of June 12, 2013.
This information is current as
Alloantigen-Specific Foxp3+ Regulatory T
Peripheral Development of
Lymph Node Occupancy Is Required for the
Ding, Sergio A. Lira, Nancy R. Krieger and Jonathan S.
Garin, Yansui Li, Peter Boros, Jaime Llodra, Yaozhong
Jordi C. Ochando, Adam C. Yopp, Yu Yang, Alexandre
2005; 174:6993-7005; ;
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The Journal of Immunology
by guest on June 12, 2013
Lymph Node Occupancy Is Required for the Peripheral
Development of Alloantigen-Specific Foxp3?Regulatory
Jordi C. Ochando,2* Adam C. Yopp,* Yu Yang,* Alexandre Garin,‡Yansui Li,†Peter Boros,†
Jaime Llodra,* Yaozhong Ding,*†Sergio A. Lira,‡Nancy R. Krieger,3†and
Jonathan S. Bromberg2*†
We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance
induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regu-
latory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-
specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion,
in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4?CD25?CD62Lhigh
T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing
conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent
immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the
regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early
allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-
independent T cell accumulation in the LNs, restored CD4?CD25?Treg in the LNs along with graft survival. These data suggest
that alloantigen-specific Foxp3?CD4?CD25?Treg develop and are required within the LNs during tolerization, and provide
compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance. The Journal of Im-
munology, 2005, 174: 6993–7005.
transplantation tolerance, in the absence of chronic immunosup-
pression, can be induced by a number of different regimens (1–4).
It is still not entirely clear by which mechanisms these regimens
induce tolerance (5), although there is increasing evidence that
tolerization protocols favor the development of regulatory or sup-
pressor T cells (6, 7), and that tolerization strategies must be di-
rected to induce, expand, or manipulate regulatory suppressive T
cells (8, 9). The most extensively studied regulatory cells are the
naturally occurring CD4?CD25?T regulatory cells (Treg)4gen-
erated in the thymus, constituting 5–10% of peripheral CD4?T
cells in mice (10). The transcription factor Foxp3 represents a
unique marker involved in the development and function of Treg
and is specifically expressed in CD4?CD25?suppressor T cells
he major goal of transplantation is the creation of clini-
cally applicable protocols to induce alloantigen-specific
tolerance. Long-term graft survival and donor-specific
(11–13). This population of suppressor T cells is thought to play an
essential protective role during tolerization to alloantigen, and
presence of Treg in transplanted recipients is tightly associated
withindefinite graft survival
CD4?CD25?Treg favors the production of alloantibodies (15)
and graft rejection (16).
Although many studies have been conducted to define the cel-
lular and molecular mechanisms by which Treg develop (17–19),
there is little knowledge about the anatomic compartments where
Foxp3-expressing Treg are activated, expanded, or display sup-
pressor function for tolerization to ensue. Distinct lymphoid and
nonlymphoid compartments may be differentially involved in the
development of Treg, because the lymphoid environment plays
essential and diverse roles in either rejection or tolerance (20).
Lymphoid architecture also plays an important role in Treg devel-
opment and differentiation, because differences in lymphoid origin
have been used to classify two distinct subsets of Treg cells: nat-
ural (thymus) vs adaptive (periphery), according to structural site
of generation (21). Naturally occurring CD4?CD25?Treg are
generated as a result of multiple selection events during T cell
development within the thymus (18, 22). Evidence for extrathymic
CD4?CD25?T cell development supports an alternative de novo
pathway for the origin of Treg in the periphery (23), and indicates
that CD4?CD25?Treg generated in the periphery are probably
different from thymically produced CD25?CD4?Treg (21).
Therefore, it is likely that particular anatomic sites provide specific
milieus that allow tolerization to occur by permitting suppressor
Treg to be activated, expanded, or function in the periphery. Thus,
defining the Foxp3-expressing CD4?CD25?T cell population in
distinct anatomic compartments may elucidate the precise sites
where tolerization takes place.
*Department of Gene and Cell Medicine,†Recanati/Miller Transplantation Institute,
and‡Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029
Received for publication November 15, 2004. Accepted for publication March
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.
1This work was supported by National Institutes of Health Grants R01 AI41428,
AI62765, and AI44929 (to J.S.B.).
2Address correspondence and reprint requests to Dr. Jonathan S. Bromberg or Dr.
Jordi C. Ochando, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box
1104, New York, NY 10029-6574. E-mail address: firstname.lastname@example.org
3Current address: Bristol-Myers Squibb, Princeton, NJ 08543.
4Abbreviations used in this paper: Treg, T regulatory cell; LN, lymph node; DST,
donor-specific transfusion; SI, stimulation index; TMN, total mononuclear cells.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
by guest on June 12, 2013
We previously demonstrated that CD62L-mediated lymph node
(LN) homing is necessary for the induction of anti-CD2 plus anti-
CD3 mAb-induced tolerance, because coadministration of anti-
CD62L mAbs prevents both LN homing and tolerance, in both
nonvascularized and vascularized cardiac transplant models (24).
Adoptive transfer of CD62L?/?T cells and the use of CD62L?/?
recipients provided complementary genetic evidence to confirm
the importance of CD62L and LN occupancy by T cells in toler-
ization. In this report, we further characterized these observations
by studying the CD4?CD25?Treg population in distinct anatomic
domains after treatment with two different tolerization protocols
(3, 4), and after manipulating T cell LN homing with anti-CD62L
mAb and the sphingosine 1-phosophate receptor modulator
FTY720 (reviewed in Ref. 25). Our results demonstrate that al-
loantigen-specific Treg occupancy in the LNs of tolerant animals is
necessary for immunological tolerance to vascularized cardiac al-
lografts. Alloantigen-specific Treg were expanded in recipient
LNs, but not other peripheral sites, under both tolerization proto-
cols. Anti-CD62L mAbs prevented T cell LN homing, resulting in
abrogation of both Treg expansion in the LNs or other lymphoid
areas and graft survival, whereas FTY720-driven T cell LN se-
questration restored both LN Treg expansion and graft survival.
Together, the results suggest an essential role of the LN for the
development and function of CD4?CD25?Treg in tolerant
Materials and Methods
BALB/c (H-2d), C57BL/6 (H-2b), and CBA (H-2k) mice, 8–10 wk of age,
were purchased from The Jackson Laboratory, and used as donors, recip-
ients, or third-party controls. All mice were housed in a specific pathogen-
free facility in microisolator cages. All experiments were performed with
age- and sex-matched mice in accordance with the Institutional Animal
Care and Utilization Committee-approved criteria and protocols.
