Low Ligand Requirement for Deletion
and Lack of Synapses in Positive Selection
Enforce the Gauntlet of Thymic T Cell Maturation
Peter J.R. Ebert,1Lauren I. Richie Ehrlich,1,2and Mark M. Davis1,*
1Howard Hughes Medical Institute and The Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford,
CA 94305, USA
2Present address: Stanford Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford,
CA 94305, USA
Immature double-positive (CD4+CD8+) thymocytes
by forming an immune synapse that sustains contact
with the antigen-presenting cell (APC). Using fluores-
cently labeled peptides, we showed that as few as
two agonist ligands could promote APC contact
and subsequent apoptosis in reactive thymocytes.
Furthermore, we showed that productive signaling
for positive selection, as gauged by nuclear translo-
cation of a green fluorescent protein (GFP)-labeled
NFATc construct, did not involve formation of a syn-
apse between thymocytes and selecting epithelial
ade of endogenous positively selecting ligands pre-
vented NFAT nuclear accumulation in such cultures
and reversed NFAT accumulation in previously stim-
ulated thymocytes. Together, these data suggest
a ‘‘gauntlet’’ model in which thymocytes mature by
continually acquiring and reacquiring positively se-
lial cells, thereby allowing them to sample many cell
surfaces for potentially negatively selecting ligands.
The mature T cell repertoire arises as the result of interactions
between immature T cell precursors (double-positive [DP] thy-
mocytes) and antigen-presenting cells (APCs) within the thymus.
A strong interaction between the thymocyte’s T cell receptor
molecules (TCRs) and self-peptide-self-MHC on any of the
APCs it encounters is likely to induce programmed cell death
(termed negative selection, or clonal deletion), whereas failure
to achieve a sufficient threshold of TCR signaling results eventu-
ally in death by neglect (von Boehmer et al., 2003). Juxtaposed
between these two outcomes is an intermediate mode of en-
gagement with self-peptide-MHC that can induce sweeping
transcriptional changes that result eventually in the thymocyte’s
maturation to the single positive (SP [CD4+or CD8+]) stage
(Huang et al., 2004). Various studies have shown that this last
process of positive selection requires prolonged interactions
with appropriate epithelial cells (Germain, 2002; Kisielow and
Miazek, 1995; Liu and Bosselut, 2004; Yasutomo et al., 2000),
but the nature of those contacts and of the TCR signaling that
accompanies them is not well understood. It is known, however,
that thymocytes are extremely sensitive to their cognate pep-
tide-MHCligands, evenmoreso thantheir matureTcellcounter-
parts (Daniels et al., 2006; Davey et al., 1998). Negative selection
of TCR transgenic thymocytes has been shown to occur in re-
sponse to far fewer peptides per APC (on average) than mature
T cells bearing the same transgenic TCRs (Peterson et al., 1999).
With respect to mature T cells, it has been known for some
time that their interactions with agonist ligands are characterized
by the formation of an immune synapse (Grakoui et al., 1999;
Huppa and Davis, 2003), a tight interface between T cells and
APCs (Davis etal., 2007)that can sustaincontact and productive
signaling for hours in the case of CD4+T cells (Huppa et al.,
2003). This led to the suggestion that immature T cells might ar-
rest their migration and form a similar structure with selecting
thymic epithelial cells (TECs) (Bousso et al., 2002; Hogquist
previously that DP thymocytes respond to negatively selecting
ligands, presented either by thymic APCs (Richie et al., 2002)
or supported lipid bilayers (Hailman et al., 2002), by adhering
to those APCs and polarizing CD3z, Lck, and CD4 into an im-
mune synapse, albeit with differences in localization of TCR
and other signaling molecules when compared to mature effec-
tor T cells. However, although thymocytes appear to alter their
pattern of motility under conditions of positive selection (Bhakta
et al., 2005; Bousso et al., 2002), whether immune synapses are
formed could not be assessed.
In this study, we first sought to define the signaling threshold
for negative selection. Using a biotinylated version of a peptide
derived from moth cytochrome C (MCC, residues 88–103) (Irvine
et al., 2002), we estimated the number of peptides bound to
APCs in bulk cultures and also precisely determined the number
of peptides bound to I-Ekon an APC by video fluorescence
microscopy. We then observed the interaction of thymocytes
derived from 5c.c7 TCR transgenic mice with these APCs and
found that as few as two MCC peptides within a thymocyte:APC
interface promoted apoptosis of the thymocyte within 1.5–4 hr.
Strikingly, however, in cultures of whole thymi in which an esti-
mated ?5%–10% of APCs bear enough peptides to promote
734 Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc.
