Cutting Edge: Regulatory T Cells Selectively Attenuate, Not
Terminate, T Cell Signaling by Disrupting NF-kB Nuclear
Accumulation in CD4 T Cells
Yu-Hui Huang, Dorothy K. Sojka, and Deborah J. Fowell
A key consequence of regulatory T cell (Treg) suppres-
sion of CD4 T cells is the inhibition of IL-2 pro-
duction, yet how Tregs attenuate IL-2 has not
been defined. Current models predict a termination
of TCR signaling, by disrupting T–APC contacts, or
TCR signal modification, through mechanisms such
as cAMP. To directly define Treg effects on TCR sig-
naling in CD4 T cell targets, we visualized changes in
nuclear accumulation of transcription factors at time
points when IL-2 was actively suppressed. Nuclear ac-
cumulation of NFAT was highly dependent on sus-
tained TCR signaling in the targets. However, in the
presence of Tregs, NFAT and AP-1 signals were sus-
tained in the target cells. In contrast, NF-kB p65 was
selectively attenuated. Thus, Tregs do not generally
terminate TCR signals. Rather, Tregs selectively mod-
ulate TCR signals within hours of contact with
CD4 targets, independent of APCs, resulting in the
specific loss of NF-kB p65 signals.
Immunology, 2012, 188: 947–951.
and release of cytotoxic components from granules. This broad
suppressive capacity is likely exerted by different mechanisms
at different stages of immune activation (1, 2).
Fundamental to initial T cell activation is the receipt of
signals that promote cytokine production, cell proliferation,
and cell survival. IL-2 is the first cytokine produced by naive
T cells and is critical for successful adaptive immunity (3).
Upon TCR engagement, the nuclear accumulation of NFAT,
NF-kB, and AP-1, in concert, drives early IL-2 transcription
(4, 5). CD28 costimulatory signaling quantitatively changes
TCR signaling, enhancing NF-kB and AP-1 to promote
transcription and stabilizing IL-2 mRNA (6, 7). Tregs sup-
press T cell activation by inhibiting cell proliferation and
cytokine production, in particular early IL-2 production (8).
The Journal of
atural regulatory T cells (Tregs) counterbalance
immunity by suppressing cell proliferation, survival,
maturation, cytokine and/or chemokine production,
We mapped Treg suppression of IL-2 at the transcript and
protein level to a tight kinetic window 6–10 h after initial
CD4 T cell activation (9). However, the mechanism by which
Tregs specifically abort IL-2 production remains unknown.
Tregs could negatively regulate T cell signals for IL-2 via
CTLA-4/B7 (10) or cAMP (11) or potentially by using E3
ligases (12, 13). Alternatively, recent visualization of Treg
suppressive events suggested that Tregs could terminate T cell
signals by disrupting the stability or duration of CD4 T cell–
APC interactions (14, 15). To define the changes in T cell
signaling in target CD4 T cells activated in the presence of
Tregs, we used multispectral imaging flow cytometry (Amnis
Imagestream) to quantify the frequency of CD4 T cells with
specific transcription factor (TF) nuclear accumulation. Tregs
did not terminate T cell signaling at the time of IL-2 inhi-
bition. Rather, signaling in targeted CD4 T cells was selec-
tively modified by attenuation of nuclear NF-kB, but not
NFAT and AP-1, through an APC-independent mechanism.
Materials and Methods
Mice and Abs
BALB/c mice (National Cancer Institute) and Thy1.1 BALB/c mice were
maintained in the pathogen-free animal facility at the University of Rochester
Medical Center (Rochester, NY). Abs used: mouse anti-NFAT1 IgG1 (Af-
finity BioReagents); mouse anti-NFAT2 IgG1, rabbit anti-p65 IgG, rabbit
anti–c-Rel IgG, rabbit anti–c-Fos IgG, and rabbit anti–c-Jun IgG (Santa Cruz
Biotechnology); FITC goat F(ab9)2anti-rabbit IgG and FITC goat anti-
mouse IgG1 (Southern Biotech); anti-Thy1.1 eFluor 450 (eBioscience);
anti-Thy1.2 PE (BD Pharmingen).
CD4 cells were isolated from spleen and lymph nodes. CD4+CD252CD44low
naive T cells were sorted by FACSAria as a source of target CD4+T cells or
control T cells (Ctrl T). Tregs were purified from a CD4-enriched population
using a CD25+MACS column (Miltenyi Biotec) (routinely .85% Foxp3+,
with .85% suppression of CD4 proliferation at 1:1 target/Treg ratio). APCs
were isolated from spleen by complement lysis of Thy1.2-expressing T cells.
