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J. Exp. Med. Vol. 208 No. 6 1279-1289
T cells encounter several checkpoints as they
develop, and their fate often relies on the strength
of signal perceived by the antigen receptor. For
example, CD4+CD8+ double-positive (DP) thy-
mocytes with low affinity for self-peptide MHC
(pMHC) ligands undergo positive selection,
whereas those with high affinity undergo nega-
tive selection (Starr et al., 2003). Multiple stud-
ies suggest that DP thymocytes are exquisitely
sensitive and exhibit a broader range of recog-
nition of pMHC than mature T cells (Davey
et al., 1998; Lucas et al., 1999). Nonetheless,
mature T cells continue to perceive low-affinity
self-pMHC ligands in the periphery, and these
interactions are essential for survival and effec-
tor function (Kirberg et al., 1997; Stefanová
et al., 2002; Lo et al., 2009). Thus, the ability of
the TCR to distinguish pMHC ligands of differ-
ent affinity is a fundamental principal of immuno-
logical tolerance and homeostasis.
Although the affinity model explains the lin-
eage commitment of a majority of T cell progeni-
tors, some T cell subsets seem to have survived
strong TCR signals during development as dis-
played by their activated phenotype (Baldwin
et al., 2004; Kronenberg and Rudensky, 2005;
Kronenberg and Gapin, 2007). CD4+Foxp3+
regulatory T cells (Treg cells), invariant NKT
cells (iNKT cells), and CD8+ intraepithelial
lymphocytes (IELs) are hypothesized to be posi-
tively selected via strong TCR signals in the
thymus (Leishman et al., 2002; Godfrey et al.,
2004; Zhou et al., 2004). In the case of Treg cells,
Jordan et al. (2001) first demonstrated that ex-
pression of a neo-self-antigen in the thymus of
mice with TCRs specific for that antigen pro-
moted the development of Treg cells. Yet use of a
TCR with an intrinsically lower affinity for the
same neo-self-antigen failed to select Treg cells,
suggesting a role for strong TCR signals. More-
over, T cells transduced with TCRs cloned from
Treg cells underwent homeostatic expansion in
lymphopenic recipients to a greater extent than
cells transduced with receptors cloned from con-
ventional T cell (Tconv cell) CD4 T cells, which
is consistent with the idea that Treg cells recog-
nize self-pMHC more avidly (Hsieh et al., 2006).
Studies of the TCR repertoire from Treg and
Kristin A. Hogquist:
Abbreviations used: -GalCer,
bacterial artificial chromosome;
DP, double positive; IEL, intra-
epithelial lymphocyte; iNKT
cell, invariant NKT cell; MFI,
mean fluorescence intensity;
mOVA, membrane OVA;
OVAp, OVA peptide; pI:pC,
pMHC, peptide MHC; RIP,
rat insulin promoter; RTE,
recent thymic emigrant; SP,
T cell receptor signal strength in Treg
and iNKT cell development demonstrated
by a novel fluorescent reporter mouse
Amy E. Moran,1 Keli L. Holzapfel,1 Yan Xing,1 Nicole R. Cunningham,3
Jonathan S. Maltzman,2 Jennifer Punt,3 and Kristin A. Hogquist1
1Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55414
2Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104
3Department of Biology, Haverford College, Haverford, PA 19041
The ability of antigen receptors to engage self-ligands with varying affinity is crucial for
lymphocyte development. To further explore this concept, we generated transgenic mice
expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated
in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells,
GFP was induced during positive selection, required major histocompatibility complex for
maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus.
Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using
this model, we show that regulatory T cells (Treg cells) and invariant NKT cells (iNKT cells)
perceived stronger TCR signals than conventional T cells during development. However,
although Treg cells continued to perceive strong TCR signals in the periphery, iNKT cells did
not. Finally, we show that Treg cell progenitors compete for recognition of rare stimulatory
© 2011 Moran et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
TCR signal strength in T cell selection | Moran et al.
A Nur77GFP transgenic mouse reports antigen receptor
activation in lymphocytes
To create a fluorescent reporter that would be activated by anti-
gen receptor signaling in lymphocytes, we inserted a GFP-Cre
fusion protein at the start codon of the Nr4a1 (Nur77) gene
in a BAC (Fig. 1 A). The Cre recombinase gene was included
for fate mapping experiments that are not reported in this
study. One B6 × SJL F1 and two C57BL/6J founders were
generated. Each founder expressed a slightly different overall
level of GFP, but the pattern of expression was identical, and
endogenous Nur77 expression was consistent with GFP ex-
pression (Fig. S1 A). All three showed normal lymphoid and
myeloid development (unpublished data). A subset of myeloid
lineage cells in the spleen expressed high levels of GFP in the
steady-state (Fig. 1 B), whereas mature T and B lymphocytes
expressed low levels of GFP (Fig. 1 C).
To determine whether TCR stimulation induced GFP
expression, we injected Nur77GFP mice with 50 µg anti-CD3
i.v. and 12 h later harvested lymphocytes. We observed robust
induction of GFP (Fig. 1 D, left) and CD69 on T cells (not
depicted) after -CD3 stimulation. We also stimulated bulk
splenocytes with 10 µg -IgM in vitro for 3 h. Again, robust
Tconv CD4 T cells illustrated that they are equally diverse but
different from each other (Hsieh and Rudensky, 2005). How-
ever, these TCR repertoires were not entirely unique; thus,
others have suggested that Treg cells are not shaped by agonis-
tic interaction with self but rather by some stochastic event
(Pacholczyk et al., 2006, 2007). In addition, when Treg TCR
transgenics were created, no overt thymic clonal deletion was
observed (Bautista et al., 2009; Leung et al., 2009), nor was
self-reactivity evident. Thus, it remains unclear precisely what
type of TCR signals are involved in Treg cell development in
iNKT cells are CD1d-restricted T cells that recog-
nize lipid antigens. In the steady-state, they have a memory
phenotype and have been proposed to develop after ago-
nist or stimulatory interaction with a lipid self-ligand in
the thymus, yet the precise ligand remains unidentified
(Kronenberg and Gapin, 2007). Finally, CD8 IELs have
an activated phenotype and were increased in transgenic
models in which the cognate stimulatory antigen was also
present (Leishman et al., 2002). Thus, the term agonist se-
lection has been applied to all three subsets, indicating en-
counter with a stimulatory (presumed high affinity) TCR
ligand during development.
