Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo.

Abstract

Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs.

Full text

Page 1

The Journal of Experimental Medicine
BRIEF DEFINITIVE REPORT
JEM © The Rockefeller University Press $8.00
Vol. 203, No. 3, March 20, 2006 505–511 www.jem.org/cgi/doi/10.1084/jem.20050783
505
Several recent studies have revealed the dy-
namics of live T cell interactions in explanted
intact LNs, or intravitally in the LNs of live mice,
using laser-scanning microscopy (1–4). T cell
interactions with antigen-presenting DCs in
LNs are diff erent from those observed in vitro,
and it is becoming increasingly apparent that an
intact lymphoid organ structure is fundamental
for the development of normal immune re-
sponses (2, 5). These studies indicated that in
the absence of immunization T cells move ex-
tensively within the T cell zones of LNs, and
this movement facilitates the encounter of rare
antigen-specifi c T cells with antigen-loaded
DCs. Upon antigen encounter, T cells partake
in a variable period of dynamic interactions and
then arrest their locomotion for several hours
before regaining motility by 36 h (2–4, 6).
Another component that may play a role,
but has not been studied in vivo thus far, is the
regulatory T (T reg) cell compartment. T reg
cells are major components of immune re-
sponses, down-modulating Th1 or Th2 re-
sponses against auto, allo, or foreign antigens
(7–10). Despite their prominent role, little is
known about how T reg cells control immune
responses in vivo. In this study, we used intra-
vital two-photon laser-scanning microscopy to
visualize the behavior of autoantigen-specifi c
T cells in the presence or absence of naturally
occurring T reg cells in intact LNs. We found
that 20 h after immunization with autoantigen-
loaded DCs, contacts between T cells and
antigen-loaded DCs are of shorter duration in
the presence of T reg cells than in their ab-
sence, indicating an early eff ect of T reg cells
during immune responses.
RESULTS AND DISCUSSION
Demonstration of T reg cell function
in the popliteal LN (PLN)
To assess how T reg cells aff ected encephalito-
genic CD4+ T cells in vivo, we used an experi-
mental system in which the host either contains
or lacks T reg cells. The experimental system is
based on myelin basic protein (MBP) Ac1-11–
specifi c TCR transgenic mice (11). Despite the
high frequency of functional MBP-specifi c
T cells, the hemizygous transgenic T/R+ mice
do not develop experimental autoimmune en-
cephalomyelitis (EAE) because of the presence
of T reg cells. Genetic ablation of T reg cells,
which can be accomplished by crossing T/R+
mice to RAG−/− mice, to TCR α−/−β−/−
mice, or by breeding the TCR transgenes to
homozygosity (Tg/Tg), leads to the sponta-
neous development of EAE (11–13). In this study,
we used hemizygous T/R+ as mice harboring
T reg cells and Tg/Tg as mice lacking T reg
cells. On a C57BL/10.PL genetic background,
EAE spontaneously develops in T reg cell–
defi cient mice (including Tg/Tg mice) beginning
Regulatory T cells inhibit stable contacts
between CD4+ T cells and dendritic cells
in vivo
Carlos E. Tadokoro,1 Guy Shakhar,1 Shiqian Shen,1 Yi Ding,1
Andreia C. Lino,1 Antonio Maraver,1 Juan J. Lafaille,1,2
and Michael L. Dustin1,2
1Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, and 2Department of Pathology,
New York University School of Medicine, New York, NY 10016
Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses,
but it is not known how they act in vivo. Using intravital two-photon laser scanning micros-
copy we determined that, in the absence of T reg cells, the locomotion of autoantigen-
specifi c T cells inside lymph nodes is decreased, and the contacts between T cells and
antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an
early effect on immune responses by attenuating the establishment of stable contacts
during priming of naive T cells by DCs.
CORRESPONDENCE
Juan J. Lafaille:
lafaille@saturn.med.nyu.edu
OR
Michael L. Dustin:
dustin@saturn.med.nyu.edu
<NOHTML>The online version of this article contains supplemental material.
</NOHTML>

