIMAd rejectors.
Spleen cells from NIMAd-acceptor mice failed to transfer a
DTH swelling response to BDF1 Ags, whereas cells from both
controls and NIMAd-rejector mice gave strong swelling responses
( p ⬍ 0.001) (Fig. 6A). Both anti-TGF-␤ and anti-IL-10 Abs, but
not isotype control IgG (data not shown), revealed a DTH response
toward maternal Ag in the NIMAd-acceptor mice that was similar
to that of both NIMAd rejector and controls (Fig. 6A). To determine the cell subset responsible for active suppression, we removed the CD4⫹CD25⫹ TR cells and found that the DTH re-
Table III. Posttransplant frequency of alloreactive splenocytes in NIMAd-exposed and NIPAd control micea
BDF1 Stimulation
NIPAd control
NIMAd rejector
NIMAd acceptor
Stimulation
IFN-␥
IL-2
IL-10
BDF1
DBA/2
B6
C3H
BDF1
DBA/2
B6
C3H
BDF1
DBA/2
B6
C3H
1355 ⫾ 343
1191 ⫾ 370
632 ⫾ 352
543 ⫾ 260
736 ⫾ 88c
752 ⫾ 379
135 ⫾ 34
265 ⫾ 161
539 ⫾ 106c
495 ⫾ 305c
233 ⫾ 63
363 ⫾ 232
1012 ⫾ 244
1460 ⫾ 533
572 ⫾ 167
395 ⫾ 320
353 ⫾ 77c
599 ⫾ 486
177 ⫾ 38
198 ⫾ 191
321 ⫾ 43c
356 ⫾ 233c
130 ⫾ 26
227 ⫾ 85
965 ⫾ 201
NTb
701 ⫾ 206
NT
947 ⫾ 307
NT
699 ⫾ 261
NT
2559 ⫾ 685c
NT
1902 ⫾ 444c
NT
a
The results presented here show the mean number of spots per 106 cells for each cytokine ⫾ SE. Days posttransplant the
assays were performed were: 70 ⫾ 47 for NIMAd acceptors, 53 ⫾ 9 for NIMAd rejectors, and 32 ⫾ 10 for NIPAd controls, and
were not significantly different; n ⫽ 4 –16 mice/group for each cytokine tested.
b
NT, Not tested.
c
For bold values, p ⬍ 0.05 vs NIPAd control using nonparametric Mann-Whitney.
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CD4⫹CD25⫹ TR cells inhibit posttransplant responses to
maternal Ags in NIMAd-acceptor mice
sponse from NIMAd-acceptor splenocytes was restored. When the
CD4⫹CD25⫹ TR cells are added back to the splenocytes being
injected with BDF1 Ag, the DTH response was again suppressed
( p ⫽ 0.03, compared with similarly treated controls) (Fig. 6B).
To characterize the distribution of CD4⫹CD25⫹ TR cells after
transplant, flow analysis of splenocytes, ILN cells, and GIC was
performed. The ILN was selected for analysis because of its proximity to the heterotopic heart allograft, and because of its noticeably increased size in the transplanted mice. Fig. 7A shows a representative flow analysis of Foxp3 vs cell surface TGF-␤
expression on CD4⫹ cells from a NIMAd-acceptor mouse.
Whereas the majority of CD4⫹TGF-␤⫹ T cells in spleen and ILN
were Foxp3 negative, most of those in the GIC were Foxp3⫹.
Overall, both cardiac acceptor and rejector NIMAd-exposed mice
contained significantly higher percentages of Foxp3-expressing
CD4⫹CD25⫹ TR cells in the spleen and ILN compared with controls (Fig. 7B, top panel). CD4⫹CD25⫹ TR cells infiltrating the
grafts of NIMAd-acceptor mice expressed significantly more
Foxp3 than NIMAd-exposed mice that had rejected their cardiac
allografts ( p ⫽ 0.03). The latter more closely resembled the control GIC obtained at the time of rejection (Fig. 7B, top panel).
