In Vitro HIV Infection Impairs the Capacity of Myeloid
Dendritic Cells to Induce Regulatory T Cells
Pietro Presicce1, Maria E. Moreno-Fernandez1, Laura K. Rusie1, Carl Fichtenbaum2, Claire A. Chougnet1*
1Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, and Department of Pediatrics, University of Cincinnati College of
Medicine, Cincinnati, Ohio, United States of America, 2Infectious Disease Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
Myeloid dendritic cells (mDCs) are the antigen-presenting cells best capable of promoting peripheral induction of
regulatory T cells (Tregs), and are among the first targets of HIV. It is thus important to understand whether HIV alters their
capacity to promote Treg conversion. Monocyte-derived DCs (moDCs) from uninfected donors induced a Treg phenotype
(CD25+FOXP3+) in autologous conventional T cells. These converted FOXP3+cells suppressed the proliferation of responder
T cells similarly to circulating Tregs. In contrast, the capacity of moDCs to induce CD25 or FOXP3 was severely impaired by
their in vitro infection with CCR5-utilizing virus. MoDC exposure to inactivated HIV was sufficient to impair FOXP3 induction.
This DC defect was not dependent on IL-10, TGF-b or other soluble factors, but was due to preferential killing of Tregs by
HIV-exposed/infected moDCs, through a caspase-dependent pathway. Importantly, similar results were obtained with
circulating primary myeloid DCs. Upon infection in vitro, these mDCs also killed Treg through mechanisms at least partially
caspase-dependent, leading to a significantly lower proportion of induced Tregs. Taken together, our data suggest that Treg
induction may be defective when DCs are exposed to high levels of virus, such as during the acute phase of infection or in
Citation: Presicce P, Moreno-Fernandez ME, Rusie LK, Fichtenbaum C, Chougnet CA (2012) In Vitro HIV Infection Impairs the Capacity of Myeloid Dendritic Cells to
Induce Regulatory T Cells. PLoS ONE 7(8): e42802. doi:10.1371/journal.pone.0042802
Editor: Derya Unutmaz, New York University, United States of America
Received February 29, 2012; Accepted July 11, 2012; Published August 13, 2012
Copyright: ? 2012 Presicce et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Public Health Service Grant R01 AI068524 (to CAC) and University of Cincinnati Postdoctoral Fellow Research Grant 2010
(to PP). The authors acknowledge the support provided in part by the Center for Excellence in Molecular Hematology Grant 1P30DK090971-01 and Digestive
Health Center Grant AR47363. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Dendritic cells (DCs) are the most potent antigen presenting
cells (APCs), endowed with the unique ability to prime naı ¨ve CD4+
T cells. They are believed to be an important target for HIV
during sexual transmission due to their presence at mucosal
surfaces, their function as antigen capturing cells and their role in
initiating adaptive immune responses . DCs can be directly
infected by HIV (cis-infection), although the frequency of in vivo
infected DCs is 10- to 100-fold lower than that of infected CD4+T
cells . However, DCs can also transmit virus to CD4+T cells
(trans-infection), without being productively infected [3,4]. HIV
induces a semiactivated phenotype in DCs and compromises their
functionality by impairing cytokine production and antigen
presentation . In vitro, myeloid dendritic cells (mDCs) are more
susceptible to R5 HIV (virus using CCR5 as co-receptor) infection
than plasmacytoid DCs (pDCs) due to their higher expression of
CCR5 . Due to the low frequency of circulating mDCs,
monocyte-derived dendritic cells (moDCs) are often used as a
model of mDCs. MoDCs can be infected by HIV similarly to
infected mDCs . MoDCs mature when exposed to lipopoly-
saccharide (LPS), which increases their ability to mediate HIV
Regulatory T cells (Tregs) are a subset of CD4+T cells critical to
maintenance of immunological self-tolerance and immune ho-
meostasis . The role that Tregs play in the context of chronic
infection such as HIV remains unclear. Some authors have
reported that in vitro removal of Tregs from HIV-infected humans
and SIV-infected macaques enhances antiviral immune responses
[7,8], and it has been proposed that excessive Treg reactivity
suppresses the function of multiple cell types and leads to faster
progression of HIV pathogenesis . On the other hand, Tregs
may protect individuals from the deleterious effects of immune
activation that are typically observed in chronic infection
(reviewed in ).