The 145-2C11 hamster anti-murine CD3? hybridoma was a gift from J.
Bluestone (University of California, San Francisco, CA), and the 12-15 rat
IgG1 anti-murine CD2 hybridoma was a gift from P. Altevogt (Immunol-
ogy and Genetics Institute, Heidelberg, Germany). The MEL-14 rat IgG2a
anti-murine CD62L hybridoma was purchased from the American Type
Culture Collection. The hybridomas were grown in culture, and superna-
tants were purified over protein G or A columns (Amersham Biosciences).
For cell surface phenotype analysis, cells were stained with CyChrome-
anti-CD4, FITC-anti-CD25, allophycocyanin-anti-CD62L, biotin-anti-
CD45RB, PE-anti-CD44, or PE-anti-CD69 (BD Pharmingen). Anti-
CD40L (MR-1) was from Bender MedSystems. FTY720 was a gift from V.
Brinkman (Novartis, Basel, Switzerland) and was dissolved in water at 0.1
Vascularized cardiac transplantation
BALB/c hearts were transplanted as fully vascularized heterotopic grafts
into C57BL/6 as described (26). Recipients received i.v. injections of dif-
ferent Abs in 0.5 ml of PBS at the indicated times. For the anti-CD2/anti-
CD3 tolerization protocol, recipients received 100 ?g of anti-CD2 mAbs
on days 0 and 1, and 100 ?g of anti-CD3 mAbs on days 2, 3, 4, 5, and 10
with respect to transplantation. Anti-CD62L was administered at 100 ?g on
days 0 and 1. For the donor-specific transfusion (DST) and anti-CD40L
mAb protocol, recipients received DST (107donor splenocytes i.v. 7 days
before transplant) and 250 ?g of anti-CD40L mAbs on days ?7, ?4, 0,
and ?4. One group of mice received no additional treatment, whereas the
other group received 100 ?g of anti-CD62L mAbs on days ?7 and ?6.
FTY720 was administered daily via oral gavage. Graft function was mon-
itored every other day by abdominal palpation, and rejection was defined
as complete cessation of a palpable beat and was confirmed by direct vi-
sualization at laparotomy. Recipients with grafts surviving ?100 days were
defined as tolerant and were used for in vitro experiments. Untreated re-
cipients rejected allograft at days 8–10 and were used as controls. Anti-
CD62L mAb-treated animals rejected at 28–39 days.
Mice were sacrificed at the indicated times, and LNs, spleens, and cardiac
allografts were removed and gently dissociated into single-cell suspen-
sions. RBCs were removed by Tris-NH4Cl lysis. Cells were placed in com-
plete RPMI medium (RPMI 1640 supplemented with 10% FCS, 1 mM
sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 ?g/ml
streptomycin, 1? nonessential amino acids, and 2 ? 10?5M 2-ME). Lym-
phocytes were isolated from peripheral blood using Lympholyte Mammal
density separation medium (Cedarlane). Homogenized heart tissue was
treated with 5% Collagenase Type II (Worthington) for 30 min at 37°C
before lymphocyte isolation.
Purification of cell subsets
CD4?T cell subsets were isolated from LN and spleen using the Mouse T
Cell CD4 Subset Column kit (R&D Systems) according to the manufac-
turer’s protocol, and the purity of the enriched CD4?cells ranged between
85 and 90%. Afterward, the enriched CD4?cells were stained with
CyChrome-anti-CD4 mAbs and FITC-anti-CD25 mAbs, and sorted into
CD4?CD25?cells and CD4?CD25?cells using MoFlo (DakoCytoma-
tion). The purity of the sorted cells was ?98%.
MLR and suppressor assay
A total of 2 ? 105responder CD4?CD25?T cells from different groups
was cocultured in triplicate with 5 ? 1051500-rad gamma-irradiated
BALB/c splenocytes that had been T cell depleted by negative selection
using Mouse Pan T Dynabeads according to the manufacturer’s protocol
(Dynal). Cells were cultured in 96-well plates for 3 days at 37°C in a
humidified atmosphere of 5% CO2. Eighteen hours before the termination
of the culture, the wells were pulsed with 1 ?Ci of [3H]thymidine, and
incorporation was quantified with a scintillation counter. Results are ex-
pressed as stimulation index (SI), determined from mean of triplicate de-
terminations ? SEM. Alternatively, CD4?CD25?or CD4?CD25?T cells
from tolerant animals were also assessed for T cell function in MLR. The
experiment was conducted as described above. To test the suppressive
properties of the CD4?CD25?T cells from tolerant animals, 4 ? 104
freshly isolated responder CD4?CD25?T cells from naive mice were
stimulated with 1 ? 105irradiated allogeneic T-depleted splenocytes for 3
days, along with 2 ? 104CD4?CD25?T cells from different groups. The
experiment was conducted as described above.
Cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C.
Flow cytometric analysis was performed on LSR II (BD Biosciences) and
analyzed with FlowJo (Tree Star). Results are expressed as percentage of
cells staining above background, and mAbs were titered at regular intervals
during the course of these studies to ensure that saturating concentrations
Total cellular RNA was extracted from 5 ? 105CD4?CD25?T cells using
TRIzol reagent (Invitrogen Life Technologies) digested with RNase-free
DNase I (Invitrogen Life Technologies), and reverse-transcribed into
cDNA using Sensiscript RT kit (Qiagen). Foxp3 random primers (Invitro-
gen Life Technologies) according to the manufacturer’s protocol, and
mRNA levels were quantified by real-time PCR using QuantiTect SYBR
Green PCR kit (Qiagen) with the LightCycler (Roche). PCR consisted of
a 15-min 95°C denaturation step followed by 45 cycles of 15 s at 94°C,
20 s at 56°C, and 15 s at 72°C. Primers were as follows: Foxp3, 5?-CCC
AGG AAA GAC AGC AAC CTT-3? and 5?-TTC TCA CAA CCA GGC
CAC TTG-3?; cyclophilin A, 5?-AGG GTG GTG ACT TTA CAC GC-3?
and 5?-ATC CAG CCA TTC AGT CTT GG-3?. Normalized values for
Foxp3 mRNA expression were calculated as the relative quantity of Foxp3
divided by the relative quantity of cyclophilin A. All samples were run in
triplicate. For CCR2, CCR5, and CCR7, total RNA was extracted using the
RNeasy Maxi kit (Qiagen) according to the manufacturer’s instructions.