Confirmatory experiments were performed using sorted CD4+CD25+Foxp3+/
GFP+cells from Foxp3/GFP reporter mice.
A total of 1 3 105Thy1.1 naive target CD4+T cells was stimulated with anti-
CD3 mAb (1 mg/ml) and APCs (1 3 105) in coculture with either 1 3 105
Department of Microbiology and Immunology, David H. Smith Center for Vaccine
Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester,
Rochester, NY 14642
Received for publication April 20, 2011. Accepted for publication November 30, 2011.
This work was supported by Grants R01 AI070826 and U19 AI56390 (to D.J.F.) from
National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Address correspondence and reprint requests to Dr. Deborah J. Fowell, David H. Smith
Center for Vaccine Biology and Immunology, University of Rochester, 601 Elmwood
Avenue, Box 609, Rochester, NY 14642. E-mail address: Deborah_Fowell@urmc.
The online version of this article contains supplemental material.
Abbreviations used in this article: Ctrl T, control T cell; TF, transcription factor; Treg,
regulatory T cell.
Thy1.2 Ctrl T or Thy1.2 Tregs. Cells were harvested for functional assays at
various time points. In some experiments, Thy1.1 responder cells were pre-
treated with 1 mM cAMP antagonist, Rp-8-Br-cAMPS (BioLog Life Science
Institute), or the Src kinase inhibitor PP1 (10 mM, Axxora) was added to
cultures. In some experiments, suppression was assayed following Ab-coated
bead stimulation (16). M450 Dynabeads (Invitrogen) were coated with anti-
CD3 (2 mg/25 ml beads) and anti-CD28 (2 mg/25 ml beads). A total of 4 3
104Ab-coated beads was used to stimulate 1 3 105naive target CD4+T cells
in coculture with 1 3 105Ctrl T or Tregs. At 12 h, cells were collected for
p65 nuclear localization analysis (Imagestream) and IL-2 secretors by
cytokine-secretion assays (Miltenyi Biotec).
IL-2 secretors were detected by a cytokine secretion assay kit (Miltenyi Biotec),
according to the manufacturer’s instructions. For phospho-flow, cells were
fixed, permeabilized, and stained with anti-pERK mAb, according to the
manufacturer’s instructions (BD Bioscience).
Nuclear translocation analysis on Imagestream
Cells were fixed (1% paraformaldehyde), surface stained for Thy1.1/1.2,
permeabilized by 0.1% Triton X-100 (Sigma), and stained for NF-kB p65,
NF-kB c-Rel, NFAT1, NFAT2, c-Fos, or c-Jun. Nuclear dye Draq5 (5 mM;
Axxora) was added before analysis. Fluorescent images were visualized
(.6000 events per condition) on Amnis Imagestream. A mask on the nucleus
was created; within this area, colocalization of TFs and nuclear dye was
measured by similarity (IDEAS software, Amnis).
Treg suppression assay was set up as described above. At 6 h, CD4+Thy1.1+
target cells were positively selected on Thy1.2 and lysed, and nuclear extracts
were prepared (Active Motif). p65 DNA binding was quantified by TransAM
NF-kB p65 Transcription Factor Assay Kit (Active Motif). Briefly, 2 mg
nuclear extracts was loaded onto a 96-well plate coated with NF-kB consensus
sequence, followed by anti-p65 Ab and HRP detection at 450-nm absor-
bance. The relative amount of p65 bound to DNA was expressed as OD.
Results and Discussion
T cell signals and nuclear accumulation of TFs
To accurately interpret Treg effects on target T cell signaling,
we first determined the relationship between continued T cell
signaling and the nuclear accumulation of TFs key to IL-2
transcription. We performed single-cell analysis of TF nu-
clear localization using multispectral imaging flow cytometry
(17). Nuclear localization was defined by a positive similarity
score, representing the correlation coefficient between two
fluorescent signals: the relative colocalization of the nuclear
dye Draq5 and the TF NFAT2 or NF-kB p65 (Fig. 1A, 1B).