Treg frequency increases in lymphoid tissues as well as
peripheral blood during chronic HIV infection [10–15], but the
underlying mechanisms have not yet been characterized. In
addition to natural Tregs that arise and mature in the thymus,
growing evidence demonstrates that Tregs can be induced from
either naı ¨ve (reviewed in ) or memory  conventional CD4+
T cells in the periphery. Recent data highlight the role of APCs,
mDCs in particular, in inducing FOXP3 expression and
suppressive function in conventional CD4+T cells [18,19].
Manches et al. reported that pDCs exposed in vitro to HIV
induced Tregs from allogeneic naı ¨ve CD4+T cells via an
indoleamine 2,3-dioxygenase (IDO)-dependent mechanism .
Recently, we showed that tissue mDCs induce the conversion of
nonTregs into Tregs in chronically SIV-infected macaques .
Thus, mDC-mediated conversion may contribute to the accumu-
lation of Tregs observed in lymphoid tissues of HIV/SIV-infected
subjects [10,11,13,22]. To investigate whether DCs in circulating
blood could mediate the accumulation of Tregs in acute HIV
infection, we examined whether in vitro infected moDCs as well as
PLOS ONE | www.plosone.org1August 2012 | Volume 7 | Issue 8 | e42802
primary mDCs from healthy donors could convert autologous
nonTregs into Tregs and whether these converted cells were
Surprisingly, our results show that in vitro infected DCs were
severely impaired in their capability to induce functional Tregs,
and that this defect was mainly associated with the increased death
of T cells cultured with the infected DCs. This death was contact-
dependent and at least partially caspase-mediated. Importantly,
similar results were obtained with primary mDCs infected in vitro.
Exposure of DC to HIV was sufficient to alter their function.
Collectively, our data suggest that Treg induction by DCs may be
defective when they are exposed to high levels of virus, such as
during the acute stage of infection or AIDS.
Materials and Methods
Cell Isolation and Culture
Peripheral blood mononuclear cells (PBMCs) were separated by
centrifugation through Ficoll–Hypaque (GE, Fairfield, CT).
CD14+monocytes were isolated by positive selection (CD14
beads, Miltenyi Biotec, Auburn, CA) and immature moDCs were
generated by culturing the isolated monocytes for 5 days in
complete medium (RPMI 1640, supplemented with 10% of heat-
inactivated fetal calf-serum, HEPES, Glutamine) with 500 U/ml
rhIL-4 (Peprotech Inc, Rocky Hill, NJ) and 1000 U/ml GM-CSF
(Peprotech). One-third of complete medium, including cytokines,
was replaced every 3 days. For some experiments primary mDCs
were purified from circulating elutriated monocyte fraction on a
MoFlo XDP Cell Sorter (Beckman Coulter, Brea, CA). Cells were
stained in PBS containing 2% FBS using fluorochrome-conjugated
antibodies and mDCs were defined as CD142HLA-DR+CD11c+
with purity .98% . MoDCs as well as mDCs were stimulated
overnight with 500 ng/ml of LPS (Sigma-Aldrich). Immature
moDCs expressed low levels of activation/maturation markers
(CD80, CD86, CD40, PDL-1, HLA-DR and CD83), while LPS-
mature moDCs expressed high levels of these markers.