apoptosis, negative selection was still >95% efficient. This
observation suggests that thymocytes must sample large num-
bers of APCs before committing to either positive or negative
selection, rather than sustain contact with individual positively
In order to address the question of whether immune-synapse
formation operates during positive selection, we expressed the
transcription factor NFATc1 as a green fluorescent protein
(GFP) fusion protein in immature DP thymocytes. This provided
us with an indicator that permits the observation of signaling in
these cells under conditions that promote positive selection of
5c.c7 TCR transgenic thymocytes in a reaggregate thymus or-
gan culture (RTOC) system (Hare et al., 1999; Jenkinson and An-
derson, 1994). We found that a large percentage of thymocytes
(?20%–30%) mobilized NFATc1 into their nuclei in the presence
of stromal cells expressing positively selecting I-Ekligands. This
response could be blocked by an antibody against I-Ekor one
which binds a small subset of endogenous I-Ekmolecules and
has previously been shown to block positive selection of 5c.c7
T cells in vivo (Baldwin et al., 1999). Mature T cells did not mobi-
lize NFATc1 under these conditions, indicating that this is
a stage-specific phenomenon. These results suggest that posi-
tive selection involves the continuous low-level stimulation of
immature thymocytes from multiple contacts with endogenous
pMHC ligands on thymic epithelial cells. In contrast to mature
T cells, this type of stimulation promotes the translocation of
NFATinto the nucleusfor prolonged periods of time, presumably
to induce genes needed for differentiation to the single-positive
Quantifying the Signal Threshold for Negative Selection
in Thymus Cultures
T cells bearing the 5c.c7 TCR recognize MCC-I-Ekas an agonist
ligand, and immature 5c.c7 thymocytes respond to this same
ligand by undergoing negative selection. Modification of MCC
with a biotin label does not disturb the interaction with TCR
and peptide-MHC (Irvine et al., 2002), and by labeling this
peptide with streptavidin (SA)-phycoerythrin (PE) (Irvine et al.,
2002), we could quantify the number of MCC-I-Ekcomplexes
presented by APCs in fetal thymic organ cultures (FTOCs)
derived from 5c.c7, invariant chain deficient (Cd74?/?) 16-day-
old embryos. Invariant chain deficiency drastically restricts
the repertoire of self-peptides bound to class II MHC and in par-
ticular prevents presentation of those peptides that promote
positive selection of I-Ek-restricted MCC-reactive T cells (Tourne
et al., 1995). Under normal conditions, these cultures contain
less than 2% CD4+SP cells and >90% DP cells after 3 days’
We first determined the relationship between the concentra-
tion of MCC-biotin added to these cultures and MCC-biotin
loaded onto the I-Ekmolecules of thymic stromal cells. 5c.c7
Cd74?/?e16 FTOC treated with varying amounts of MCC-biotin
were trypsinized after 3 days’ culture, and the resulting cell
dure disturbs cell-surface MCC-I-Ek(data not shown). The
median fluorescent intensity of I-Ek+cells was determined by
flow cytometry and was related to the absolute number of PE
molecules with PE Quantibrite beads (Figure 1A). Bydetermining
peptide numbers on CH27 cells by flow cytometry at high doses
and by microscopy at low doses, we showed that this relation
remains approximately linear, down to a regime of one peptide
per APC (Figure S1 available online). We then used flow cytom-
etry to determine the proportion of living DP cells remaining in
of MCC-biotin (Figure 1B). Negative selection of MCC-reactive
thymocytes still occurred at doses of MCC-biotin that equate
to an average of less than one peptide per APC. This estimate
was surprisingly low, and so we sought to refine it further.
Enumerating Peptides Required for Negative Selection
in Thymocyte:APC Couples
FTOC studies gave us an estimate for the number of peptides
that provoke negative selection, but this estimate was based
on extrapolation from large peptide numbers in bulk cultures
that do not possess the full repertoire of endogenous peptides,
some of which may be important for facilitating T cell signaling.
This estimate is thus unlikely to be precise. We therefore sought
to determine the number of peptides required to induce thymo-
cyte deletion on a single-cell level.
We chose CH27 cells as APCs for these experiments because
their intrinsic fluorescence is low, and therefore single PE mole-
cules can be reliably and precisely detected on their surface by
Figure 1. Number of Peptides Required for
Negative Selection in FTOC
(A) Thymi from day 16 5c.c7 Cd74?/?embryos
were cultured for 3 days in the presence of varying
concentrations of biotin-MCC. Thymi were then
dispersedinto single-cellsuspensions with trypsin
and EDTA, and cells were stained with SA-PE and
then anti-I-Ekand analyzed by flow cytometry.
I-Ek+cells were gated and we converted their
median PE fluorescence intensity to number of
SA-PE molecules using a standard curve gener-
ated with QuantiBRITE PE beads (BD Biosci-
a relation between peptide concentration and peptides loaded per APC (number of biotin-MCC per APC = 115 3 [biotin-MCC], in mM, r2= 0.979).
(B) Thymi from day 16 5c.c7 Cd74?/?embryos were cultured for 3 days in the presence of varying concentrations of biotin-MCC. Thymocytes were then recov-
ered and stained for CD4 and CD8, and the percentage of surviving DP cells was determined by FACS analysis. The percentage of surviving DP cells is plotted
against both concentration of biotin-MCC and the number of biotin-MCC per I-Ek+cell according to the correspondence shown in part (A). The depicted results
represent three independent experiments.
Immune-Synapse Formation in Thymic Selection
Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc. 735
video fluorescence microscopy (Irvine et al., 2002; Li et al., 2004;
Purbhoo et al., 2004). CH27 cells were pulsed with MCC-biotin,
washed, and labeled with SA-PE. We carried out both steps at
4?C in the presence of azide to prevent internalization and repre-
sentation of unlabeled peptide-MHC complexes. Labeled CH27
cells were then allowed to interact with 5c.c7 Cd74?/?thymo-
cytes. Because these thymocytes failed to receive TCR signals
in vivo, they were >95% preselection DP cells. We could then
observe their interaction by fluorescence video microscopy in
medium doped with 2mMCa2+(which does notincrease thymo-
cytes’ dose response to MCC, see Figure S2) and fluorescein-
labeled Annexin V. Annexin V binds to phophatidylserine, whose
exposure on the cell surface is a hallmark of programmed cell
death. This allowed us to identify thymocytes that had commit-
ted to apoptosis by their accumulation of the fluorescein label.
Figure 2 shows the response of 5c.c7 Cd74?/?thymocytes to
one (Figures 2A–2C) or two (Figures 2D–2F) biotin-MCC pre-
sented in the interface between the thymocyte and a CH27
APC. A single peptide was unable to provoke apoptosis even
after 6 hr in any of these experiments, whereas two or more
peptides were sufficient to induce thymocyte apoptosis within
2 hours in a large majority of cell couples observed (Figure 2G).
Negative selection occurred in response to two peptides in the
thymocyte-APC interface even when no other peptides were
present on the APC (e.g., as depicted in Figure 2E). In light of
these results, it is striking that deletion was so efficient in
FTOC. Under conditions in which APCs possess an estimated
average of 0.2 peptides, APCs presenting two or more peptides
would be very rare (less than one in 20 APCs under a normal dis-
tribution, or less than one in ten APCs in a distribution in which
an APC either presents zero or two peptides), and yet 90% of
thymocytes still undergo negative selection in such cultures
sess two or more MCC-I-Ek, over 50% of reactive thymocytes
were deleted from our thymus cultures. It seems likely then
that thymocytes sample a large number of thymic APCs before
committing to their fate, an idea that is incompatible with the no-
tion of positive selection via any long-lasting synapse formation
with one epithelial cell. To explore this issue, we set out to ob-
serve thymocyte:epithelial cell interactions that lead to positive
selection using NFAT as an indicator for productive signaling.
