This information is current as
of July 28, 2017.
Enhanced Engagement of CTLA-4 Induces
Antigen-Specific CD4 +CD25+Foxp3+ and
CD4 +CD25− TGF-β1+ Adaptive Regulatory
T Cells
Ruobing Li, Nicolas Perez, Subha Karumuthil-Melethil,
Bellur S. Prabhakar, Mark J. Holterman and
Chenthamarakshan Vasu
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:5191-5203; ;
doi: 10.4049/jimmunol.179.8.5191
http://www.jimmunol.org/content/179/8/5191
The Journal of Immunology
Enhanced Engagement of CTLA-4 Induces Antigen-Specific
CD4ⴙCD25ⴙFoxp3ⴙ and CD4ⴙCD25ⴚ TGF-␤1ⴙ Adaptive
Regulatory T Cells1
Ruobing Li,* Nicolas Perez,* Subha Karumuthil-Melethil,* Bellur S. Prabhakar,†
Mark J. Holterman,* and Chenthamarakshan Vasu*2
P
ositive and negative coreceptors of the CD28 family have
powerful modulatory effects on the activation, proliferation, and cytokine production of T cells. T cell activation
requires both Ag-specific triggering of the TCR complex and costimulation mediated through the positive coreceptor CD28 (1, 2).
Ligation of CD28 on T cells by its ligands CD80 (B7.1) and CD86
(B7.2), which are primarily found on APCs, leads to IL-2 production and T cell proliferation. The negative coreceptor, CTLA-4,
plays a primary role in T cell homeostasis and peripheral tolerance
through the inhibition of T cell activation, IL-2 production, and
cell cycle progression as evidenced by the lethal lymphoproliferation seen in CTLA-4 deficient mice (3).
CTLA-4 can mediate negative regulation of T cell responses
through several mechanisms. Although its competition with
CD28 for CD80/86 binding results in lowered activation, signaling through the cytoplasmic tail of CTLA-4 and modulation
of TCR signaling by activating phosphatases such as SHP-2
leads to cell cycle disruption and suppression of IL-2 production. Recently, it has been shown that the binding of CTLA-4 to
CD80 and CD86 can lead to modulation of dendritic cell (DC)3
function by inducing the release of IDO (4, 5). CTLA-4 medi-
*Department of Surgery and †Department of Microbiology and Immunology, College
of Medicine, University of Illinois, Chicago, IL 60612
Received for publication April 13, 2007. Accepted for publication August 8, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the National Institutes of Health Grant R21 A1059745
and Juvenile Diabetes Research Foundation Regular Grant 1-2005-27.
2
Address correspondence and reprint requests to Dr. Chenthamarakshan Vasu, Department of Surgery, University of Illinois at Chicago, 909 South Wolcott, College of
Medicine Research Building, Room 7113, M/C790, Chicago, Illinois 60612. E-mail
address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; BiAb, bispecific antibody; BM,
bone marrow; BMDC, BM-derived DC; Foxp3, foxhead box p3 transcription factor;
GITR, glucocorticoid-induced tumor necrosis factor receptor; HEL, hen egg lywww.jimmunol.org
ated interference of lipid raft formation on the T cell surface
and disruption of CD28 localization at the immunological synapse have also been implicated in the suppression of T cell
response (6, 7). Recently, it was shown that CTLA-4 can reduce
the contact period between T cells and APCs and lead to a
decrease in proinflammatory cytokine production and proliferation (8). These properties indicate a pivotal role for CTLA-4 in
regulating T cell function. Therefore, manipulating CTLA-4
signaling could be an effective strategy to modulate the immune
response for treating various immune-mediated clinical conditions (9 –13).
While the blockade of CTLA-4 signaling using an anti-CTLA-4
Ab resulted in protection against tumors and viral and bacterial
infections, the blockade of CD28 signaling using CTLA-4-Ig has
shown promise in treating autoimmunity and transplant rejection
(9, 10, 12, 13). Although a role for CTLA-4 in the negative regulation of T cells is well recognized, its contribution to Treg function remains controversial (14 –17). More importantly, although
the requirement of CTLA-4 in TGF-␤1-mediated adaptive Treg
generation has been reported (18), a direct role for CTLA-4 signaling in Treg induction is not known. Earlier, we showed that
coating an autoimmune or alloimmune target with anti-CTLA-4
agonistic Ab can result in an increase in the memory regulatory T
cell (Treg) numbers in vivo (19, 20). However, the direct effect of
enhanced CTLA-4 signaling in the context of Ag-specific TCRengagement on T cell differentiation into adaptive Tregs has not
been understood.
The ability of B7.1 and B7.2 to bind to CD28 has precluded
examination of the outcome of selective engagement of CTLA-4 in
vivo. In this study, we examined the effect of dominant CTLA-4
engagement by Ag-presenting DCs coated with an agonistic Ab on
sozyme; LN, lymph node; MFI, mean fluorescence intensity; mTg, mouse thyroglobulin; Treg, regulatory T cell.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this
article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the
induction of Ag-specific CD4ⴙCD25ⴙFoxp3ⴙ and CD4ⴙCD25ⴚTGF-␤1ⴙ adaptive Tregs. These cells were CD62Llow and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response
compared with their CD62Lhigh counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag
presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further,
the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results
showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that
allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy. The Journal
of Immunology, 2007, 179: 5191–5203.
5192
T cell response and differentiation in vivo and found that it can
result in the induction of Ag-specific adaptive Tregs. Our results
also demonstrate that CTLA-4 engagement by DCs coated with an
agonistic anti-CTLA-4 Ab can produce long-lasting protection
from autoimmunity through the suppression of thyroglobulin-specific T cell and Ab responses.
Materials and Methods
Mouse strains
Six- to 8-wk-old female wild-type BALB/c, MHC class II OVA(323–339)
epitope-specific TCR-transgenic (D011.10 in BALB/c background), and
wild-type CBA/J mice were purchased from The Jackson Laboratory. All
animal studies were approved by the animal care and use committee of the
University of Illinois, Chicago IL.
Cell lines, Ags, and antibodies
Priming mice with Ags
Wild-type BALB/c mice were i.v. injected with 50 ␮g of OVA or HEL and
5 ␮g LPS at least 10 days before being used in experiments. In some
experiments, OVA was emulsified in CFA and s.c. injected. Cells from
draining lymph nodes (LNs) were obtained from these mice 15 days after
priming.
DCs and coating with Abs
DCs were either isolated from the spleens of wild-type BALB/c mice or
generated in vitro from BM cells. DCs from spleens were isolated using
magnetic bead labeled anti-CD11c Ab (Miltenyi Biotec) according to
the manufacturer’s directions. The percentage of CD11c⫹ cells in the
enriched population was generally ⬎90%. For generating DCs in vitro,
BM cells were cultured in complete RPMI 1640 medium containing
10% heat-inactivated FBS in the presence of 20 ng/ml GM-CSF at 37°C
in 5% CO2 for 2 days and then for a further 4 days in fresh complete
RPMI 1640 medium containing 20 ng/ml GM-CSF and 5 ng/ml IL-4.
The nonadherent cells from 6-day cultures were used.
