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J. Exp. Med. Vol. 207 No. 8 1701-1711
CD4+ T cells recognize and respond to peptide
antigens in the context of MHCII. The nature of
TCR–peptide MHC (pMHC) interactions de-
termines the stimulation threshold for positive
and negative selection of T cells in the thymus,
and it has also been shown to influence the lin-
eage decisions of the developing cells (Singer
et al., 2008). For example, strong TCR signals
have been proposed to guide double-positive
thymocytes toward the CD4 fate (Itano et al.,
1996). In peripheral T cells, the potency of TCR
ligand can have a profound effect on the extent
of activation; higher affinity TCR–pMHC in-
teractions generally lead to increased signaling
downstream of the TCR and, subsequently,
more robust proliferation and cytokine produc-
tion (Davis et al., 1998; Germain and Stefanová,
1999). In addition to influencing the magnitude
of the T cell response, the potency and density
of pMHC affinity may also instruct CD4+
helper differentiation (Constant et al., 1995;
Hosken et al., 1995; Tao et al., 1997; Rogers and
Regulatory T cell (T reg cell) differentiation
and function is also dependent on TCR stimula-
tion (Josefowicz and Rudensky, 2009; Shevach,
2009). T reg cells are defined by their expression
of the winged helix/forkhead transcription fac-
tor forkhead box p3 (Foxp3) and have been
shown to suppress both pathological and healthy
immune responses (Sakaguchi, 2004; Fontenot
and Rudensky, 2005; Belkaid, 2007). Foxp3 is
required for the development, maintenance, and
suppressive function of these cells, as indicated
by the multiorgan autoimmunity resulting from
its loss of function in both mice and humans
(Fontenot et al., 2003; Hori et al., 2003; Khattri
et al., 2003; Gavin et al., 2007; Zheng and
Rudensky, 2007). Foxp3+ T reg cells can be di-
vided into two categories based on their site of
origin: thymic T reg cells and induced T reg
cells, which leave the thymus as naive CD4+
Foxp3-negative T cells but then acquire Foxp3
expression and suppressor function in the pe-
riphery (Curotto de Lafaille and Lafaille, 2009).
The requirement for TCR stimulation in
the thymic development of T reg cell is illus-
trated by the failure of TCR transgenic T cells
to express Foxp3 in the absence of endogenous
James P. Allison:
Abbreviations used: Foxp3,
forkhead box p3; MCC,
moth cytochrome c; pMHC,
TCR ligand density and affinity determine
peripheral induction of Foxp3 in vivo
Rachel A. Gottschalk,1,3 Emily Corse,1 and James P. Allison1,2
1Department of Immunology, Howard Hughes Medical Institute, and 2Ludwig Center for Cancer Immunotherapy,
Memorial Sloan-Kettering Cancer Center, New York, NY 10021
3Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, New York, NY 10021
T cell receptor (TCR) ligation is required for the extrathymic differentiation of forkhead box
p3+ (Foxp3+) regulatory T cells. Several lines of evidence indicate that weak TCR stimulation
favors induction of Foxp3 in the periphery; however, it remains to be determined how TCR
ligand potency influences this process. We characterized the density and affinity of TCR ligand
favorable for Foxp3 induction and found that a low dose of a strong agonist resulted in
maximal induction of Foxp3 in vivo. Initial Foxp3 induction by weak agonist peptide could be
enhanced by disruption of TCR–peptide major histocompatibility complex (pMHC) interactions
or alteration of peptide dose. However, time course experiments revealed that Foxp3-positive
cells induced by weak agonist stimulation are deleted, along with their Foxp3-negative coun-
terparts, whereas Foxp3-positive cells induced by low doses of the strong agonist persist. Our
results suggest that, together, pMHC ligand potency, density, and duration of TCR interactions
define a cumulative quantity of TCR stimulation that determines initial peripheral Foxp3
induction. However, in the persistence of induced Foxp3+ T cells, TCR ligand potency and
density are noninterchangeable factors that influence the route to peripheral tolerance.
© 2010 Gottschalk et al. This article is distributed under the terms of an
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The Journal of Experimental Medicine
TCR ligand density and affinity in Foxp3 induction | Gottschalk et al.
peptides in our system. However, Foxp3+ cells induced by the
weak agonist peptides did not persist compared with those
generated by the higher affinity ligand. Our data suggest that it
is the cumulative level of TCR stimulation that determines
initial induction of Foxp3 in the periphery but that TCR li-
gand density and potency are noninterchangeable factors in the
persistence of the induced Foxp3+ population.
There is an inverse relationship between TCR/pMHC affinity
and optimal peptide concentration for Foxp3 induction in vitro
To explore the impact of TCR/pMHC affinity on peripheral
Foxp3 induction we used 5C.C7 T cells, which recognize a
peptide from moth cytochrome c (MCC; 88–103) in the con-
text of the MHCII molecule I-Ek (Davis et al., 1998). The
5C.C7 TCR is ideal for addressing this question because, in
addition to the natural ligand MCC, a wide variety of related
peptide ligands have been characterized for their TCR binding
properties and in vitro activating potencies (Reay et al., 1994;
Rabinowitz et al., 1996; Wilson et al., 1999; Li et al., 2004;
Krogsgaard et al., 2005). In addition to MCC, we assessed
Foxp3 induction by the weak agonist peptide 102S (Reay
et al., 1994; Rabinowitz et al., 1996) and the superagonist K5
(Krogsgaard et al., 2003, 2005; Li et al., 2004), both of which
TCR rearrangement, unless their cognate antigen is present
(Olivares-Villagómez et al., 1998; Itoh et al., 1999; Jordan
et al., 2001; Apostolou et al., 2002; Kawahata et al., 2002).
The selection of T reg cells upon encounter of transgenically
expressed neo-autoantigens suggests that TCR specificity for
self could play a role in T reg cell development, which is con-
sistent with a study demonstrating that T reg cell TCRs are
more self-reactive than their non–T reg cell counterparts
(Hsieh et al., 2004). Thymic T reg cell selection may be associ-
ated with relatively strong TCR stimulation because thymo-
cytes expressing a TCR more weakly stimulated by its antigen
were not selected to be T reg cells (Jordan et al., 2001).
