Differentiation of regulatory Foxp3?T cells
in the thymic cortex
Adrian Liston*†‡, Katherine M. Nutsch†, Andrew G. Farr§, Jennifer M. Lund†, Jeffery P. Rasmussen†, Pandelakis A. Koni¶,
and Alexander Y. Rudensky†?‡
*John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 0200, Australia;†Departments of
Immunology and§Biological Structure and?Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195; and¶Immunotherapy and
Cancer Centers, Department of Medicine, Medical College of Georgia, Augusta, GA 30913
Edited by Christophe Benoist, Harvard Medical School, Boston, MA, and approved June 6, 2008 (received for review February 14, 2008)
Regulatory Foxp3?T cells (TR) are indispensable for preventing
differentiation T cell receptor (TCR)–ligand interactions within a
certain increased affinity range, in conjunction with ?c-containing
cytokine receptor signals, induce Foxp3 expression and thereby com-
mit developing thymocytes to the TR lineage. The contribution of
distinct MHC class II–expressing accessory cell types to the differen-
tiation process of Foxp3?thymocytes remains controversial, because
a unique role in this process has been ascribed to either thymic
dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC).
Furthermore, it was suggested that the thymic medulla, where the
provides a specialized microenvironment supporting TRdifferentia-
tion. Here, we report that the cortex, as defined by cortical thymic
epithelial cells (cTEC), is sufficient for supporting TRdifferentiation.
MHC class II expression restricted to both cTEC and mTEC or to cTEC
alone did not significantly affect the numbers of Foxp3?thymocytes.
Furthermore, genetic or pharmacologic blockade of thymocyte mi-
gration resulted in a prominent accumulation of Foxp3?thymocytes
in the cortex, demonstrating that secondary signals required for
or tDC do not serve as a cell type singularly responsible for TR
provides an environment suitable for Foxp3 induction. Instead, mul-
tiple accessory cell types probably contribute to the thymic genera-
tion of regulatory Foxp3?T cells.
immune tolerance ? selection ? thymus
peripheral TRcells arise in the thymus, where up-regulation of the
transcription factor Foxp3 is necessary for a subset of thymocytes
to commit to the regulatory T cell lineage (2, 3). Foxp3 functions
by regulating a broad set of genes required for TR suppressor
activity and for proliferative and metabolic fitness (3, 4) and by
repressing alternative T cell differentiation fates (5). TR cells
originate from thymocytes expressing T cell antigen receptors
(6). Although activated Foxp3?TR cells suppress immune re-
sponses in an antigen-nonspecific fashion, induction of the sup-
pressor function by TR cells seems to require antigen-specific
stimulation through their TCR (7). These observations suggest that
TCR specificity for tissue-restricted ‘‘self’’ antigens confers on the
corresponding tissue (8).
Different types of antigen-presenting cells (APC) in the thymus
display distinct repertoires of endogenous peptide–MHC com-
plexes, in part because of differences in proteolytic processing
machinery. For example, cortical thymic epithelial cells (cTEC), a
cell type responsible for the bulk of positive selection but thought
proteinase cathepsin L (CatL) for MHC class II maturation and
I antigen processing (9). In contrast, tDC and medullary thymic
egulatory T cells (TR) are indispensable for suppression of
autoimmunity mediated by self-reactive T cells (1). Most
epithelial cells (mTEC) key APC-mediating negative selection of
self-reactive thymocytes, and peripheral APC rely primarily on
cathepsin S but not on CatL for MHC class II maturation (9).
Importantly, mTEC, but not cTEC or tDC, are capable of express-
ing a broad range of tissue-restricted antigens via a poorly defined
transcriptional mechanism dependent on nuclear factor Aire (10).
This feature of mTEC led to the idea that selection of Foxp3?TR
precursors on tissue-specific self-antigens displayed by mTEC is
requisite for preventing tissue-specific autoimmunity (11). In sup-
port of this idea, thymocytes co-expressing a transgenic TCR
differentiate into Foxp3?TRcells on encounter with its cognate
ligand encoded by a transgene expressed in Aire?mTEC (12).