Reverse transcription was performed from 2 ?g of RNA. Quantitative real-
time PCR was conducted in duplicate from 25 ng of cDNA with 0.4 ?M
each primer in a 30-?l final reaction volume of 1? SYBR Green PCR
Master Mix (Applied Biosystems). PCR cycling conditions were as follows:
50°C for 2 min, 95°C for 15 min and 40 cycles of 95°C for 15 s, and 60°C for
1 min. Relative expression levels were calculated as 2(Ct ubiquitin RNA ? Ct gene)
(for details see ABI PRISM 7700, User Bulletin No. 2) using ubiquitin RNA
as an endogenous control. The following were used: CCR2-forward, GTT
TAG TC; CCR5-forward, TTG CAA ACG GTG TTC AAT TTT C; CCR5-
reverse, TCT CCT GTG GAT CGG GTA TAG AC; CCR7-forward, CAC
6994 LN Treg IN TRANSPLANT TOLERANCE
by guest on June 12, 2013
GCT GAG ATG CTC ACT GG; CCR7-reverse, CCA TCT GGG CCA CTT
GGA; ubiquitin-forward, TGG CTA TTA ATT ATT CGG TCT GCA T;
ubiquitin-reverse, GCA AGT GGC TAG AGT GCA GAG TAA.
Total cellular RNA was extracted from 5 ? 105cells using TRIzol reagent
(Invitrogen Life Technologies) digested with RNase-free DNase I (Invitro-
gen Life Technologies), and reverse-transcribed into cDNA using Sensis-
cript RT kit (Qiagen) and random primers (Invitrogen Life Technologies)
according to the manufacturer’s protocol. Foxp3 mRNA levels were quan-
tified by RT-PCR (Applied Biosystems). PCR consisted of a 15-min 95°C
denaturation step followed by 35 cycles of 1 min at 94°C, 2 min at 56°C,
and 2 min at 72°C. Foxp3 primers were as follows: 5?-CCC AGG AAA
GAC AGC AAC CTT-3? and 5?-TTC TCA CAA CCA GGC CAC TTG-3?.
All samples were run in triplicate.
For B220 and Thy1.2, LNs and spleens were harvested, subdivided, frozen
directly in OCT (Fisher), and stored at ?80°C in preparation for immu-
nological studies. Sections of 8 ?m were cut using a Leica 1900CM cryo-
microtome, fixed, and mounted with Gel/Mount (Biomeda) in polylysine-
coated slides. FITC-anti-B220and
purchased from BD Pharmingen and were developed by HRP-conjugated
rabbit anti-FITC (DakoCytomation), and by alkaline phosphatase-conju-
gated streptavidin (Zymed). For H&E sections, LNs, spleens, and grafts
were fixed, embedded in paraffin, and cut into 10-?m-thick sections using
a Finesse Microtome (Shandon).
Fresh tissue was harvested, subdivided, frozen directly in OCT (Fisher),
and stored at ?80°C. Sections of 8 ?m were cut using a Leica 1900CM
cryomicrotome. Endogenous peroxidase, FcRs, and biotin binding sites
were blocked as previously described (27). Biotinylated rat anti-mouse
CD4 (H129.19) and biotinylated rat IgG2a, were purchased from BD
Pharmingen. Anti-FOXP3 rabbit anti-sera were a gift from Novus Biologi-
cals. The Cy3-conjugated donkey anti-rabbit IgG, and 7-amino-4-methyl-
coumarin-3-acetic acid-streptavidin were purchased from Jackson Immu-
noResearch.All slideswere mounted
Laboratories) to preserve fluorescence. Images were acquired using a Leica
DMRA2 fluorescence microscope and a digital Hamamatsu close circuit
device camera. Separate green, red, and blue images were collected and
analyzed with Openlab software (Improvision). Captured image layers
were sliced to standard density values, and total cell numbers per square
millimeter of tissue were measured as single independent intensity objects
with the Openlab cell measurement module. Twelve-bit grayscale objects
with an area ?0.01 U were ignored. Final image processing was performed
using Volocity software (Improvision).
For graft survival, one-way ANOVA was performed. For CD4?CD25?T
cell growth, t test and F test were performed to compare variances. For cell
proliferation, one-way ANOVA was performed at each time point. The
group effects were all significant at p ? 0.05. To examine individual dif-
ferences, comparison between every pair of groups was performed. For
Foxp3?T cell counts and chemokine receptor gene expression, one-way
ANOVA and Dunnett test were performed, to examine individual differ-
ences compared with the naive control.
LN occupancy is required to induce tolerance to vascularized
We have previously shown that lymphocyte LN homing and lo-
calization are required for anti-CD2 plus anti-CD3 mAb-induced
tolerance to nonvascularized and vascularized heart allografts (24).
We next investigated whether this finding held true using another
well-defined tolerizing regimen, DST plus anti-CD40L mAb (4).
BALB/c donor vascularized cardiac grafts were transplanted into
C57BL/6 recipients, which received either anti-CD2 plus anti-CD3
mAbs, DST plus anti-CD40L mAbs, or were left untreated. Both
tolerance protocols induce long-term graft survival with normal
histology in vascularized allografts (3, 28) (Fig. 1A). Third-party
CBA (H-2k), but not donor strain BALB/c (H-2d), second-set al-
lografts placed ?60 days following the initial transplant were re-
jected (Refs. 29 and 30, and data not shown), demonstrating donor
alloantigen-specific tolerance. Administration of anti-CD62L mAb
prevented allograft survival in animals given DST plus anti-
CD40L mAb (Fig. 1B). Coadministration of anti-CD62L mAb de-
creased mean survival time from ?70.6 ? 13.2 to 28.0 ? 6.8 days
in the anti-CD2 plus anti-CD3 mAb-treated group, and from
?88.2 ? 1.8 to 39.6 ? 0.9 days in the DST plus anti-CD40L
mAb-treated group (Fig. 1B). These results demonstrate that both
the anti-CD2 plus anti-CD3 mAb and the DST plus anti-CD40L
mAb tolerogenic regimens require CD62L-dependent LN homing
to induce prolonged survival and tolerance in a vascularized car-
diac allograft model. Furthermore, administration of FTY720 to
anti-CD62L mAb plus tolerogen-treated mice (Fig. 1C) restored
graft survival with normal allograft histology, and supports the
hypothesis that LN occupancy is required for tolerance.
Intact lymphoid architecture is necessary for long-term allograft
To further determine the significant role of distinct anatomic com-
partments during tolerance and acute rejection, we examined the
structure of lymphoid organs and grafts of the tolerized anti-CD2
plus anti-CD3 mAb-treated, rejecting anti-CD62L mAb-treated,
tolerized FTY720-treated, and rejecting untreated animals (Fig. 2).
Allografts, spleens, and LNs (inguinal, axillary, and paraortic; n ?