There was no significant difference between Foxp3 expression on
CD4⫹CD25⫹ TR GIC between NIMAd-acceptor and controls, although the trend favored the NIMAd acceptors (Fig. 7B, top
panel).
As previously noted in the in vivo MLR, the major difference
between NIMAd-exposed and control mice was the expression of
cell surface TGF-␤, an expression that was sustained in NIMAdexposed mice that became tolerant, but lost in those that rejected
their graft. CD4⫹CD25⫹ TR cells from NIMAd-acceptor mice expressed significantly more cell surface TGF-␤ in all three compartments compared with both controls and NIMAd-rejector mice
( p values ⬍.01 for all comparisons) (Fig. 7B, center panel). These
same results were obtained when looking at CD4⫹CD25⫹ TR cells
expressing both Foxp3 and TGF-␤ ( p ⬍ 0.01 for all comparisons)
(Fig. 7B, bottom panel). TGF-␤⫹ and TGF-␤/Foxp3 dual-positive
cells represented a higher proportion of total CD4⫹CD25⫹ TR
cells in the graft and ILN as compared with spleen. This trend was
not seen in all rejector mice, whose levels of TGF-␤⫹ and TGF␤/Foxp3 dual-positive cells remained low in all compartments
(Fig. 7B, center and bottom panels).
To confirm the findings obtained with flow cytometry, immunohistochemical analysis was performed on tolerant and rejected
CD4⫹ TR CELLS TO NIMA
6758
the NIMAd-acceptor mice posttransplant as shown by ELISPOT,
we stained for IL-10 and cell surface TGF-␤ in a cardiac acceptor
NIMAd-exposed mouse at 5 wk posttransplant which was injected
with BFA 6 h before harvesting the spleen, ILN, and GIC. Fig. 9
(top panels) shows the staining of CD4⫹ cells for IL-10 vs cell
surface TGF-␤ vs isotype controls (bottom panels). In the spleen,
very few IL-10⫹ cells were detected ex vivo. In contrast, the ILN
contained an increased percentage of IL-10⫹ T cells. These IL10⫹ T cells were present in both the TGF-␤-negative and TGF-␤⫹
CD4 T cell populations. Although very few cells infiltrating the
accepted graft could be obtained for analysis, most of the CD4⫹IL10⫹ cells present were also positive for cell surface TGF-␤
(Fig. 9).
Discussion
hearts, which were stained for CD4, CD8, Foxp3, and TGF-␤ latency associated peptide (LAP). Fig. 8 shows representative immunohistological staining in which NIMAd-acceptor hearts contained more LAP⫹ cells compared with NIMAd-rejector and
control hearts (Fig. 8, top row). Furthermore, the cardiac acceptor
NIMAd-exposed hearts displayed LAP staining in the interstitium,
a staining pattern of which none of the rejected hearts displayed.
The number of Foxp3⫹ cells was also elevated in the NIMAdacceptor heart compared with NIMAd-rejected and control hearts
(Fig. 8, second row), consistent with flow cytometric analysis of
GIC (Fig. 7). Furthermore, the NIMAd-acceptor heart had more
CD4⫹ cells infiltrating the heart compared with both NIMAd-rejector and control hearts (Fig. 8, third row). The opposite was true
for CD8⫹ cells, with rejected hearts containing more CD8⫹ cells
than hearts from NIMAd-acceptor mice (Fig. 7, bottom row). Table
II summarizes the histological data showing that the NIMAd-acceptor hearts contained more LAP⫹, Foxp3⫹, and CD4⫹, and
lower CD8⫹ cells per high-powered field compared with mice that
rejected their allografts.