Short of cloning TCRs and identifying the selecting
ligand in the thymus, it is difficult to know if a given T cell
perceives a strong or weak TCR signal during develop-
ment. Therefore, we sought to make a reporter mouse in
which the level of a fluorescent protein reflects the strength
of antigen receptor signal. We generated a transgenic mouse
in which we inserted GFP into the Nr4a1 (Nur77) locus
of a bacterial artificial chromosome (BAC). Nur77 is an
immediate early gene up-regulated by TCR stimulation in
thymocytes and T cells (Osborne et al., 1994). It is an or-
phan nuclear receptor whose function in T cells is not
completely understood, although data suggest it may play
a role in thymocyte apoptosis (Cainan et al., 1995; Cho
et al., 2003). In a microarray screen, we showed that thy-
mocytes undergoing both positive and negative selection
induced Nr4a1 but to different expression levels (Baldwin
and Hogquist, 2007). Thymocytes undergoing positive
selection showed a twofold increase in Nr4a1 expression,
whereas those undergoing negative selection showed a
10-fold increase. Together, these observations suggested
that a Nur77 reporter mouse might be a useful system for
understanding the role of TCR signal strength during
T cell development.
In this study, we report that GFP is up-regulated by anti-
gen receptor stimulation in Nur77GFP mice, but unlike CD69,
another common marker of T cell activation, it is not in-
duced by inflammatory stimuli. Furthermore, the level of GFP
expressed during acute activation reflects the strength of
TCR stimulation, and the low basal level of GFP expressed
in mature naive T cells is dependent on continued inter-
action with MHC. We applied this novel tool to study the
TCR signal strength perceived by different T cell subsets dur-
Figure 1. A Nur77GFP BAC transgenic mouse expresses GFP upon
TCR activation. (A) A GFP-Cre fusion protein was inserted at the start
site of the Nr4a1 (Nur77) gene of a BAC construct and used to generate
B6 or B6.SJLF1 transgenic lines. (B) GFP was highly expressed in a subset
of myeloid cells of the spleen but not lymph node. (C) T and B lympho-
cytes expressed a low level of GFP. Three founder lines showed similar
cell-specific patterns of GFP expression, but higher levels were observed
in the B6-820 line (n = 5 mice). (D) GFP was up-regulated in T cells 12 h
after anti-CD3 injection in vivo or in B cells after 3 h of anti-IgM treat-
ment in vitro (n = 4 mice and three experiments).
JEM Vol. 208, No. 6
(Hogquist et al., 1994; Santori et al., 2002) and in vivo
(Stefanski et al., 2001).
GFP up-regulation was transient after TCR stimulation
with maximum expression observed between 12 and 24 h
(Fig. 2 B). The up-regulation of endogenous Nur77 protein
was also determined in parallel, and endogenous levels also
correlated with strength of stimulus (Fig. S1 B), although peak
induction of endogenous Nur77 occurred earlier than GFP,
presumably reflecting the time required for maturation of a fully
fluorescent GFP and the greater stability of GFP (Fig. S1 C).
Analogous experiments were performed in vivo using
OT-I/Nur77GFP cells transferred into mice and infected with
strains of Listeria monocytogenes expressing variants of the OVAp.
As seen with peptides in vitro, the level of GFP in OT-I T
cells in vivo reflected the stimulatory strength of the variant
peptide ligand, even in the context of an infection (Fig. S2).
Thus, the Nur77GFP mouse has the potential to be a sensitive re-
porter of TCR signal strength both in vitro and in vivo.
GFP expression is induced by positive selection
and maintained by tonic MHC signals
Because Nr4a1 message was up-regulated during positive
selection (Baldwin and Hogquist, 2007) and GFP could be
induced by low-affinity TCR ligands (Fig. 2 A), we sought to
determine whether GFP was up-regulated by positive selec-
tion in vivo. In the thymus of Nur77GFP mice, only a fraction
of cells expressed GFP (Fig. 3 A). Further analysis revealed
that the GFP+ population was enriched for DP dull, CD4,
and CD8 single-positive (SP) cells (Fig. 3 A, right). Among
DP thymocytes, the GFP+ cells were high for CD69 and the
TCR- chain (Fig. 3 B, dot plot), suggesting that induction
of GFP occurred at the time of positive selection (Fig. 3 B,
histogram). Furthermore, we observed very low expression of
GFP in DP thymocytes from OT-I Tapo (nonselecting) mice
compared with OT-I (selecting) control mice (Fig. 3 C), dem-
onstrating that positive selection induced GFP in vivo. Con-
sistent with the induction of GFP expression during positive
selection, the majority of GFP bright cells were located in the
medulla with only a few GFP-positive thymocytes found in
the cortex (Fig. 3 D).