3 in each group) were harvested from tolerant recipients at ?100
days following transplantation, from rejecting recipients on days
8–10 at the time of acute rejection, and from naive controls. Tissue
sections were stained with H&E for light microscopy, and for
Thy1.2 and B220 for staining of T and B cells, respectively. The
allografts from the acutely rejecting and anti-CD62L mAb-treated
recipients have significant interstitial infiltrates with severe myo-
cardial necrosis and disrupted architecture, consistent with graft
failure due to acute cellular rejection. In contrast, the allografts
from the tolerogen-treated and FTY720-treated groups contain
minimal or no cellular infiltrates, with no evidence of vascular
intimal hyperplasia. In untreated and rejecting anti-CD62L mAb-
treated mice, the LNs are expanded with disrupted germinal center
architecture, with scattered T and B cell populations forming dis-
persed groups of large cells, indicative of an unmodified rejection
response. On the contrary, the LN architecture in mice treated with
the tolerogenic regimen or FTY720 is similar to that of naive mice,
with preserved structure containing small lymphocytes and dis-
cernible T cell areas with little dispersion of the B cell population.
Furthermore, FTY720 treatment alone in naive or transplanted an-
imals had no effect in tissue architecture (data not shown). Com-
pared with naive mice, the spleen of the acutely rejecting groups
maintains organized architecture, with well-defined white and red
splenic pulp areas. In addition, the splenic white pulp is expanded
containing markedly enlarged lymphocytes and germinal centers.
In contrast, the splenic architecture of the mice treated with the
tolerogenic regimens is disorganized, with a significant decrease in
size of the splenic white pulp. These results suggest not only that
the systemic lymphoid organs to which lymphocytes migrate are
important determinants of rejection vs tolerance, but also that the
histologic organization of microdomains are key factors regulating
CD4?CD25?T cells expand in the LN during tolerization
The immunohistochemistry showed differences in lymphoid struc-
ture and organization among the rejecting and tolerant groups. To
characterize the relationship of lymphoid compartment occupancy
and structure during tolerization, we further examined whether
these alterations were associated with a shift in the distribution of
6995 The Journal of Immunology
by guest on June 12, 2013
CD4?CD25?T cells in the blood, LN, spleen, and grafts of tol-
erant and rejecting mice. Total mononuclear cells (TMN), CD4?T
cells, and CD4?CD25?T cells were examined in various lym-
phoid and nonlymphoid organs of the tolerized anti-CD2 plus anti-
CD3 mAbs, and DST plus anti-CD40L mAb-treated mice; and
compared with untreated rejecting mice at different time points.
Fig. 3A shows that the TMN cell number decreases in the LN of
rejecting mice, whereas there is a progressive increase of LN cells
in the tolerant mice. In contrast, the number of mononuclear cells
increases sharply in the spleens of the rejecting recipients while
increasing only slightly in the tolerogen-treated group. Therefore,
the tolerogen-treated group has a higher number and percentage of
total lymphocytes in the LN compared with the rejecting group. As
expected, the graft has an increase in mononuclear cells in the
rejecting animals, whereas the tolerant group does not. The blood
TMN counts do not show significant differences among any of the
treatment groups. Paralleling the results of the TMN cells, the total
CD4?T cell population decreases in the LN and graft, but in-
creases over time in the spleen and blood during rejection. Con-
versely, during the establishment of tolerance, the CD4?T cells
increase in the LN and graft, but remain the same in the spleen and
blood. Focusing further on the putative Treg subset, the
CD4?CD25?T cell population in the rejecting animals is low in
all compartments, and decreases further as rejection ensues.
allograft survival after transplantation into C57BL/6 (H-2b). Recipients were injected i.v. with 100 ?g of anti-CD2 mAbs on days 0 and 1 and 100 ?g
anti-CD3 mAbs on days 2, 3, 4, 5, and 10 for tolerance (f), or 1 ? 107donor splenocytes i.v. 7 days before transplant, and 250 ?g of anti-CD40L mAbs
on days ?7, ?4, 0, and ?4 (?). Control rejecting mice received hamster Ig in PBS (E) (n ? 6 for each group). Rejection of cardiac allografts was
determined by cessation of heartbeat. B, Mice were treated as in A, along with 100 ?g of anti-CD62L mAb administered on days 0 and 1 in the anti-CD2
plus anti-CD3 mAbs group, or on days ?7 and ?6 in the DST plus anti-CD40L mAb group. C, Mice were treated as in B plus FTY720 administered orally
via gavage at 0.1 mg/kg/day. Representative allograft images of H&E staining of the above groups are shown. Tolerant animals ?100 days; untreated
control recipients on day 10 (time of acute rejection); rejecting anti-CD62L mAb-treated animals, 28.0 ? 6.8 days in the anti-CD2 plus anti-CD3 mAbs,
and 39.6 ? 0.9 days in the DST plus anti-CD40L mAb-treated group; and tolerant FTY720-treated animals ?100 days. Magnification, ?100.
LN occupancy is required to induce tolerance to vascularized cardiac transplants. A, Fully mismatched vascularized BALB/c (H-2d) cardiac
6996 LN Treg IN TRANSPLANT TOLERANCE
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CD4?CD25?T cells increase in the LNs of tolerized recipients,
which is significant by day 30, and persists for ?100 days, indi-
cating that it takes time for the CD4?CD25?T cells to develop.
There is also a less significant increase in the CD4?CD25?T cells
in the grafts of tolerant recipients, accompanied by an overall de-
crease in these cells in the spleen. More detailed analysis of these
cells in Fig. 3B shows that the CD4?CD25?T cell population
increases dramatically in the LNs of tolerant animals, eventually
constituting ?50% of the total CD4?T cell population, compared
with 5–10% of CD4?LN cells in naive or rejecting mice. The
CD4?CD25?T cell population also increases in the grafts of tol-
erant mice, although less dramatically, constituting ?20% of the
total CD4?T cell population. Fig. 3B also shows an increase in the
CD4?CD25?LN cell population to similar levels (21–23%) in
both the tolerant and rejecting groups, indicating that other cell
subtypes arealso activated.
CD4?CD25?cell population increases in the graft during rejec-
tion, when compared with tolerant or naive animals.
As expected, theactivated
LN CD4?CD25?T cells from tolerant animals express a
Because the results in Fig. 3 suggest that Treg might be generated
and/or expanded in the LNs, it was important to further character-
ize these T cells and, in particular, to distinguish them from acti-
vated effector T cells. Because the CD25 molecule is a surface
marker for either activated or suppressor CD4?T cells, we there-
fore analyzed in parallel additional cell surface markers expressed
on CD4?CD25?T cells that delineate the regulatory phenotype.