By ELISPOT analysis, splenocytes of NIMAd-exposed mice,
both cardiac acceptor and rejector, contained significantly fewer
IFN-␥ ( p ⫽ 0.006, p ⫽ 0.038, respectively) and IL-2 ( p ⫽ 0.043,
p ⫽ 0.015, respectively) producing cells reactive to B6D2F1 stimulators as compared with controls (Table III). Splenocytes from
NIMAd-acceptor mice contained significantly more IL-10-producing cells compared with both controls ( p ⫽ 0.024) and NIMAdrejector ( p ⫽ 0.034) splenocytes when stimulated with B6D2F1
cells (Table III). When stimulated with fully allogeneic DBA/2
splenocytes, only NIMAd acceptors produced significantly less
IFN-␥ ( p ⫽ 0.01) and IL-2 ( p ⫽ 0.03) compared with NIPAd
controls. The same trends in all three cytokine responses can be
seen when splenocytes were stimulated with irradiated B6 cells,
suggesting ongoing responses in vivo after heart transplant. Because the rejected or tolerated DBA/2 heart was still present, it is
possible that continued activation of TE and TR cells is occurring
in vivo resulting in this elevated cytokine response seen immediately ex vivo.
Because IL-10 has been shown to be involved in the regulatory
mechanism using DTH analysis, and production was elevated in
In this study, we found that the mechanism underlying the tolerogenic NIMA effect seen in this murine model is associated with the
development of NIMAd-specific TR cells that mediate suppression
through the production of immunosuppressive cytokines IL-10 and
TGF-␤. Several types of TR cells have been reported in the mouse.
Natural CD4⫹CD25⫹ TR cells which are specific for self Ags, are
dependent on intrathymic encounter with Ag for their development
(22). However, it has been shown that TR cells can be induced
from naive conventional CD4⫹ T cells in the periphery, and these
adaptive TR cells are most likely important in the induction of
tolerance to nonself Ags (23, 24). TR1 (25) and Th3 (26) cells are
examples of peripherally induced adaptive TR cells with regulatory
properties that share many phenotypic characteristics with natural
thymus-derived TR cells, but unlike natural TR cells which usually
work in a cell-cell contact-dependent manner, their suppressive
ability seems to depend primarily on the secretion of cytokines
such as IL-10 and TGF-␤ (27). Matsuoka et al. (14), using the
same F1 backcross model of NIMAd exposure, also found that the
tolerogenic NIMA effect seen in their model of bone marrow transplantation was mediated with CD4⫹CD25⫹ TR cells showing that
the favorable NIMA effect suppressing GVHD disappeared when
the NIMAd-exposed donor cells were depleted of CD4⫹CD25⫹
TR cells. Unlike our studies, the authors did not study the mechanism of suppression by the CD4⫹CD25⫹ TR cells. Similar to our
results, dependency on TR cell production of IL-10 and TGF-␤
have been demonstrated in colitis models (28, 29), lung allergic
responses (30), prevention of experimental autoimmune encephalomyelitis (31), and modulation of graft acceptance in liver and
kidney recipients (19, 32).
The development of NIMA-specific natural and adaptive TR
cells in NIMAd-exposed mice could occur through numerous
mechanisms and routes of exposure. We have shown previously
that both in utero and oral exposure are required for the NIMA
effect seen in this highly immunogenic murine heart allograft
model (13). In utero exposure to maternal soluble Ags and cells,
and the establishment of maternal microchimerism, could allow for
NIMA presentation in the thymus, thus allowing for the development of natural TR cells (8, 33, 34). Oral administration of Ag has
previously been shown to expand CD4⫹CD25⫹ TR cells which are
able to suppress DTH reactions (35), and under more physiological
conditions like nursing, it has been shown that oral tolerance is
largely mediated through mucosal induction of adaptive CD4⫹ TR
cells producing IL-10 and TGF-␤ that can suppress the systemic
response to the same Ag (36, 37). Furthermore, the development of
NIMA-specific adaptive TR cells could occur through anterior
chamber-associated immune deviation-like mechanisms (38 – 40).
Like the anterior chamber of the eye, the pregnant uterus is thought
of as an immunologically privileged site (41) containing large
amounts of TGF-␤ (42). It has been hypothesized that drinking
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FIGURE 9. IL-10 vs TGF-␤ staining of cells harvested from the spleen,
ILNs, and heart from a NIMAd-acceptor mouse. BFA was administered i.v.