Resting autologous CD4+T cells were first purified from the
CD142cell population by negative selection (CD4 separation kit,
Miltenyi Biotec), as described above. Tregs and nonTregs were
further separated by cell sorting using a FACSAria (BD), with
Tregs defined as CD8negCD25hiCD127lowand nonTregs defined
as CD8negCD25lowCD127hi. In some experiments, nonTregs were
further separated into naı ¨ve defined as CD8negCD25lowCD127-
hiCD45RAposand memory defined as CD8negCD25lowCD127hiC-
D45RAneg. The purity of Tregs and nonTregs was evaluated post-
sorting by intracellular detection of FOXP3 (clone PCH101, e-
Bioscience, San Diego, CA). Purified Tregs were .90% FOXP3+,
whereas nonTregs were less than 0.5% FOXP3+. Where
indicated, CD252nonTregs (,1.3% FOXP3+cells post-isolation)
and CD25+Tregs (.80% FOXP3+cells post-isolation) were
purified from CD4+T cells using CD25 magnetic beads (Miltenyi
Treg Phenotypic Characterization
The following antibodies (Abs) were used for phenotypic
characterization of induced Tregs and DCs. Anti-CD14 (61D3)
FITC-, anti-CD1a (HI149) PE-, anti-CD40 (5C3) FITC-, anti-
CD86 (FUN-1) FITC-, anti-HLA-DR (L243) PerCP-, anti-CD11c
(3.9) APC-, anti-CD83 (HB15e) APC-, anti-CD80 (2D10) PE-,
anti-PDL-1 (MIH1) PE-, anti-CD3 (SK7) PerCPCy5.5-, anti-CD4
(RPA-T4) PB-conjugated were purchased from BD Biosciences
(San Diego CA). Anti-FOXP3 (PCH101) PB- and AF647-
conjugated were obtained from eBioscience. Anti-CTLA-4
(14D3) PE-, anti CD45RA (MEM-56) PE-Cy7-, anti-CD45RO
(UCHL-1) PB, anti-CD25 (M-A251) APC-H7-conjugated were
purchased from BD Biosciences (San Diego, CA). Anti-CD127
(R34.34) PE-conjugated was purchased from Beckman Coulter
(Fullerton, CA). Anti-CD4 (RPA-T4) AF700-conjugated was
purchased by Biolegend (San Diego, CA). Unconjugated anti-
GARP (Plato-1) was purchased from Alexis Biochemicals (San
Diego, CA) and conjugated using Zenon PE mIgG2b labeling kit
(Invitrogen; Carlsbad, CA) following manufacturer’s instructions.
All antibodies were titrated for optimal detection of positive
populations and mean fluorescent intensity (MFI) prior to use.
Cells were treated with 20 mg/ml of human IgG to block Fc
receptors and stained for surface markers for 30 min at 4uC, in
PBS containing 2% fetal calf serum and 0.1% sodium azide. Cells
were then washed and fixed with Fixation/Permeabilization Buffer
(eBioscience). After 30 minutes of incubation at 4uC, cells were
(eBioscience) and incubated with rat serum for 15 min at 4uC to
block non-specific binding of antibodies. Cells were finally stained
with FOXP3 and CTLA-4 mAbs for 30 min at 4uC and analyzed
on FACS LSR-II (BD). At least 150,000 events were recorded for
each sample. Doublets were excluded on the basis of forward- and
side-scatter properties and dead cells were gated out using LIVE/
DEAD Viability kit (Invitrogen). Data were analyzed using
FACSDiva (BD) and FlowJo software (TreeStar Inc., Ashland,
with Permeabilization Buffer
Virus Production and DC Infection
Simian Immunodeficiency Virus (SIV)mac-Virion Like-Parti-
cles (VLPs) and HIV viruses were prepared by transfection of
293 T cells with plasmids encoding the R5-tropic (YK-JRCSF)
HIV lab strain and SIV-mac VLP, using FuGENE (Roche) .