Like mature SP thymocytes, naive T cells in the periphery
expressed GFP, although at slightly lower levels (Fig. 4 A,
right), suggesting a decay of GFP with maturation. In fact,
there was a modest stepwise decrease in GFP expression dur-
ing development with semimature SP thymocytes expressing
the highest level of GFP, followed by mature SP, then recent
thymic emigrants (RTEs; HSAhiQa2lo), and finally naive non-
RTE T cells (Fig. S3). Nonetheless, naive non-RTE T cells ex-
pressed a level of GFP in the steady-state that was significantly
above background and did not vary with age. Interestingly,
this basal level of GFP was higher in CD4 than CD8 T cells,
which is consistent with some models that have proposed that
the CD4 coreceptor delivers a stronger signal than the CD8
coreceptor (Veillette et al., 1988; Legname et al., 2000).
Memory phenotype CD4 and CD8 T cells did not express sig-
nificantly different levels of GFP when compared with their
GFP expression was observed in B cells (Fig. 1 D, right) but
not T cells. Thus, we conclude that GFP expression can be
induced after lymphocyte antigen receptor activation both
in vitro and in vivo.
Initial microarray experiments showed differential expres-
sion of Nr4a1 in thymocytes undergoing positive versus neg-
ative selection. In light of this, we asked whether the level of
GFP induced in T cells would correlate with the strength of
TCR signal perceived. We used Kb/OVA-specific OT-I TCR
transgenic mice, for which many variant peptide ligands have
been characterized (Hogquist et al., 1994; Daniels et al.,
2006). DP thymocytes from OT-I/Nur77GFP mice lacking
the transporter associated with antigen process 2 gene (Tapo)
were stimulated with APCs pulsed with OVA peptide (OVAp)
variants in vitro. In Fig. 2 A, these are listed according to
stimulatory strength, with the cognate OVAp on the left and
the weakest variant (E1) on the right. The level of GFP in-
duced by each directly correlated with its stimulatory activity
(Fig. 2 A). Interestingly, even the low-affinity variant E1 and
the self-peptide -CAT induced GFP above the background
level (control peptide p815; Fig. 2 A, inset). Neither of these
weak peptides stimulates OT-I T cells to proliferate, but
they support positive selection of OT-I in organ cultures
Figure 2. The level of GFP expression reflects TCR signal strength
and is transient. (A) OT-I/Tapo/Nur77GFP thymocytes were co-cultured for
3 h with B6 splenocytes pulsed with the peptide SIINFEKL (OVAp) or the
indicated altered peptide ligands, listed in order of decreasing potency.
The MFI of GFP was normalized to the level observed with OVAp stimula-
tion (n = 5). cntl, control. (B) Splenocytes from OT-I/Nur77GFP mice were
cultured with the indicated peptides for various lengths of time. The data
represent the mean normalized GFP levels from six different experiments
of at least six mice. Error bars indicate standard deviation.
TCR signal strength in T cell selection | Moran et al.
Thymocytes undergoing negative selection express
high levels of GFP
If GFP levels reflect the strength of the TCR signal perceived,
one would predict higher GFP expression in thymocytes under-
going negative selection compared with positive selection. To
test this, we used the OT-I/rat insulin promoter (RIP)–mem-
brane OVA (mOVA) system, in which negative selection
occurs via clonal deletion in CD8 SP thymocytes (Kurts
et al., 1997). However, cells undergoing clonal deletion are
rapidly cleared by thymic macrophages (Surh and Sprent,
1994). Thus, we created OT-I/Bimo/Nur77GFP transgenic
mice, in which deficiency of the proapoptotic molecule Bim
prevented apoptosis. Accordingly, bone marrow chimeras
were created using OT-I/Nur77GFP or OT-I/Bimo/Nur77GFP
mice as donors and B6 or RIP-mOVA mice as recipients
(Fig. 5). As expected, we observed efficient positive selection
of OT-I in B6 recipients and efficient deletion in RIP-
mOVA recipients (Fig. 5 A, left). Moreover, OT-I/Bimo cells
underwent efficient positive selection in B6 recipients, but
Bim deficiency completely rescued OT-I cells from deletion
in RIP-mOVA recipients (Fig. 5 A, right; and Fig. S5). Inter-
estingly, GFP expression was substantially higher in OT-I/
Bimo thymocytes rescued from deletion in RIP-mOVA re-
cipients compared with cells undergoing positive selection
in B6 recipients (Fig. 5 B). Therefore, we conclude that GFP
levels were induced to higher levels by negative selection sig-
nals as compared with positive selection stimuli.
Induction of GFP is TCR specific
Many immunological studies use the expression of CD69 as a
read-out for TCR activation. One caveat to this is that CD69
expression can be induced by inflammatory stimuli such as
type I interferons (Sun et al., 1998; Shiow et al., 2006), thereby
limiting its use as a marker of TCR stimulation in infection
or inflammatory settings. We sought to determine whether
inflammatory stimuli would also induce GFP expression in
Nur77GFP mice. Neither polyinosinic:polycytidylic acid (pI:pC;
Fig. 6 A) nor LPS (Fig. 6 B) induced GFP expression, although
both induced CD69
confirm this obser-
vation in the context
of an infection, we
Nur77GFP T cells into
lymph node T cells (right; defined as
CD44loCD69CD25). Data are representative
of >10 mice. (B) 1–2 × 106 polyclonal
Nur77GFP CD4 T cells were transferred into B6
or I-Ab–deficient (MHC IIo) recipients and
analyzed 6 or 9 d later. Bar graph shows the
mean GFP level on cells adoptively transferred
into I-Ab–deficient recipients normalized to
the level on CD4 T cells in B6 recipients. Data
are representative of 11 mice from four inde-
pendent experiments. Error bars indicate stan-
dard deviation. NTg, nontransgenic;
naive counterparts (unpublished data). Finally, in the absence
of the co-stimulatory molecule CD28, T cells expressed identical
levels of GFP compared with CD28-sufficient T cells (Fig. S4).