Another study implicated superior survival of Foxp3+ thymo-
cytes in contributing to the increased frequency of T reg cells
observed in TCR transgenic systems where the cognate anti-
gen was expressed (van Santen et al., 2004). Differences in the
strength of TCR–pMHC interactions could determine T reg
cell selection versus deletion of self-reactive thymocytes. That
TCRs preferentially used by T reg cells in wild-type mice are
also present in the repertoires of Foxp3-deficient mice is con-
sistent with the notion that these TCR–self-pMHC interac-
tions fall between the avidity ranges resulting in positive and
negative selection (Hsieh et al., 2006).
TCR specificity has also been implicated in Foxp3 expres-
sion by induced T reg cells (Lathrop et al., 2008). Stimulation
of adoptively transferred TCR transgenic T cells demonstrated
that peripheral Foxp3 induction is associated with subop-
timal activation and inversely correlates with proliferation
(Kretschmer et al., 2005). Consistent with these in vivo find-
ings, more recent in vitro studies have suggested a mechanism
by which extensive TCR stimulation is detrimental for the
generation of induced T reg cells; constitutive or prolonged
signaling through the Akt–PI3K–mTor pathway, which is
downstream of the TCR, antagonizes the induction of Foxp3
(Haxhinasto et al., 2008; Sauer et al., 2008). Another possible
explanation for the negative impact of robust TCR signaling
on induced T reg cell generation is cell cycle–dependent main-
tenance of a silenced state of the Foxp3 locus (Josefowicz et al.,
2009). Collectively, these data suggest that weak TCR stimu-
lation is favorable for the peripheral induction of Foxp3
(Kretschmer et al., 2005; Haxhinasto et al., 2008; Sauer et al.,
2008; Josefowicz et al., 2009).
Although it is clear that the level of TCR stimulation influ-
ences Foxp3 induction, several factors impact the cumulative
TCR stimulation a cell receives, such as TCR ligand density
and the affinity and duration of TCR–pMHC interactions.
In this study, we sought to define the TCR ligand characteristics
ideal for in vitro and in vivo induction of Foxp3 using TCR
transgenic T cells recognizing a panel of peptide ligands with
varying affinities for the TCR. Given the current assumption
that weaker TCR stimulation favors peripheral Foxp3 induc-
tion, we were surprised to find that a low-affinity agonist was
greatly diminished in its ability to induce a persistent popula-
tion of Foxp3-expressing cells in vivo. Modulating the dura-
tion of TCR–pMHC interactions and the density of pMHC
resulted in efficient initial Foxp3 induction by weak agonist
Figure 1. There is an inverse relationship between TCR/pMHC
affinity and the optimal peptide concentration for Foxp3 induction
in vitro. (A) EC50 values for the experiment shown were determined as the
peptide concentration resulting in 50% of maximal proliferation. LN cells
from 5C.C7 TCR transgenic RAG2/ mice were stimulated with irradiated
splenocytes and the indicated peptide for 60 h. 3H-methyl-thymidine was
used to assess proliferation. (B and C) 5C.C7 RAG2/ LN cells were cultured
with irradiated splenocytes in the presence of IL-2 and the indicated pep-
tide for 4 d before flow cytometry analysis of the frequency (B) or number
(C) of Foxp3+ cells. (C) Percentage of maximum cell number is shown, with
Foxp3+ 5C.C7 represented as a solid line and Foxp3 cells as a dashed line.
In A and B, error bars show mean ± SD, with n = 3 or n = 2 wells, respec-
tively. Data are representative of at least three independent experiments.
JEM VOL. 207, August 2, 2010
response to two peptides differing in
their EC50 values (Turner et al., 2009),
and with in vivo data indicating that
Foxp3 induction is favored by condi-
tions suboptimal for proliferation of re-
sponding cells (Kretschmer et al., 2005).
The addition of exogenous TGF-,
a factor known to facilitate TCR-
dependent Foxp3 induction (Chen
et al., 2003; Selvaraj and Geiger, 2007),
widened the range of TCR stimulation over which Foxp3
induction occurred in vitro (Fig. S1). Furthermore, the su-
peragonist K5 resulted in the most Foxp3+ cells, peaking at
concentrations of peptide higher than favorable for Foxp3
induction in the absence of exogenous TGF-. Thus, in
terms of Foxp3 induction, TGF- reduces the sensitivity to
strong stimulation through the TCR. This is consistent with
the requirement of TGF- and other negative regulators for
Foxp3 induction in systems relying on relatively strong TCR
stimulation (Chen et al., 2003; Zheng et al., 2006; Selvaraj
and Geiger, 2007). Our in vitro data suggest that the combi-
nation of ligand density and affinity determine an optimal
range of TCR stimulation conducive for expression of Foxp3.
Sensitivity to variations in these parameters is decreased in the
presence of exogenous TGF-.
The weak agonist 102S yields diminished frequency
and number of induced Foxp3+ 5C.C7 T cells in vivo
To dissect the effects of antigen density and affinity on Foxp3
induction in vivo, we opted for a single intravenous dose of
peptide, which has previously been shown to tolerize a mono-
clonal population of antigen-specific T cells (Thorstenson and
Khoruts, 2001). Low doses of MCC peptide were able to
induce Foxp3 expression in adoptively transferred 5C.C7
T cells (Fig. 2 A). Over a 100-fold titration of peptide, we found
0.1 µg MCC to be optimal for peripheral Foxp3 induction,
contain mutations in TCR contact residues that result in a
shorter or longer half-life of 5C.C7 TCR–pMHC interac-
tions, respectively (Corse et al., 2010; Huppa et al., 2010).
All of the peptides used in our study bind to MHC with compara-
ble affinity (Krogsgaard et al., 2003), which is essential to sepa-
rate the influences of TCR ligand potency versus density of
pMHC ligand upon T cell responses. To confirm the rank
order of the peptides in our experimental setting, we stimulated
5C.C7 T cells with irradiated splenocytes and the indicated ti-
tration of peptide (Fig. 1 A). As expected, at equivalent doses
of peptide 102S induced less 5C.C7 proliferation and K5 more
proliferation, relative to the natural ligand MCC. This differ-
ence in in vitro potency is apparent in the higher and lower
EC50 values for 102S and K5, respectively (Fig. 1 A).