After two days, supernatants were harvested, and the viruses were
precipitated using polyethyleneglycol. YK-JRCSF virus titer was
determined using TZM-bl indicator cells as previously described
. SIVmac-VLPs titers were determined using the Reverse
Transcriptase colorimetric assay kit (Roche). In addition, for some
experiments, YK-JRCSF-containing supernatants were treated
with 1 mM 2,29-aldrithiol (AT-2) for 1 hour at 37uC in agitation,
as previously described . This treatment has been shown to
eliminate infectivity of retroviral virions while preserving the
conformation of envelope glycoproteins. Microvesicles, isolated
from uninfected cultures of 293 T cells following the same
procedures as those used to prepare AT-2 HIV, were included
in the experiments as negative control.
LPS-mature DCs were infected at different multiplicities of
infection (MOI, from 0.01 to 3). Briefly, DCs were incubated with
YK-JRCSF plus SIVMAC-VLPs for 6 hours at 37uC, washed
twice with complete RPMI, and cultured for 24 hours  before
culture with nonTregs. For some experiments DCs were treated
with Zidovudine (AZT, 1 mM) at the time of HIV infection and
during the co-cultures.
To quantify integrated HIV DNA in DCs, infection of LPS-
matured moDCs was carried out with viruses pre-treated with
DNase I at 20 U/ml in 10 mM MgCl2to eliminate cellular DNA
carryover from virus production. After 18 hours at 37uC, cells
were collected and suspended in lysis buffer containing 10 mM
Tris HCl (pH 9), 0.1% Tween 20-NP40 and 400 mg/ml
Proteinase K (Invitrogen). Cellular lysates were used to quantify
cell-associated HIV-1 DNA by nested real-time PCR . Briefly,
the first round of amplification used Alu specific primers and the
HIV-1 long terminal repeat (LTR). In the same reaction, the CD3
gene was quantified to precisely determine the number of input
cells. This reaction was followed by a second round of
amplification with specific primers and a labeled probe specific
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for the HIV LTR, performed in a Light Cycler (Roche). In
parallel, CD3 was re-amplified and detected using SYBR Green.
The ACH-2 cell line, a line of human T-lymphocytic leukemia
that contains a single copy of HIV-1 proviral DNA, was used to
determine the efficiency of the primers (NIH AIDS Research and
Reference Reagent Program) (detection limit=3 copies of HIV
Treg Suppression Assays
Cells from nonTreg/DC co-cultures (hereafter referred to as
‘‘bulk’’, containing converted Tregs) were harvested after 5 days of
CD4+CD25lowCD127hinonTregs (i.e. responder cells) at a 10:1
ratio in presence of immobilized anti-CD3 Ab (96 well U-bottom
plates were coated with 1 mg/ml Ab, BD Biosciences, at 37uC for
2 hours) and soluble anti-CD28 Ab (0.5 mg/ml, BD Biosciences).
Responder nonTregs were CFSE-labeled before stimulation. In
one well, responder cells were cultured alone as negative control.
As positive control, purified Tregs (CD4+CD25hiCD127low) were
mixed with autologous nonTregs at different ratios (0.1:1, 0.5:1,
1:1, 1:2, 1:4). After 4 days, cells were harvested and the
suppression ability of Tregs was determined by analyzing the
percentage of responder cells having divided at least once
(CFSElow) in the different cultures.
In vitro Modulation of Treg Induction
Recombinant human IL-10 (rIL10) was purchased from
Peprotech (Rocky Hill, NJ) and used at 10 ng/ml (after titration).
Recombinant human TGF-b (rTGF-b) and rPDL-1 were
purchased from R&D Systems (Minneapolis, USA) and used at
10 ng/ml (after titration). Neutralizing antibodies against human
IFN- c-receptor 1 (IFN-cR1), IL-4R (R&D Systems), IL-10 and
IL-17 (BD Bioscences) were used at 10 mg/ml, while neutralizing
antibodies against human TGF-b (Sigma Aldrich) was used at
10 ng/ml. Recombinant IL-2 (rIL-2) was used at 100–1000 IU/
ml. All the above mentioned reagents were added at the beginning
of the co-cultures. The pan-caspase inhibitor Z-VAD-FMK was
purchased from R&D Systems and added twice daily starting at
day 0 at a final concentration of 50 mM.