NFAT Activity as a Hallmark of Positive Selection
Prior work had indicated that calcineurin activity is necessary for
positive but not for negative selection (Neilson et al., 2004), and
for positively selecting signals in live thymocytes. Although there
are four NFAT family members expressed in T cells, NFATc1
expression was easily detected (Figures 3A and 3B) and is suffi-
cient to allow T cell maturation in the absence of the other thymic
NFAT family members (Ranger et al., 1998). Constitutively active
NFAT family members have also been shown to promote posi-
tive selection (Amasaki et al., 2002; Hayden-Martinez et al.,
To correlate endogenous NFAT activity with positive selection
in vivo in our 5c.c7 transgenic TCR system, we fixed freshly
isolated 5c.c7 thymocytes from selecting and nonselecting
backgrounds for immunofluorescent staining and analysis.
Figure 2. Thymocyte Apoptosis in Response to a Defined Number of Peptide-MHC Ligands
CH27 cells were loaded with biotin-MCC, extensively washed and labeled with SA-PE, and imaged together with 5c.c7 Cd74?/?thymocytes in the presence of
2 mM Ca2+and Annexin V-FITC. SA-PE signal was acquired within 5 min of interaction in 1 micron sections and then reconstructed with Metamorph software so
that the number of SA-PE molecules present could be determined. FITC fluorescence was assessed every 15 min for 6.5 hr.
(A and B) A DIC image (A) and PE fluorescence image (B) of a thymocyte-CH27 interaction. PE fluorescence images are rotated 90?so that the thymocyte-APC
interface faces the viewer. This interface contains a single peak of PE fluorescence corresponding in fluorescence intensity to a single SA-PE molecule.
(C) DIC (top) and Annexin V-FITC fluorescence images (bottom) of this cell couple over time. Frames are taken 30 min apart; the last frame represents 6 hr after
(D and E) A DIC image (D) and PE fluorescence image (E) of a thymocyte-CH27 interaction in which the interface contains a single peak of PE fluorescence with
double the intensity of that in (B), corresponding to 2 SA-PE molecules.
(F) A time-lapse of DIC and Annexin V-FITC shows that the thymocyte has committed to apoptosis after 1.5–2 hr of interaction (frames are 30 min).
(G) Summary of data represented in (A)–(F), n = 12–18 cell couples for each group.
Immune-Synapse Formation in Thymic Selection
736 Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc.
More than 20% of DP cells from positively selecting 5c.c7
B10.BR thymi had predominantly nuclear NFATc1 (Figures 3A–
3C). Only a tiny fraction of DP thymocytes from the nonselecting
5c.c7 B6 background showed such NFAT activation, suggesting
that NFATc1 nuclear localization might be a useful indicator for
positively selecting signals.
To further establish NFAT as a faithful indicator of positive-
selection events, we generated a self-inactivating retrovirus
(Kripke-Skillern and Nolan, 2002) that directed the expression
of GFP under the control of an NFAT-binding cassette (Fig-
ure S3A). A small but substantial population of mature T cells
(8%–10%) infected with this construct upregulated GFP in
response to antigen stimulation or ionomycin-induced calcium
hibitor FK506, demonstrating the reporter’s fidelity (Figure S4).
We then intrathymically injected reporter-infected 5c.c7 DP cells
into B10.BR hosts. By gating on the small proportion of GFP-
positive cells on subsequent days after injection, we could
show that thymocytes displaying NFAT-DNA-binding activity
positive selection in vivo (Correia-Neves et al., 2001; Sant’An-
gelo et al., 1998) (CD4intCD8intand CD4hiCD8intsubsets; Fig-
ure 3D, and see also Figure S5).
Expression and Localization of NFATc1-GFP
In order to observe NFATc1 mobilization during thymocyte
selection in RTOC, we made a retroviral construct of NFATc1,
tethered by a 21 amino acid linker to GFP (Figure S3B). As ex-
of resting cells (see the first panels of Figures 4A and 4B). To ver-
ify that overexpression of the altered NFAT had no deleterious
effects on thymic selection, we generated RTOC containing DP
thymocytes transduced with NFATc1-GFP and analyzed them
by flow cytometry after 3 days. DP thymocytes were selected
into the CD4+SP lineage in similar proportions regardless of
whether they expressed NFATc1-GFP, and a negatively select-
ing concentration of agonist MCC peptide resulted in a complete
absence of DP thymocytes that was also independent of
NFATc1-GFP expression (Figures S6A–S6D).
NFATc1-GFP Nuclear Translocation as a Sensor
for TCR Signaling
We first observed the dynamics of NFATc1-GFP localization in
mature T cells interacting with APCs. Not surprisingly, mature
5c.c7 T cells exhibited rapid nuclear import of NFATc1-GFP
upon engagement with CH27 B cells presenting agonist MCC-
upon initiation of calcium flux and was accompanied by tight
apposition of T cell and B cell membranes. NFAT was retained
iment (>30 min in many cases). When CH27 B cells presented
only their endogenous peptide-MHC ligands, no NFAT mobiliza-
tion occurred (Figure 4C).
To examine NFAT mobilization in immature thymocytes, we
generated RTOC composed of NFATc1-GFP-transduced 5c.c7
aging by live-cell video microscopy (Richie et al., 2002). As ex-
pected, the vast majority of DP thymocytes displayed a nuclear
localization of NFATc1-GFP when thymic APCs presented the
negatively selecting ligand MCC-I-Ek(Figure 4C). Although the
initiation of cell-cell contact could not accurately be determined
in RTOC, NFAT nuclear import was rapid because NFAT was
present in the nuclei of nearly all thymocytes, even when MCC
was added only 5 min prior to the onset of imaging. Consistent
with previous work, DP thymocytes under these conditions
Figure 3. Nuclear Accumulation of Thymocytes’ Endogenous
NFATc1 in Positively Selecting Thymi
(A) Thymi were dissected from 5c.c7 B6 mice and thymocytes were liberated
directly into 4% paraformaldehyde. Fixed cells were then permeabilized and
stained with anti-CD8a PE and mouse anti-NFATc1 and then goat anti-mouse
Alexa 488. The left panel shows CD8a staining, the middle panel shows
NFATc1 staining, and the right panel shows an overlay with CD8a staining in
red and NFATc1 staining in green.