Unstimulated CD4+T cells had a negative similarity score,
corresponding to the absence of nuclear NFAT2 and NF-kB
(Fig. 1A, 1B). After 6 h of activation, most CD4+T cells were
positive for nuclear NFAT2 (Fig. 1A, 1B) and a proportion
of CD4+T cells exhibited nuclear localization of NF-kB
(Fig. 1A, 1B). The Src kinase inhibitor PP1 ablated the nuclear
localization of NFAT2 and NF-kB p65, showing the de-
pendency on TCR/CD28 signaling (Fig. 1A). Kinetically,
nuclear NFAT2 and p65 peaked at 6 h; NFAT2 was sustained
through 12 h, whereas p65 declined (Fig. 1C). To understand
the dynamics of TF nuclear localization and TCR signaling,
we blocked Src kinase signaling at the peak of TF nuclear
accumulation (6 h) and followed the frequency of cells with
a nuclear pool of NFAT2 and p65 (Fig. 1D). Nuclear NFAT2
was very sensitive to termination of TCR signaling: an 80%
loss in nuclear NFAT2+cells was noted within 1 h of PP1
addition. In contrast, p65 was relatively stable in the nucleus,
with only 10.36% loss of nuclear NF-kB+cells within 1 h of
PP1 addition, possibly reflecting non-TCR signals sustaining
nuclear NF-kB p65. This single-cell assay for detection of
nuclear TFs provides a sensitive platform for the detection of
possible perturbations in TCR signaling mediated by Tregs.
Sustained NFAT and AP-1 signaling in the presence of Tregs
To determine whether Tregs broadly extinguish target T cell
signaling at the time of IL-2 inhibition, we first examined
TCR-dependent nuclear NFAT in the target CD4+T cells in
coculture with Tregs or Ctrl T, non-Treg CD4 T cells (Fig.
2A, 2B). The presence of Tregs had no effect on the magni-
tude or timing of NFAT nuclear accumulation (Fig. 2A, 2B)
at time points when IL-2 production was suppressed (Fig. 2C)
(9). Thus, IL-2 is downregulated, despite ongoing TCR-
dependent signals that support NFAT nuclear accumula-
tion. Tregs also did not change nuclear accumulation of AP-1
components c-Fos and c-Jun (Fig. 2A, 2B). The AP-1 com-
plex is largely regulated at the protein level; therefore, we also
analyzed c-Fos or c-Jun total protein in target T cells in co-
culture with Tregs and found no change compared with tar-
gets cultured with Ctrl T (Supplemental Fig. 1A).
The sensitivity of nuclear NFAT to perturbations in TCR
signaling (Fig. 1D) and the absence of Treg-induced changes
in nuclear NFAT (Fig. 2) both suggested that CD4+target
T cell signaling remains largely intact in the presence of Tregs
at the time of IL-2 regulation (Fig. 2C). To confirm ongoing
upstream signaling, we used phospho-flow to measure kinase ac-
panels) or NFAT2 (upper panels). Graphs gated on CD4+T cells and percentages of activation-induced nuclear accumulation are shown. B, Representative cell
images using Imagestream. Stained with anti-NFkB or anti-NFAT mAb and the nuclear dye Draq5. Original magnification 340. C, Kinetic analysis. Graphs show
mean and SEM of the percentage of cells with similarity scores .0.5 from three independent experiments. D, CD4 cells were stimulated for 6 h, as in A, before
addition of PP1. Cells were analyzed for nuclear TF and normalized to target cells in the absence of PP1; mean and SEM from three independent experiments.
TCR-dependent nuclear localization of TFs. CD4 T cells were stimulated with anti-CD3/APCs. A, At 6 h, cells were stained for NF-kB p65 (lower
948CUTTING EDGE: REGULATORY T CELLS SELECTIVELY BLOCK NF-kB
tivation. Under our stimulation conditions, we were unable to
detect increases in p-PLCg or p-Zap70 in the 6–12-h time-
frame. However, ERK signaling was readily detectable from
1 h after anti-CD3/APC stimulation (Fig. 2D). The presence
of Tregs did not interfere with ERK signaling in the target
T cells, despite concomitant inhibition of IL-2 (Fig. 2C, 2D).
Tregs attenuate nuclear accumulation of NF-kB in CD4+target
In contrast to NFAT and AP-1, Tregs significantly attenuated
the frequency of target T cells with nuclear NF-kB p65 at 6 h
after activation and frequencies had returned to unstimulated
levels by 12 h (Fig. 3A, 3B). The reduction in nuclear p65 was
not due to a decrease in total cellular p65 (Fig. 3C), indicating
that differential generation or targeted degradation of NF-kB
p65 was unlikely. An NF-kB DNA-binding assay confirmed
a Treg-induced loss in nuclear NF-kB activity in CD4 targets
(Fig. 3D). Interestingly, Treg attenuation of NF-kB was se-
lective for p65 and was not seen for c-Rel (Supplemental Fig.