Activated T cells express high levels of CD44, CD69, and CD25,
and low levels of CD45RB; naive T cells express low levels of
CD44 and CD69 and high levels of CD62L and CD45RB; memory
T cells express low levels of CD45RB and high levels of CD44;
and Treg express high levels of CD25 and low levels of CD45RB
(31–35). T cell activation is followed by CD69 up-regulation and
gated CD4?CD25?T cells from the LNs of rejecting mice show
markedly increased CD69 expression compared with groups re-
ceiving the tolerogenic regimen or naive animals (Fig. 4A, first
panel). CD62L mediates homing of leukocytes to the LNs through
binding to ligands on high endothelial venules, and it is shed after
T cell activation. Fig. 4A (second panel) indicates that LN
CD4?CD25?T cells from tolerant and naive animals continue to
express high levels of CD62L, whereas LN CD4?CD25?T cells
from rejecting animalshave
CD45RBhighis expressed in Ag-inexperienced T cells, whereas
CD45RBlowis expressed in Ag-experienced T cells (activated/
memory), and transition from the naive to the Ag-experienced state
is accompanied by down-regulation of CD45RB. CD45RB was
expressed at high levels on most CD4?CD25?T cells from naive
mice and at low levels on most CD4?CD25?T cells from both the
tolerant and rejecting animals (Fig. 4A, third panel). CD44 is also
a marker for T cell activation, but in contrast with CD69, it was
highly expressed on CD4?CD25?T cells from both tolerant and
rejecting animals (Fig. 4A, fourth panel). Taken together, these
results indicate that the expanded CD4?CD25?T cell population
in the LNs of both groups of tolerogen-treated animals express a
distinct phenotype compared with the CD4?CD25?T cells in the
rejecting group. The results suggest that the majority of the
CD4?CD25?T cells from all groups are Ag experienced
(CD44highCD45RBlow), but that T cells from the tolerogen-treated
groups are only partially activated (CD62LhighCD69low) in com-
parison to T cells from the rejecting group, which are more fully
We next assessed Foxp3 expression to confirm further the reg-
ulatory phenotype of the CD4?CD25?LN T cells. As shown in
Fig. 4B, CD4?CD25?T cells from tolerant animals up-regulated
the expression of Foxp3 transcripts in the LNs but not the spleen.
Assuming a relatively constant level of transcript per Treg cell,
thequantitative real-time PCR
CD4?CD25?Foxp3?Treg are 4- to 10-fold more frequent in tol-
erant compared with naive or rejecting groups, consistent with the
flow cytometry data (Fig. 3). Fluorescent immunohistochemistry
was performed to enumerate the number of CD4?Foxp3?cells.
These results are also consistent with the real-time PCR and flow
cytometry, and indicate that Foxp3?cells are more frequent in the
LNs, but not the spleen, of tolerant animals (Fig. 4C). Because the
results in Fig. 3A suggest that Treg might be generated in the LNs
vival is associated with intact lym-
phoid architecture. Animals trans-
planted as in Fig. 1 and tissues
harvested on days 8–10 for untreated
rejected, day ?100 for anti-CD2 plus
anti-CD3-tolerant, day 28 for anti-
CD2 plus anti-CD3 plus anti-CD62L-
rejected, and day ?100 for anti-CD2
plus anti-CD3 plus anti-CD62L plus
FTY720-tolerant animals. H&E stain-
ing, Heart, spleen, and LN. Immuno-
histochemical staining for T (Thy1.2,
blue) and B (B220, brown) lympho-
cytes. A total of five sections per tis-
sue were performed from each mouse
(n ? 3 animals/group). Magnification,
6997 The Journal of Immunology
by guest on June 12, 2013
over time, we further characterized the Foxp3 expression of
CD4?CD25?T cells at different time points. Fig. 4D shows that,
by day 5 after transplantation, Foxp3 was increased in
CD4?CD25?T cells from tolerogen-treated animals compared
with untreated rejecting animals. The results also suggest a sub-
stantial increase in the Foxp3 expression of CD4?CD25?LN T
cells from tolerant animals by day 30, indicating that it takes time
for the CD4?CD25?T cells to develop.
The Foxp3?CD4?CD25?LN T cells are anergic and
The results demonstrate differences in CD4?CD25?T cell distri-
bution in distinct lymphoid and nonlymphoid compartments, and
phenotypic variations within the LNs of anti-CD2 plus anti-CD3
mAbs or DST plus anti-CD40L mAb-treated recipients, compared
with rejecting control or naive mice. In vitro proliferation assays
were next performed to prove that CD4?CD25?T cells possess
CD4?CD25?T cells from tolerant LNs exhibited ?4-fold more
The resultsshow that
potent suppression of the MLR when compared with tolerant
CD4?CD25?LN or splenic T cells from the naive or rejecting
groups (Fig. 5A). Further characterization of the CD4?CD25?T
cells shows that the suppressive activity is alloantigen specific
when evaluated ?100 days after transplantation (Fig. 5B). Inter-
estingly, at earlier time points, CD4?CD25?Treg suppressive ac-
tivity is alloantigen specific for LN-derived, but not splenic Treg.
This indicates that Treg may expand first in the LN, and then
migrate to different lymphoid and nonlymphoid organs, because
Fig. 3A shows a significant increase in CD4?CD25?LN T cells by
day 30. Additionally, we analyzed the MLR response of freshly
isolated Treg, along with CD4?CD25?and CD4?CD25?T cells
from tolerant LNs (Fig. 5C). These latter cells constitute ?15 and
?22%, respectively, of the total LN population from tolerant an-
imals (Fig. 3B). The results demonstrate that, as expected, the
CD4?CD25?T cells are anergic, whereas isolated CD4?CD25?
and CD4?CD25?(mostly CD8?, not shown) T cells from the
tolerant animals are alloreactive. When these latter subsets are
T cells in LNs is associated with long-
term allograft survival. A, TMN, total
CD4?T cells, and total CD4?CD25?T
cells are shown. Five mice per group
were sacrificed on days 5, 10, and 30,
and ?100 days after vascularized car-
diac transplantation in the anti-CD2 plus
anti-CD3 mAb-treated group (?), and
DST plus anti-CD40L (?); and from
untreated control recipients (E) on days
5 and 10 (time of acute rejection).
Splenocytes, LNs (aortic, axillary, cer-
vical, and mesenteric
mouse)), graft-infiltrating lymphocytes,
and blood (1 ml/group) were harvested
and analyzed by cell counts and flow cy-
tometry. B, Phenotypic analysis of the
CD4?CD25?T cell population from un-
treated mice, tolerant animals, and re-
jecting animals (8–10 days). Separate
treatment groups are noted in the figure.