To assess the ability of the various ligands to induce Foxp3
in vitro, 5C.C7 T cells were stimulated in the presence of exog-
enous IL-2 and a titration of peptide. Interestingly, to reach peak
in vitro Foxp3 induction for each ligand, the peptide concentra-
tion had to be adjusted to compensate for affinity. Relative to
MCC, a higher concentration of 102S and a lower concentra-
tion of K5 was required (Fig. 1 B). For all three peptides, the
optimal concentration for in vitro Foxp3 induction is below the
concentration of peptide required for expansion of the Foxp3-
negative compartment within the same cultures (Fig. 1 C).
These results are consistent with a recently published study,
comparing in vitro Foxp3 induction and proliferation in
Figure 2. A single low dose of intra-
venous MCC peptide results in efficient
peripheral induction of Foxp3. B10.A mice
containing 106 naive adoptively transferred
5C.C7 RAG2/ CD45.1 T cells were injected
intravenously with the indicated dose of MCC
peptide. CD4+CD45.1+ cells were assessed for
expression of Foxp3 (A and B) and CD44 (in
the Foxp3 population; C). (D) A time course
was performed to assess Foxp3 expression
and CFSE dilution of adoptively transferred
5C.C7 at each indicated day after peptide
injection. The frequency of Foxp3+ cells, as
a percentage of total 5C.C7, is shown on the
plots. Error bars show mean ± SD of two mice
per group, and data are representative of at
least three independent experiments. Dot
plots in A are gated on CD4+ cells, whereas all
other plots and histograms are gated on
CD4+CD45.1+, unless otherwise indicated.
TCR ligand density and affinity in Foxp3 induction | Gottschalk et al.
Foxp3 induction (Kretschmer et al., 2005; Haxhinasto et al.,
2008; Sauer et al., 2008; Josefowicz et al., 2009), we were sur-
prised to find that the weak agonist 102S appeared diminished
in its ability to induce expression of Foxp3. Although intrave-
nous administration of 102S peptide did result in some Foxp3
expression, the percentage of 5C.C7 expressing Foxp3 was
fourfold less than that observed with the optimal dose of MCC
8 d after peptide injection (Fig. 3, A and C). Furthermore,
normalization to remove the effect of the Foxp3 5C.C7
T cells from the calculation revealed a drastically smaller number
Foxp3+ 5C.C7 in the 102S-treated mice, compared with both
high-affinity ligands (Fig. 3 B). We detected even less Foxp3
induction upon further increase of the dose to 100 µg (Fig. 3,
A and B). This indicates that optimal Foxp3 induction by the
102S ligand cannot be accomplished by further increasing the
concentration of peptide. This could not be attributed to de-
creased in vivo stability of 102S compared with MCC, as both
peptides appeared to be cleared at a comparable rate (Fig. S3).
Disruption of TCR–pMHC interactions in vivo can inhibit
proliferation while enhancing Foxp3 induction
To better understand the diminished ability of 102S to in-
duce Foxp3 in vivo, we examined proliferation of Foxp3+
and Foxp3 5C.C7 T cells in response to the various peptides
and doses. In contrast to stimulation that favored Foxp3 in-
duction (0.1 µg MCC; Fig. 2D), 10 µg 102S resulted in pro-
liferation by the majority of 5C.C7 T cells (Fig. 4 A, left).
We hypothesized that decreasing the level of cumulative
TCR stimulation in conditions where the majority of 102S-
stimulated cells were dividing would favor Foxp3 induction.
We sought to do this by manipulating the duration of TCR
stimulation. It has previously been demonstrated using two-
photon imaging that T cell–APC interactions can be dis-
rupted in vivo by injection of an MHCII antibody after
peptide injection (Celli et al., 2007). Using this approach, we
interfered with TCR–pMHC interactions at various times
after peptide administration by injecting a monoclonal anti-
body to the MHCII molecule I-Ek (Fig. 4).
Strikingly, injection of anti-MHCII between 6 to 16 h
after peptide administration resulted in enhanced Foxp3
induction by the weak agonist 102S (Fig. 4, A and B), which
was assayed 6 d after peptide injection. This was true both in
terms of the frequency and number of Foxp3+ 5C.C7 T cells
(Fig. 4, B and C). Disrupting TCR–pMHC interactions
during the hours in which formation and persistence of sta-
ble T cell–APC contacts dictate subsequent effector function
(Hugues et al., 2004; Miller et al., 2004; Celli et al., 2007;
but not 36 h after peptide injection; Fig. 4, A and B), low-
ered the level of cumulative stimulation, as assessed by CFSE
dilution, and changed the response of the 5C.C7 T cells. This
resulted in an increased frequency of Foxp3-expressing cells,
whereas proliferation of the entire 5C.C7 population was
reduced (Fig. 4, D and E). In contrast, administration of anti-
MHCII subsequent to stimulation with the optimal dose of
MCC results in fewer Foxp3+ 5C.C7 T cells (Fig. S4),
suggesting that the reduced level of TCR stimulation
both in terms of the percentage of 5C.C7 expressing Foxp3
and the number of 5C.C7 Foxp3+ cells (Fig. 2 B). This dose
was below the threshold of activation required for complete
CD44 up-regulation of the 5C.C7 Foxp3 cells (Fig. 2 C)
and resulted in only partial CFSE dilution (Fig. 2 D). This
suggests that in our system, induction of Foxp3 expression is
coincident with a weaker TCR signal (in terms of a lower
peptide dose). A time course after injection of the optimal
dose showed that although Foxp3 can be detected in some
5C.C7 T cells as early as 2.5 d after immunization, peak accu-
mulation of Foxp3+ 5C.C7 T cells occurs between 4 and 10 d
after immunization (Fig. 2 D). 5C.C7 T cells induced to ex-
press Foxp3 under our experimental conditions express high
levels of CD25 and CTLA-4 and display phenotypic charac-
teristics common to T reg cells (Fig. S2). Consistent with pre-
vious studies (Kretschmer et al., 2005; Josefowicz et al., 2009),
these Foxp3+ 5C.C7 T cells had undergone only a few cell
divisions, with the majority remaining undivided at early time
points (Fig. 2 D, days 2.5 and 4; and Fig. S2 D).