1B), suggesting that Tregs may target the p65 complexed to
IkBa, rather than the c-Rel–IkBb complexes, predominant in
naive cells (18). IL-2R signaling can regulate NF-kB; thus,
Tregs could modulate NF-kB by limiting IL-2 availability
(19, 20). Addition of exogenous IL-2 failed to rescue the
Treg-mediated change in p65 (Supplemental Fig. 1C). cAMP
is also a known inhibitor of NF-kB and a potential mediator
of Treg suppression (11); however, cAMP antagonists failed
to block Treg suppression of p65 or IL-2 in our system
(Supplemental Fig. 1D–F). In contrast, increased CD28 sig-
naling potently upregulated NF-kB and was able to override
Treg attenuation of NF-kB p65 (Supplemental Fig. 2A–C),
consistent with CD28 signals bypassing Treg suppression (9).
Rapid NF-kB downregulation independent of APCs
Full effector function requires continued signaling for $10 h
(21). To determine whether Tregs can acutely regulate T cell
activation, we initiated CD4+T cell cultures in the absence of
Tregs and then added freshly isolated Tregs or Ctrl T at 6 h.
Strikingly, Tregs attenuated nuclear NF-kB and IL-2 pro-
duction within 2 h of addition to the culture (Fig. 4A–C),
which was not due to acute IL-2 consumption (data not
shown). It is not known how the availability of individual TFs
correlates with the magnitude of IL-2 production in CD4
T cells. Therefore, we dual labeled cells to examine the rela-
tionship between the loss of nuclear NF-kB and IL-2 atten-
uation (Fig. 4D). IL-2 production early after activation was
found in a small fraction of CD4+T cells within the pop-
ulation showing an activation-induced increase in nuclear p65
APCs in coculture with Thy1.2+Ctrl T or Thy1.2+Tregs. A, At 6 h, cells were analyzed for nuclear NFAT1, NFAT2, c-Fos, and c-Jun by Imagestream. Graphs
gated on Thy1.1+target CD4+T cells; percentages are the frequency of target T cells with nuclear TFs. Representative plots from at least three experiments. B,
Kinetics of nuclear NFAT and AP-1 in coculture, frequency of Thy1.1+targets. C, Frequency of IL-2 producers. D, pERK frequency and MFI, gated on Thy1.1+
targets. Mean and SEM from three independent experiments. *p , 0.05, paired Student t test.
CD4+T cells sustained NFAT, AP-1, and ERK signaling in the presence of Tregs. Thy1.1+naive CD4+T cells were stimulated with anti-CD3/
Ab, and Draq5. Representative histograms gated on the Thy1.1+targets with percentage of targets with nuclear NF-kB p65. B, Activation-induced nuclear NF-kB
in target CD4+T cells in coculture. C, Total fluorescence intensity of NF-kB in the whole cell. B and C show mean and SEM from three independent
experiments. *p # 0.05, paired Student t test. D, DNA-binding assay for p65. Thy1.1 target cells were isolated from Ctrl T or Treg coculture at 6 h. Nuclear
extracts were assayed for amount of p65 bound to NF-kB–binding consensus sequence. Representative data from one of two independent experiments.
Tregs attenuate nuclear NF-kB in CD4+T cells. Cultures as in Fig 2. A, At indicated time points, cells were stained with anti-Thy1.1, NF-kB p65
The Journal of Immunology 949
(positive similarity score, Fig. 4D). However, there was no
positive correlation between the degree of nuclear p65 (sim-
ilarity score) and the amount of IL-2 produced (Fig. 4D). The
data suggested that a threshold amount of nuclear NF-kB is
required for IL-2 gene competency but that the degree of
nuclear NF-kB does not control the magnitude of the IL-2
response. Therefore, by reducing the number of cells that
reach this nuclear NF-kB threshold (Fig. 4D), Tregs appear to
limit the number of IL-2–producing cells.
Mechanistically, Tregs could modulate NF-kB directly in
the target T cell or indirectly via the APC. A Transwell ex-
periment confirmed that NF-kB was only modulated when
Tregs were in close proximity to targets and APCs (Fig. 4E).
To test the requirement for APCs, we stimulated target T cells
with anti-CD3/CD28–coated beads (16) with or without
Tregs, using conditions in which Tregs successfully inhibited
IL-2 (Supplemental Fig. 2D, 2E). In the absence of APCs,
Tregs retained the ability to inhibit nuclear NF-kB in target
T cells (Fig. 4F). Tregs also retained the ability to attenuate
NF-kB in cultures with fixed APCs (data not shown). Thus,
Tregs rapidly and selectively attenuated NF-kB T cell acti-
vation signals in CD4 targets, independent of the APCs.