Doses and regimens as in Fig. 1. Results
represent mean of the values ? SEM.
Three sets of experiments were per-
formed at each time point (??, p ? 0.01
by t and F test).
Increase in CD4?CD25?
6998LN Treg IN TRANSPLANT TOLERANCE
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examined using five-color flow cytometry for the cell surface markers CD69, CD62L, CD45RB, and CD44 in untreated mice rejecting mice (f) (8–10
days), anti-CD2 plus anti-CD3 (solid line), or DST plus anti-CD40 mAbs (gray line) tolerant mice (?100 days), and naive animals (u). Histograms are
representative of three independent studies. B, Foxp3 expression is up-regulated in the LNs of tolerant animals (?100 days) as measured by real-time
RT-PCR from freshly isolated CD4?CD25?T cells, when compared with naive or rejecting mice. Results representative of three independent studies are
shown (?, p ? 0.05; ??, p ? 0.01 by unpaired Student’s t test compared with naive control). C, Fluorescent immunohistochemistry of LN and spleen from
different groups, showing CD4?T cells (CD4–7-amino-4-methylcoumarin-3-acetic acid, blue) and Foxp3-expressing cells (Foxp3-Cy3, red) as merged
(pink) images. The optical thickness of the image is 8 ?m. Magnification, ?400. Numbers represent number of Foxp3?CD4?cells per square millimeter
(?, p ? 0.01 by Dunnett test compared with naive control). D, Foxp3 expression of CD4?CD25?T cells over time measured by RT-PCR.
CD4?CD25?T cells in the LN of tolerant animals express a regulatory phenotype. A, The gated CD4?CD25?T cells from Fig. 3B were
6999The Journal of Immunology
by guest on June 12, 2013
stimulated in the presence of the CD4?CD25?Foxp3?T cells, the
proliferative response is suppressed (Fig. 5C). These results dem-
onstrate that the CD4?CD25?Treg actively suppress alloreactive
T cells, which have not been deleted, in the tolerant state.
Treg LN occupancy is required for tolerance
Because we previously demonstrated that T cell LN occupancy is
required to induce alloantigen-specific tolerance (24), we hypoth-
esized that Treg development within the LNs of tolerant animals is
necessary for tolerance to ensue. We administered anti-CD62L
mAb, which alters the distribution of T cells among secondary
lymphoid organs (36) by inhibiting LN homing, alters the second-
ary lymphoid organ architecture (Fig. 2), and prevented allograft
survival in animals receiving either anti-CD2 plus anti-CD3 or
DST plus anti-CD40L mAbs (Fig. 1B). Because anti-CD62L mAb
administration prevented tolerance in both groups, we examined
whether this treatment also prevented the development or accu-
mulation of Treg within the LN. The results in Fig. 6A when com-
pared with those in Fig. 3B show that the CD4?T cell population
is markedly reduced in the LN, with a simultaneous decrease in
CD4?CD25?T cells, after treatment with anti-CD62L mAbs.
Remarkably, whereas the total CD4?T cell population increases
in the spleen, there is no concomitant increase in the CD4?CD25?
splenic T cell population, suggesting that Treg failed to develop or
expand in this compartment. The rejected grafts also accumulated
CD4?T cells and activated CD25?cells. Taken together, the
CD4?CD25?T cells from anti-CD2 plus anti-CD3 mAb (?), or DST plus anti-CD40L mAb (?) (?100 days), untreated rejecting (E) (8–10 days), and
naive animals (‚). CD4?CD25?effector T cells were cocultured at indicated ratios with CD4?CD25?suppressor T cells and irradiated BALB/c T
cell-depleted splenocytes. Results are expressed as SI, determined from mean of triplicate determinations ? SEM. Three sets of experiments were
performed at each time point. B, Alloantigen specificity of the CD4?CD25?T cells from tolerant animals at different indicated time points. Freshly isolated
LN and splenic CD4?CD25?T cells from naive and tolerant animals were cultured with naive CD4?CD25?(ratio 1:4), and with either alloantigen-specific
BALB/c or third-party CBA T cell-depleted stimulator splenocytes. Results are expressed as SI, determined from mean of triplicate determinations ? SEM (??,
p ? 0.01 by one-way ANOVA). C, The CD4?CD25?T cell population inhibits in vitro proliferation of other alloreactive populations. CD4?CD25?,
CD4?CD25?, and CD4?CD25?T cells from ?100-day tolerant animals (2 ? 105) were cultured with alloantigen-specific BALB/c T cell-depleted stimulator
splenocytes (5 ? 105). MLR results are expressed as SI, determined from mean of triplicate determinations ? SEM (??, p ? 0.01 by one-way ANOVA).
Foxp3?CD4?CD25?T cells in the LNs of tolerant animals suppress alloreactive cells. A, Suppressor function of LN and splenic
7000 LN Treg IN TRANSPLANT TOLERANCE
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results indicate that after anti-CD62L mAb treatment, CD4?T
cells fail to enter or accumulate in the LNs. Furthermore,
CD4?CD25?Treg are absent in recipient LNs and other periph-
eral lymphoid and nonlymphoid sites, even though tolerogenic
treatment had been administered, and rejection rapidly ensues.
Phenotypic analyses of the CD4?CD25?T cells within the LNs of
anti-CD62L mAb-treated recipients reveal an activated CD44high
CD45RBlowCD62LlowCD69highphenotype, rather than a regula-
tory CD62LhighCD69lowone (Fig. 6B). Further evidence for the
absence of Treg development in the periphery of these recipients is
the relative lack of Foxp3 expression in the CD4?CD25?T cells
from LNs and spleens, measured by both fluorescent immunohis-
tochemistry and quantitative real-time RT-PCR (Fig. 6, C and D).
Enforced lymphoid homing restores LN Treg and graft survival
To further address the requirement for Foxp3-expressing CD4?
CD25?Treg within the LNs of tolerant animals, we hypothe-
sized that if CD4?T cells could be driven back into the LNs,
group at time of rejection. B, Five-color flow cytometry for the cell surface markers CD69, CD62L, CD45RB, and CD44 of the gated CD4?CD25?T cells.