(B) 5c.c7 B10.BR thymocytes were isolated and stained as in (A).
(C)Summary ofdatafrom(A)and (B),andfor5c.c7B6thymocytestreated with
the ionophore A23187 for 10 min prior to fixation, or thymi allowed to rest in RT
PBS for 5 min prior to thymocyte liberation and fixation. Data are expressed as
the proportion (mean ± standard deviation) of CD8+cells with nuclear NFAT
accumulation (>50% nuclear Alexa 488 staining, n > 300) from two mice for
(D) NFAT transcriptional activity correlates with thymocyte maturation in vivo.
5c.c7 DP thymocytes transduced with NFAT-promoter-driven GFP were
injected intrathymically into B10.BR recipients; 5 3 107thymocytes were
prepared from recipient mice at the indicated days after injection, stained for
surface expression of CD4 and CD8, and analyzed by flow cytometry. Shown
are the CD4-CD8 profiles of total ungated cells (first panel) or cells gated for
expression of NFAT-driven GFP on day 2 (second panel), day 3 (third panel),
and day 4 (fourth panel) after intrathymic injection. Each profile is representa-
tive of two to three mice.
Immune-Synapse Formation in Thymic Selection
Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc. 737
Figure 4. Characterization of the NFATc1-GFP Retrovirus
middle row shows Fura 340/380 ratio in a false-color scale, and the bottom row shows NFATc1-GFP. Times in seconds are given relative to the onset of Ca2+flux
above the DIC images, and the ratio of nuclear to cytosolic gfp signal is indicated below the fluorescence images.
(B)Video time-lapsemicroscopyof5c.c7DPthymocytesexpressingNFATc1-GFPinRTOCwithB10.BRthymicstromalcellswithnopeptide added.Thetoprow
showsDIC,andthebottomrowshowsNFATc1-GFP. Times inminutesaregivenrelative totheonsetofimaging, and theratioofnucleartocytosolicGFPsignalis
indicated below the fluorescence images.
(C) Summary of video microscopy data, presented as the average number of NFATc1-GFP+cells displaying nuclear accumulation (>50% of total GFP signal
confined to the nuclear region in consecutive time points) of NFAT, among >100 cells in each case. For mature T cells, only cells displaying Ca2+flux (fura
2am 340/380 ratio > 1.5) were considered for analysis. RTOC were composed of B10.BR thymic stromal cells unless otherwise indicated.
Immune-Synapse Formation in Thymic Selection
738 Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc.
TCR, Lck (Richie et al., 2002), and CD4 (data not shown). Given
the stability of cell-cell interactions, it is not surprising that
responding DP thymocytes retained NFAT in their nuclei for
upward of 30 min.
However, we also found that a smaller but substantial propor-
tion of DP thymocytes mobilized NFAT in a stable fashion in
response to thymic APCs presenting only endogenous pep-
tide-MHC ligands (Figures 4B and 4C). This observation was
not just a reflection of the difference in microenvironment or
APCs because mature T cells did not translocate NFAT when
placed in these cultures (Figure 4C). It is also not an artifact of
the NFATc1-GFP construct because treatment with the calci-
from thymocyte nuclei within 5–15 min (Figure S6E). These ob-
servations suggest that sustained thymocyte signaling occurs
in these positively selecting RTOC cultures.
TCR Signaling Despite the Lack of Stable
Thymocyte-Stromal Cell Interactions in RTOC
We previously showed that thymocytes form immune synapses
in positively selecting RTOC at a much lower frequency and with
different cell-surface-molecule dynamics than in negatively se-
lecting RTOC (Richie et al., 2002). To determine whether these
rare synapses represented positively selecting interactions, we
generated RTOC containing CD3z-GFP-transduced 5c.c7 DP
thymocytes together with thymic stromal cells and a small pop-
ulation of PKH26-labeled B10.BR thymic dendritic cells (DCs) or
interface with APCs presenting either negatively selecting MCC-
I-Ekor endogenous peptide-MHC ligands and find out whether
the thymocytes accumulated their TCRs in that interface (Fig-
ure 5). Dendritic cells presenting MCC-I-Ekadhered to and pro-
moted TCR accumulation in nearly half of the DP thymocytes
available to them (Figure 5B). In contrast, DCs presenting only
endogenous ligands bound fewer than 5% of available thymo-
cytes (Figure 5A). Similarly, thymic epithelial cells presenting
positively selecting ligands did not promote stable contacts
with CD3z accumulation, unlike TECs presenting negatively
selecting MCC-I-Ek(Figures 5C and 5D, and enumerated in
Figure 5E), although the lower efficiency with which TECs
provoke synapses relative to DCs may also contribute to this
observation. The small number of stable contacts formed in
RTOC containing B10.BR epithelial cells was formed toward
I-E-deficient APCs and was thus unlikely to represent positively
selecting interactions (Figure 5E, white bar). Further analyses of
synapse formation are presented in Figure S7.
Despite this lack of synapse formation, thymic epithelial cells
that this NFAT mobilization occurred in response to endogenous
(14.4.4) generally or a subset of self-peptide-I-Ekcomplexes
(G35) that includes those necessary for the positive selection
tibodies inhibit the progression of 5c.c7 thymocytes to the CD4+
SP lineage in B10.BR mice (Baldwin et al., 1999) and in FTOC
sponse of 5c.c7 thymocytes in B10.BR RTOC when added 4 hr
prior to imaging (shown in Figures 5F and 5G, and quantified in
Figure 5. Paucity of Stable Thymocyte-
Thymic APC Contacts in Positively Select-
(A) A representative image of an RTOC composed
GFP (in green), with unlabeled B10.BR TECs, and
PKH26-labeled B10.BR thymic DCs (in red).