Page 2

506 INTRAVITAL MICROSCOPY ANALYSIS OF T REG CELL ACTION | Tadokoro et al.
at ?45 d of age, with 50% EAE incidence reached at 60 d
and 100% by 120 d of age (12, 13).
We fi rst characterized the cellularity and organization of
the PLN in T/R+ and Tg/Tg mice. At 5 wk old or younger,
both T/R+ and Tg/Tg mice harbor an equally high number
of MBP-specifi c T cells, the vast majority of which display
naive phenotype (Fig. S1 a, available at http://www.jem.org/
cgi/content/full/jem.20050783/DC1;
and Tg/Tg mice also have similar proportions of DC
subpopulations (Fig. S1 c). The most striking diff erence is the
presence of Foxp3-expressing T reg cells in T/R+ mice but
not Tg/Tg mice (Fig. S1 b). The naive status of MBP- specifi c
T cells in the 5-wk-old T/R+ and Tg/Tg mice (11), despite
the presence of MBP in the central nervous system, suggests
that DCs in PLNs are not presenting endogenous MBP
autoantigen (14). Immunohistology was used to confi rm that
in both types of recipient mice, PLNs were structurally
normal and the transferred CFSE-labeled T cells homed to
the T cell areas (Fig. S1 d).
Both T reg cell–defi cient and –suffi cient MBP-specifi c
TCR transgenic mice undergo fulminant EAE upon immu-
nization with MBP emulsifi ed in complete Freund’s adjuvant
and pertussis toxin, indicating that T reg cells can be over-
whelmed or disengaged by strong infl ammatory signals (12).
Therefore, we immunized with MBP Ac1-11 in IFA to pro-
vide an in situ depot for autoantigen delivery to DCs without
overwhelming T reg cells. This was an eff ective strategy
because subcutaneous injection in the foot pad with MBP
<CIT>reference 11</CIT>). T/R+
Ac1-11 in IFA triggered IL-2 production by MBP-specifi c
T cells in the PLNs in the Tg/Tg mice, but not the T/R+
mice, at 20 h (Fig. S2, available at http://www.jem.org/cgi/
content/full/jem.20050783/DC1). Thus, MBP Ac1-11 pep-
tide in IFA triggered an immune response in the PLN that
was suppressed by endogenous T reg cells.
Infl uence of T reg cells on the movement of CD4 effector
cells in vivo
We next used two-photon intravital microscopy to study the
movement of MBP-specifi c T cells in the presence or absence
of T reg cells. CFSE-labeled MBP-specifi c CD4 T cells puri-
fi ed from Tg/Tg mice (naive eff ector precursor T cells) were
transferred i.v. into T/R+ or Tg/Tg recipient mice (scheme
depicted in Fig. 1 a).[ID]FIG1[/ID] PLNs from anesthetized mice were
exposed and imaged by two-photon laser-scanning intravital
microscopy 20 h after T cell transfer. We analyzed the images
to determine the speed and arrest coeffi cient of MBP-specifi c
T cells under both conditions. The arrest coeffi cient is the
proportion of time in which a T cell does not move (thresh-
old <2 μm). The arrest coeffi cient is generally low when
T cells are not engaged in stable contacts with DCs (6).
In the absence of immunization, MBP-specifi c T cells
migrated at a mean speed of 6.72 μm/min in T/R+ mice
and 6.34 μm/min in Tg/Tg mice, but this diff erence was not
signifi cant (Fig. 1, b and c, and Videos S1 and S2, available at
http://www.jem.org/cgi/content/full/jem.20050783/DC1).
The mean arrest coeffi cients were also similar in the presence
Figure 1. In the absence of immunization, T reg cells do not
infl uence the movement of MBP-specifi c CD4+ T cells. (a) Experi-
mental protocol. T/R+ or Tg/Tg mice, which have or lack endogenous
T reg cells, respectively, received 107 CFSE-labeled MBP Ac1-11–specifi c
CD4+ T cells by tail vein injection. 20 h later, PLNs were imaged.
(b) Representative tracks of MBP-specific T cells in T/R+ and Tg/Tg
recipients. (c) Mean speeds of MBP-specific CD4+ T cells in both types
of recipient mice. (d) Arrest coefficient for CD4+ T cells in both
types of recipient mice. Results are representative of three
independent experiments.