Histograms are representative of three independent studies. C and D, Foxp3 expression in CD4?CD25?T cells from the LNs and spleens of anti-CD62L
mAb-treated animals, measured by quantitative real-time RT-PCR and fluorescent immunohistochemistry (?, p ? 0.01 by Dunnett test compared with naive
Treg LN occupancy is required for tolerance maintenance. A, Flow cytometric examination of the CD4?CD25?T cell population from each
7001 The Journal of Immunology
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this could restore the presence of an expanded Treg population
in the LN and graft survival. FTY720 is a sphingosine lipid with
agonist and antagonist properties on sphingosine 1-phosphate
receptors, that drives T cells into LNs in a CD62L-independent
manner (24, 37). When FTY720 was administered to rejecting
anti-CD62L-treated animals, the CD4?CD25?T cell popula-
tion was restored to the LNs of FTY720-treated animals (Fig.
7A compared with Fig. 6A). Analysis of cell surface phenotype
(Fig. 7B) and Foxp3 expression (C and D) of the CD4?CD25?
T cell populations demonstratedthat these cellsare
CD44highCD45RBlowCD62LhighCD69lowand Foxp3?, suggest-
ing that CD4?CD25?Treg developed and/or expanded in the
LNs of tolerized animals, and are necessary for the induction
and maintenance of tolerance.
CD4?CD25?T cells express high levels of LN-homing
chemokine receptors during tolerance
To address the mechanisms by which Treg may develop within the
LNs of tolerant animals, we studied the chemokine receptor ex-
pression in the CD4?CD25?T cells from the distinct animal
each different group ?100 days after transplantation. B, Five-color flow cytometry for the cell surface markers CD69, CD62L, CD45RB, and CD44 of the
gated CD4?CD25?T cells. Histograms are representative of three independent experiments. C and D, Foxp3 expression is up-regulated in CD4?CD25?
T cells from the LN of the FTY720-treated animals, measured by quantitative real-time RT-PCR (??, p ? 0.01 by one-way ANOVA) and fluorescent
immunohistochemistry (?, p ? 0.01 by Dunnett test compared with naive control).
FTY720 restores graft survival and lymphoid Treg occupancy. A, Flow cytometric examination of the CD4?CD25?T cell population from
7002 LN Treg IN TRANSPLANT TOLERANCE
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groups. Table I shows that higher levels of the LN homing receptor
CCR7 are expressed in the LN CD4?CD25?T cells after treat-
ment with DST plus anti-CD40L, when compared with naive or
rejecting mice. Addition of anti-CD62L, which abrogates toler-
ance, resulted in a significant decrease in the LN CD4?CD25?T
CCR7 levels; an increase in the CCR5, expressed in activated
cells; and no significant differences in CCR2 levels. Furthermore,
addition of FTY720 re-established high levels of CCR7 RNA in
LN CD4?CD25?T cells, increased CCR2, and decreased CCR5
in a fashion similar to the tolerant group. Together, the data sug-
gest active recruitment of T cells to the LNs during tolerance via
CCR7 and CCR2 but not CCR5. Further genetic evidence support-
ing these results is shown in Fig. 8, where CCR2 knockout or
paucity of LN T cells mice, lacking the expression of CCR7 li-
gands CCL21 and CCL19, rejected their allografts in both un-
treated recipients or after treatment with DST plus CD40L.
We report for the first time that alloantigen-specific Foxp3?Treg
accumulate in vivo in the LNs of tolerant animals as a result of
either expansion of naturally occurring Treg and/or development
of de novo Treg. Treg accumulating in the LN under two distinct
tolerogenic treatments (anti-CD2 plus anti-CD3 mAbs, DST plus
anti-CD40L mAb) meet many of the criteria established for their
CD69low, Ag experienced, partially activated, anergic, suppressive,
the induction and maintenance of tolerance, because interfering with
T cell LN occupancy with anti-CD62L mAb prevents localization of
Treg to the LN and tolerance, whereas sphingosine 1-phosphate re-
ceptor modulation restores both Treg presence in lymphoid tissues
and tolerance. These observations suggest that Treg occupancy of the
LN, but not the spleen, is a generalized phenomenon in the establish-
ment of tolerance, and reveals the peripheral sites where Treg develop
in vivo in response to alloantigen, and where they likely exercise
functional effector activities.
Several laboratories, including our own (38–42), have reported
the induction of transplantation tolerance using a variety of distinct
approaches that interfere with signals 1, 2, and/or 3. The mecha-
nisms that prevent graft rejection following these therapies have
not been fully characterized, although evidence for anergy, igno-
rance, deletion, immune deviation, and immune regulation has
been reported (43–47). Recent studies have also implicated Treg in
tolerance induction (7, 48) and have shown that CD4?CD25?
Treg can also develop in the periphery through thymus-indepen-
dent pathways (48, 49). To further address the role of Treg in
tolerance, we induced tolerance to vascularized cardiac transplants
by using two distinct tolerant protocols, and characterized the
CD4?CD25?T cell population in distinct anatomic compartments
at different time points. In the anti-CD40L plus DST model, other
investigators have provided evidence that anergy, activation-in-
duced cell death, immune deviation, and Treg contribute to the
induction and maintenance of tolerance (29). In the anti-CD2 plus
anti-CD3 mAb model, graft survival and tolerance were related to
mechanisms that included partial T cell activation, anergy, im-
mune deviation, and partial T cell depletion (3, 30). In this report,
we now provide evidence that both of these tolerance protocols
also require the activity of Foxp3?CD4?CD25?Treg and that
these cells must be localized to the LNs but not the spleen.
Table I. CD4?CD25?T cell chemokine receptor expression by PCRa
Anti-CD2 ? Anti-CD3
? Anti-CD62L (day 30)
Anti-CD2 ? Anti-CD3
? Anti-CD62L ?
FTY720 (day 30)
Anti-CD2 ? Anti-CD3
? Anti-CD62L ?
FTY720 (day ?100)
26 ? 0.7
0.0 ? 0.0
0.0 ? 0.0
164 ? 3?
0.2 ? 0.1
0.0 ? 0.0
353 ? 1?
18 ? 10
118 ? 38?
17 ? 4
52 ? 22
1938 ? 35??
0.0 ? 0.0??
0.4 ? 0.2
164 ? 11?
105 ? 4?
0.0 ? 0.0
0.0 ? 0.0
29 ? 5
62 ? 16??
38 ? 0.1
5 ? 1
0.0 ? 0.0
0.0 ? 0.0
88 ? 3?
2 ? 0.2
1.2 ? 0.1
27 ? 1
51 ? 42
464 ? 159?
2 ? 1
11 ? 3
1080 ? 136?