(B) RTOC generated as in (A), but DCs additionally
(C) A representative image of an RTOC composed
GFP (in green), with unlabeled B6 TECs, and
PKH26-labeled B10.BR TECs (in red).
(D) RTOC generated as in (C), but TECs addition-
ally present MCC-I-Ek.
(E) Summary of data from (A)–(D). Data are ex-
pressed as the average percentage of thymocytes
with >1.3-fold polarized accumulation of CD3z-
GFP toward an unlabeled B6 APC (white bars),
or >1.3-fold polarized accumulation of CD3z-
GFP toward a contact with a labeled B10.BR
APC (gray bars) for more than 3 min; data are
averages (± SD) over three experiments, with >50
cells per experiment.
(F–H) Positively selecting ligands are responsible
for NFAT mobilization in 5c.c7 thymocytes in
selecting RTOC. (F) and (G) are representative
images of RTOC composed of 5c.c7 DP thymo-
cytes expressing NFATc1-GFP and B10.BR stro-
mal cells, treated with (F) 20 mg/ml 14.4.4 or (G) 500 mg/ml G35 antibody 4 hr prior to imaging. (H) shows a summary of data from RTOC containing 5c.c7 DP
thymocytes expressing NFATc1-GFP and treated with the indicated antibodies. Data are expressed as the average percentage (± SD) of GFP+ thymocytes
displaying a sustained (>5 min) nuclear accumulation (>50% nuclear GFP signal) of NFATc1-GFP over three experiments, with n > 50 for each experiment.
Immune-Synapse Formation in Thymic Selection
Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc. 739
Figure 5H). An isotype control antibody, D4 (which binds MCC-I-
or FTOC [Baldwin et al., 1999]), did not have a marked effect on
NFAT mobilization. 5c.c7 thymocytes in nonselecting B6 RTOC
also did not mobilize NFAT into their nuclei (2.8% thymocytes
with nuclear NFAT, Figure 4C). Thus, a fairly large proportion of
DP 5c.c7 thymocytes (20%–30%) experience self-peptide-
MHC-dependent signaling, whereas few if any of them form an
immune synapse. As additional evidence, we find that an
antibody that blocks LFA-1 in FTOC did not prevent positive se-
lection despite inhibiting immune-synapse formation (Figure S8).
Transient and Serial Encounters Lead to Sustained
NFAT Nuclear Translocation
Directvisualization alsosupported the ideathatthymocytessus-
ure 6 shows the progression over time of 5c.c7 DP thymocytes
through reaggregate cultures composed of B10.BR thymic
stromal cells. In these representative time-lapse images, we
see NFATc1-GFP translocated to the thymocyte nucleus upon
encounter with a thymic APC. However, thymocytes did not alter
their morphology to increase their contact area with that APC
(i.e., no flattened or cupped interface as in Figure 5D). In fact,
Figure 6. Sustained Nuclear NFAT in DP Thymocytes Results from Intermittent, Transient Contact with Selecting Ligands
(A) Time-lapse video microscopy of 5c.c7 DP thymocytes expressing NFATc1-GFP in RTOC with B10.BR stromal cells. RTOCs were allowed to cool to room
temperature to quench cell signaling prior to imaging at 37?C. The putatively stimulating epithelial cell is marked by a red asterisk.
(B) Time-lapse video microscopy of 5c.c7 DP thymocytes expressing NFATc1-GFP in RTOC with B10.BR stromal cells. We added 20 mg/ml anti-I-Ekantibody
14.4.4 immediately prior to imaging.
(C) Percentage of DP thymocytes with nuclear NFATc1-GFP (>50% nuclear GFP signal) over time in selecting RTOC.
(D) Percentage of DP thymocytes with nuclear NFATc1-GFP (>50% nuclear GFP signal) over time in RTOC with 14.4.4 added at 0 min. Individual traces in (C) and
(D) are separate experiments tracking 12–20 cells each.
Immune-Synapse Formation in Thymic Selection
740 Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc.
the thymocyte, although rounded in morphology, was not teth-
ered to any particular epithelial cell and could immediately
move away from the putatively stimulating APC (marked with
a red asterisk in the panel in which NFAT translocation occurs,
and again in the last panel of Figure 6A). We identified the stim-
ulating APC on the basis of its initiating contact with the thymo-
cyte 30–60 s prior to NFAT translocation (because it is difficult to
identify the stimulating APC precisely under these conditions,
real-time imaging of thymocytes leaving contact with labeled
stimulating epithelial cells is shown further in Movies S1 and
S2). Nonetheless, NFAT remains in the nucleus of these cells.
Note that the percentage of responding thymocytes increased
strictly with time (Figure 6C) with only one exception (n > 50) be-
cause once confined to the nucleus, NFAT was not re-exported
to the cytoplasm in the course of these experiments (>30 min).
However, if we blocked calcineurin activity after ionophore treat-
ment, NFAT returned to the thymocyte cytoplasm in well under
30 min (?10 min, Figure S6E).
To test whether continued contact with MHC ligands is re-
quired for NFAT retention in the nucleus (as opposed to some
thymocyte-specific mechanism), we used the 14.4.4 antibody
to block endogenous peptide-I-Ekimmediately prior to video
microscopic visualization. We found a reduction in the number
of responding thymocytes after 5 min of antibody treatment,
and those thymocytes that had nuclear NFAT at the onset of
imaging released it into their cytosol over the course of several
minutes (Figures 6B and 6D). Thus, transient but repeated
engagements with MHC ligands presented by thymic APCs
seem to be required for sustained NFAT activity in immature
We have shown here that immature thymocytes initiate pro-
grammed cell death in response to contact with as few as two
agonist peptide-MHC ligands on an APC. Importantly, this is
fewer peptides than are required to elicit an effector response
from mature T cells of the same antigen specificity (Irvine et al.,
2002; Peterson et al., 1999) and also agrees very closely with
earlier estimates of the range of peptides required (Peterson
et al., 1999). Although CH27 B cells possess the costimulatory
andadhesion molecules thoughtto beimportant fornegative se-
lection (Graham et al., 2006), thymic APCs may of course be dif-
ferent in terms of the number of peptides they require to delete
self-reactive DP T cells, although it is hard to imagine that the
threshold could be placed much lower. Negative selection can
also occur at the semimature SP stage (Kishimoto and Sprent,
1997), although we and others (Villunger et al., 2003) find that
5c.c7 DP and semimature SP thymocytes are similarly sensitive
to agonist stimuli. This property of heightened sensitivity at the
DP and semimature SP stage (Davey et al., 1998; Li et al.,
2007) presumably enforces the stringency of central tolerance.