We addressed the influence of TCR/pMHC affinity on
in vivo Foxp3 induction by comparing varying doses of the
weak agonist 102S and the superagonist K5 with MCC 8 d
after peptide injection (Fig. 3). All three peptides, including
the weak agonist 102S, are able to stimulate canonical in vivo
T cell responses, including proliferation, cytokine production,
and memory, upon subcutaneous immunization with LPS
(Corse et al., 2010). In response to intravenous peptide injec-
tion, we found K5 to induce comparable numbers of Foxp3+
5C.C7 T cells as MCC at the same low doses of peptide
(Fig. 3 B). Given results that low levels of TCR stimulation favor
Figure 3. The weak agonist 102S results in a diminished frequency
and number of induced Foxp3+ 5C.C7 T cells in vivo. B10.A recipients
received 5C.C7 RAG2/ CD45.1 T cells and were subsequently injected
with the indicated dose of 102S, MCC, or K5 peptide. After 8 d, LN cells
were assessed for the percentage (A) and number, as normalized to
the endogenous CD4+CD45.2+ population (B), of 5C.C7 T cells expressing
Foxp3. Error bars show mean ± SD of two mice per group and data are
representative of at least three independent experiments. (C) Below the
graphs are representative CD4+CD45.1+ gated dot plots, with the percent-
age of Foxp3+ cells shown on the plot.
JEM VOL. 207, August 2, 2010
Foxp3+ 5C.C7 induced by injection of weak agonist peptide
do not persist
Considering that disruption of TCR–pMHC interactions
in vivo leads to reduced proliferation coincident with Foxp3
induction, we examined whether there was a dose of 102S
that would stimulate cell division, which is comparable to
low doses of MCC. In this more refined in vivo titration,
we also included the peptide 102N, another weak agonist
that induces comparable or slightly less in vitro IL-2 pro-
duction and conjugate formation than 102S (Egen and
Allison, 2002). A titration of MCC, 102S, and 102N
in vivo showed that dose could compensate for pMHC po-
tency in stimulating comparable amounts of proliferation.
There was a dose of each peptide that resulted in a similar
intermediate level of CFSE dilution 5 d after peptide injec-
tion (0.3 µg MCC, 3 µg 102S, and 10 µg 102N; Fig. 5,
A and B [boxed data points]; and Fig. S6 A, full titration).
This readout suggests that all three peptides are capable of
achieving a similar level of TCR stimulation. Consistent
with this, 2 d after stimulation with these doses of MCC
and 102S we detected equivalent levels of ki67 expression
and Akt phosphorylation, which represents the degree of
activity of Akt kinase and mTORC2 complex (Fig. 5 E).
This comparable amount of Akt phosphorylation is of
under these conditions falls below the range conducive for
Slightly reduced proliferation and increased Foxp3 ex-
pression upon blockade was seen in mice injected with 10 µg
MCC, with the effect being less drastic than for 102S (Fig. 4,
D and E). This may be explained by the observation that
102S-stimulated cells express the proliferation antigen ki67
with a temporal delay, compared with MCC at the same dose
of peptide, indicating that the cells responding to stimulation
with the lower affinity ligand may require more time to enter
the cell cycle (Fig. S5 A). At 10 µg, the majority of MCC-
stimulated cells are ki67+ by 24 h, whereas ki67 expression is
not detected in most 102S-stimulated cells until after 48 h
(Fig. S5 A). Thus, disruption of TCR–pMHC interactions
after 102S injection resulted in an increased portion of 5C.C7
T cells below the threshold of stimulation required for ki67
expression, whereas cells stimulated with 10 µg MCC are all
ki67+ by 48 h, regardless of antibody treatment (Fig. S5 B).
This is consistent with the modest effect of MHC blockade
on the proliferative capacity and Foxp3 expression of 5C.C7
T cells responding to MCC (Fig. 4, D and E) but does not
exclude the possibility that there are doses of MCC at which
Foxp3 induction could be more efficiently enhanced by
Figure 4. Disruption of TCR–pMHC interactions in vivo can inhibit proliferation while enhancing Foxp3 induction. B10.A recipients were adop-
tively transferred with naive CFSE-labeled CD45.1+ 5C.C7 T cells and subsequently injected with the indicated peptide followed by intravenous anti-MHCII
at the indicated times. CD45.1+ LN cells were analyzed 6 d after peptide injection. (A and B) Antibody to MHCII was injected at varying time points after
injection with 10 µg 102S, and the frequency of Foxp3+ 5C.C7 T cells was assessed. Representative dot plots (A) are shown beside data pooled from two
independent experiments. Each point represents one mouse and horizontal bars indicate the mean (B). (C) The influence of anti-MHCII injection on the
number of Foxp3+ 5C.C7 T cells was addressed by normalizing to the endogenous CD4+CD45.2+ population. n = 2 with SD. (D and E) Anti-MHCII was in-
jected between 8 and 10 h after injection of either MCC or 102S, as indicated. 5C.C7 CFSE dilution and Foxp3 expression was assessed (D). Percentage of
5C.C7 expressing Foxp3 versus the proliferative capacity of the total 5C.C7 population are analyzed in a scatter plot. Data are pooled from at least three
independent experiments, each data point representing one mouse (E). Dot plots are gated on CD4+CD45.1+ cells, and the percentages that are positive
for Foxp3 are shown. All data are representative of at least three independent experiments.
TCR ligand density and affinity in Foxp3 induction | Gottschalk et al.