To determine whether the GFP levels in naive T cells re-
flect tonic TCR stimulation by self-pMHC, we adoptively
transferred Nur77GFP CD4 T cells into congenic WT or
MHC II–deficient hosts and analyzed them after 6 or 9 d.
The GFP level in naive phenotype, non-RTE CD4 T cells
was maintained after adoptive transfer into WT recipients
(Fig. 4 B, red line). In contrast, GFP was lost from CD4 T cells
in MHC II–deficient recipients. These data suggest that TCR
signals maintain GFP expression in peripheral T cells.
Figure 3. GFP expression is induced during positive selection.
(A) Flow cytometric analysis of GFP in total thymocytes (left). Dot plots
(right) show CD4 and CD8 expression on total or GFP-positive thymocytes
from Nur77GFP mice. (B) The GFP+ DP population was enriched for
CD69+TCR-+ cells (dot plot). CD69+TCR-hi (postselection) DP thymocytes
expressed higher levels of GFP compared with CD69TCR-lo (preselection)
DP thymocytes (histogram overlay). (C) GFP expression of DP thymocytes
from WT or Tap-deficient mice. NTg, nontransgenic. (D) Immunofluores-
cence analysis of GFP in the Nur77GFP thymus, with the cortical region de-
fined by staining for the 5t proteasome subunit. Data are representative of
>10 mice from at least three independent experiments. Bars, 100 µm.
Figure 4. GFP expression is maintained
in the steady-state by tonic MHC signals.
(A) Analysis of GFP levels in mature CD4 and
CD8 SP thymocytes (left; defined as HSA-
loQa2hi) or naive phenotype CD4 and CD8
JEM Vol. 208, No. 6
CD4+ T cells (Fig. 7 and Fig. S6). These data together suggest
that Treg cells perceive stronger TCR signals than Tconv cells
during development and that this perception continues in the
periphery. Interestingly, the GFP histograms for Treg and Tconv
cells are not completely distinct, but overlap. This is consistent
with TCR repertoire studies (Hsieh et al., 2006; Pacholczyk
et al., 2006), which showed that some clones are unique to
Treg cells, some are unique to Tconv cells, and some are shared.
STAT5 signaling does not increase GFP levels
in Treg or Tconv cells
Although it is proposed that avid interactions with self-ligands
are required for Treg cell development, c cytokines (IL-2 and to
a lesser extent IL-15 and IL-7) are also known to be crucial
(Burchill et al., 2007; Vang et al., 2008). To address the poten-
tial contribution of cytokine signaling to GFP expression, we
isolated thymocytes and lymphocytes from Nur77GFP mice and
cultured them for 3–12 h with 25 ng/ml IL-2 and observed no
increase in GFP expression (unpublished data). A previous study
showed that constitutive expression of STAT5 (Stat5b-CA) in-
creased the frequency and number of Foxp3+ Treg cells (Burchill
et al., 2003). Therefore, we generated Nur77GFP/Stat5b-CA mice.
B6 recipient mice and infected them with L. monocytogenes that
did or did not express OVA. Only when the pathogen ex-
pressed the OVA antigen was GFP up-regulation observed in
OT-I T cells (Fig. 6 C). Together, these results suggest that GFP
expression is driven by antigen receptor signaling in T cells in
Nur77GFP mice and not by other homeostatic or inflam-
Treg cells express higher levels of GFP than Tconv cells
Because GFP levels reflected TCR signal strength in Nur77GFP
mice, we sought to use these mice to test whether Treg cells
perceive stronger TCR signals compared with Tconv cells dur-
ing development. Thymic Foxp3+ Treg cells expressed approxi-
mately twofold higher mean fluorescence intensity (MFI) for
GFP than conventional CD4SP (Fig. 7). A higher level of
GFP in Treg cells might arise if they were developmentally
younger than Tconv cells because we observed a slight decrease
in GFP as cells matured in the thymus (Fig. S3). This is un-
likely because a previous study showed that thymic Treg cells
are, on average, developmentally more mature than conven-
tional CD4SP (McCaughtry et al., 2007). Furthermore, thy-
mic Treg cell progenitors (defined as CD4SP, CD25+, CD122hi,
Foxp3; Burchill et al., 2008) had an even higher GFP ex-
pression level (Fig. 7). Finally, a twofold higher GFP level was
also observed in peripheral Treg cells compared with Tconv
Figure 5. Thymocytes undergoing negative selection express
higher levels of GFP compared with those undergoing positive se-
lection. OT-I/Nur77GFP mice or OT-I/Bimo/Nur77GFP mice were generated
and used as bone marrow donors. 5–10 × 106 bone marrow cells were
injected into lethally irradiated B6 or RIP-mOVA recipients. (A) Expression
of CD4 and CD8 on thymocytes from the indicated chimeric mice. (B) GFP
expression on V2+ CD8SP from the indicated chimeric mice. Representa-
tive data are from five experiments with more than five mice.
Figure 6. Induction of GFP is TCR specific. (A and B) Nur77GFP mice
were injected i.v. with pI:pC (A) or LPS (B). After 6 h, cells were analyzed
for GFP (left) and CD69 (right) expression. (C) 5 × 106 OT-I/Nur77GFP lymph
node cells were adoptively transferred into B6 recipients and infected
with L. monocytogenes expressing the OVAp (LM-OVA) or not (LM). GFP
expression on V2+CD8+ transferred cells was evaluated after 12 h. Histo-
grams show representative data from three independent experiments
with at least three mice.