If the densities of particular determinants in the thymus and
periphery are similar (Marrack et al., 1993), and immature
T cells delete themselves in response to fewer peptides than
an effector T cell can respond to, it becomes unlikely that an
autoreactive T cell clone could both fail to find negatively select-
ing ligands in the thymus and yet find enough ligands in the
periphery to provoke an effector response.
This result is also interesting in light of our results in whole-
thymus cultures. Even under conditions in which only a few
APCspresentsufficiently manyligandsto inducenegativeselec-
tion, negative selection was still remarkably efficient. This sug-
gests that the efficiency of deletion must stem not only from
the sensitivity of thymocytes for their ligands but also from
some ability of thymocytes to seek out those rare APCs within
the thymus that are competent to delete them. Such an interpre-
tation would be at odds with a single-niche model of positive
selection (Merkenschlager et al., 1994) or with models of stable
contacts during the course of positive selection (Starr et al.,
2003). If a thymocyte could complete its maturation (either to
the fully mature SP stage or to the medulla-bound semimature
SP stage) by contacting the first epithelial cell it encountered, it
wouldfail toexperience a largeportion ofthethymic self-peptide
repertoire (Anderson et al., 2002; Barton and Rudensky, 1999a).
Instead, we show here that transient but repeated encounters
with selecting ligands are necessary and sufficient to promote
sustained NFAT signaling in immature DP thymocytes and that
these encounters did not involve immune-synapse formation in
a reaggregate culture system that supports positive selection.
When we also saw that sustained NFAT activity correlates with
progression toward the mature T cell stage, we inferred from
these observations that maturing thymocytes run a gauntlet of
repeated engagement with many different APCs as they move
through the thymus. This mode of signal acquisition would also
help to explain the elegant data of Merkenschlager et al.
(1994), who showed that negative selection could remain effi-
cientwith asfew asonein 10–20thymicAPCs presenting appro-
priate ligands—in fact, we arrive at a very similar estimate from
our peptide-counting studies.
NFAT is a particularly informative marker for TCR signaling
(Hooijberg et al., 2000) because its nuclear import rapidly indi-
cates the initiation of TCR signaling, whereas its nuclear resi-
dence indicates the duration of that signaling, and because its
nuclear translocation closely correlates with its transcriptional
activity (Neal and Clipstone, 2001), which includes the regulation
of thousands of genes in mature T cells (Diehn et al., 2002). The
sustain their activity is of particular interest given the role that
tion and also in the lineage decision of selected T cells (Kisielow
and Miazek, 1995; Liu and Bosselut, 2004; Yasutomo et al.,
2000). Here, we have shown that NFAT activity was found pref-
erentially in those thymocytes undergoing positive selection
in vivo, whereas in RTOCs a large fraction of DP thymocytes
translocate NFAT to their nuclei in a manner that can be inhibited
by blocking selecting ligands (Baldwin et al., 1999). This sug-
gests that the thymocytes that mobilize NFAT into the nucleus
are likely to progress toward full maturation.
Although effector T cells seem to rely on the tight T cell-APC
contact mediated by immune-synapse formation to sustain
signaling, and DP T cells display a similar behavior when
responding to negatively selecting signals, DP thymocytes
displayed neither behavior in response to positively selecting li-
gands. Indeed they did not even adhere tightly enough to thymic
epithelium presenting such ligands to remain in contact with
those cells over the course of minutes in a RTOC. Nonetheless,
thymocytes can maintain their downstream NFAT response
Immune-Synapse Formation in Thymic Selection
Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc. 741
through re-exposure to selecting ligands on nearby epithelial
cells. It is likely then that thymocytes achieve the duration of
signaling required for maturation through many transient en-
counters, potentially with many different epithelial cells, rather
than the prolonged contact with a single epithelial cell that im-
re-encountered selecting ligands within the 5–15 min required
for NFAT nuclear export, NFAT transcriptional activity could be
maintained by many separate thymocyte:stromal cell liaisons,
similar to how mature T cells appear to acquire and accumulate
signals from T cell:DC interactions in 3D collagen matrices
(Gunzer et al., 2000) and lymph nodes (Miller et al., 2004; Miller
et al., 2002).
Recent work has shown that calcium signaling promotes an
arrest of thymocyte motility in thymus tissue slices (Bhakta
et al., 2005). These pauses in motility were generally brief
(10–20 min) and were followed by subsequent cycles of motility
and arrest. The duration of thymocyte-epithelial cell contact we
observed is even shorter (<5 min), possibly because of the less
compact RTOC structure. Nonetheless, the reacquisition of
motility in thymus slices (Bhakta et al., 2005) and intact thymus
lobes (Witt et al., 2005) imply that thymocytes are not forming
stable synapse-mediated contacts (which can last for hours
in mature T helper cells [Faroudi et al., 2003; Huppa et al.,
2003]). Because NFAT is re-exported from the nucleus slowly
after the cessation of signaling, NFAT could act to ‘‘remember’’
recent encounters with selecting ligands (Tomida et al., 2003).