Before being used in the experiments, DCs (1 ⫻ 106/ml) were incubated
for 48 h at 37°C in the presence of OVA or mTg (20 ␮g/ml), or OVA
peptide (2 ␮g/ml) with or without LPS (5 ␮g/ml), washed, incubated with
control or test BiAb (10 ␮g/107 cells/ml) for 30 min on ice, washed further,
and used as control Ab or anti-CTLA-4 Ab-coated DCs. Bound Ab levels
on DCs were tested by FACS analysis before every experiment after staining with FITC-labeled anti-hamster IgG Abs. Maturation status of LPStreated DCs was also tested in comparison with untreated DCs by using
Abs against the activation markers CD80, CD86, CD40, and MHC II by
FACS before every experiment.
Testing of BiAb
Control and test BiAbs were tested for their ability to bind to respective
Ags and stay on the DCs upon coating. ELISA was conducted to test the
binding efficiency of the CTLA-4 portion of the BiAb to recombinant
CTLA-4-Ig as described earlier (19, 20). Binding efficacy of anti-CD11c
portion of the BiAb to CD11c expressed on DCs was tested by FACS. The
persistence of BiAb on coated DCs was tested by analyzing aliquots of
cells obtained at different time points in a FACSCalibur flow cytometer
(BD Biosciences). The persistence of Abs on a DC surface was also tested
using a Zeiss LSM 510 confocal microscope.
In vitro T cell assay
Ag-pulsed, Ab-coated DCs (1 ⫻ 105 cells/well) were plated in 96-well
flat-bottom tissue culture plates in triplicate along with purified T cells
(5 ⫻ 105 cells/well) from OVA-primed mice in RPMI 1640 medium containing 2% mouse serum. After 48 h, cells were pulsed with l ␮Ci/well
[3H]thymidine for 18 h. Thymidine incorporation was measured as described previously (19, 20).
Treatment with Ab-coated DCs
OVA-primed mice were injected i.v. with 5 ⫻ 106 OVA or OVA-peptide
pulsed DCs and control or anti-CTLA-4 Ab-coated mature DCs once or
twice at a 10-day interval and sacrificed on day 15 postinjection to test for
T cell response to ex vivo challenge with the Ag. To test the Ag specificity
of T cell suppression, HEL-primed mice were adoptively transferred (i.v.)
with CD4⫹ T cells from DO11.10 TCR-transgenic mice, followed by
OVA323–339 peptide-pulsed and Ab-coated DCs (5 ⫻ 106 cells/mouse) after
24 h. These mice were sacrificed on day 15 posttreatment to test for Agspecific T cell responses.
Ex vivo T cell response
T cell proliferation against ex vivo antigenic challenge (10 ␮g/ml OVA, 10
␮g/ml HEL, 1 ␮g/ml OVA peptide, and 20 ␮g/ml mTg) was tested either
by [3H]thymidine incorporation as described above or a CFSE dilution
assay as described earlier (20). CFSE dilution was measured by FACS
analysis after 5 days of culture. In some assays, saturating concentrations
of neutralizing anti-mouse IL-10 (1 ␮g/ml), anti-mouse TGF-␤1 (1 ␮g/ml),
and/or isotype-matched control Abs were added. In some assays, enriched
T cell subpopulations were cultured in the presence of OVA (50 ␮g/ml) or
anti-CD3 and CD28 Abs (5 ␮g/ml each) in the presence or absence of rIL-2
(50 U/ml).
FACS analysis
Cells were washed with PBS supplemented with 2% FBS (pH 7.4) and
blocked with anti-CD16/CD32 Fc block Ab on ice for 15 min. For surface
staining, cells were incubated with FITC-, PE-, and PE-Cy5- or PE-Texas
Red-labeled appropriate Abs in different combinations on ice for 30 min.
For intracellular staining, surface-stained cells were fixed, permeabilized
using fixation/permeabilization kits (Ebioscience), and incubated with fluorochrome-labeled Abs. Stained cells were analyzed using a FACSCalibur,
LSR, or CyAn analyzer, and the data were analyzed using the CellQuest,
WinMDI, Weasel, or Summit applications. Specific regions were marked,
and the gates and quadrants were set while analyzing the data based on
isotype control Ab background staining.
Cytokine analysis
Cell-free supernatants collected after 48 h were tested for cytokine levels
by ELISA or Luminex multiplex assays as per the manufacturer’s directions (BD Pharmingen and BioSource International). The amount of cytokine was determined using an appropriate cytokine-specific standard curve.
Background cytokine levels of effector cell cultures in the absence of Ag
were subtracted from test values to calculate the actual cytokine response.
Isolation of T cell subpopulations
CD4⫹ and CD4⫹CD25⫹ T cell subpopulations were isolated using the
appropriate kits and magnetic separation columns (Miltenyi Biotec). T cell
subpopulations were also purified by high speed FACS. Cells were stained
with PE-Texas Red-anti-CD4, PE-Cy5-anti-CD62L, PE-anti-CD25 (clone
74D; noncytotoxic IgM Ab), and anti-TGF-␤1-biotin/streptavidin-FITC
(clone A75-3; nonneutralizing), and the CD4⫹CD62LhighCD25⫹,
CD4⫹CD62LlowCD25⫹, CD4⫹CD62LhighTGF-␤1⫹, CD4⫹CD62Llow
TGF-␤1⫹, and CD4⫹CD25⫺TGF-␤1⫺ subpopulations were sorted using
the MoFlo high speed sorter (DakoCytomation).
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Hamster anti-mouse CTLA-4 (clone UCI0-4-F-I0-11) and CD11c (clone
N418) hybridomas were purchased from American Type Culture Collection and grown in serum-free/protein-free medium (BD Biosciences) and
the Abs were purified using protein L or A (Sigma-Aldrich) columns. AntiCTLA-4-anti-CD11c bispecific Ab (BiAb) was prepared and purified as
described earlier (19 –21). Purified hamster IgG (Fitzgerald International)
linked to the anti-CD11c Ab served as a control BiAb. Ag binding efficiencies of BiAbs were tested by FACS using bone marrow (BM)-derived
DCs (BMDCs) and ELISA using recombinant CTLA-4-Ig (R&D Systems)
as described earlier (21).
Type VI OVA and hen egg lysozyme (HEL), LPS from Salmonella
enterica, and CFA were purchased from Sigma-Aldrich. OVA(323–339) peptide was synthesized in the Research Resources Center at the University of
Illinois (Chicago, IL). Mouse thyroglobulin (mTg) was prepared as described earlier (19, 22). Purified anti-mouse-TGF-␤1 (clone A75-2; nonneutralizing) and anti-CD16/CD32 (Fc block) Abs were purchased from
Caltag Laboratories. FITC-, PE-, PE-Cy5- and PE-Texas Red-conjugated
Abs were from BD Pharmingen, eBioscience, R&D Systems, and Biolegend Laboratories. Paired Abs and the required cytokine standards for detecting mouse IL-2, IL-4, IFN-␥, and IL-10 (eBioscience) and TGF-␤1
(R&D Systems or BD Pharmingen) were used in ELISA. Neutralizing Ab
to mouse IL-10 (clone JES5-2A5) was purchased from eBioscience. Recombinant mouse IL-2, neutralizing Ab to mouse TGF-␤1 (clone 1D11),
and normal rat IgG1 Ab were purchased from R&D Systems. Magnetic
bead-based cell isolation kits were purchased from Miltenyi Biotec.
Multiplex cytokine assay reagents were purchased from BioSource
International.