TCR signal strength in T cell selection | Moran et al.
precursory frequency, suggesting that Treg cell progenitors com-
pete for a limited factor during development (Bautista et al.,
2009; Leung et al., 2009). This factor might be a cytokine,
given the profound requirement for c cytokine signaling dur-
ing Treg cell development (Burchill et al., 2007). Alternatively,
Treg cell progenitors might compete for recognition of rare
high-affinity self-ligands during development. In the Nur77GFP
mouse, the level of GFP reflects TCR signal strength; thus, we
postulated that if Treg cell development were limited by compe-
tition for high-affinity ligands, GFP would be increased at low
precursor frequency of a TCR specific for high-affinity ligands.
Alternatively, if Treg cell development were limited by non-
TCR factors, GFP would not increase with less competition
for the selecting ligand. To test this, using a mixed bone marrow
chimera strategy, we generated animals with varying frequen-
cies of Nur77GFP/G113 TCR transgenic precursors. G113 was
cloned from a naturally occurring Treg cell, although its TCR
specificity is unknown (Hsieh et al., 2006). Similar to what
was previously reported (Bautista et al., 2009), we observed
that monoclonal G113 mice have an almost undetectable fre-
quency of CD25+ Treg cells in the thymus (Fig. 9 A) or periph-
ery (not depicted). Interestingly, the level of GFP was not
higher on intact G113 CD4SP compared with the polyclonal
CD4SP population (Fig. 9 A, top row). This suggests that
Treg cell–encoded TCRs do not have an intrinsically higher
affinity for ubiquitous self-antigens and that both Treg and
conventional CD4 precursors are positively selected through
similar (low) affinity interactions in the cortex. In contrast to
monoclonal G113 mice, when chimerism was <1%, the fre-
quency of CD4+CD25+ cells dramatically increased, as pre-
viously reported (Bautista et al., 2009). Surprisingly, the level
of GFP increased on all G113 precursors (Fig. 9 A) when they
were at low precursor frequency, with an inverse relationship
between percent chimerism and the GFP MFI (Fig. 9 B). This
was true for both CD25 and CD25+ G113 CD4SP thymo-
cytes. GFP did not increase on non-Treg cell (OT-II)
precursors in analogous control chimeras (Fig. 9 B).
These data demonstrate that thymic Treg cell precursors
compete for interactions that lead to strong TCR stim-
ulations and imply that the high-affinity self-antigens
that support Treg cell development are rare.
NKT cells express high levels of GFP
during thymic selection
iNKT cells have been described as autoreactive by de-
sign with a preponderance of indirect data suggesting
Thymocytes and lymphocytes were harvested and analyzed for
Foxp3 expression and total GFP. As previously described
(Burchill et al., 2003), we observed an increase in Treg cells in
Nur77GFP/Stat5b-CA mice in both the thymus and the periph-
ery when compared with WT littermate controls (Fig. 8 A).
However, the total MFI of GFP in Treg cells from Nur77GFP/
Stat5b-CA thymocytes did not increase but rather decreased in
both the thymus (42 ± 2% decrease) and the periphery (32 ± 8%
decrease; Fig. 8 B), whereas GFP expression in conventional
CD4 thymocytes and lymphocytes did not change (Fig. 8 C).
These data suggest that c cytokine signaling via Stat5 does not
account for the increased expression of GFP observed in the
CD4+Foxp3+ population. Rather, the decrease in the total GFP
MFI in the Treg cell population of Stat5b-CA mice likely reflects
the recruitment of low-affinity TCR clones normally found in
the naive repertoire into the Treg cell population as previously
suggested (Burchill et al., 2008).
Treg cells compete for strong TCR ligands in the thymus
Previous studies with Treg TCR transgenic mice showed that
development of the Foxp3+ Treg cell lineage is impaired at high
Figure 7. Treg cells express higher levels of GFP compared with
Tconv cells. Lymphoid organs from Nur77GFP mice were analyzed by flow
cytometry. Treg cells were defined as CD4SP CD25+Foxp3+. Treg cell progeni-
tors were CD4SP CD25+CD122hiFoxp3. Data are representative of more
than eight mice from eight experiments. NTg, nontransgenic.
Figure 8. Stat5 signaling does not increase GFP in Treg or
Tconv cells. Stat5b-CA/Nur77GFP and Nur77GFP mice were generated.
(A) Increased frequency of CD4+Foxp3+ Treg cells in Stat5b-CA/
Nur77GFP mice in both the thymus and periphery. (B) Histogram
overlays of CD4+Foxp3+ cells of Stat5b-CA/Nur77GFP and Nur77GFP
control thymocytes (left) and lymphocytes (right). (C) Histogram
overlays of CD4+Foxp3 CD4 T cells from Stat5b-CA/Nur77GFP and
Nur77GFP mice. Data are representative of three experiments with
four mice per group. NTg, nontransgenic.
JEM Vol. 208, No. 6
that precursors interact with a stimulatory self-lipid ligand, which
remains incompletely identified (Bendelac et al., 2001; Gapin,
2010). Surprisingly then, both thymic and splenic iNKT cells
expressed very low levels of GFP in Nur77GFP mice (Fig. 10 A).
To confirm that iNKT cells could in fact up-regulate GFP
after TCR stimulation, we injected mice i.p. with 5 µg
-galactosylceramide (-GalCer). There was robust induc-
tion of GFP in splenic CD1d tetramer–positive cells (Fig. 10 B).