As expected, the dose for each peptide that triggered
partial division of the responding 5C.C7 population was also
the peak of Foxp3 induction, based on the percentage of
Foxp3+ 5C.C7 T cells (0.3 µg MCC, 3 µg 102S, and 10 µg
102N; Fig. 5, B and C). Although the peak of Foxp3 induc-
tion by 102S peptide in this more refined titration (3 µg)
produced slightly more Foxp3 induction than 10 µg, both
doses still resulted in diminished numbers of Foxp3+ 5C.C7
T cells compared with MCC. The numbers of 102S-induced
Foxp3+ 5C.C7 T cells were lower at day 8 (Fig. 3) than at
day 5 (Fig. 5 D). Thus, to gain more insight into the fate of
cells stimulated by these strong and weak agonists, we quan-
titated the number of Foxp3+ and Foxp3 5C.C7 T cells
over time (Fig. 6 and Fig. S7).
We found that intravenous injection with either MCC or
102S resulted in a dose-dependent expansion and sharp con-
traction, just after the peak of the response (Fig. S7, A and B).
As a control, we immunized with the same dose of peptide
subcutaneously, at the base of tail with LPS, which generates
a more typical effector response and does not result in such
drastic cell loss (Fig. S7 A). The contraction of 5C.C7 T cells
responding to intravenous peptide injection is consistent with
studies reporting deletion of T cells stimulated by intravenous
peptide as a mechanism of tolerance (Kearney et al., 1994;
Liblau et al., 1996). When comparing the doses of MCC,
102S, and 102N that result in a comparable level of prolifera-
tion, we observed a similar decline in number of 5C.C7
T cells in both the LNs and the spleen (Fig. S7, C and D).
This apparent deletion was coincident with the presence of
a fraction of 5C.C7 T cells positive for active caspases, which
was not present in recipients that did not receive peptide in-
jection and is consistent with cell death in response to toler-
izing antigen administration (Fig. S7 E).
The two weak agonist peptides assessed in our system
are able to induce a substantial frequency of Foxp3+ 5C.C7
T cells if the right dose of peptide is administered. At early
time points, all three peptides induce a comparable percentage
of Foxp3+ 5C.C7 (Fig. 6, days 2.5 and 5). However, Foxp3+
5C.C7 induced by the low-affinity ligands disappear quickly
after the peak of the response, within the same time frame as
the Foxp3 compartment, whereas a low dose of MCC re-
sults in increased frequencies and numbers of Foxp3+ 5C.C7
throughout the course of the experiment (Fig. 6). Consider-
ing the loss of the Foxp3 5C.C7 T cells in all of these condi-
tions (Fig. 6 A), it is the persistence of the Foxp3+ 5C.C7
population after low-dose MCC stimulation that is specific,
rather than the deletion of either 5C.C7 compartment after
stimulation with weak ligand. We do not attribute the differ-
ence in Foxp3+ 5C.C7 persistence to an inability of 5C.C7
T cells responding to 102S peptide to produce IL-2 (Fig. S8).
Our measurements of the degree of proliferation and initial
Foxp3 induction suggest that increasing the dose of 102S re-
sults in the same quantity of stimulation as received by MCC-
stimulated cells. However, time course experiments revealed
that a low dose of the strong agonist peptide, but not a high
dose of weak agonist, was able to induce a persistent population
particular interest considering recent studies reporting that
the Akt–mTor pathway, which is essential for T cell activa-
tion and proliferation, may antagonize peripheral Foxp3
induction (Haxhinasto et al., 2008; Sauer et al., 2008). The
level of phospho-Akt correlated with ki67 expression, re-
gardless of stimulating peptide (Fig. 5 F).
Figure 5. In vivo, peptide dose compensates for potency to reach
comparable levels of proliferation and Akt phosphorylation, but not
numbers, of Foxp3-expressing 5C.C7 T cells. Naive 5C.C7 RAG2/
CD45.1 T cells were adoptively transferred and stimulated in vivo by intra-
venous injection of the indicated peptide and dose. All dot plots and histo-
grams are gated on CD4+CD45.1+ LN cells. (A) Day-5 representative dot
plots for each peptide at the doses yielding comparable frequencies of
divided cells and peak of Foxp3 induction: 0.3 µg MCC, 3 µg 102S, and
10 µg 102N. The percentage of divided (B), percentage of Foxp3+ (C), and
number of Foxp3+ 5C.C7 T cells (D) 5 d after injection of the indicated pep-
tide and dose are shown with SD. n = 2. The dashed rectangle in B indi-
cates the optimal peptide doses. (E and F) 2 d after injecting doses of MCC
and 102S that yield comparable CFSE dilution (0.3 µg MCC and 3 µg 102S),
ki67 expression and Akt phosphorylation in total 5C.C7 T cells were mea-
sured by flow cytometry. Bar graphs in E show data pooled from three
independent experiments with SD. As a control, Akt phosphorylation was
compared with the maximum resulting from immunization at the base of
tail with MCC and LPS, which results in a substantial portion of pAkt+ 5C.
C7 T cells. Histograms in F separate CD4+CD45.1+ cells by ki67 expression,
adjacent to linear regression analysis of the percentage of 5C.C7 express-
ing ki67 versus the phospho-Akt MFI for the total 5C.C7 population, for
individual samples (best fit line shown: R2 = 0.647; P = 0.016). All data are
representative of at least three independent experiments.
JEM VOL. 207, August 2, 2010
density and the strength of TCR–pMHC interactions on the
peripheral induction of Foxp3 in vivo.
Upon comparing ligands of varying affinities across an
in vitro peptide titration, we found that there was a limited range
of TCR stimulation over which Foxp3 induction occurred.
Relative to the natural ligand MCC, peak Foxp3 induction by
the superagonist K5 occurred at lower concentrations of pep-
tide, whereas the peak induction by the weak agonist 102S oc-
curred at higher concentrations. All three ligands were able to
induce a comparable magnitude of in vitro conversion of 5C.
C7 T cells, so long as peptide concentration was altered to
compensate for a higher or lower affinity ligand. For each pep-
tide, the TCR stimulation range favoring conversion was
below the concentration of peptide required for expansion of
the nonconverted cells. The addition of exogenous TGF-
widened the range of TCR stimulation over which conversion
occurred, which allowed for Foxp3 induction at concentra-
tions of peptide that were otherwise too high. This is consis-
tent with previously published reports demonstrating the
requirement for TGF- and other negative regulators of T cell
activation in scenarios where T cells are receiving high levels
of TCR stimulation (Chen et al., 2003; Zheng et al., 2006;
Selvaraj and Geiger, 2007).