Page 3

JEM VOL. 203, March 20, 2006
507
BRIEF DEFINITIVE REPORT
and absence of T reg cells (0.35 and 0.39, Fig. 1 d). Thus,
baseline T cell migration is similar in the presence and absence
of T reg cells.
We next asked if the eff ect of autoantigen on MBP-
specifi c CD4+ T cell movement is altered in the presence
or absence of T reg cells. Mice were immunized in the hind
footpad with MBP Ac1-11 emulsifi ed in IFA, and, at the
same time, naive T cells from Tg/Tg mice were transferred
i.v. (Fig. 2 a).[ID]FIG2[/ID] We then imaged the draining PLNs at 20 h, as
this has been shown to be appropriate to observe foreign an-
tigen eff ects on CD4+ T cells (2, 6, 15). Immunization slowed
the MBP-specifi c T cells substantially only in the Tg/Tg
mice (Fig. 2 b and Videos S3 and S4, available at http://
www.jem.org/cgi/content/full/jem.20050783/DC1). In the
presence of T reg cells, MBP-specifi c T cells did not signifi -
cantly change their speed in response to autoantigen, which
remained at 6.72 μm/min (Fig. 2 c), but signifi cantly
increased their arrest coeffi cient to 0.49 (Fig. 2 d, P = 0.0011).
Earlier studies with foreign antigen–specifi c T cells showed
larger eff ects of antigen on migration in this time frame, but
autoantigens are often weaker agonists than foreign antigens,
which may account for the smaller eff ect. In contrast, in the ab-
sence of T reg cells, MBP-specifi c T cells signifi cantly decreased
their speed to 3.13 μm/min (Fig. 2 c, P < 0.0001) and
increased their arrest coeffi cient to 0.70 (Fig. 2 d, P < 0.0001).
To have an internal control for the integrity of the
PLN preparations in Tg/Tg mice where antigen-specifi c
T cells sometimes were fully arrested, we cotransferred and
imaged WT polyclonal (WT) CD4+CD25− T cells from
nontransgenic syngeneic chicken β-actin promoter cyan
fl uorescence protein (CFP) transgenic mice (16). 20 h after
transfer, we imaged CFSE-labeled MBP-specifi c T cells and
CFP-expressing WT T cells in the draining PLNs of Tg/Tg
mice immunized with MBP Ac1-11 peptide in IFA. The
same fi elds that contained arrested MBP-specifi c cells had
actively moving WT T cells. The latter cells displayed a speed
of 5.51 μm/min (Fig. 2 c and Video S5, available at http://
www.jem.org/cgi/content/full/jem.20050783/DC1) and
an arrest coeffi cient of 0.50, values that are similar to MBP-
specifi c T cells in unimmunized mice. Thus, we conclude
that the presence of T reg cells signifi cantly increased the
speed and decreased arrest of antigen-specifi c CD4+ T cells
in the presence of autoantigen.
Interactions between CD4 effector cells and DCs
in the presence or absence of T reg cells
One possible explanation for our results could be that in the
presence of T reg cells the stable contact between antigen-
specifi c CD4+ naive T cells and DCs was diminished. To di-
rectly test this possibility, we transferred CFSE- labeled
Figure 2. T reg cells release antigen-specifi c CD4+ T cells in the
presence of antigen. (a) Experimental protocol for b–d. T/R+ or Tg/Tg
mice, which have or do not have endogenous T reg cells, respectively,
received 107 CFSE-labeled MBP Ac1-11–specifi c CD4+ T cells via the tail
vein. As endogenous control, Tg/Tg animals received, by tail vein injec-
tion, 1–2 × 107 CFSE-labeled MBP-specifi c T cells from Tg/Tg mice to-
gether with 107 CD4+CD25− T cells from syngeneic CFP-expressing mice.
On the same day, mice received 50 μg MBP Ac1-11 peptide emulsified
in IFA in the footpad. 20 h after immunization, draining PLNs were
imaged. (b) Representative tracks of MBP-specifi c T cells (green) in T/R+
and Tg/Tg immunized recipients. (c) Mean speeds of MBP-specifi c CD4+
T cells in both types of recipient mice. (d) Arrest coeffi cient for CD4+
T cells in both types of recipient mice. Results are representative of three
independent experiments. Representative tracks of WT CD4+CD25−
T cells (blue) are shown in b. Arrest coeffi cients for WT CD4+ T cells (blue)
are shown in d.