REGULATORY T CELLS THROUGH CTLA-4 ENGAGEMENT
The Journal of Immunology
5193
Coculture assay
Effector T cells (CFSE labeled) from OVA-primed mice were mixed with
enriched subpopulations of T cells from tolerant mice at various ratios.
These mixtures or individual cell populations were used in T cell proliferation assays (total of 0.6 ⫻ 105 cells/well) either in the presence or
absence of OVA. For Transwell assays (BD Biosciences), Treg subpopulations from tolerant mice and APCs in the well insert (upper compartment)
and CFSE-labeled T cells from OVA-primed mice and APCs (2.5 ⫻ 106)
in the well (lower compartment) in a 24-well plate were cultured in the
presence or absence of OVA. CFSE dilution was measured by FACS analysis after 5 days of incubation.
Induction of autoimmune thyroiditis and treatment using Abcoated DCs
Statistical analysis
Mean, SD, and statistical significance ( p value) were calculated using a
Microsoft Excel or SSPS statistical application. In most cases, values of the
test group (mice that received anti-CTLA-4 Ab-coated DCs) were compared with that of control group (mice that received isotype control Abcoated DCs) unless specified. P ⱕ 0.05 was considered significant.
Results
FIGURE 1. Characterization of cross-linked Abs. Hamster anti-mouse
CTLA-4 mAb and purified hamster IgG were chemically cross-linked to
hamster anti-mouse CD11c mAb as described in Materials and Methods.
A, Fate of DC-bound Abs was tested by confocal microscopy. Immature
(untreated) or mature (LPS-treated) BMDCs were incubated on ice with
and anti-CD11c Ab that was linked to PE-labeled hamster IgG, washed,
and distributed to chamber slide wells. These cells were fixed at different
time points after incubation at 37°C and processed for microscopy. B, Fate
of DC-bound cross-linked Abs was tested by FACS. BMDCs were coated
with hamster IgG (10 ␮g/1 ⫻ 107/ml), washed, and incubated in complete
medium at 37°C and aliquots were collected at different time points and
stained with anti-hamster-IgG-FITC (open histograms) and tested by
FACS. Filled histograms represent background staining of cells not coated
with BiAb but incubated with anti-hamster IgG- FITC. Representative panels from experiments conducted in triplicate are shown. Assays were repeated at least three times.
Initial characterization of anti-CTLA-4 Ab-coated DCs
To generate an effective method for enhanced CTLA-4 engagement upon Ag presentation, we adopted a BiAb approach that we
have thoroughly tested in our earlier studies (19 –21). In this study,
agonistic anti-CTLA-4 Ab (as test) or purified hamster IgG (as
control) was chemically linked to DC-specific anti-mouse CD11c
Ab. Because both anti-CD11c and anti-CTLA-4 Abs were of hamster origin and there is no reliable isotype-specific anti-hamster Ab
available, we adopted the following approaches to test the crosslinked Ab. We confirmed the efficiency of the BiAb to bind to
CTLA-4 and CD11c by using CTLA-4 Ig as the Ag in an ELISA
and BMDCs as CD11c-expressing cells in a FACS assay (not
shown). As expected, confocal microscopy and FACS analyses
showed that mature DCs are better able to retain Abs on the surface compared with immature DCs (Fig. 1). Further characterization indicated that presence of optimum amounts of Abs (1 ⫻ 107
DCs coated with ⱕ10 ␮g of BiAb in 1 ml) on the DC surface has
no significant effect on their Ag-presenting function (not shown).
Therefore, 10 ␮g BiAb was used for coating every 1 ⫻ 107 DCs
in a 1-ml volume throughout this study.
The effect of Ag-pulsed, anti-CTLA-4 Ab-coated DCs on T
cell activation was tested in vitro using naive and Ag-primed
CD4⫹ T cells. Anti-CTLA-4 Ab-coated DCs suppressed Agexposed but not naive T cell responses (Fig. 2A). This suggested
either a lack of or slower recruitment of CTLA-4 on these T
cells compared with Ag-primed T cells. To examine whether
anti-CTLA-4 Ab-coated untreated and LPS-treated DCs can
suppress T cell response, OVA-pulsed Ab-coated DCs were incubated with CD4⫹ T cells from OVA-primed mice. As anticipated, LPS-treated DCs induced significantly higher activation
of T cells compared with untreated DCs (Fig. 2B). Although the
early activation of T cell response was not significantly different, anti-CTLA-4 Ab-coated, LPS-treated DCs induced a sig-
nificant suppression of T cell proliferation and IL-2 production
and higher amounts of IL-10 and TGF-␤1 production compared
with control Ab-coated DCs, indicating a dominant CTLA-4mediated signaling (Fig. 2C). This suggests that the initial activation, an essential step required for the up-regulation of
CTLA-4 on T cells, is unaffected by the presence of agonistic
anti-CTLA-4 Ab on the DC surface but that the subsequent
enhanced engagement of CTLA-4 efficiently reduces the proliferation of these T cells. In addition, the suppression of T cell
proliferative response was induced only by anti-CTLA-4 Abcoated and LPS-treated, and not by untreated, DCs. This could
be due to the superior ability of LPS-treated DCs to induce T
cell activation and up-regulation of CTLA-4 and to maintain
sufficient amounts of Ab on the surface for a longer period for
an effective enhanced engagement of CTLA-4. Therefore, LPStreated DCs were used for rest of the study.
DC-directed, enhanced CTLA-4 engagement in vivo results in T
cell hyporesponsiveness
To examine the effect of treatment with Ag-pulsed, anti-CTLA-4
Ab-coated DCs on T cell activation in vivo, OVA-primed mice
were treated i.v. with OVA-pulsed DCs that were coated with either hamster IgG (control group) or anti-CTLA-4 Ab (test group).
CD4⫹ T cells from these mice were tested for surface expression
of the Treg marker CD25 and the memory-related marker CD62L
and their response to ex vivo challenge with OVA. As seen in Fig.
3A, although the test group of mice demonstrated a significant
increase in CD4⫹CD25⫹ T cell numbers compared with control
mice ( p ⫽ 0.032), they appeared to have relatively lower, although
not statistically significant, CD62Llow memory T cell numbers. To
examine the effect of challenge exposure to OVA, these T cells
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For inducing experimental autoimmune thyroiditis, 8-wk-old CBA/J mice
were i.v. injected with 100 ␮g of mTg along with 25 ␮g of bacterial LPS
on day 0 and 100 ␮g of mTg and 5 ␮g of LPS on day 10. Mice were treated
with mTg-pulsed Ab-coated DCs on days 15 and 25 and sacrificed on day
40 for disease evaluation. In some experiments, mice were rechallenged
with 100 ␮g of mTg and 5 ␮g LPS on day 85 and sacrificed on day 100.
Thyroids were fixed and 5-␮m paraffin sections were made and stained
with H&E to examine lymphocyte infiltration. Cellular infiltration and follicular damage were scored as grades 0 – 4 as described earlier (19, 22).
mTg-specific T cell proliferative and cytokine responses were tested using
spleen and LN cells, and serum samples were tested for mTg-specific Ab
response by ELISA as described earlier (19, 22, 23).
5194
were maintained in the presence of OVA ex vivo and tested for the
early activation marker CD69 after 16 h, for cytokines after 48 h,
and for proliferation after 5 days. The numbers of cells expressing
the early activation marker CD69 were not significantly different in
the test and control groups. Importantly, CD4⫹ T cells from the
test group of mice showed a significantly lower number of T cells
proliferating compared with control mice (7.19 vs 20.14%) (Fig.