6 h before harvesting of splenocytes, ILN, and GIC from a NIMAd-acceptor mouse 33 days posttransplant. Cells were stained with Abs against
CD4, IL-10, cell surface TGF-␤, rat IgG2b isotype, and mouse IgG1 isotype. Shown are dot plots of IL-10 and cell surface TGF-␤ staining of
CD4⫹ cells.
The Journal of Immunology
some NIMAd-exposed mice reject their allografts, the exposure to
NIMA still had some imprint on their immune response. This decrease in TE and increase in TR is perhaps why the NIMAd-rejector
mice display delayed rejection and have lower DTH responses
compared with controls. The key differences between the NIMAdacceptor mice and all rejectors appears to lie in the ability to induce adaptive TR cells producing IL-10 and cell surface TGF-␤,
and for these cells to migrate to the graft itself. This increased
homing of adaptive TR cells to peripheral LNs and the graft suggest that the TR cells are suppressing the immune response locally
and depends on a delicate balance between TE and TR cells (47–
49). Natural TR cells gaining access to the graft could set up the
appropriate noninflammatory conditions to promote the peripheral
induction of adaptive TR cells and work in concert as seen in the
NIMAd-acceptor mice.
There are several possible explanations as to why some NIMAdexposed mice experience tolerance to their allograft, and some do
not. First, it is important to mention that although collectively all
NIMAd-exposed mice were able to bystander suppress a TT/DT
response pretransplant, they did so at varying levels, indicating
that not all NIMAd-exposed mice contain the same amounts of
regulation. Second, the condition of the allograft is very important
to the transplant outcome. Differences in the amount of damage to
the donor heart caused by slightly longer ischemia times has been
shown to cause MHC class II hyperexpression and increased production of inflammatory cytokines and chemokines (50 –52). Such
an environment will cause the preferential recruitment of TE over
TR setting up the graft for rejection. The grafts harvested from the
NIMAd-rejector mice had significantly more TE cells present compared with TR cells and such an inflammatory environment could
explain why the NIMAd-rejector mice were unable to mobilize
their natural TR cells from the ILN to the graft itself, thus preventing the induction of adaptive TR cells.
Other possible explanations as to why some NIMAd-exposed
mice experience allograft tolerance and some do not include differences in minor Ag inheritance and/or exposure, and the presence of microchimerism establishment or amount of exposure to
NIMA. Whether tolerance or priming is achieved in early life appears to be highly dependent on the exposure dose. The “switch”
from tolerance to sensitization has been shown to occur in mouse
neonates being exposed to allogeneic cells over a fairly narrow
range (2– 4x) of cell numbers (16, 53). Besides dose, distribution
of maternal cells locally or systemically may be important. Although it is well-established in mouse models that transmission of
maternal cells into the fetus during pregnancy can occur, microchimerism establishment is not universal, typically resulting in
⬃50% of the fetus having detectable maternal microchimerism in
varying locations (7). Thus, it is possible that the different outcomes seen following transplantation may be a reflection of fetal
or neonatal exposure to different numbers, types, and/or location of
maternal cells. We have recently begun to precisely quantitate maternal microchimerism using quantitative PCR. Preliminary results
with this technique shows that 1) there is considerable variation in
the amount of maternal microchimerism present in NIMAd-exposed mice, and 2) there is a correlation of degree of donor-Ag
induced bystander suppression of a recall DTH responses with the
amount of maternal microchimerism present in a given animal (P.
Dutta, M. L. Molitor-Dart, and W. J. Burlingham, manuscript in
preparation).
It is also important to note that besides intrastrain variability,
there is also interstrain variability in the NIMA effect. Using the
same backcross breeding strategy used to create the NIMAd model
described here, we have tested five additional NIMA models using
different strain combinations. So far, only when the tolerizing Ags
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amniotic fluid containing NIMA-bearing maternal cells and soluble Ags together with TGF-␤ and other potential immune modulating factors thereby modulates the APCs in the Peyer’s patches
inducing adaptive TR cells (41).