TRcells do not differentiate solely in response to a certain TCR
14). The insufficiency of the TCR signal alone is demonstrated by
studies in transgenic mice featuring a single TCR specificity and in
and Foxp3?cells in the thymus and in the periphery (15). The
constitutive activation of the ?c-cytokine signaling target Stat5b
(16). A requirement for TCR and an accessory signal suggests that
particular thymic microenvironments or accessory cell types might
be needed to support TR lineage commitment. This issue has
become further complicated by recent studies showing a 2-step
process for TRcommitment, whereby ?c-cytokine signals can be
received after TCR stimulation and still lead to induction of Foxp3
expression (16, 17).
Most Foxp3?thymocytes are localized in the medulla in unma-
nipulated mice (18). Based on these observations and the afore-
mentioned mTEC-driven up-regulation of Foxp3 in TCR-
transgenic TR cells, mTEC were proposed to serve as the key
accessory cell for TRdifferentiation. Alternatively, another recent
in the medulla did not result from reliance on mTEC but rather
from the dense network of tDC. In this model, tDC gain the
capacity to induce expression of Foxp3 in differentiating thymo-
and Foxp3?CD4?T cells with a suppressive capacity were found
(20, 21). A caveat for these studies, however, was that they did not
Author contributions: A.L. and A.Y.R. designed research; A.L., K.M.N., A.G.F., and J.M.L.
performed research; J.P.R. and P.A.K. contributed new reagents/analytic tool; A.L., K.M.N.,
A.G.F., and J.M.L. analyzed data; and A.L. and A.Y.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
‡To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
August 19, 2008 ?
vol. 105 ?
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TRlineage occurred in the cortex; in line with the aforementioned
2-step process of TRdifferentiation, one can envision a scenario in
which TCR stimulation occurs in the cortex, but Foxp3 expression
requires medullary production of cytokines. Additional studies
have demonstrated that, on early expression of a TCR transgene
with a high affinity to self, some double positive (DP) cells can be
these studies did not address the anatomical location of these cells
or whether these observations can be extended to physiological
of TRlineage commitment and the roles of mTEC, cTEC, or tDC
as essential accessory cells in this process remained obscure.
Here, we revisit the issue of the location of TRlineage commit-
ment (i.e., Foxp3 up-regulation) within the thymus to test a model
of a dedicated role for thymic medulla in this process. Our studies
demonstrated the competence of the thymic cortex to provide both
TCR-dependent and TCR-independent signals to facilitate TR
lineage commitment and support the idea that the cortex is a site
of generation of a substantial proportion of Foxp3?thymocytes in
MHC Class II Expression by Thymic Dendritic Cells Is Dispensable for
Foxp3?TR Lineage Commitment. Recent in vitro studies suggested
that tDC exposed to TSLP in the thymic medulla gain the ability to
However, genetic evidence in support of a unique role for TSLP in
TRdifferentiation is lacking, because murine TLSP deficiency does
not result in a detectable reduction in the numbers of Foxp3?
TR-cell deficiency (24). To test the numerical contribution of bone
marrow (BM)-derived APC cells to the generation of the Foxp3?