0.0 ? 0.0
0.0 ? 0.0
87 ? 7
0.0 ? 0.0
0.7 ? 0.3
0.0 ? 0.0
0.3 ? 0.1
3 ? 0.2
1.3 ? 0.3
aExperiments were performed in duplicate (n ? 3). Results are expressed as mean ? SEM. One-way ANOVA to compare samples against the naive: ??, p ? 0.01, ?, p ? 0.05.
tolerance. Fully mismatched vascularized BALB/c (H-2d) cardiac allograft
survival after transplantation into CCR2 (A) or plt (B) knockout (KO)
C57BL/6 (H-2b). Recipients were injected i.v. with 1 ? 107donor splenocytes
i.v. 7 days before transplant, and 250 ?g of anti-CD40L mAbs on days ?7,
?4, 0, and ?4. Control rejecting mice received hamster Ig in PBS (n ? 2 for
each group). Rejection of cardiac allografts was determined by cessation of
Genetic evidence that lymphoid occupancy is required for
7003The Journal of Immunology
by guest on June 12, 2013
Evidence from a variety of autoimmune studies suggests that the
LN is a critical site for Ag presentation that determines either
priming or tolerization (21). These models suggest that natural
Treg expand in the pancreatic LN as a consequence of exposure to
self-Ag, and failure to generate CD4?CD25?T cells correlates
with accelerated diabetes progression in the nonobese diabetic
mouse (50, 51). The increase in CD4?CD25?T cells occurs in the
draining LN, but not the spleen or other peripheral LNs (6), sup-
porting the idea that distinct anatomic sites play different roles in
autoimmunity. Transplant studies are significantly different from
the autoimmune model in many respects. With regard to the role of
secondary lymphoid organs, recent reports demonstrated that al-
loantigen is presented not in a localized fashion, but systemically
in all recipient lymphoid organs very soon after allografting (52,
53). As a result, multiple lymphoid tissues participate simulta-
neously in alloantigen presentation, which makes it more difficult
to define the role of each secondary lymphoid organ and its mi-
crodomains in transplant rejection or tolerance. Our results here
show at the structural level, that the LN architecture in the toler-
ized animals is preserved, and is similar to that of naive mice,
whereas in the spleen the architecture is significantly altered (Fig.
2). Conversely, the architecture in the spleen of the acutely rejecting
untreated mice is preserved, but is disorganized in the LNs. Thus, as
ture where lymphocytes encounter and interact with alloantigen under
the influence of systemic immunosuppression are critical determi-
nants for the development of either immunity or tolerance. In partic-
ular, LNs are important for actively supporting tolerization.
Secondary lymphoid tissues have a highly organized architec-
ture, regulating complex T cell-APC interactions (54, 55) that de-
termine the development of either rejection or tolerance (20). Lak-
kis et al. (20) reported that asplenic Hox11?/?mice reject cardiac
and skin allografts, whereas splenectomized aly/aly mice that lack
all secondary lymphoid organs do not reject either skin or vascu-
larized heart allografts. These results were interpreted as showing
that secondary lymphoid organs are required for priming and ini-
tiation of an alloresponse, and indicate that the spleen is not ab-
solutely necessary for rejection, as long as other lymphoid tissues
are intact. Aly/aly mice reject heart but not skin grafts, arguing that
it is the draining lymphoid tissues that are responsible for proper
Ag presentation and T cell priming; and that the designation of
“draining” is dependent on the type of allograft, its anatomic lo-
cation, and the surgical manipulation required for its placement. In
contrast, Zhou et al. (56) showed that splenectomized lympho-
toxin-? and lymphotoxin-? receptor gene knockout mice, that also
lack secondary lymphoid organs, reject skin and heart allografts,
demonstrating that secondary lymphoid organs are not always nec-
essary for initiating the alloimmune response. These contradictory
results on the role of secondary lymphoid organs in transplant
rejection may relate to the use of genetically altered mice, which
have immunologic and developmental differences due to both gene
knockout and background strain variations. We also suggest that
experimental manipulations not only cause differences in T cell
priming that lead to graft rejection, but also cause differences in T
cell priming that lead to Treg development, with subsequent effects
on suppressive and tolerogenic responses to alloantigen. There-
fore, the study of lymphoid organs and domains that are respon-
sible for priming and rejection must now be coupled to studies that
analyze these same domains for the induction or maintenance of
tolerance (57). This conclusion is supported by a recent report that
shows that autoimmune diabetes is the result of the balance be-
tween effector and regulatory T cells in the pancreatic LN, and that
evaluation of only a single T cell subset in the LN may lead to
incomplete conclusions (58).
Finally, the mechanisms by which Treg accumulate or develop
in the LN of tolerant animals may be related to chemokine com-
partmentalization. Table I shows that CCR7 is up-regulated in LN
CD4?CD25?T cells during tolerance, but not during rejection,
which suggests that CD4?T cells may migrate to the LNs where
they further develop during tolerance. Therefore, it is possible that
under the cover of immunosuppressive therapy such as anti-CD2
plus anti-CD3 mAb or DST plus anti-CD40L mAb, CD4?T cells
home to the LNs, where altered TCR signal transduction, partial T
cell activation, and/or low-affinity Ag binding in the context of
persistent allo- and tissue-specific Ag exposure, favor the devel-
opment of Treg. Furthermore, chemokine-dependent LN T cell
migration may represent a mechanism directly responsible for pre-
venting CD4?T cell-mediated rejection. Ho ¨pken et al. (59) re-
cently described a key role for CCR7 in alloreactive T cell priming
within the LNs, and specific localization of Treg within the lym-
phoid compartment may interfere with successful effector T cell
priming that leads to graft rejection. This implies that anti-CD62L-
treated T cells become primed to alloantigen, yet are not subject to
tolerogenic influences because of their inability to home to the
LNs, whereas FTY720 induces homing and colocalization of both
effector and Treg cells to the LN.
We conclude that, whereas alloantigen can prime effector T cells
either in the LNs or the spleen to initiate an immune response that
leads to graft rejection, Treg can only be activated and/or expanded
in an alloantigen-dependent manner in the LNs under systemic
tolerization protocols. Our results reveal the location where thy-
mic-derived CD4?CD25?Treg may expand, and/or where CD4?
T cells may develop de novo into alloantigen-specific Treg in the
peripheral lymphoid environment. CD4?CD25?T cells localized
near the high endothelial venule conduits within the LN microen-
vironment may help to maintain a tolerant environment by regu-
lating multiple interactions between naive T cells with APC. This
finding may help to unmask the factors that promote the differentia-
tion and activation of regulatory suppressor cells and allow the gen-
eration of extrathymic Treg for the control of immune responses.
We acknowledge the technical contributions of Minwei Mao, Dan Chen,
Italas George, and Patricia Rebollo, and the helpful discussion with Dr.
The authors have no financial conflict of interest.
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