When MCC, K5, and 102S were titrated in vivo, we did
not observe the same simple relationship between ligand affin-
ity and optimal Foxp3 induction dose seen in vitro. The super-
agonist peptide K5, which peaked at concentrations of peptide
100-fold lower than MCC in vitro, was comparable to MCC
in vivo, resulting in efficient conversion of 5C.C7 T cells at the
same low dose. Strikingly, stimulation with the weak agonist
102S resulted in drastically reduced numbers of Foxp3+ 5C.C7
T cells 8 d after peptide injection, compared with either of the
strong ligands. This was surprising given the studies reporting
of Foxp3+ T cells. Thus, there are two phases that must be
considered. In the first phase, antigen dose can compensate for
potency to achieve the optimal quantity of TCR stimulation
for initial Foxp3 induction, whereas in the second phase, high
doses of peptide are detrimental to the persistence of the stimu-
lated 5C.C7 T cells, including the induced Foxp3+ T cells in
the case of the weak agonist peptide. This highlights limitations
of comparing in vivo and in vitro systems, the latter of which
do not address the issue of persistence.
Foxp3+ T reg cell suppression of self-reactive effector T cells is
critical for the prevention of autoimmunity (Sakaguchi, 2004).
Peripheral induction of Foxp3 in mature CD4+ T cells may be
an important mechanism of maintaining this self-tolerance.
The partial overlap of TCR repertoires of induced T reg cells
and thymic T reg cells indicates that peripheral Foxp3 induc-
tion could potentially offer a second chance for self-reactive
cells that escape thymic selection to enter the T reg cell lineage
(Lathrop et al., 2008). Alternatively, cells could convert into
T reg cells in response to non–self-antigens presented in toler-
izing conditions (Curotto de Lafaille and Lafaille, 2009). Under-
standing the conditions optimal for peripheral Foxp3 induction
in vivo could provide insight into how this process may be
manipulated in a therapeutic setting.
Several studies altering antigen dose and degree of Akt
and/or mTor activity have shown that efficient Foxp3 induc-
tion occurs in conditions suboptimal for activation (Kretschmer
et al., 2005; Haxhinasto et al., 2008; Sauer et al., 2008), sug-
gesting that a weaker TCR stimulation favors peripheral con-
version. It was less clear how defined TCR/pMHC binding
parameters, such as affinity and off rate, influence this process.
In this study, we address the cumulative influence of antigen
Figure 6. Foxp3+ 5C.C7 induced by in-
jection of weak agonist peptide do not
persist, compared with the high-affinity
ligand MCC. After adoptive transfer of 5C.C7
RAG2/ CD45.1 T cells, recipient mice were
injected with varying doses of the indicated
peptides, which have been previously deter-
mined to give equivalent CFSE dilution and
peak Foxp3 induction (0.3 µg MCC, 3 µg 102S,
and 10 µg 102N), and then sacrificed at the
indicated time points after peptide injection.
(A) The percentage of Foxp3+ and numbers of
Foxp3+ and Foxp3 5C.C7 in LN were assessed
at each time point. Error bars show SD of two
mice per group. Representative dot plots
gated on CD4+CD45.1+ cells are shown in B,
with the percentage of Foxp3-positive 5C.C7
shown on the plot. Data are representative of
at least three independent experiments.
TCR ligand density and affinity in Foxp3 induction | Gottschalk et al.
Our system suggests that the range of TCR stimulation favor-
ing this process is very narrow, as the dose response for all
three peptides is very steep. Based on our in vitro data, where
the addition of TGF- allowed for Foxp3 induction in re-
sponse to higher concentrations of peptide, we would expect
that a particular cytokine environment could also widen the
range of TCR stimulation over which Foxp3 induction could
occur in vivo. In addition, our results suggest that it may be
conceivable to expand the range of antigenic conditions over
which Foxp3 induction can occur in a therapeutic setting
through manipulation of the duration of TCR–pMHC inter-
actions. Disrupting T cell–APC interactions allowed us to de-
crease the cumulative TCR stimulation, resulting in Foxp3
induction when ligand density would otherwise be unfavor-
able. In doing so, it may be possible to alter the frequency of
antigen-specific T reg cell, which could skew a potentially
pathogenic T cell response toward tolerance.
Despite finding doses of 102S and 102N that yielded sim-
ilar proliferation and initial Foxp3 induction compared with
optimal doses of MCC, these weak agonists still generated
diminished numbers of Foxp3+ 5C.C7 T cells at longer times
after peptide injection. Time course experiments revealed
that at the doses of each peptide yielding optimal initial Foxp3
induction, injection of weak agonist peptides resulted in sim-
ilar deletion of both the Foxp3 negative and positive popula-
tions, whereas stimulation with MCC resulted in specific
persistence of the induced Foxp3+ population of 5C.C7
T cells. Thus, there are two phases influencing the numbers
of Foxp3+ 5C.C7 observed after in vivo stimulation. During
initial induction of Foxp3, antigen dose could compensate
for potency to achieve the optimal quantity of TCR stimula-
tion, whereas in the second phase, maintenance versus dele-
tion of this population appeared to be determined by unique
stimulation criteria. This highlights the importance of study-
ing in vivo systems, as persistence is not addressed in short
term in vitro assays. Low-potency ligands are efficient at
achieving the weak TCR signals favoring Foxp3 induction
both in vitro and in vivo. However, our data demonstrate
that these interactions do not necessarily induce a persistent
population of Foxp3+ T cells in vivo.