thymocytes, we transferred MHC class II–sufficient Ab1WT
Foxp3GFPor MHC class II–deficient Ab1nullFoxp3GFPBM into
irradiated RAG-1–deficient recipient mice. Flow cytometric anal-
ysis of MHC class II expression 8–10 weeks after BM transfer
confirmed essentially complete reconstitution of BM-derived APC
by cells of donor origin. Analysis of Ab1nullFoxp3GFPBM-
reconstituted mice showed a characteristic increase in the propor-
tion of single-positive (SP) thymocytes resulting from deficient
a minor reduction in the proportion of Foxp3?cells was observed
(Fig. 1 a and b). However, because of the numerical increase in size
of the SP thymocyte subset, there no was reduction in the absolute
numbers of Foxp3?thymocytes in the absence of MHC class II
expression by BM-derived APC (Fig. 1c). Based on the expression
shown). These results suggested that thymic BM-derived APC,
primarily tDC, are dispensable for thymic differentiation of TR
cells. This conclusion is conditional upon the assumption that the
requirements for thymic TRdifferentiation are similar in irradiated
crossed mice harboring a conditional IAb flx/flxallele with recently
described mice expressing Cre recombinase transgene under the
control of the CD11c promoter to induce an MHC class II deletion
in DC (25, 26). We found essentially complete deletion of MHC
class II in thymic dendritic cells (tDC), but its expression on thymic
epithelial cells and thymic B cells was spared (Fig. 1d; data not
shown). In agreement with the analysis of BM chimeras, we found
a numerical increase in SP numbers in IAb flx/flxCD11c-Cre mice
(Fig. 1e) with a concomitant decrease in the percentage, but not in
the absolute number, of Foxp3?SP (Fig. 1 f and g). Together, these
results indicate that presentation of MHC class II by tDC was
dispensable for Foxp3?TRlineage commitment.
Subset of Foxp3?Thymocytes Localized to the Thymic Cortex. Previ-
ously, cTEC-restricted expression of MHC class II molecules was
(K14) promoter into MHC class II–deficient Abnullmice (K14-??b
pensable for commitment to the Foxp3?lineage or
gain of suppressor function. (a) B6 mice were irradi-
ated and reconstituted with either Foxp3GFPor MHC
class II–deficient Foxp3GFPBM. Eight weeks after re-
constitution, commitment to the Foxp3?lineage was
files are shown (n ? 10,6). (b) Percentages of SP thy-
mocytes that express Foxp3 and (c) absolute number
of Foxp3?SP thymocytes from Foxp3GFP3 B6 and
Ab1nullFoxp3GFP3 B6 chimeras (mean ? standard
CD4 SP thymocytes, (f) the percentage of CD4 SP thy-
ber of Foxp3?SP cells (mean ? standard deviation).
Hemopoietic MHC class II expression is dis-
www.pnas.org?cgi?doi?10.1073?pnas.0801506105 Liston et al.
to support differentiation of CD25?CD4?TRcells (20). However,
the proportion of Foxp3?cells within the thymic and peripheral
CD25?CD4?T cell subset was not determined. A more recent
study used K14-driven transgenes to drive expression of an MHC
class II–bound self-mimicking arthritogenic bovine type I1 collage
(21). However, neither study excluded a reliance on TCR-
independent signals in the thymic medulla for generation of regu-
latory T cells. Furthermore, a recent study suggested that TR
differentiation occurs primarily in the medulla. To assess defini-
tively the ability of the thymic cortex to support the differentiation
of Foxp3?TRcells, we first analyzed the intrathymic distribution of
Foxp3?thymocytes in WT MHC class II–deficient Abnulland
observed (18), in WT mice the highest proportion (?3%) of
Foxp3?thymocytes was found within the CD4 SP subset (Fig. 2 a
and b). By contrast, only rare CD4?CD8?DP cells were Foxp3?,
information (SI) Fig. S1]. In a normal thymus, DP and SP thymo-
cyte subsets exhibit an overwhelmingly cortical and medullary
localization, respectively. Accordingly, Foxp3?cells were frequent
(0.9 Foxp3?cells per 100 ?m2) (Fig. 2c, Table S1). Although
Foxp3?thymocytes were comparatively rare as a proportion of
cortical DP cells, in absolute numbers they amounted to approxi-
mately one third of total Foxp3?thymocytes according to flow
cytometric analysis (Fig. 2d) and one fourth by immunofluores-
cence analysis (Table S1), because of the high absolute number of
DP cells and the greater overall volume of the cortex. The differ-
ence in the proportion of Foxp3?cells detected in the cortex
Foxp3?cells (?25% of the total population) that have migrated to
the medulla as DP cells, whereas the majority are localized in the
The existence of a sizeable population of Foxp3?DP cells
Foxp3?lineage that may coincide with or follow positive selection.