Our results suggested that models of Treg action whereby
Tregs modulate the frequency or duration of T–APC conju-
gation (14, 15) cannot fully account for the early inhibition
of IL-2. Rather, qualitative changes in T cell signaling, with
a decrease in available nuclear NF-kB, appear to underlie
early suppressive events. Interestingly, an in vivo study using
an NF-kB luciferase reporter also showed decreased pathogen-
induced NF-kB activation with Tregs present (22). The tar-
geting of such a fundamental signaling pathway by Tregs
suggested that attenuated NF-kB may account for the ability
of Tregs to modulate the activities of many cell types, from
mast cells to B cells (1, 23, 24). Lymphocytes may be par-
ticularly sensitive to Treg downregulation of NF-kB p65 if
costimulation or inflammatory cytokines are limited (self-Ag)
but would override suppression when costimulatory signals,
particularly CD28/NF-kB or TNF-a, are upregulated.
The downstream consequence of reduced NF-kB in CD4
T cells has not been established. We do not know whether
there is a direct linkage between Treg-attenuated NF-kB and
loss of IL-2. Once a threshold level of nuclear NF-kB is
achieved, the amount of nuclear NF-kB p65 does not impact
the magnitude of the IL-2 response. Therefore, Tregs appear
to limit the number of T cells reaching that nuclear NF-kB
threshold. Loss of NF-kB could modify the expression of a
number of antiapoptotic molecules, such as Bcl-2, and cell
cycle-promoting molecules, like CDK (25), leading to the
indirect loss of early IL-2 producers as a result of a failure to
support their survival. Alternatively, reduced NF-kB could
lead to an alteration in the balance of TFs, with a predomi-
nant NFAT signal. NFAT signals trigger an anergy-related
gene profile, with the upregulation of E3 ligase-associate
genes (26). Interestingly, we showed that Tregs induced a
unique transcriptional program in CD4 targets that showed
the most overlap with ionomycin-induced anergy (27).
We have shown that Tregs do not terminate T cell-
activation signals but instead induce a unique signaling pro-
gram in CD4+target T cells with sustained NFAT/AP-1 but
significantly reduced nuclear NF-kB p65. This early NF-kB
modulation is independent of the APCs. We propose that
Treg suppression occurs in distinct mechanistic phases, with
early modulation that can occur in an APC-independent
fashion, qualitatively altering T cell signaling, and a later
phase during which modulation of the APCs may terminate
T–APC interactions. Thus, the context in which CD4+T cells
encounter Tregs may make them differentially sensitive to
these two phases and, hence, account for the heterogeneity
and controversy in the timing and proposed mechanisms of
Thy1.2+Tregs were added to the culture, and the targets were analyzed for nuclear NFAT2 (A) and NF-kB (B). Percentage of control cultures that did not have
cells added at 6 h; mean and SEM from three independent experiments. C, Frequency of IL-2 secretors after addition of Tregs or Ctrl T at 6 h; mean and SEM
from three independent experiments. D, Costaining of p65 with IL-2 secretors at 12 h, gated on Thy1.1+target cells, and the frequency of IL-2 secretors with
nuclear p65. Representative plots from one of three independent experiments. E, Target cells were loaded into lower chambers with anti-CD3/APCs. Ctrl T or
Tregs were added to upper chamber or lower chambers. Frequency of targets cells with nuclear p65 at 12 h. Mean and SEM from three independent experiments.
F, CD4 cells were stimulated by either anti-CD3/APCs or anti-CD3/CD28–coated beads with Ctrl T or Tregs. Representative data from one of two independent
experiments. *p # 0.05, **p # 0.005, two-tailed Student t test.
Acute regulation of NF-kB independent of APCs. Naive Thy1.1+CD4 cells were stimulated with anti-CD3/APCs. At 6 h, Thy1.2+Ctrl T or
950 CUTTING EDGE: REGULATORY T CELLS SELECTIVELY BLOCK NF-kB
We thank the Fowell Lab for helpful discussion and Jim Miller for careful
review of the manuscript. We also appreciate technical assistance from
Angie Hughson, T. Bushnell (University of Rochester Flow Core) and
T. George and R.A. DeMarco (Amnis).
The authors have no financial conflicts of interest.
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