This would explain why we observed continuous nuclear NFAT
accumulation, whereas Bhakta et al. observed punctuated cal-
The efficiency with which TCR transgenic thymocytes are
positively selected correlates with the number of stromal cells
expressing selecting MHC in chimeric reaggregate thymus cul-
tures (Merkenschlager et al., 1994). This might indicate that thy-
mocytes compete for cellular niches within the thymus in which
they can continually engage their selecting ligands (Merkenschl-
ager, 1996). However, the efficiency of positive selection has
also been shown to correlate with the expression of selecting
MHC in a genetically homogeneous thymus (Berg et al., 1990;
Wong et al., 2000). Thus, the density of MHC determines the
efficiency of positive selection even under conditions of equal
cellular space. The requirement that thymocytes continually re-
encounter selecting ligands in order to maintain productive sig-
naling might explain both observations. A thymocyte’s chance
of reencountering ligands compatible with their TCR would
decline in both cases, whether because the requisite MHC was
limited to fewer cells, or because it was expressed at a lower
density on all cells.
These findings bear particularly on how thymocytes access
peripheral mechanisms can help to ameliorate autoimmune
responses, immune tolerance is critically reliant on the thymus’
ability to remove immature T cells carrying self-reactive TCRs
(Anderson et al., 2002; Daniels et al., 2006; Sprent and Kishi-
that thymic APCs are most effective as a group. Because the
number of potential self-determinants outnumbers the MHC
complexes that can be expressed by any one APC, with a large
variation in the abundance of any particular determinant (Barton
and Rudensky, 1999a, b; Chmielowski et al., 2000), no one APC
can present the entire array of self-peptides. A self-reactive thy-
mocyte might therefore have to scan many such cells before
finding one that can promote negative selection or TCR editing.
A lack of synapse-sustained contact might also enhance the
efficacy of positive selection. If thymocytes need to continually
scan (Miller et al., 2004; Wu et al., 2002) epithelial cells for pep-
tide-MHCs that promote positively selecting signals, this would
ensure a lower error rate in this critical transition, preventing
the maturation of thymocytes that are ‘‘restricted’’ only to very
rare peptide-MHC. This also raises the possibility that T cells
might preferentially recognize the ligands that selected them
once they reach the periphery. Prior work has shown that mature
CD4+T cells need to interact with endogenous peptide-MHC in
theperiphery inordertomaintainoptimal responsiveness (Stefa-
nova et al., 2002) and that particular endogenous peptide-MHC
can synergize with agonist ligands to activate T cells, whereas
other self peptides can’t (Krogsgaard et al., 2005). Positive
selection might therefore select T cells not only for their ability
to recognize self-MHC but also more specifically for their ability
to utilize particular synergistic self-peptide-MHC complexes
(Davis et al., 2007).
5c.c7 mice on the B10.BR background were obtained from Taconic Farms.
We obtained 5c.c7 B6 mice by crossing 5c.c7 onto the C57Bl/6 background
for more than seven generations (Richie et al., 2002). We obtained 5c.c7
Cd74?/?mice by crossing 5c.c7 onto Cd74?/?B10.BR mice (a kind gift of
C. Benoist and D. Mathis). All mice were bred and maintained at the Stanford
University Department of Comparative Medicine Animal Facility (protocol
3540) in accordance with National Institutes of Health guidelines.
Anti-I-Ek(14.4.4 s) and anti-LFA-1 (anti-CD11a, M17/4) were obtained from
BD Biosciences. Anti-NFATc1 (7A6) was a kind gift of G. Crabtree. The gener-
ation of anti-MCC/I-Ekantibodies G35 and D4 is described in (Baldwin et al.,
Retroviral Constructs and Virus Preparation
CD3z-GFP in a modified form of the pIB vector was as previously described
mel (UCSF) and cloned into a modified pMSCV vector (see Figure S1B). The
minimal NFAT promoter (Graef et al., 1999) (see also http://crablab.stanford.
edu) driving eGFP was cloned into the CKHGppSin vector (a gift of G. Nolan
and K. Kripke-Skillern) to generate the Npro construct (see Figure S1A).
Constructs were transfected into Phoenix-ecotropic cells (a gift of Dr. Nolan)
and selected for construct integration by treatment with Blasticidin, Zeocin,
or Puromycin (Invitrogen).
Infection and Isolation of Thymocytes and T Cells
For reaggregate cultures, e16 thymic lobes were dissociated and thymocytes
wereresuspendedinvirus-containing supernatant,thencentrifuged at32?Cat
750 G for 1 hr. Cells were then transferred to 37?C overnight. For intrathymic
injection, thymi from 4- to 6-week-old 5c.c7 mice were dissociated, then DP
and SP cells were depleted with anti-CD4 dynalbeads (Dynal Biotech). DN
cells were then transduced as above. In both cases, cells were allowed to
mature to the DP stage 12–16 hr at 37?C. For mature cells, lymph nodes
by addition of 5 mM MCC peptide (day 0). Lymph node preparations were
additionally treated with 250 U/ml IL-2 after 12–16 hr and were transduced
by 1.5 hr centrifugation in viral supernatant 6 hr later. T cells were used for
in vitro and microscopy studies on day 5 of culture.
Immune-Synapse Formation in Thymic Selection
742 Immunity 29, 734–745, November 14, 2008 ª2008 Elsevier Inc.
Cell-surface molecules were stained with anti-CD4 (GK1.5 or RM4-5), anti-
CD8a (53-6.7), anti-I-Ek(14.4.4 s), streptavidin-PE, and/or anti-Vb3 (KJ25),
all from BD Biosciences. FITC-Z-VAD-FMK (CaspACE) was from Promega.
Live cells were identified by their forward- and side-scatter profiles and exclu-
sion of propidium iodide. Samples were run on a FACStar (Becton Dickinson)
and analyzed with Flowjo software (Treestar).
Immunofluorescent Staining of Fresh Ex Vivo Thymocytes
Thymi were dissected from 4- to 6-week-old 5c.c7 B10.BR or 5c.c7 B6 mice
and were either immediately dissociated in 4% paraformaldehyde (Sigma) in
PBS or treated 10 min with 0.5 mM A23187 and then fixed for 30 min. Cells
were then plated onto poly-L-lysine treated chambers (Lab-Tek), washed
33 PBS, permeabilized with 0.1% Triton X-100 for 10 min, blocked with
TNB 1 hr, and stained with anti-NFATc1 (7A6), with subsequent staining
with biotinylated goat anti-mouse IgG and finally streptavidin Alexa 488 and
anti-CD8 PE (BD Biosciences).