The lack of GFP in thymic iNKT cells was seemingly contra-
dictory to data implicating agonistic TCR stimulation in thy-
mic selection of iNKT cells. However, it is well known that
iNKT cells undergo cell division after selection (Benlagha
et al., 2002) and that mature iNKT cells can be retained in the
thymus for extremely long periods of time (Berzins et al.,
2006). Thus, to inquire more specifically about the intensity
of TCR stimulation during iNKT cell selection, we sought to
evaluate the earliest iNKT cell precursors, previously named
stage 0 precursors, which can be identified as binding CD1d/
-GalCer tetramer, and are HSAhi, CD44lo, NK1.1 (Benlagha
et al., 2005). Because such cells are rare in the thymus, we
performed CD1d/-GalCer tetramer–based magnetic enrich-
ment. As expected, the majority of thymic iNKT cells were
stage 3 mature cells (CD44hiNK1.1+), with a smaller subset of
stage 2 and 1 cells (NK1.1; Fig. 10 C). CD44 iNKT cells
were further defined as stage 1 (HSAlo) or stage 0 (HSAhi).
Interestingly, stage 0 iNKT cell progenitors expressed a higher
level of GFP than age-matched conventional CD4SP, sug-
gesting that they are indeed selected on stronger TCR ligands
than Tconv CD4 T cells. However, unlike Tconv and Treg CD4
T cells, most iNKT cells lost GFP expression after matura-
tion in the thymus and persisted like this in the periphery
(Fig. 10 A).
In this study, we introduced a unique BAC transgenic mouse
useful for studying T cell activation in vivo. We showed that
antigen receptor signaling was a major inducer of GFP in
lymphocytes in Nur77GFP mice. GFP was not induced by
Figure 9. Treg cells compete for strong TCR ligands during develop-
ment. G113/Rag1o/Nur77GFP (CD45.2+) and WT (CD45.1+) bone marrow
were mixed at various ratios and used to reconstitute lethally irradiated
recipients (CD45.1+). Chimerism was determined by analyzing the G113+
T cell fraction from lymphoid tissue. 8 wk after transplant, lymphoid or-
gans were harvested and analyzed by flow cytometry. (A) Dot plots on the
far left show percent chimerism from select animals ranging from 97 to
0.032% G113 donor–derived T cells. Histograms on the right show GFP
after gating on CD45.2+V2+V6+CD4SP that were either CD25 negative
or positive. (B) Similar mixed bone marrow chimeras were set up with
OT-II/Nur77GFP bone marrow. Graph shows the GFP MFI on V6+ CD4SP
thymocytes (for G113) or V5+ CD4SP (for OT-II). Data are representative
of three experiments with more than five mice. n.d., not determined.
Figure 10. NKT cells express higher
levels of GFP immediately after selection
but do not maintain it in the periphery.
(A) Histograms show the level of GFP on con-
ventional CD4SP or CD1d/-GalCer tetramer–
binding iNKT cells in the thymus or spleen of
Nur77GFP mice. Data are representative of
more than five mice in at least three indepen-
dent experiments. (B) GFP levels on splenic
iNKT cells 6 h after injection with -GalCer or
solvent control. (C) CD1d/-GalCer tetramer–
binding iNKT cells were enriched from adult
thymus using magnetic beads. Dot plots show
the gating strategy used to identify subsets of
thymic iNKT cells. (D) GFP levels on various
staged iNKT cell populations compared with
conventional CD4SP thymocytes. Data are
representative of four experiments and more
than nine mice. NTg, nontransgenic.
TCR signal strength in T cell selection | Moran et al.
the failure of IL-2 to increase GFP expression in vitro or
Stat5b-CA to increase GFP in vivo suggested otherwise.
In fact, Treg cells from Stat5b-CA mice showed an overall de-
crease in GFP MFI. This is consistent with the TCR reper-
toire analysis performed by Burchill et al. (2008) in the
Stat5b-CA mice, in which they observed that overexpression
of Stat5 diverted TCR clones from the naive population into
the Treg cell repertoire. Because naive T cells expressed lower
GFP when compared with Treg cells, this resulted in a de-
crease in the total GFP MFI of the Treg cell population in
Using a Treg TCR transgenic model (G113) at low precur-
sor frequency, we were able to provide evidence that Treg cells
compete for strong TCR ligands during development. We
observed that when there was high competition, as seen in
the 100% G113 chimeras, there was no increase in the overall
level of GFP on Tconv cells, suggesting that the G113 TCR
does not have an intrinsically higher affinity for ubiquitous
(presumably positive selecting) self-antigens. However, the
level of GFP in G113 cells was higher when the progenitor
was present at low precursor frequencies. This finding implies
that G113 precursors compete for rare higher affinity ligands,
either because the proteins they are derived from are low
abundance or because the APCs that process and present such
ligands are not numerous. The Nur77GFP mouse may provide
a useful tool to distinguish between these possibilities in the
future. Interestingly, even at very low precursor frequencies,
where all G113+ thymocytes were GFPhi, not all were con-
verted to the Treg cell lineage. This may suggest that there are
other factors that also limit Treg cell development. Alterna-
tively, it may reflect the delay between time of TCR stimula-
tion and CD25 up-regulation and Foxp3 induction or clonal
deletion of some of this population. A delay is consistent with
work suggesting that Foxp3 is not required for the initial lin-
eage decision in the thymus, but is downstream of a TCR sig-
nal and thus a delay in lineage differentiation (Gavin et al.,
2007; Lin et al., 2007).
Finally, we show that iNKT cells also perceive a stronger
TCR stimulus than Tconv cells upon selection in the thymus.
However, unlike Treg cells, iNKT cells do not continue to
perceive this stimulus as they mature and emigrate to the pe-
riphery. Interestingly, the level of GFP on iNKT cells in the
spleen and liver was so low that it suggests they receive very
weak if any TCR stimulation in the steady-state. Given this,
it is unclear why iNKT cells express intermediate levels of
the T cell activation marker CD69, although it is well estab-
lished that other stimuli can induce CD69 (Shiow et al., 2006).