This idea was supported by an analysis of the expression of
phenotypic markers of thymocytes that were being positively se-
lected or already had passed this checkpoint. We found that most
a phenotype that identifies a subset of positively selected DP
thymocytes (Fig. 3 a and b). Following positive selection, DP
thymocytes up-regulate chemokine receptor CCR7, which is essen-
tial for the migration of the postselection transitional DP-SP
necessary for Foxp3?TRcell commitment. Foxp3?TR
cell differentiation was compared in wild type,
Ab1null, and Ab1nullK14-A?btransgenic mice. (a) Rep-
resentative flow cytometric profiles showing wild
type, Ab1null, and Ab1nullK14-A?btransgenic CD4,
CD4?splenocyte populations. (b) Percentage of thy-
mocytes expressing Foxp3 at the DP, early SP, and SP
white bar), and Ab1nullK14-A?btransgenic (n ? 12,
green) in the thymic cortex (CDR1/6C3, blue) and me-
dulla (unlabeled) of wild type (top), Ab1null(middle),
are representative of four experiments. (d) Average
absolute number of thymocytes expressing Foxp3 at
the DP, early SP, and SP stages.
Liston et al.
August 19, 2008 ?
vol. 105 ?
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thymocytes from the thymic cortex to the medulla (28, 29). Al-
though only 0.03% of CCR7locells expressed Foxp3, the frequency
of Foxp3?cells within the CCR7hiDP subset was increased by
?70-fold, reaching a proportion similar to that of Foxp3?cells
within the CD4 SP subset (Fig. 2 c and d). Furthermore, the level
of CCR7 expression by Foxp3?DP thymocytes was as high as that
of SP thymocytes (Fig. 3 e and f). By contrast, the level of CD25 on
DP Foxp3?cells was intermediate between naı ¨ve thymocytes and
SP Foxp3?thymocytes (Fig. 3g). These results suggest that DP
Foxp3?cells in the cortex are enriched within the small fraction of
DP thymocytes that have up-regulated CCR7 enabling their mi-
gration from the cortex to the medulla.
Cortical Microenvironment Is Sufficient for Induction of Foxp3 Expres-
sion in Thymocytes. ThenotionofefficientdifferentiationofFoxp3-
expressing thymocytes in the cortex was seemingly at odds with the
observation that in both K14-A?bAb1nullmice with the cTEC-
restricted MHC class II expression and in WT mice most Foxp3?
thymocytes were found in the medulla (Fig. 2). A possible expla-
nation for this discrepancy was that the up-regulation of Foxp3 in
thymocytes in K14-A?bAb1nullmice was induced in the cortex, but
thereafter these Foxp3?thymocytes migrated rapidly to the me-
dulla. This migration would produce a scenario similar to the step
of DP-SP differentiation in positive selection, which is known to
occur in the cortex, but SP thymocytes localize exclusively in the
medulla because of the tight coupling of differentiation and CCR7
up-regulation (28, 29). Alternatively, in a model analogous to
negative selection, the up-regulation of Foxp3 in thymocytes in
K14-A?bAb1nullmice might occur in 2 steps, in which TCR-MHC
interactions in the cortex are required but are not sufficient to
induce Foxp3 expression, and in which the medullary microenvi-
ronment is required to ‘‘complete’’ the process initiated in the
cortex by providing a required second signal (17).
To distinguish formally between these 2 models, we inhibited G
protein–coupled receptor signaling including chemokine receptors
at a concentration capable of blocking the migration of newly
treated with PT showed no increase in Foxp3?DP cells (Fig. 4a)
but had a dramatic increase in the number of Foxp3?thymocytes
in the cortex (Fig. 4b), indicating that the transition of late DP to
the Foxp3?SP thymocytes was not impaired in the presence of PT.
Thus, these results demonstrate that the second step of the sug-
gested 2-step TRcell differentiation process (16, 17) is not limited
to the medulla.