Our findings demonstrate that ligand potency and den-
sity are noninterchangeable factors. Increasing the dose of
low-potency ligand appeared to achieve a similar level of
cumulative TCR stimulation (comparable proliferation, Akt
phosphorylation, and Foxp3 induction at early time points) but
failed to generate persistent Foxp3+ 5C.C7, compared with the
strong agonist MCC. Therefore it is not simply a matter of
getting the same quantity of TCR stimulation; there are quali-
tative differences that must be considered. This difference in
cell fate suggests that there may be mechanisms allowing
T cells to discriminate between signals comings from a few
strong TCR–pMHC interactions, versus many complexes
with weaker interactions. One can imagine the triggering of
survival signals specifically downstream of a strong TCR–
pMHC interaction, as opposed to proapoptotic signals which
are induced by the triggering of many separate TCRs. There is
that weaker TCR stimulation favors Foxp3 induction
(Kretschmer et al., 2005; Haxhinasto et al., 2008; Sauer et al.,
2008). Thus, we went on to modulate multiple parameters
influencing the level of TCR stimulation received by 102S-
stimulated cells to optimize Foxp3 induction, and subsequently
assessed the frequencies and numbers of Foxp3+ 5C.C7 T cells
induced by the strong and weak agonists over time.
Upon assessing proliferation of cells responding to 10 µg
102S, the dose where we had observed a detectable frequency
of Foxp3+ 5C.C7, it appeared that induction of Foxp3 by
5C.C7 T cells responding to 10 µg 102S was accompanied by
proliferation more robust than that in conditions that are
optimal for Foxp3 induction (i.e., 0.1 µg MCC peptide).
The idea that such a strong stimulation would be detrimental to
Foxp3 induction is consistent with studies demonstrating that
Akt activity can antagonize expression of Foxp3 (Haxhinasto
et al., 2008; Sauer et al., 2008). In addition, proliferation may
oppose Foxp3 induction by cell cycle–dependent recruitment
of maintenance DNA methyltransferase, which results in a
silenced state of the Foxp3 locus (Josefowicz et al., 2009).
Considering these findings, we sought to decrease the cu-
mulative TCR stimulation in response to this high density of
102S pMHC complexes by injecting antibodies to MHCII 6–
16 h after injection of peptide. During this time period, T cell–
APC contacts form and persist, and antibodies to MHCII have
been shown to result in disruption of T cell–APC interactions
(Hugues et al., 2004; Miller et al., 2004; Celli et al., 2007). By
blocking TCR–pMHC interactions, we were able to both de-
crease the frequency of 102S-stimulated cells entering cell
cycle and greatly lower their overall proliferative capacity, which
correlated with increased initial induction of Foxp3. These re-
sults highlight the influence of pMHC potency, density, and
duration of interactions with TCR on the overall quantity of
TCR stimulation that a cell receives and, thus, its decision to
divide or express Foxp3.
Together, our data are in agreement with the idea that
weak TCR stimulation that is suboptimal for proliferation
favors initial Foxp3 induction. Indeed, a refined titration of
peptides in vivo revealed that both MCC and the two weak
ligands 102S and 102N could produce this favorable quantity
of TCR stimulation if the dose was adjusted to compensate
for ligand strength. At these doses, which span nearly two or-
ders of magnitude between MCC and 102N, stimulation by
all three peptides resulted in equivalent Foxp3 induction,
based on the frequency of Foxp3+ 5C.C7 at early time points.
In conditions favoring optimal Foxp3 induction, there was
no difference in the ability of strong and weak ligands to trig-
ger Akt phosphorylation, which simply correlated with ki67
expression, regardless of stimulating peptide.
Considering only the initial induction of Foxp3, our data
varying ligand dose, potency, and duration of TCR–pMHC
interactions in vivo would suggest that perhaps it is simply the
quantity of TCR stimulation that is important. This would be
consistent with a model in which all of these factors contrib-
ute to a cumulative signal, which determines T cell responses
(Rosette et al., 2001; Rachmilewitz and Lanzavecchia, 2002).
JEM VOL. 207, August 2, 2010
effects of maintaining T reg cells that recognize ubiquitous
self-ligands, such as bystander suppression (Masteller et al.,
2005; Tang and Bluestone, 2008).
In summary, initial in vivo Foxp3 induction can be
achieved by optimizing the quantity of cumulative TCR
stimulation, which can be thought of as a sum of the factors
of ligand potency, density, and duration of TCR–pMHC in-
teractions. However, TCR ligand potency and density are
clearly noninterchangeable in determining the persistence of
the cells induced to express Foxp3. These findings provide
important insight into how affinity and prevalence of antigen
may determine the route to peripheral tolerance.
MATERIALS AND METHODS
Mice. 5C.C7 TCR transgenic RAG2/ mice (Taconic) were bred to B10.
A CD45.1 (provided by W. Paul via the National Institute of Allergy and
Infectious Diseases contract facility at Taconic) to generate 5C.C7 RAG2/
CD45.1 mice, which were used as donor mice in adoptive transfer experi-
ments. Male B10.A mice (Taconic) were used as adoptive transfer recipients
at between 6 and 9 wk of age. All mice were maintained in microisolator
cages, and treated in accordance with the regulations of the National Insti-
tutes of Health and the American Association of Laboratory Animal Care.
Experiments in this study were approved by the Memorial Sloan-Kettering
Cancer Center Institutional Animal Care and Use Committee.
In vitro T cell activation and Foxp3 induction. Cells were cultured in
a 37°C humidified chamber with 5% CO2 in complete RPMI1640 (supple-
mented with 10% FCS, 2 µM glutamine, 100 U/ml penicillin and strepto-
mycin, and 2 µM 2-mercaptoethanol). Single cell suspensions were prepared
from LNs harvested from 5C.C7 RAG2/ mice (routinely >90%
Vll+V3+ by flow cytometry) and stimulated in duplicate or triplicate,
as indicated, in 96-well round bottom plates. In T cell proliferation assays,
3 × 104 5C.C7 cells were stimulated with 2 × 105 irradiated splenocytes and
the indicated peptides for 60 h. 1 µCi/well 3H-methyl-thymidine was used
to monitor T cell proliferation. Cells were harvested onto glass-fiber filters
using a Tomtec harvester, and filters were counted using a MicroBeta scintilla-
tion counter (PerkinElmer). For in vitro T reg cell conversion assays,
105 5C.C7 T cells were cultured with 4 × 105 irradiated splenocytes that had
been depleted of T cells using CD90.2 microbeads (Miltenyi Biotec), the in-
dicated peptide, and 100 U/ml of recombinant human IL-2 (PeproTech),
with or without human TGF-1 (PeproTech). Cells were cultured for
90–96 h before cells were stained for flow cytometry analysis. In some cul-
tures the absolute number of cells was determined using an automated cell
counter (Guava Technologies Inc.).