Npro-transduced DP cells were injected into 4- to 6-week-old B10.BR recipi-
ents as previously described (Jerabek and Weissman, 2001). A total of 107
cells were extensively washed and resuspended in 10 ml PBS for injection
with a 10 ml Hamilton syringe after anesthetization with ketamine and xylazine
or with Avertin. Mice were euthanized and 5 3 106thymocytes were analyzed
by flow cytometry after 2–7 days.
Generation of Reaggregate Cultures
e15 thymi were cultured for 6–8 days on 0.8 micron filters (Millipore) floated on
culture medium containing 0.36 mg/ml 2-deoxyguanosine (Sigma) for removal
of thymocytes and dendritic cells, and thymic stromal cells (TSC) were pre-
pared by dissociation of thymic lobes with trypsin and EDTA. Residual bone
marrow-derived cells were removed with streptavidin Dynalbeads coated
with biotinylated anti-CD45 (BD Biosciences). Thymic dendritic cells were iso-
lated by collagenase D (Boeringer) treatment of 4- to 6-week-old B10.BR
thymi, followed by density separation on a gradient of 14.5% metrizamide
(Sigma) and purification with anti-CD11c miltenyi beads (Miltenyi Biotech).
Stromal cells or dendritic cells were labeled with a PKH26 linker kit (Sigma).
Transduced thymocyte preparations were enriched to >97% DP cells with
streptavidin Dynalbeads coated with biotinylated anti-CD8 (53-6.7, BD Biosci-
ences), and cells were released from the beads by treatment with trypsin and
EDTA (Sigma). A total of 1.5 to 3 3 105TSCs were mixed with 1 to 2 3 105DP
cells and centrifuged 750 G in nontissue-culture-treated V-bottom 96-well
plates. Plates were then transferred to a sealed chamber containing 70% 02,
25% N2, and 5% CO2(Praxair Bioblend), and cells were allowed to reaggre-
gate 4–12 hr prior to imaging or transfer to 0.8 micron filters for culture at 37?C.
In Vitro Responses
Atotalof53105day 5poststimulation5c.c7T cells weremixed with2.5 3105
CH27 cells with or without 2 mM MCC peptide. Cells were harvested and
analyzed by flow cytometry at indicated times, gating on live CD4+cells. Cells
were treated with 0.5 mM A23187, 1 nM FK506, and/or 50 ng/ml Cyclosporin A
RTOC cultures were transferred with a cut 200 ml pipette tip to an FCS2 cham-
ber (Bioptechs) and sandwiched between a slide and coverslip separated by
a 0.25 mm gasket. Mature T cells and thymocyte suspensions were loaded
with fura 2am (Molecular Probes) for 20 min and washed prior to imaging in
eight-well borosilicate chambers (Lab-Tek), and CH27 B lymphoma cells
were used as APCs. Cells were imaged at 37?C and 5% CO2with a Zeiss
Axiovert-100TV microscope as described (Richie et al., 2002). NFATc1-GFP
was considered nuclear when the ratio of nuclear GFP signal to cytosolic
GFP signal was greater than one in consecutive frames (nuclear accumulated
thymocytes: 1.68 ± 0.543; cytosolic accumulated thymocytes: 0.543 ± 0.212).
For FTOC, e16 thymi were dissected and cultured on 0.8 micron filters (Milli-
pore) in standard culture medium supplemented with biotinylated MCC (Irvine
et al., 2002). Cells were dispersed after 3 days with trypsin and EDTA, then
stained with 14.4.4-Fitc and streptavidin-PE. A standard curve of PE fluores-
cence was generated with QuantiBRITE PE beads (BD Biosciences). For
microscopy studies, CH27 cells expressing H-2Kb(Purbhoo et al., 2004)
were incubated with biotinylated MCC at 4?C for 1 hr, then washed extensively
in ice-cold PBS/5% FCS/0.1% NaN3before staining with 10 mg/ml SA-PE at
4?C in PBS/5% FCS/0.1% NaN3for 40 min. CH27 cells were then washed ex-
tensively in PBS/5% FCS for removal of free SA-PE. Thymocytes and CH27
cells were then incubated for 5 min at 37?C to allow cell couples to form,
then spun gently onto a Lab-Tek chambered coverglass (Nalge Nunc) pre-
coated withanti-H-2Kbtoimmobilize CH27 cells.SA-PEwasvisualizedacross
the whole cell at 1 micron intervals with 100 ms exposure times, and the im-
ages were reconstructed into a 3D image with Metamorph software (Universal
peak of fluorescence intensity of ?70 ± 12 units; peaks of fluorescence inten-
were thus identified as single peptides. Multiple peptides could occur as
multiple peaks of 60–80 unit fluorescence, or as single peaks of 60–80, 120–
160, or 180–240 units. Peaks of greater than 250 units of fluorescence were
counted as four or greater peptides. For apoptosis determination, the medium
was spiked with Annexin V (1:20, BD Biosciences) and 2 mM CaCl2.
Supplemental Data include nine figures and two movies and can be found
with this article online at http://www.immunity.com/supplemental/S1074-
We thank M.F. Krummel for the original NFATc1-GFP construct; G. Nolan,
K. Kripke-Skillern, and R. Wolkowitz for the CKHGppSin vector; and M. Kuhns
for the modified pMSCV vector. We also thank J.J.T. Owen for his guidance,
Q.-J. Li and F. Tynan for critical reading and J.R. Neilson, M. Winslow, and
E. Gallo for productive discussions. This work was supported by Howard
Hughes Medical Institute predoctoral fellowships (P.J.R.E. and L.I.R.E.) and
by the US National Institutes of Health (R01 AIO22511) and Howard Hughes
Medical Institute (M.M.D.).
Received: September 18, 2007
Revised: July 18, 2008
Accepted: September 9, 2008
Published online: November 6, 2008
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