These NIMA-specific TR cells were not readily detectable pretransplant. In vitro experiments, including ELISPOT, MLR, and
FACS analysis, did not reveal any differences between normal F1
backcross NIMAd-exposed and NIPAd control mice. In a previous
publication, we showed a marked reduction in cytokine production
by NIMAd-exposed T cells in response to allogeneic targets relative to B6 strain mice (13). However, when comparing responses
from NIMAd-exposed splenocytes to their NIPA control counterparts, we no longer see differences pretransplant when stimulated
in an ELISPOT. This parallels the human studies in which no
apparent influence of NIMA exposure on the in vitro alloreactive
T cell repertoire in healthy individuals was found using similar in
vitro assays (43– 45). Matsuoka et al. (14) reported that NIMAd
exposure resulted in a hyporesponsiveness characterized by decreased IFN-␥ in vitro compared with NIPAd controls. In their
MLR assay, specially cultured and pretreated dendritic cells were
cocultured with sorted CD4⫹ responder cells, and under these conditions, a NIMA effect on IFN-␥ production was discovered. Those
conditions were very different from our in vitro culture system, in
which a simple 1:1 ratio of unseparated splenocytes was used,
probably accounting for the differences seen between the two assays. Upon the use of in vivo assays, effects of NIMA exposure
started to become evident, leading us to believe that in vivo reexposure to NIMA might be essential for the induction of NIMAspecific tolerance (46). In in vivo MLR assays, NIMAd-exposed
mice displayed increased cell surface TGF-␤ production compared
with controls. This data is in line with studies from Nakamura et
al. (24) who postulated that membrane-bound TGF-␤ might be
responsible for the inhibitory activity of murine CD4⫹CD25⫹ TR
cells. The fact that NIMAd-exposed mice regulated DTH responses
through TGF-␤ indicates that these cell surface TGF-␤⫹ cells are
likely to be important in the adaptive TR cell response to NIMA in
NIMAd-exposed offspring.
The confirmation of the presence of NIMA-specific TR cells
came when we discovered that the NIMAd-exposed mice exhibited
bystander suppression of a recall DTH response to TT/DT. These
results (bystander suppression in NIMA-exposed but not NIPA
control mice) are clinically relevant, because tolerogenic mismatches could potentially by revealed before renal transplantation,
allowing for better donor selection. Indeed, the mouse studies were
inspired by our initial evidence for pretransplant regulation to maternal but not to paternal alloantigens in patients with end-stage
renal disease, using the trans-vivo DTH assay system (E.
Jankowska-Gan and W. J. Burlingham, manuscript in preparation).
Interestingly, no NIMAd TE DTH response was revealed upon the
addition of neutralizing Abs to cytokines in F1 backcross NIMAdexposed mice indicating that these mice lack NIMA-specific TE
cells. This is in contrast to the post-DST situation, where controls
readily produce a DTH response to BDF1 Ag, while NIMAd-exposed mice do not respond unless anti-TGF-␤ or anti-IL-10 Abs
are coadministered to neutralize the TR cells. This suggests that TE
cell numbers for NIMA are kept at a low level in adult mice, until
rechallenge with a high maternal Ag dose.
Results found posttransplant revealed a clear difference between
the NIMAd exposed and controls. Both cardiac acceptor and rejector NIMAd-exposed mice displayed a decreased production of
IFN-␥ and IL-2 posttransplant compared with controls. Similarly,
both cardiac acceptor and rejector NIMAd-exposed mice had significantly more natural CD4⫹CD25⫹Foxp3⫹ TR cells present in
their spleens and ILNs posttransplant. This means that even though
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Acknowledgments
This work is the subject of Melanie L. Molitor-Dart’s doctoral thesis and
we thank her thesis committee members Drs. Zsuzsa Fabry, James Malter,
Ted Golos, and M. Suresh for helpful comments.
Disclosures
The authors have no financial conflict of interest.
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