3B). In addition, as indicated by the CFSE dilution, T cells from
the test group of mice showed profoundly slower proliferation
and/or less number of divisions compared with the control T cells.
Consistent with the proliferation response, T cells from the test
group of mice produced significantly less IL-2 and IFN-␥ com-
FIGURE 3. In vivo suppression of T cell response by DC-directed, enhanced CTLA-4 engagement. OVA-primed mice were left untreated or
treated once i.v. with 5 ⫻ 106 OVA-pulsed LPS-treated BMDCs that were
coated with either control (control group) or anti-CTLA-4 Ab (test group).
Naive mice were also included as an additional control group. A, Spleen
cells were tested for CD25⫹ (upper panel) and CD62L⫺ (lower panel)
subpopulations among CD4⫹ T cells by FACS. B, Spleen cells were incubated with OVA, tested for the activation marker CD69 after 16 h (upper
panel), and tested for CFSE dilution after 5 days (lower panel) in cells
gated for CD4⫹ population by FACS. The MFI values for cells with CFSE
dilution are shown for this panel. Representative graphs are shown for both
A and B with the range of values obtained from five individual mice in
parentheses. C, Spent medium collected from 48-h cultures were tested for
IL-2, IL-10, and IFN-␥ by multiplex assay and for TGF-␤1 by ELISA.
Mean ⫾ SD values of cells from five mice tested in triplicate are shown.
Assays were repeated twice with similar results (a total of 15 mice per
group were tested). ⴱ, p ⬍ 0.05. Cells obtained from peripheral LNs also
produced similar results (not shown).
pared with cells from control mice ( p ⬍ 0.013) (Fig. 3C, left
panels). However, cells from these mice produced considerably
higher IL-10 and TGF-␤1 than the controls ( p ⬍ 0.0094) (Fig. 3C,
right panels). Collectively, these results suggest that DC-directed,
enhanced CTLA-4 engagement during Ag presentation resulted in
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FIGURE 2. DC-directed CTLA-4 engagement suppresses T cell response in vitro. A, T cells from naive or OVA-primed mice were incubated
with OVA-pulsed, LPS-treated DCs that were left untreated or coated with
hamster IgG or anti-CTLA-4 Abs and tested for T cell proliferation by the
[3H]thymidine incorporation method. B, CD4⫹ T cells enriched from
OVA-primed mice were incubated with OVA-pulsed untreated or LPStreated BMDCs that were left untreated or coated with hamster IgG or
anti-CTLA-4 Ab for 16 h and tested for an early activation marker, CD69,
by FACS or incubated for 48 h, pulsed with [3H]thymidine for 18 h, harvested, and tested for thymidine incorporation. C, Cell-free supernatants
collected from these wells after 48 h were tested for IL-2, IL-10, and
TGF-␤1 by ELISA. Representative panels or mean ⫾ SD of the triplicate
values are shown. Assays were repeated thrice with similar results. ⴱ, p ⬍
0.05; statistically significant value.
REGULATORY T CELLS THROUGH CTLA-4 ENGAGEMENT
The Journal of Immunology
5195
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FIGURE 4. Role of CD4⫹CD25⫹ T cells and cytokines in the hyporesponsiveness of T cells to OVA. OVA-primed mice were treated twice with
OVA-pulsed, LPS-treated BMDCs that were coated with either control or anti-CTLA-4 Ab at a 10-day interval. A, Spleen and LN cells obtained
15 days after the second treatment were pooled and CD4⫹ or CD4⫹CD25⫺ T cells were enriched as described in Materials and Methods. B,
CFSE-labeled total CD4⫹ and CD4⫹CD25⫺ T cells were tested for proliferation upon exposure to OVA (10 ␮g/ml) for 5 days by FACS (upper two
panels). Total CD4⫹ T cells from mice that received anti-CTLA-4 Ab-coated DCs were stimulated with OVA also in the presence of neutralizing
Abs to IL-10 and TGF-␤1 (lower panel). Representative graphs of three individual mice tested in triplicate are shown with the range of values in
parenthesis. C, Cytokine levels in the 48-h culture supernatants from the above cultures were tested by Luminex multiplex assay (IL-2, IL-10, and
IFN-␥) or by ELISA (TGF-␤1). Spleen cells from naive mice depleted of T cells were used as APCs. Right bars of left panels (separated by vertical
lines) show the cytokine response by total CD4⫹ T cells from the test group of mice in the presence of neutralizing Abs. These assays were repeated
twice with similar results (a total of nine mice per group were tested). ⴱ, p ⬍ 0.05 relative to control. D, Total CD4⫹CD25⫹ and CD4⫹CD25⫺ T
cells were used in a T cell suppression assay. CFSE-labeled, purified total T cells from OVA-primed mice acting as effector T cells and T
cell-depleted splenocytes from naive mice acing as APCs were used in this assay. On day 5, cells were stained using a PE-labeled anti-CD4 Ab and
the CFSE dilution in CD4⫹ cells was tested by FACS. Shown is the CFSE dilution in CD4⫹ T cells from the coculture assay (all three populations
were cultured together in the same chamber) (upper panels) or from a Transwell system (effectors and suppressors were cultured in separate
chambers of the same well along with APCs in both of the chambers) (lower panel). The representative percentage and the MFI value of cells
showing the CFSE dilution at a 2:1 effector:Treg ratio are shown. Enriched Tregs from three individual mice were tested separately in triplicate and
the assay was repeated twice with similar results (a total of nine mice per group was tested). ⴱ, p ⬍ 0.05.
5196
REGULATORY T CELLS THROUGH CTLA-4 ENGAGEMENT
the induction of Ag-specific hyporesponsive T cells. As evident
from CD69, IL-10, and TGF-␤1 expressions, these T cells are, in
fact, responsive to cognate Ag but hyporesponsive in terms of their
proliferative and proinflammatory cytokine responses. This observation and the presence of a higher number of CD25⫹ T cells
further suggest the presence of Ag-specific regulatory/suppressor
T cells in the test group of mice and that these cells can be functionally activated upon challenge with the Ag.
CD4⫹CD25⫹ T cells are partially responsible for the T cell
hypoproliferative response
FIGURE 5. TGF-␤1⫹ T cells may be responsible for the regulatory
property of CD25⫺ T cells from the test group of mice. Spleen cells from
control and anti-CTLA-4 Ab-coated LPS-treated DC recipient and naive
(none) mice were tested for the surface expression of TGF-␤1 before and
after the depletion of CD25⫹ T cells. Representative values (A) and
mean ⫾ SD of duplicate samples from three to four individual mice (B) are
shown. T cells from three mice per group were tested individually in duplicate and the assay was repeated twice with similar results (a total of at
least nine mice per group were tested). ⴱ, p ⬍ 0.05.