It was thought likely that PT treatment inhibits the migration of
newly developing SP cells to the medulla through the inhibition of
CCR7 signaling. To test this notion, we examined the localization
of Foxp3?thymocytes to the thymic cortex and medulla in CCR7-
in the cortex (Fig. 4c). To exclude potential effects of CCR7
deficiency on thymic epithelial cells and to examine the cortico-
medullary distribution of CCR7-deficient and -sufficient Foxp3?
thymocytes in the same environment, we generated a series of BM
chimeras. Specifically, irradiated Rag1nullrecipients were reconsti-
or with their mixture at a 1:1 ratio. This combination of BM donors
allowed discrimination between Ccr7wtand Ccr7?/?thymocytes by
flow cytometric analysis of Ly5.1 and Ly5.2 expression. Immuno-
fluorescence analysis of Foxp3 and GFP expression distinguished
between WT Foxp3?cells (Foxp3?GFP?) and Ccr7?/?Foxp3?
cells (Foxp3?GFP?). Although Ccr7?/?thymocytes exhibit dimin-
ished migration to the medulla (28, 29), flow cytometric analysis
revealed overall normal development of Ly5.1 and Ly5.2 thymo-
cytes and a comparable size of Foxp3?thymocyte subsets origi-
nating from WT and Ccr7?/?BM (Fig. 4c). To validate our
approach, we next examined the presence of Foxp3- and GFP-
expressing cells in control chimeric mice reconstituted with only
and GFP antibody staining was essentially overlapping, whereas in
the latter Foxp3?cells were present, but GFP?cells were not (Fig.
4e). Similar analysis of GFP and Foxp3 immunofluorescence of
thymuses in the mixed BM chimeras revealed that the rates of
Ccr7?/?Foxp3?cells in the cortex were 10-fold higher than the
rates of Ccr7wtFoxp3?cells (Fig. 4 e and f). Together, the analyses
of K14-AbbAbnullmice, PT-treated mice, and Ccr7?/?mice dem-
onstrated that the cortex is sufficient, whereas the medulla is
dispensable for differentiation of Foxp3?thymocytes and that
predominant medullary localization of Foxp3?thymocytes proba-
bly results from rapid CCR7-dependent migration following Foxp3
Our observation that cortical Foxp3?DP cells represent one
fourth of all Foxp3?thymocytes (Table S2) and express high levels
of CD69 and CCR7 is consistent with the idea that in WT animals
Foxp3?DP thymocytes also can differentiate into Foxp3?SP
thymocytes and contribute substantially to the Foxp3?TRpopula-
tion. Previous studies of the differentiation of Foxp3?thymocytes
in neonates demonstrated that Foxp3 induction is delayed signifi-
reconstitution of an irradiated thymus (Fig. S2). Because this time
frame is much longer than the DP or SP thymic dwell time,
of three experiments. (c) Proportion of Foxp3?cells among CCR7?DP cells (Left)
and CCR7?DP cells (Right). (d) Percentage of CCR7?DP and CCR7?DP cells that
are Foxp3?(mean ? standard deviation, n ? 5). (e) Representative histograms
DP cells (solid gray area), Foxp3?DP cells (black line), Foxp3?SP cells (blue line),
SP (blue line), Foxp3?SP (black line), and Foxp3?DP (solid gray area) cells
(representative of n ? 10).
DP Foxp3?cells are postselection. (a) Foxp3?DP cells (line) compared
www.pnas.org?cgi?doi?10.1073?pnas.0801506105Liston et al.
