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.