DC-directed, enhanced CTLA-4 engagement also induces
CD25⫺ T cells with surface-bound TGF-␤1
The above described results demonstrated three important aspects
of T cell hyporesponsiveness in anti-CTLA-4 Ab-coated DC
treated mice. These are: 1) an incomplete restoration of Ag-specific T cell response upon depletion of CD4⫹CD25⫹ T cells; 2) the
ability of CD4⫹CD25⫺ T cells to suppress effector T cells in a
contact-dependent manner; and 3) the reversal of the hypoproliferative response of T cells from the test group of mice by neutralizing anti-TGF-␤1 Ab. These observations prompted us to test
CD4⫹ T cell populations from treated mice for surface-bound
TGF-␤1. T cells from the test mice showed a significantly higher
number of TGF-␤1⫹ T cells compared with control and untreated
naive mice. Importantly, depletion of CD4⫹CD25⫹ T cells (middle panel of Fig. 5A) did not significantly affect the overall percentage of CD4⫹TGF-␤1⫹ T cells in the test group (lower panel
of Fig. 5A and also B). This suggested that the regulatory function
of CD4⫹CD25⫺ T cells from the test group is mediated by cells
with surface-bound TGF-␤1⫹ (CD4⫹CD25⫺TGF-␤1⫹) T cells.
CD4⫹CD25⫹ and CD4⫹TGF-␤1⫹ T cells induced upon
DC-directed, enhanced CTLA-4 engagement are
predominantly CD62Llow
Because the CD62Llow memory T cell levels were relatively
lower in the test mice (Fig. 3A), we examined them for memory
and naive subpopulations of Tregs. Although the total number
of CD4⫹ T cells with a memory phenotype (CD62Llow) in the
test group was lower compared with that of the control group
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Because the test group of mice showed significantly higher
numbers of CD4⫹CD25⫹ T cells (Fig. 3A) and the frequency
was boosted by an additional dose of DCs (not shown), we
speculated that these CD4⫹CD25⫹ T cells may have a role in
the Ag-specific hypoproliferative response of effector T cells in
the test mice. To examine this possibility, total CD4⫹ and
CD4⫹CD25⫺ T cells from both test and control mice were isolated (Fig. 4A) and tested for their proliferative responses
against OVA. T cells from both test and control mice showed a
small increase in the proliferative response to OVA challenge
when CD4⫹CD25⫹ T cells were depleted (Fig. 4B, upper two
panels). Although CD4⫹ T cells from the test group of mice
showed a significant reduction in TGF-␤1 and IL-10 responses
when CD4⫹CD25⫹ cells were depleted ( p ⬍ 0.0021), a noticeable change in the level of IL-10, but not TGF-␤1, was observed with control T cells (Fig. 4C, right panel). In a recall
response assay, neutralization of TGF-␤1 alone or together with
IL-10 caused a profound increase in T cell proliferative (23.8
and 26.1%, bottom panel, compared with 11.9%, middle panel
of Fig. 4B) and inflammatory cytokine (i.e., IFN-␥ and IL-2; left
panels of Fig. 4C) responses in cells from the test group
of mice.
To further examine the role of CD4⫹CD25⫹ and CD4⫹CD25⫺
T cells from the Ab-coated DC-treated mice, they were cocultured with CFSE-labeled, purified effector T cells from OVAprimed mice along with Ag-pulsed APCs. Ag-specific T cell
proliferative response was significantly suppressed by both
CD4⫹CD25⫹ and CD4⫹CD25⫺ subpopulations of T cells, with
a more potent inhibition of proliferation by the enriched
CD4⫹CD25⫹ population from the test group (12.4 vs 22.2% of
cells with CFSE dilution) at a 2:1 effector:suppressor ratio.
(Fig. 4D, upper panel). Further, the effector T cell suppression
induced by these subpopulations was also higher in terms of
their rate of proliferation (mean fluorescence intensity (MFI) of
482 and 592 vs 325 in the absence of suppressor cells). Importantly, only the CD4⫹CD25⫹ population from the control group
of mice demonstrated a considerable ability to suppress the effector T cell response, albeit significantly lower compared with
that of CD4⫹CD25⫹ cells from the test group. When the assay
was performed in a Transwell system (Fig. 4D, lower panel),
CD4⫹CD25⫺ T cells from the test group of mice showed no
significant suppression of effector T cell proliferation. However, CD4⫹CD25⫹ cells from these mice retained some ability
to suppress Ag-specific T cell proliferation (CFSE dilution in
22.8% of cells compared with 31.9% in the culture with no
suppressor cells), albeit lower compared with when the cells
were in the same chamber. This suggested that the effector T
cell suppression by CD4⫹CD25⫺ cells is contact dependent,
whereas suppression by CD4⫹CD25⫹ cells is most likely
through secreted cytokines and the effect is probably diluted
when the suppressors are not in close proximity to the effectors.
The Journal of Immunology
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FIGURE 6. Adaptive Tregs are generated upon DC-directed, enhanced CTLA-4 engagement. OVA-primed mice were treated twice with OVApulsed LPS-treated BMDCs that were coated with either control or anti-CTLA-4 Ab at a 10-day interval, and spleen cells obtained 15 days after
the second dose of DCs were tested. A and B, Spleen cells were stained with anti-CD4-PE-Texas Red, anti-CD62L-PEcy5, and anti-CD25-FITC and
analyzed using three-color FACS analysis. CD4⫹ cells were gated for CD62L analysis and CD4⫹ and/or CD62Lhigh or CD62low populations were
gated for CD25 analysis. C, Percentage of CD62Lhigh or CD62low populations among CD4⫹ T cells is presented as mean ⫾ SD. D, Cells were also
analyzed after staining with the above Abs for surface markers and with PE-labeled anti-mouse Foxp3 Ab after permeabilization. Total, CD62Lhigh,
and CD62Llow CD4⫹CD25⫹ T cells were gated for the histograms shown. E, Cells from the test group of mice (ant-CTLA-4 Ab-coated DC
recipients) were also stained for surface CD4, CD25, and CD62L and GITR or CTLA-4 and intracellular CTLA-4, IL-10, or TGF-␤1 and tested by
FACS. Histograms were generated using the indicated populations identified by four-color FACS analysis and gated based on isotype control Ab
staining. F and G, Spleen cells from test and control mice were stained for CD4, CD62L, and TGF-␤1 for a three-color analysis by FACS.
Percentages of CD62Lhigh or CD62low TGF-␤1⫹ populations among CD4⫹ T cell are presented as mean ⫾ SD. H, Spleen cells from control and
ant-CTLA-4 Ab-coated DC recipient mice were stained for CD4, TGF-␤1, and other markers. CD4⫹CD62LhighTGF-␤1⫹ and CD4⫹CD62LlowTGF␤1⫹ T cells tested for the expression of indicated markers are shown. For D, E and H, filled and open histograms show staining using isotype control
and marker-specific Abs, respectively. Percentage of cells with specific staining is also shown on each histogram for these panels. Representative
results from cells obtained from three mice per group analyzed individually in triplicate are shown for A–H. Assays were repeated three times using
three to four mice in each group with similar results (at least 12 mice/group were tested). ⴱ, p ⬍ 0.05.
5198
REGULATORY T CELLS THROUGH CTLA-4 ENGAGEMENT
(47.19 vs 54.14%) even after a second dose of DC treatment
(Fig. 6A), ⬃150% more memory CD4⫹CD25⫹ (CD4⫹CD25⫹
CD62Llow) cells were detected in test mice (12.57 vs 26.02%).