reconstitution of an irradiated thymus cannot be used to determine
populations. To establish the temporal relationship between the
appearance of Foxp3-expressing DP and SP thymocyte subsets, we
monitored the kinetics of their homeostatic regeneration following
ablation in Foxp3DTRmice. These knockin mice harbor ‘‘ablatable’’
thymic and peripheral Foxp3?T cell populations because of the
expression of a human diphtheria toxin receptor (DTR)-GFP
fusion protein (1). In heterozygous female Foxp3wt/DTR-GFPmice,
random X chromosome inactivation leads to generation of 2
subset expressing DTR and, therefore, sensitive to diphtheria toxin
(DT)-induced ablation, and a GFP?Foxp3?TR subset lacking
DTR which is resistant. In agreement with our previous report,
Foxp3wt/DTR-GFPmice lost thymic and peripheral GFP?Foxp3?TR
cells within 48 h of DT treatment, but these mice were fully
protected from immune-mediated inflammation by DT-resistant
GFP?Foxp3?TRcells because the expression of T cell activation
markers or cytokine production remained unchanged (data not
shown). Interestingly, we observed restoration of normal numbers
of DP GFP?Foxp3?cells 1 day after cessation of DT treatment,
and the SP Foxp3?subset was fully restored to its original size 2 to
3 days later (Fig. 5). The rebound was not caused by a niche-filling
mechanism, because compensation does not occur in Foxp3?/?
notion that Foxp3?DP thymocytes make a substantial numerical
contribution to the Foxp3?SP subset.
To test the precursor–product relationship between Foxp3?DP
and SP directly, we exploited a Foxp3 reporter enabling magnetic
treated with two doses of DT at day ?1 and day 0 and traced for reconstitution
of GFP?cells. (b) Mean ? standard deviation (n ? 3, 4, 4, 4, 3, 2) for the absolute
DT at day ?1 and at ?2 h. Short and long dashes indicate numbers of SP and DP
GFP?cells, respectively, in uninjected mice. (c) Expression of CD8 (Left) and
Foxp3Thy1.1(Right) on Ly5.1?Ly5.2-thymocytes (shaded) and purified Ly5.2 DP
Foxp3Thy1.1?thymocytes 18 h after intrathymic injection into Lys5.1 mice.
does not impede Foxp3 commitment. The thy-
muses of PT-treated mice and CCR7-deficient
mice (representative sections, n ? 6). (b) Thymic
sections from untreated and PT-treated mice
stained for Foxp3 (green) and CDR1/6C3 (cortex,
blue) (representative sections, n ? 6). (c) Thymic
sections from wild type and Ccr?/?mice stained
Number of Foxp3?thymocytes in Ly5.1 Foxp3GFP
BM, Ly5.1 Foxp3GFP? Ly5.2 Ccr7?/?mixed BM,
and Ly5.2 Ccr7?/?BM chimeras, for Ly5.1 (wild
type, black bar) and Ly5.2 (Ccr7?/?, white bar)
cells, corrected for degree of BM chimerism. (e)
Number of Foxp3?cells in the cortex of Ly5.1
Foxp3GFPBM, Ly5.1 Foxp3GFP? Ly5.2 Ccr7?/?
Foxp3 (Left) or GFP (Right). n ? 5. (f) Thymic
sections from Ly5.1 Foxp3GFP? Ly5.2 Ccr7?/?
mixed BM chimeras, stained for CDR1/6C3 (cor-
tex, blue) and either GFP (green; left) or Foxp3
(green; right) (representative sections, n ? 5).
Liston et al.
August 19, 2008 ?
vol. 105 ?
no. 33 ?
bead enrichment of Foxp3?CD25lowDP thymocytes. Thy1.1 was Download full-text
expressed on the surface of Foxp3?TRcells on insertion of the
corresponding DNA sequence equipped with an internal ribosome
entry site into the 3? UTR of the Foxp3 sequence (Fig. S4a).
We found tight coexpression of Foxp3 and the Thy1.1 reporter
in both the thymus and peripheral tissue (Fig. S4 b–d). The
combination of anti-Thy1.1 bead enrichment followed by FACS
sorting allowed efficient purification of DP Foxp3Thy1.1?cells
(?90%) (Fig. S4e). Sorted DP Foxp3Thy1.1?cells were injected into
congenic Ly5.1 mice and 18 h later were found to have progressed
to CD4?CD8?Foxp3Thy1.1?SP thymocytes, in agreement with a
precursor–product relationship between DP Foxp3?cells and SP
Foxp3?cells. Taken together, our results strongly suggest that DP
Foxp3?cells differentiate into SP Foxp3?thymocytes.