Peptides. Peptides were synthesized and HPLC purified (≥95%) by
Flow cytometry and antibodies. Flow cytometry was done on an LSRII
(BD). Antibodies to surface markers were obtained from BD, eBioscience, or
BioLegend. Foxp3 was detected using antibodies (eBioscience) and fixation/
permeabilization reagents. For detection of phosph-Akt (Ser473), we used
an Alexa Fluor 647–conjugated antibody (Cell Signaling Technology; DE9),
after immediate fixation with 2% paraformaldehyde and subsequent permea-
bilization with cold methanol. CTLA-4 and ki67 antibodies (BD) were de-
tected after fixation and permeabilization as required for Foxp3 or phospho-
Akt analysis, depending on the experiment.
In vivo Foxp3 induction. Between 5 × 105 and 1 × 106 5C.C7 RAG2/
CD45.1 T cells were transferred into B10.A recipients by tail vein injection.
Mice were subsequently immunized intravenously the same or next day with
0.03–100 µg of peptide diluted in PBS. When disruption of T cell–APC in-
teractions was desired, 500 µg of monoclonal antibody to the mouse MHC
precedent for cell-intrinsic mechanisms of ligand discrimina-
tion via negative signaling feedback loops, which is proposed
to be important in the discrimination of self- and foreign li-
gands (Stefanová et al., 2003). In addition, it has been sug-
gested that the density of antigen may influence TCR signal
inhibition, based on an in vivo model comparing tolerance in
response to high and low levels of self-antigen (Singh and
Although is probable that cell-intrinsic mechanisms could
be in place to aid in discrimination between high-potency
versus high-density ligands, it is also possible that cell-extrinsic
factors play a role in this process. This seems especially im-
portant considering that the maturation state of APCs is
known to play an important role in T cell fate under toleriz-
ing conditions. Low levels of costimulation favor Foxp3 in-
duction (Benson et al., 2007), anergy, and deletion (Hawiger
et al., 2001). Both dose and potency of peptide ligand have
been reported to influence the kinetics and type of effector
T cell–APC contact formation (Bousso and Robey, 2003;
Skokos et al., 2007; Henrickson et al., 2008). Thus, it is pos-
sible that low doses of MCC and high doses of 102S achieve
comparable cumulative TCR stimulation, but in the context
of different types of T cell–APC interactions. For example,
very low concentrations of peptide may only result in short
interactions, which, given a potent ligand, may be sufficient
to provide the minimal stimulation required for Foxp3 in-
duction. In this scenario, more stable T cell–APC interac-
tions resulting from higher concentrations of peptide could
play a role in the subsequent deletion of these cells. Transient
TCR–pMHC interactions upon low-dose injection of pep-
tide could explain the similarity in levels of Foxp3 induction
by MCC and K5 we observed, as the increased potency of
the high-affinity superagonist may be minimized in the ab-
sence of more stable conjugate formation.
We propose that a low density of a high-affinity TCR li-
gand is optimal to achieve the quantity and quality of stimu-
lation required to induce a persistent population of Foxp3+
T cells in vivo. In fact, considering that the Foxp3 5C.C7
T cells are deleted in these same conditions, and that injec-
tion of high doses of either MCC or 102S results in compa-
rable loss of 5C.C7 T cells, specificity is most apparent in the
survival of the Foxp3+ 5C.C7 in response to small amounts
of strong ligand. Deletion and Foxp3 induction are two pro-
posed mechanisms of peripheral tolerance. However, how
the strength and density of TCR ligand steers a self-reactive
T cell between these fates is understudied. TCR repertoire
analysis has suggested that T reg cells may recognize self-
antigens with low avidity (Hsieh et al., 2004). We suggest
that low concentrations of specific self-pMHC complexes
could contribute to peripheral Foxp3 induction in response
to self-antigens, even if the TCR–pMHC interaction is of a
high affinity. In the case of more abundantly expressed self-
pMHC complexes, our data suggest that deletion of the cog-
nate T cells is a more likely mechanism of tolerance, regardless
of the strength of the ligand and extent of Foxp3 induction.
It is interesting to speculate that there could be detrimental
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In some experiments cells were CFSE labeled before transfer. To enrich for
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Analysis. Flow data were analyzed using FlowJo (version 8.8.6; Tree Star,
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Online supplemental material. Fig. S1 shows the influence of exogenous
TGF- on in vitro Foxp3 induction by peptides of varying potency. Fig. S2
shows characteristics of Foxp3+ 5C.C7 T cells that are common to T reg
cells. Fig. S3 demonstrates comparable stability of 102S and MCC peptides
in vivo. Fig. S4 shows the impact of MHCII blockade on Foxp3 induction
by an optimal low dose of MCC. Fig. S5 contains analysis of ki67 expression
after in vivo stimulation with varying doses of MCC and 102S, with and
without disruption of TCR–pMHC interactions. Fig. S6 includes additional
peptide doses from titration and timecourse experiments shown in Figs. 5
and 6. Fig. S7 characterizes loss of 5C.C7 T cells after injection of intrave-
nous peptide, including dose dependence and expression of active caspases.
Fig. S8 shows ex vivo IL-2 production by 5C.C7 T cells, harvested 6 d after
in vivo peptide stimulation. Online supplemental material is available at
We thank A. Trumble-Koncelik and J. Geddes for technical assistance and M. Huse,
S.Z. Josefowicz, P.A. Savage, S.A. Quezada, Y. Zheng, R. Niec, T. Pentcheva-Hoang,
M.A. Sepulveda, and W. Walkowicz for helpful discussion and critical reading of
R.A. Gottschalk is a predoctoral fellow of the Cancer Research Institute.
E. Corse was supported by a postdoctoral fellowship from the Irvington Institute of
Immunological Research. J.P. Allison is an investigator of the Howard Hughes
Medical Institute and is supported by grants from the National Institutes of Health.
The authors have no conflicting financial interests.
Submitted: 16 September 2009
Accepted: 29 June 2010
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