In contrast, the percentage of naive CD4⫹CD25⫹ T cells
(CD4⫹CD25⫹CD62Lhigh) was similar in both groups (10.36%
and 11.89%) (Figs. 6B and 4C). Intracellular staining revealed
that a majority of the CD4⫹CD25⫹CD62Llow cells from the test
group were foxhead box p3 transcription factor (Foxp3) positive (Fig. 6D). However, part of the CD4⫹CD25⫹CD62Llow T
cells from the control group were Foxp3⫺, indicating that a
significant number of these T cells are perhaps in a state of
activation. Further, CD4⫹CD25⫹CD62Llow cells from the test
group showed relatively higher CTLA-4, TGF-␤1, IL-10, and
glucocorticoid-induced tumor necrosis factor receptor (GITR)
expression compared with CD4⫹CD25⫹CD62Lhigh cells (Fig.
6E). Whereas CD4⫹CD25⫹CD62Lhigh cells from control mice
showed expression of these markers at levels comparable to that
of the test group, control CD4⫹CD25⫹CD62Llow cells ex-
pressed significant levels of GITR but not the other markers
(not shown).
TGF-␤1⫹ T cells from the test and control groups of mice
were examined for the expression of various markers. As shown
in Fig. 6, F and G, these T cells from the test group are primarily of the memory type (CD62Llow). Although the percentages of CD62LlowTGF-␤1⫹ cells ranged between 6.6 and 9.7,
CD62LhighTGF-␤1⫹ T cells were significantly lower (between 0.8
and 1.7%) in these mice ( p ⫽ 0.0039). Examination of resting
CD62Llow and CD62Lhigh subpopulations of TGF-␤1⫹ T cells
from the test group for Treg markers revealed that CD62Llow, but
not CD62Lhigh, population expressed significant levels of GITR
(Fig. 6H). Although only a fraction of these cells was positive for
Foxp3, both subpopulations demonstrated high levels of intracellular CTLA-4. IL-10 production and surface expression of
CTLA-4 were not detectable in either population. In agreement
with the above observations (Fig. 4), these results suggested that
CD4⫹TGF-␤1⫹ adaptive Tregs function through surface-bound
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FIGURE 7. Both CD4⫹CD25⫹ as well as CD4⫹TGF-␤1⫹ memory populations can effectively suppress an Ag-specific T cell response. OVA-primed
mice were treated twice with anti-CTLA-4 Ab-coated LPS-treated DCs at a 10-day interval as described in Materials and Methods. A, Pooled spleen and
LN cells were stained using fluorochrome-labeled Ab reagents (anti-CD4-PE-Texas Red, anti-CD62L-PE-Cy5, anti-CD25-PE, and anti-TGF-␤1-biotin/
streptavidin-PE), and CD62Lhigh or CD62low fractions of CD4⫹CD25⫹ and CD4⫹TGF-␤1⫹ T cells were FACS-sorted simultaneously as shown. FSC,
forward scatter. B and C, CFSE-labeled CD4⫹ T cells from OVA-primed mice were cocultured with the purified Tregs subpopulations, OVA (10 ␮g/ml),
and APCs from naive mice. The CFSE dilution in effector T cells gated for CD4⫹ population was tested on day 5. The assay was conducted using different
effector: suppressor ratios. Representative values (B) of assay using 0.5:1 suppressor:effector ratio and the mean of triplicate values of the assay using
different ratios (C) are shown. ⴱ, p ⬍ 0.05; statistically significant level of suppression of effector T cells in comparison with cells cultured in the absence
of Tregs. This assay was conducted using pooled spleen and LN cells from at least six mice and the experiment was repeated with a similar result. D,
Purified subpopulations of Tregs were labeled with CFSE and cultured in the presence of OVA and APCs or soluble anti-CD3 and CD28 Abs. A set of
similar wells received 50 U/ml rIL-2. CFSE dilution was tested after 5 days, and the percentage of cells with diluted CFSE is shown. This assay was
conducted using varying concentrations of OVA and CD3/CD28 Abs and representative values using 50 ␮g/ml OVA or 5 ␮g/ml CD3 and CD28 Abs each
are shown. Representative values from the assay conducted using pooled cells from six mice in triplicate are shown and this experiment was repeated with
similar result.
The Journal of Immunology
5199
TGF-␤1 in a contact-dependent manner. Fig. 6 also shows that
enhanced CTLA-4 engagement upon Ag presentation resulted in
the generation of at least two distinct populations of Tregs. Examination of CD62Llow and CD62Lhigh subpopulations from the
control group of mice showed that CD62Lhigh, but not CD62Llow,
population expressed moderate levels of intracellular CTLA-4. Expression levels of GITR, IL-10, Foxp3, and surface CTLA-4 were
not significant in either population.
Memory CD25⫹ and TGF-␤1⫹ T cell subpopulations are more
efficient in suppressing an Ag-specific T cell response than their
naive counterparts
Both CD25⫹ and TGF-␤1⫹ T cells are anergic but can
proliferate in the presence of IL-2 and strong stimuli
One of the major characteristics of Tregs is their hyporesponsiveness to cognate Ag. Therefore, we tested whether the enriched
population of Tregs from the test group of mice can proliferate
upon stimulation with OVA-pulsed APCs or anti-CD3/CD28 Abs
in the presence or absence of excess IL-2 (50 U/ml). Although
stimulation using OVA failed to induce the proliferation of all four
subpopulations, the addition of IL-2 induced the division of a considerable number (24.3 and 30.5% of cells with CFSE dilution) of
memory (CD62Llow) Tregs (Fig. 7D). Further, similar numbers of
these memory Tregs underwent cell division(s) when stimulated
with anti-CD3/CD28 Abs, which was further increased in the presence of exogenous IL-2 (35.6 and 58.0% of cells with CFSE di-
FIGURE 8. DC-directed CTLA-4 engagement induces Ag-specific T
cell suppression. HEL-primed mice were adoptively transferred with
CFSE-labeled or unlabeled CD4⫹ T cells from DO11.10 TCR-transgenic mice, left untreated (none), or treated with OVA(323–339) peptidepulsed control or anti-CTLA-4 Ab-coated LPS-treated BMDCs. A, Mice
that received CFSE-labeled CD4⫹ cells were sacrificed on day 5 posttransfer and spleen and LN cells were tested for proliferated cells by
FACS. B, Mice that received unlabeled CD4⫹ cells were sacrificed on
day 15 posttransfer and spleen cells were stained with CFSE, cultured
with either HEL (10 ␮g/ml) or OVA(323–339) peptide (1 ␮g/ml) for 5
days, and tested for CFSE dilution after staining with fluorochrome
labeled anti-mouse CD4 and DO11.10 TCR (KJ-126) specific Abs.
CFSE dilution patterns of CD4⫹ DO11.10 TCR⫺ (for HEL) or CD4⫹
DO11.10 TCR⫹ (for OVA(323–339)) populations are shown. Cells from
hamster IgG group were used as unstimulated control cells. Percentage
of cells with CFSE dilution among KJ-126⫹ cells and their MFI values
are shown. Results are representative of three individual experiments,
and these assays were repeated with similar results (at least six mice per
group were tested. ⴱ, p ⬍ 0.05.
lution). The proliferative response of Tregs to strong anti-CD3/
CD28 Ab stimulation alone and to cognate Ag in the presence of
excess IL-2 revealed that strong activation signals can induce the
expansion of adaptive Tregs.