Contrary to models ascribing a dedicated role for the thymic
medulla, and for mTEC or tDC in particular, in inducing Foxp3
expression and, therefore, TR lineage commitment (12, 19), we
found the thymic cortex to be fully capable of supporting TR
differentiation. Indeed, a very modest decrease in the overall
number of Foxp3?thymocytes was observed when MHC class II
as cortical DP thymocytes. Because the expression of CD69 and
CCR7Foxp3?was increased in DP thymocytes, it seems likely that
the TCR signaling leading to Foxp3 up-regulation in DP thymo-
cytes was either coincident with or subsequent to positive selection.
Although these results show that the thymic cortex is sufficient
for Foxp3 induction, they by no means argue against the ability of
medulla and MHC class II?tDC and mTEC to support differen-
TCR transgenic thymocytes are able to commit to the Foxp3?
lineage on direct presentation of the transgene-encoded HA anti-
ability of temporally discrete signals to induce Foxp3 (16, 17) also
raise the possibility that some DP cells are primed through TCR
signaling in the cortex and become Foxp3?only upon later TCR-
independent stimulation as SP cells in the medulla. Therefore, it is
likely that Foxp3 induction is not limited to a single anatomical
location and that multiple APC types including cTEC, mTEC, and
tDC are able to support the generation of Foxp3?thymocytes and
contribute to the peripheral TRcell pool in normal animals. These
findings raise a question about the TCR specificity of TR cells
selected by different APC types and their potency in preventing
require the development of new genetic models .
Materials and Methods
Mice. Foxp3GFP(18), Foxp3KO(2), Foxp3GFP-DTR(1), Foxp3Thy1.1, CD11c-Cre (25),
the B6 background. Foxp3GFPmice also were used on the B6.Ly5.1 background.
BM chimeras were constructed using 7 ? 106BM cells per recipient, injected i.v.
of PT administered i.p. 2.5 days before analysis. Intrathymic injection was per-
formed on mice under tribromoethanol anesthesia. Thymocytes for intrathymic
injection were enriched with MACS using anti-Thy1.1 with the magnetic acti-
vated cell sorting LS column system (Miltenyi Biotec) followed by FACS of
(20 ?l/lobe) and extracted for analysis at 18 h. Experimental mice were age- and
dance with guidelines from the Institutional Animal Care Committee of the
University of Washington.
Flow Cytometry and Immunofluorescence. Five to 10-week-old mice were ana-
lyzed using the following antibodies: CD4-PerCP (PharMingen), CD8-PE-Cy7,
CD25-PE, CD69-PE, Ly5.1-PE, Ly5.2-APC, MHC class II-APC, Foxp3-APC, and CCR7-
APC (eBioscience). For CCR7 staining, cells were incubated for 60 min at 37°C
before staining. For tDC staining, the thymus was minced and treated with 2
with 5 mM EDTA/5% FBS/HBSS for 5 min at 37°C.
Thymic sections were prepared and stained as described in ref. 18, using
polyclonal IgG anti-Foxp3 antibodies (2), polyclonal anti-GFP antibodies (Rock-
land), and anti-CDR1/6C3 (cortex). Estimations of cortex: medulla ratios were
performed by analysis of serial sections (every 10th section through the thymus)
using immunohistochemical staining with ER-TR5 supernatant. Estimations of
frequency of cortical and medullary Foxp3?cells were performed by immuno-
sections (every 25th section).
ACKNOWLEDGMENTS. We thank A. Chervonsky for providing CD11c-Cre mice,
by grants from the National Institutes of Health and the Juvenile Diabetes
Health and Medical Research Council, and the Menzies Foundation. A.G.F. is
supported by National Institute of Allergy and Infectious Diseases AI059575 and
AI024137. A.Y.R. is a Howard Hughes Medical Institute investigator.
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