Page 4

508 INTRAVITAL MICROSCOPY ANALYSIS OF T REG CELL ACTION | Tadokoro et al.
MBP-specifi c T cells into T/R+ or Tg/Tg animals and,
24 h later, transferred 5-(and-6)-(((4-chloromethyl) ben-
zoyl) amino) tetramethylrhodamine (CMTMR)-labeled
antigen-loaded DCs into the foot pad of the same recipi-
ent mice (Fig. 3 a).
the naturally occurring MBP Ac1-11 is highly unstable, an
MBP Ac1-11 mutant peptide (referred to as Ac1-11[4Y])
was used (14, 17). The PLNs were then imaged 20 h later,
which allows time for DC migration and therefore should
provide similar timing of T–DC interaction as the IFA im-
munization experiments described above. Antigen-specifi c
T cell movement in PLNs that received antigen-pulsed DCs
was qualitatively (Fig. 3 b vs. Fig. 2 b) and quantitatively
(Fig. 3 d vs. Fig. 2 d) similar to the movement observed in
peptide-immunized animals. Labeling antigen-loaded DCs
with a vital dye allowed us to determine the duration of
their interaction with MBP-specifi c T cells in the presence
and absence of T reg cells. Contact time was measured by
analyzing frame-by-frame individual T cell–DC interactions
while viewing the rendered three-dimensional datasets from
the most optimal angles. The mean contact time in the ab-
sence of T reg cells was 995 s, which was close to the entire
time of the datasets, whereas this was reduced to 340 s in
the presence of T reg cells (65% reduction, P = 0.0001;
Fig. 3 c; see also Videos S6 and S7, available at http://www.
jem.org/cgi/content/full/jem.20050783/DC1). Thus, the
diff erence in duration of interactions in the presence or
[ID]FIG3[/ID] Because the complex between I-Au and
absence of T reg cells was about threefold. The arrest coef-
fi cient for antigen-specifi c T cells was signifi cantly lower in
the T/R+ recipients compared with the Tg/Tg+ recipients
with transferred DCs (36%, P < 0.0001; Fig. 3 d). CFP-
expressing polyclonal CD4+ T cells used as internal controls
had a contact time and arrest coeffi cient similar to MBP-
specifi c cells transferred to animals that harbor T reg cells
(Fig. 3, c and d).
Reduction of CD4+ T cell–DC interaction after adoptive
transfer of T reg cells
To exclude other possible diff erences between T/R+ mice
and Tg/Tg mice, we transferred T reg cells into Tg/Tg
mice. We previously showed that both CD4+CD25+ and
CD4+CD25− T cells from T/R+ mice suppressed EAE de-
velopment, but later it was found that in T reg cell–defi cient
experimental systems some of the transferred CD4+CD25−
T cells acquire the CD4+CD25+Foxp3+ T reg cell pheno-
type and function in vivo, in an IL-2–dependent manner
(18, 19). Because this process of peripheral T reg cell in-
duction from CD25− T cells takes several days, it is possible
to examine acute (<36 h) eff ects of T reg cells by transfer
of CD4+CD25+ cells with CD4+CD25− cells as a negative
control. To prove that T reg cells were responsible for the
reduction in the duration of contacts between naive antigen-
specifi c CD4+ T cells and antigen-loaded DCs, we trans-
ferred purifi ed WT splenic CD4+CD25+ T cells (T reg cells)
Figure 3. The presence of endogenous T reg cells results in shorter
contact time between CD4+ T cells and peptide-pulsed DCs. (a) Ex-
perimental protocol. T/R+ or Tg/Tg mice, which have or lack endogenous
T reg cells, respectively, received 107 CFSE-labeled MBP Ac1-11–specifi c
CD4+ T cells together with 107 CD4+CD25− T cells from syngeneic CFP
mice by tail vein injection and, 24 h later, received 5–10 × 105 CMTMR-
labeled DCs pulsed with MBP Ac1-11[4Y] peptide in the footpad.
20 h after DC transfer, PLNs were imaged. (b) Representative tracks of
MBP Ac1-11–specifi c CD4+ T cells in T/R+ and Tg/Tg DC-transferred
recipients. Trackings of CFP-expressing WT CD4 T cells are represented in
blue. (c) Contact time between MBP-specifi c or WT CD4 T cells and
antigen-loaded DCs. (d) Arrest coeffi cient for transgenic (green or red) or
WT (blue) CD4+ T cells in T/R+ and Tg/Tg DC-transferred recipients. Results in
b–d are representative of three independent experiments.