DC-directed, enhanced CTLA-4 engagement-induced T cell
hyporesponsiveness is Ag specific
To test the Ag specificity of DC-directed, CTLA-4 engagementinduced T cell hypoproliferation, an adoptive transfer experiment was conducted. HEL-primed mice were adoptively transferred with CFSE-labeled CD4⫹ T cells from OVA(323–339)
peptide-primed DO11.10 TCR-transgenic mice and treated with
OVA(323–339) peptide-pulsed control or anti-CTLA-4 Ab-coated
DCs. Spleen and LN cells from recipient mice, obtained on day
4 posttransfer, were tested for CFSE dilution. The test group of
mice showed significantly lower T cell proliferation compared
with the controls ( p ⬍ 0.0084) (Fig. 8A). In a parallel experiment, unlabeled DO11.10 CD4⫹ T cell recipient mice were
treated with Ab-coated DCs. Lymphocytes obtained on day 15
posttreatment were labeled with CFSE and further tested for T
cell proliferation upon challenge with Ags ex vivo. CD4⫹ T
cells from both groups responded similarly to HEL challenge ex
vivo (Fig. 8B, upper panel). However, the recall response of
DO11.10 T cells from the test group of mice was significantly
lower in terms of the number of proliferating cells ( p ⫽ 0.0048)
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CD62Llow and CD62Lhigh subpopulations of CD4⫹CD25⫹ and
CD4⫹TGF-␤1⫹ T cells from the test group of mice were sorted
by high speed FACS (Fig. 7A) and tested for their ability to
suppress an Ag-specific effector T cell recall response. As
shown in Fig. 7, B and C, TGF-␤1⫹CD62Llow T cells suppressed effector T cell proliferation as effectively as
CD25⫹CD62Llow Tregs (percentages of proliferated T cells
were 8.5 and 10.4, respectively, compared with 32.7 in the presence of CD25⫺TGF-␤1⫺ control T cells and 35.2 in the absence
of Tregs). However, CD62Lhigh fractions of both CD25⫹ and
TGF-␤1⫹ T cells showed comparable efficacy (percentages of
proliferated cells were 16.5 and 17.4, respectively), albeit lower
compared with CD62Llow fractions, in suppressing the recall
response. In addition, as evident from the MFI values, effector
T cells cultured in the presence of CD62Llow adaptive Tregs
failed to undergo multiple divisions compared with cells cultured in the presence of control T cells or naive Tregs. Although
the adaptive Treg (CD62Llow) population demonstrated a significant level of suppression even at a suppressor:effector ratio
of 0.125:1 ( p ⬍ 0.021), relatively larger numbers of CD62Lhigh
cells (at least 0.25:1 suppressor:effector ratio; p ⬍ 0.043) were
needed to induce a significant level of suppression (Fig. 7C).
These observations demonstrated a higher suppressive efficacy
of adaptive Tregs, presumably due to their specificity toward
the cognate Ag. Although CD25⫹CD62Llow T cells from control mice failed to suppress the effector T cell response, the
suppressive abilities of naive CD25⫹ T cells from control and
test mice were comparable (not shown). This suggests that
CD25⫹CD62Llow T cells from control mice, at least in part, are
activated effector T cells and, therefore, do not suppress effector
T cells efficiently. Significantly lower levels of various Tregs
markers on these T cells from control mice compared with
CD25⫹CD62Llow T cells from test mice as evident from Fig. 6
support this notion. Collectively, these observations suggest
that the suppressive ability of CD4⫹CD25⫹ T cells from control mice observed in Fig. 4 is primarily mediated by the
CD62Lhigh population.
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REGULATORY T CELLS THROUGH CTLA-4 ENGAGEMENT
as well as the rate of proliferation (Fig. 8B, lower panel). This
demonstrated the Ag specificity of T cell hyporesponsiveness
induced upon DC-directed enhanced CTLA-4 engagement.
These observations suggest that adaptive Tregs also played a
key role in the suppression of thyroiditis.
DC-directed CTLA-4 engagement suppresses mTg-specific T cell
response and experimental autoimmune thyroiditis
Thyroiditis suppression induced upon DC-directed, CTLA-4
engagement is long lasting
To examine whether the Treg inducing potential of enhanced
CTLA-4 engagement can be exploited to treat autoimmunity,
CBA/J mice that were immunized with mTg to induce thyroiditis were injected with mTg-pulsed anti-CTLA-4 or control Abcoated DCs and tested for immune response and disease outcome 15 days posttreatment. Significantly reduced lymphocyte
infiltration into thyroids and minimal follicular destruction were
noted in treated mice compared with controls (Fig. 9A). Although 90% of the control mice developed grade 2–3 thyroiditis, only 30% of the mice from the test group showed grade 2
thyroiditis while the remaining 70% showed grade 1 thyroiditis.
Importantly, as observed above using OVA as the candidate Ag
(Fig. 6), mice from the test group showed a significant increase
in Foxp3⫹ and TGF-␤1⫹ adaptive Tregs compared with control
mice (Fig. 9B). Consistent with the results shown in Fig. 3, ex
vivo challenge of spleen cells from Ab-coated DC-treated mice
with mTg demonstrated that CD4⫹ T cells from these mice
were not only hyporesponsive to mTg but could also produce
significantly higher IL-10 and TGF-␤1 and reduced IFN-␥ and
IL-4 compared with T cells from control mice (not shown).
Next, we examined whether DC-directed CTLA-4 engagement
confers a long-lasting therapeutic effect. Mice were immunized
with mTg and treated with control or anti-CTLA-4 Ab-coated,
mTg-pulsed DCs and rested for 60 days before rechallenging
them with mTg. As shown in Fig. 10A, whereas the control mice
demonstrated grade 2 or 3 thyroiditis, the test group showed
grade 0 or 1 thyroiditis 15 days postchallenge. Although T cell
proliferative (Fig. 10B) and adaptive Treg responses (not
shown) remained more or less similar to that observed above in
Fig. 9, the test group showed significantly reduced titers; mean
IgG1 and IgG2a Ab titers were 8 ⫻ 104 and 4 ⫻ 104, respectively ( p ⬍ 0.01) against mTg compared with 1.6 ⫻ 105 and
2.4 ⫻ 105 for the control group. Interestingly, challenge exposure to Ag did not appear to boost the Ab response. These
observations led us to conclude that Ab response is also significantly suppressed, perhaps due to a decrease in the IFN-␥
and IL-4 levels. The above results showed that disease suppression induced by DC-directed, enhanced CTLA-4 engagement is
persistent even after rechallenge with the Ag and the inflammatory agent LPS.
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FIGURE 9. DC-directed CTLA-4 engagement suppresses ongoing autoimmune thyroiditis. Eight-week-old CBA/J mice were immunized with mTg
plus LPS on days 0 and 10, left untreated, or treated with mTg-pulsed control or anti-CTLA-4 Ab-coated LPS-treated BMDCs on days 15 and 25
as described in Materials and Methods. These mice were sacrificed on day 40 and tested for thyroiditis and T cell properties. A, H&E-stained thyroid
sections were examined for lymphocyte infiltration. Representative images are shown in the left panel. The severity of lymphocyte infiltration was
graded and the percentages of mice (n ⫽ 10/group) with different thyroiditis severity are plotted in the right panel. Unlike the other three groups
that received both LPS and mTg, the LPS control group received only LPS. B, Spleen cells were examined by FACS after staining for CD4, CD62L,
CD25, and TGF-␤1 (for surface expression) or Foxp3 (intracellular expression) in a four-color assay. Foxp3 and CD25 (left panel) and TGF-␤1 and
CD25 (right panel) expressions by total CD4⫹ T cells or cells gated for CD62Lhigh and CD62Lhigh populations are shown. Representative values
from the assay conducted using five individual mice in triplicate are shown for B. Assays for B were repeated with the remaining five mice (described
in A) with similar results.