However, our findings are consistent with a published re-
port that iNKT cells can persist long term in the absence of
CD1d (McNab et al., 2005).
Many cell types in the body express CD1d (Bendelac
et al., 1997). The glycosphingolipid iGb3 was identified as a
potential self-lipid ligand for NKT cells, although it is not
clear that it is the sole endogenous antigen that stimulates
iNKT cells (Zhou et al., 2004; Gapin, 2010). There is emerg-
ing evidence that stimulatory lipids are continually catabolized
TLR (toll-like receptor) ligands or other inflammatory
stimuli, did not require co-stimulation, and was dependent on
MHC for induction and maintenance in T cells. This finding
is surprising in light of evidence that mechanical force, hor-
mones, growth factors, and cytokines could induce Nur77
expression at the transcriptional level in nonlymphoid tissues
(Pei et al., 2005; Pols et al., 2007). The only other stimulus re-
ported to induce Nur77 in T cells is the thymotoxic plastics
stabilizer DBTC (di-n-butylin dichloride; Gennari et al., 2002),
whose biochemical effect is not understood.
We observed that lymphocytes in the steady-state expressed
a low level of GFP that was nonetheless consistently above
the nontransgenic background. Polyclonal T and B lympho-
cytes expressed similar levels of GFP, and both responded to
antigen receptor activation with rapid induction of GFP.
Moreover, the thymic and peripheral expression pattern of
GFP was consistent with antigen receptor regulation of sig-
naling and TCR “tuning” and suggests this reporter mouse
may be useful for imaging selection events in the thymus.
Consistent with the idea that Nur77 expression is tightly reg-
ulated by the TCR, we found that adoptive transfer of CD4
T cells into an MHC II–deficient environment resulted in a
loss of GFP expression that was otherwise maintained in the
presence of MHC. This suggests that the tonic TCR signals
perceived by T cells sustain the elevated GFP expression in
the steady-state. Whether GFP expression in B cells requires
tonic BCR signals is unknown. Naive CD4 T cells expressed
higher basal levels of GFP compared with naive CD8 T cells.
These data may suggest that CD4 T cells as a population ex-
press TCRs that perceive stronger self-pMHC signals through
the TCR/coreceptor than CD8 T cells, but more experi-
ments are required to address this hypothesis.
Using the Nur77GFP mouse as a reporter of TCR signal
strength, we tested the idea that CD4+Foxp3+ Treg cells perceive
a stronger signal during thymic development than Tconv CD4
T cells. We showed that polyclonal Treg cells expressed a higher
level of GFP, implying they perceived stronger TCR stimulation
upon selection and continued to do so in the periphery. These
data are consistent with previous work with TCR transgenic
mice in which coexpression of a neo-self-antigen and specific
TCRs skewed the T cell repertoire to a higher frequency of
CD4+Foxp3+ T cells (Jordan et al., 2001; Kawahata et al., 2002).
It is also consistent with the fact that mutations in LAT (linker
of activated T cells) led to Tconv cell development but not
CD4+Foxp3+ cell development (Koonpaew et al., 2006). Inter-
estingly, we noted that there was distinct overlap in the GFP
levels between Treg and non-Treg cells. This pattern is conceptu-
ally reminiscent of the repertoire analysis of Treg and non-Treg
cells, which showed distinct receptor specificities that were
found predominantly in one population or the other and some
receptor specificities that were shared (Hsieh et al., 2006;
Pacholczyk et al., 2006).
Stat5 signaling is known to be required for CD4+Foxp3+
Treg cell development (Burchill et al., 2007). In light of this, it
was possible that increased cytokine sensitivity and signaling
in Treg cells accounts for the increased GFP expression. However,
JEM Vol. 208, No. 6
Immunofluorescence. Tissue was harvested and immediately placed in 4%
PFA in PBS overnight. Tissue was washed three times with PBS before being
placed in 15% sucrose in PBS overnight. Tissue was then embedded in OCT
compound and frozen with 2-methylbutane with dry ice and stored at
80°C for long-term use. After cutting tissue sections, slides were dried for
30 min and then submerged in 0.1% Triton X-100 in PBS at room tempera-
ture for 5 min. Blocking was performed with 3% BSA in PBS before general
antibody staining or endogenous GFP detection.
Tetramer-based enrichment of thymic iNKT cells. Enrichment of
CD1d+ cells was performed using an adult thymus as previously described
(Matsuda et al., 2000). After tetramer enrichment, cell surface stains were
performed, and a dump strategy (including B220, CD11c, Gr1, and CD25)
was used to eliminate nonspecific events. The smallest gate (stage 0) included
a mean of 115 events.
In vivo and in vitro stimulation. For stimulation with -CD3, 50 µg
-CD3 was injected i.v., mice were euthanized, and tissues were harvested
and analyzed 12 h later. Stimulation of NKT cells was performed by i.v. in-
jection of 5 µg -GalCer 4–6 h before harvesting the spleen and liver. 50 µg
LPS and 100 µg pI:pC were administered i.v., and tissues were harvested
6 h or 12 h later, respectively. For stimulation with -IgM, 106 bulk spleno-
cytes were cultured with 10 µg soluble -IgM for 3 h at 37°C and then
stained for FACS analysis. Plate-bound stimulation was performed by pre-
coating 48-well plates with 10 µg -CD3 or 10 µg -CD2/CD3 and 50 µg
-CD28 O/N at 4°C and then culturing thymocytes and lymphocytes at
106 cells/well for 3 h at 37°C. OT-I Tapo stimulation was performed by
peptide pulsing APCs with saturating concentrations of peptides and then
adding thymocytes at a 1:4 ratio. Cells were incubated for 3 h at 37°C
before FACS analysis.