Page 5

JEM VOL. 203, March 20, 2006
509
BRIEF DEFINITIVE REPORT
or control CD4+CD25− T cells into Tg/Tg animals and ob-
served the interactions between CFSE-labeled CD4+ naive
MBP-specifi c T cells and CMTMR-labeled Ac1-11[Y4]-
loaded DCs 12 h after DC injection (Fig. 4 a). [ID]FIG4[/ID] Transfer of
T reg cells to Tg/Tg mice recapitulated the qualitative (Fig.
4 b vs. Fig. 2 b and Videos S8 and S9, available at http://
www.jem.org/cgi/content/full/jem.20050783/DC1) as well
as quantitative (Fig. 4, c and d, vs. Fig. 3, c and d) features of
T/R+ mice. Tg/Tg animals that received T reg cells displayed
signifi cantly shorter CD4–DC interaction time compared
with Tg/Tg mice that received CD4+CD25− T cells (29%,
P = 0.004). Note that the data shown in Fig. 4 correspond
to a 20-min observation experiment (maximum interaction
time, 1,200 s). It was not unexpected that the eff ect of T
reg cells transferred into Tg/Tg mice was not as great as the
eff ect of T reg cells endogenously generated in the T/R+
mice (Fig. 4 c vs. Fig. 3 c) because of issues related to the pu-
rifi cation of T reg cells with antibodies, followed by transfer
and engraftment. These issues notwithstanding, WT T reg
cell transfer into Tg/Tg mice decreased the arrest coeffi cient
of antigen-specifi c T cells compared with CD4+CD25− cell
transfer (34%, P < 0.0001; Fig. 4 d). Observation at later
time points in CD25+ cell–transferred Tg/Tg mice, but not
CD25− cell–transferred mice, showed a decreased number of
DCs (not depicted), suggesting that antigen+ DCs may have
a shorter half-life in the PLN in the presence of T reg cells,
an observation that requires further experimentation.
One of the most sought-after answers in the T reg cell
fi eld is to the question of how these powerful cells exert their
function in vivo. Our real-time intravital microscopy imag-
ing showed that one of the ways in which T reg cells aff ect
immune responses is by decreasing the contact time between
DCs and the precursors of CD4 eff ector cells. This was ob-
served in two ways, one in which T reg cells were present in
the host (T/R+ mice), and one in which WT T reg cells
were transferred into T reg cell–defi cient Tg/Tg recipients.
Absence of T reg cells always resulted in prolonged, more
stable contacts between CD4 cells and DCs. This phenom-
enon required autoantigen, as T reg cells did not change the
movements of MBP-specifi c T cells in the absence of the auto-
antigen (Fig. 1). Furthermore, T reg cells did not aff ect the
speed or arrest coeffi cient of polyclonal CD4+ cells (Fig. 2,
c and d). While our paper was under review, a complementary
study by Tang et al. (20) obtained similar fi ndings in vitro.
Using two-photon imaging of explanted pancreatic LNs in
the nonobese diabetic mouse model, they found that the
presence of endogenous T reg cells decreased the duration of
autoantigen-specifi c naive and T reg cell interactions with
pancreatic islet emigrant DCs. Direct imaging of T reg cells
and pancreatic islet emigrant DCs demonstrated the capacity
for long-lived interactions of T reg cells with DCs.
An important distinction between T/R+ mice and T reg
cell–transferred Tg/Tg mice is that T/R+ mice harbor sev-
eral MBP-specifi c CD4+CD25+ T reg cells. In this and other
Figure 4. Reconstitution of Tg/Tg animals with T reg cells
diminishes the contact time between CD4+ T cells and peptide-
pulsed DCs. (a) Experimental protocol. Tg/Tg mice received 107 CFSE-
labeled MBP-specifi c CD4+ T cells via tail vein and, on the same day,
5–10 × 105 CD4+CD25+ T cells (T reg cells) or CD4+CD25− T cells
(non–T reg cells) from a syngeneic WT animal in the footpad. 24 h
later, CMTMR-labeled DCs pulsed with MBP Ac1-11[4Y] peptide were
injected in the footpad, and PLNs were imaged 12 h after DC transfer.
(b) Representative tracks of CD4 effector T cells in Tg/Tg animals
reconstituted with T reg or non–T reg cells. (c) Contact time between
CD4 effector T cells and DCs in Tg/Tg mice that received T reg or non–
T reg cells. (d) Arrest coeffi cient of CD4+ T cells in Tg/Tg mice that
received T reg or non–T reg cells. Results in b–d are representative of
three independent experiments.