The Journal of Immunology
5201
Discussion
Although a role for CTLA-4 in Treg function and TGF-␤1 induced
Treg generation has been suggested (14 –18), understanding its
direct role in Treg generation has remained elusive. Considering
that CTLA-4 plays an important role in T cell negative regulation,
we hypothesized that enhancing the CTLA-4 signaling strength at
the immunological synapse upon Ag presentation may affect T cell
differentiation. Therefore, we examined the ability of an agonistic
anti-CLTA-4 Ab-coated Ag presenting matured DCs to suppress
Ag-specific T cell response in vitro and in vivo. Our study demonstrated that the dominant engagement of CTLA-4 induced not
only Ag-specific T cell hyporesponsiveness but, more importantly,
a strong adaptive Treg response. In vivo studies showed that the
ability of T cells from anti-CTLA-4 Ab-coated DC recipients to
respond to challenge Ag exposure was dramatically reduced and
that a majority of the responding T cells failed to undergo multiple
cell divisions compared with T cells from control mice. Interestingly, anti-CTLA-4 Ab-coated DC treated mice showed a significant increase in the CD4⫹CD25⫹ T cell numbers and T cells from
these mice produced appreciably higher amounts of suppressor
cytokines such as IL-10 and TGF-␤1 upon re-exposure to the Ag,
suggesting their Ag specificity and potential mode of action.
Intriguingly, treatment with Ag-pulsed and anti-CTLA-4 Abcoated DCs resulted in a significant increase in the number of
memory (CD62Llow) CD4⫹CD25⫹ T cells. However, enhanced
CTLA-4 engagement in vivo had no effect on the overall number
of naive (CD62Lhigh) CD4⫹CD25⫹ T cells. Expression of Foxp3
by a large proportion of CD4⫹CD25⫹CD62Llow T cells from the
test group compared with the control group provided further credence to their identity as Tregs. In agreement with this observation, T cells depleted of a CD4⫹CD25⫹ population showed an
enhanced proliferative response and produced more inflammatory
cytokines (e.g., IL-2 and IFN-␥) but showed reduced levels of
the regulatory cytokines IL-10 and TGF-␤1. Importantly,
CD4⫹CD25⫹D62Llow Tregs more profoundly suppressed effector
T cell response compared with CD4⫹CD25⫹D62Lhigh Tregs.
Studies have shown that CD4⫹CD25⫹ T cells originate in the
thymus after self-Ag-mediated selection and that these T cells can
suppress self-Ag-specific T cells effectively in the periphery (24 –
26). Both thymic and peripheral naive CD4⫹CD25⫹ T cells express significant levels of CD62L on the surface (27, 28). Further,
studies have also shown that the CD62Lhigh, but not the CD62Llow,
population is more effective in suppressing effector T cell response
(29, 30). Although the origin of CD4⫹CD25⫹D62Llow T cells is
not well understood, they could be activated effector T cells, CD4⫹
CD25⫹D62Lhigh Tregs that were activated through TCR ligation,
and/or induced Tregs of CD4⫹CD25⫺ T cell origin. Therefore,
despite the possible heterogeneity, the CD4⫹CD25⫹D62Llow T
cell population with suppressor function was considered as adaptive Treg in this study.
Production of significantly higher amounts of IL-10 and
TGF-␤1 upon exposure to Ag by adaptive Tregs and reversal of
their suppressive effects upon neutralization of these cytokines indicated that they are functionally activated upon cognate Ag binding and exert their function primarily through IL-10 and/or TGF␤1. Importantly, our results also showed that CD25⫹, but not the
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FIGURE 10. DC-directed CTLA-4 engagement produces long-lasting effect on disease suppression. Mice were immunized and treated as described
above for Fig. 9. These mice were challenged with mTg plus LPS on day 85, scarified on day 100, and tested for anti-Tg immune response and thyroiditis.
H&E-stained thyroid sections were examined for lymphocyte infiltration. Unlike the other two groups that received both LPS and mTg, LPS control group
received only LPS. A, Representative images are shown in the top panel, and the severity of infiltration and follicular damage was graded and the
percentages of mice (n ⫽ 10/group) with different thyroiditis severity are plotted in the bottom panel. B, CFSE-stained spleen cells were incubated with
mTg (20 ␮g/ml) for 5 days, cells were stained using anti-CD4 PE Ab, and the proliferation was examined by FACS in a CD4⫹ population. Representative
scatter plots of five individual mice tested in triplicate are shown. C, Serum samples collected from the test and control mice were tested for anti-mTgspecific Abs by ELISA. Means ⫾ SD of OD (450 nm) values from five individual samples tested at 1/10,000 dilution were plotted as histograms and the
mean Ab titers are shown. Assays for B and C were repeated with the remaining five mice (described in A) with similar results. ⴱ, p ⬍ 0.05.
5202
ment of CTLA-4 by B7.1 and B7.2 present on the DCs alone may
not be sufficient to induce IL-10 and TGF-␤1 responses and Tregs.
However, controlling T cell activation by increasing the CTLA-4
specific ligand strength on APCs as demonstrated here can skew
the cytokine response from the inflammatory to the suppressive
type, which, in turn, can influence Treg induction.
If these adaptive Tregs are truly Ag specific, we wondered
whether CTLA4 can be selectively engaged to treat autoimmunity.
Because the Ag-specific TCR and the enhanced T cell repressor
signal are concomitantly delivered by the DCs, the resulting hyporesponsiveness most likely will be Ag specific. In this context,
when OVA peptide-specific T cells were introduced to HELprimed mice followed by treatment with OVA peptide-pulsed, antiCTLA-4 Ab-coated DCs (Fig. 8), only OVA peptide-specific, but
not HEL-specific T cells, showed significant hypoproliferative response to the Ag. More importantly, the ability of mTg-pulsed
anti-CTLA-4 Ab-coated DCs to suppress thyroiditis, characterized
by a profound hypoproliferative response of T cells against mTg,
showed that dominant engagement of CTLA-4 upon Ag presentation is an effective way to suppress autoimmunity. In addition,
enhanced CTLA-4 engagement by anti-CTLA-4 Ab-coated DCs
could produce long-lasting disease suppression, and rechallenge
with mTg failed to overcome the treatment-induced T cell hyporesponsiveness. Intriguingly, dominant CTLA-4 engagement affected both Th1 and Th2 T cells as evident from the cytokine and
subclass Ab levels.
Collectively, we have not only shown that selective CTLA-4
signaling can play an important role in adaptive Treg induction,
but also that the DC-directed, selective CTLA-4 engagement approach is an effective way to treat autoimmunity. This approach
exploits the unique Ag-presenting properties of DCs to harness the
potential of CTLA-4-mediated active T cell inhibition to induce
Ag-specific Tregs. This approach is highly flexible and can be
readily adapted to a wide range of Ags for treating autoimmunity
and transplant rejection.
Disclosures
The authors have no financial conflict of interest.
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TGF-␤1 can suppress effector T cells in both the free (secreted)
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