L. monocytogenes infection. 5 × 106 OT-I (CD45.2+) lymphocytes were
adoptively transferred in B6.SJL (CD45.1+) hosts. 24 h after transfer, mice
were infected i.v. with 5 × 103 CFU of L. monocytogenes or variants expressing
either the OVAp or one of the OVA altered peptide ligands (Zehn et al.,
2009) provided by M. Bevan (University of Washington, Seattle, WA). Mice
were euthanized, and spleens were harvested 24 h after infection. Tissue was
incubated with 5% collagenase D in serum-free HBSS for 30 min with mild
agitation before performing cell surface staining.
Statistical analysis. Prism software (GraphPad Software) was used for sta-
tistical analysis. Paired and unpaired, two-tailed Student’s t tests were used for
data analysis and generation of p-values.
Online supplemental material. Fig. S1 shows that GFP and endog-
enous Nur77 reflect strength of antigen receptor signal but that GFP de-
cays more slowly. Fig. S2 shows that GFP expression correlates with TCR
signal strength during infection in vivo. Fig. S3 shows that GFP expression
changes with developmental age. Fig. S4 shows that GFP expression is in-
dependent of CD28. Fig. S5 shows that peripheral OT-I T cells that escaped
deletion in RIP-mOVA recipients expressed a high level of GFP. Fig. S6
shows the normalized MFI of GFP in various lymphocyte subsets. Online
supplemental material is available at http://www.jem.org/cgi/content/full/
We thank C.-S. Hsieh for providing G113 transgenic mice and helpful feedback,
X.-J. Ding, J. Vevea, C. Mora-Solano, and S. Perry for technical assistance, and
S. Jameson and M. Farrar for Stat5b-CA mice and discussions. J. Olson provided
This work was supported by the National Institutes of Health (grant RO1
AI39560 to K.A. Hogquist and grant T32 AI007313 to A.E. Moran).
The authors have no competing financial interests.
Submitted: 9 February 2011
Accepted: 15 April 2011
in lysosomes, and it was recently shown that when the cata-
bolic enzyme -galactosidase is absent, CD1d+ cells are able
to activate iNKT cells (Bendelac et al., 1995; Zhou et al.,
2004; Darmoise et al., 2010). Importantly, TLR signaling
seems to inhibit -galactosidase activity, thereby allowing for
iNKT cell activation in the context of infection (Darmoise
et al., 2010). The Nur77GFP mice may therefore be useful in
determining what types of infections and stimuli activate
APCs to display self-lipids that then stimulate iNKT cells.
Historically, CD69 has been used to study T cell activation.
However, CD69 expression is up-regulated by inflammatory
stimuli (Shiow et al., 2006), whereas GFP in Nur77GFP mice
was not. Therefore, this difference may make the Nur77GFP
tool useful for determining whether certain populations of
T cells, such as CD8 IELs that express high levels of CD69,
are being activated through their antigen receptor or whether
the environmental stimuli cause the activated phenotype.
In light of the tight regulation of GFP expression by TCR
ligation and the differential expression of GFP based on TCR
signal strength, we propose that the Nur77GFP mouse may be
a novel model for studying TCR signal strength in vivo. In
addition, because inflammatory stimuli that induce CD69
expression fail to up-regulate GFP expression, we expect that
these mice will be a useful tool for tracking activated T cells
in several different experimental contexts such as acute and
chronic infection, cancer, and transplantation.
MATERIALS AND METHODS
Mice. A Nur77GFP targeting construct was created by insertion of a GFP-
Cre fusion protein cDNA into the start site of the Nr4a1 gene on a 167-kb
BAC vector. An 135-kb fragment from this vector was purified via BsiWI
restriction sites and microinjected into C57BL/6J (B6) embryos at the
Mouse Genetics Laboratory at the University of Minnesota. Alternatively, a
167-kb linearized DNA fragment was injected into B6 × SJL F1 embryos
at the Transgenic and Chimeric Mouse Core Facility at the University
B6 and B6.SJL (CD45.1 congenic B6) mice were obtained from the
National Cancer Institute. MHC I-Ab–deficient mice were obtained from
Taconic. CD28-deficient mice were obtained from The Jackson Laboratory.
G113 TCR transgenic mice were provided by C.-S. Hsieh (Washington
University in St. Louis, St. Louis, MO), and Stat5b-CA mice were pro-
vided by M. Farrar (University of Minnesota, Minneapolis, MN). All ani-
mal experimentation was approved by and performed according to
guidelines from the Institutional Animal Care and Use Committee at the
University of Minnesota.
Flow cytometry. Cell surface staining was performed with antibodies
from eBioscience, BD, or BioLegend. For intracellular Foxp3, cells were
stained with the Foxp3 Staining Buffer set (eBioscience). For endogenous
Nur77 detection, cells were fixed with fresh 4% PFA, vortexed well, and
permeabilized with 0.1% Triton X-100. Antibody was used at 1:50 for
staining. Biotinylated CD1d/-GalCer monomers were obtained from the
tetramer facility at the National Institutes of Health. Isolation of CD1d/
-GalCer binding cells via tetramer enrichment was performed as previ-
ously described (Matsuda et al., 2000). Samples were analyzed on an LSR
II (BD). Data were processed with FlowJo software (Tree Star).
Bone marrow chimeras. Bone marrow was depleted of T cells with anti-
Thy1.2 antibody and complement. Bone marrow was injected into lethally
irradiated (1,000 rad) recipient mice. Chimeras were euthanized and ana-
lyzed at 8–12 wk after transplant.
TCR signal strength in T cell selection | Moran et al.
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