Cellular RNA was isolated using TRIZOL. TGF-b and
Ubiquitin ligase (UBI) mRNA were quantified by RT-PCR using
the Light Cycler (Roche). All reactions were performed in
duplicate using a SYBR green PCR mix (Qiagen, Valencia,
CA), according to the following thermal profile: denaturation at
95uC for 15 seconds, annealing at 60uC for 15 seconds, and
extension at 72uC for 15 seconds (data collection was performed
during the extension step). The following primers were used: TGF-
b (QT00000728, Qiagen, Santa Clarita, CA) and UBI forward
(QT02306724, Qiagen). The threshold level was determined by
the software according to the optimization of the baseline and the
standard curve. Results are presented as ratios between the target
gene mRNA and the UBI mRNA.
Statistical analysis was performed using Prism (GraphPad
Software 5). Paired t-test was used in the comparison between
different conditions. Linear regression analysis was used to test the
correlation between percentage of death in CD3+CD4+T cells
and percentage of induced CD25+FOXP3+Tregs as well as the
correlation between percentage of memory cells and percentage of
induced CD25+FOXP3+Tregs, age and percentage of induced
CD25+FOXP3+Tregs. A p value of less or equal to 0.05 was
considered to be significant.
Uninfected Mature moDCs Induce Tregs but their
Infection with HIV Impairs this Process
Myeloid DCs are reported to be better inducers of Tregs than
other APCs . As DCs can be infected with HIV, we
investigated the impact of their infection on Treg conversion.
To this end, we compared the ability of in vitro infected moDCs
and uninfected moDCs to induce CD25 and FOXP3 expression in
purified nonTregs (defined as CD4+CD25lowCD127hiT cells,
which contained less than 0.5% FOXP3+cells). The optimal ratio
of DCs to T cells for the FOXP3 induction was 1:10 (0.056106
DCs: 0.56106nonTregs), as determined in preliminary experi-
ments using either autologous or allogeneic culture conditions. In
addition, kinetic experiments showed that FOXP3 induction
peaked after 5 days of culture. These experimental conditions were
thus used throughout the study. LPS-activated moDCs induced
higher expression of FOXP3 in nonTregs compared to unstimu-
lated immature moDCs (Figure 1A). This increase was not specific
to LPS stimulation as Staphylococcus aureus Cowan-activated moDCs
induced similar percentages of FOXP3+cells as LPS-moDCs (data
To achieve high levels of DC infection, LPS-activated moDCs
(mature moDCs) were infected with the R5 HIV strain YK-
JRCSF in presence of non-infectious virion-like particles derived
from the Simian Immunodeficiency Virus (SIVmac VLPs) (both at
3 MOI). This method has been shown to increase the efficiency of
transduction by HIV-1 lentiviral vectors. The effect is restricted to
DCs and likely mediated by providing additional viral accessory
protein Vpx that counteracts the restriction factor SAMHD1
[24,28]. Consistent with these previous reports, addition of
SIVmac VLPs significantly increased the levels of DC infection
compared to infection with YK-JRCSF alone (Figure 1B).
Notably, a similar viability .88% was observed in both uninfected
and infected DCs.
Induction of both CD25 and FOXP3 expression was severely
impaired when nonTregs were cultivated with in vitro HIV-infected
moDCs (mean 6 SE % of CD25+FOXP3+cells: 10.02%61.64%
vs. 0.14%60.05% in uninfected vs. infected co-cultures, respec-
tively; p,0.001, n=18, Figure 1C). Notably, LPS stimulation
after infection did not affect the ability of DC to induce Tregs (not
shown). Analysis of CFSE dilution indicated that FOXP3
expression appeared early after activation and persisted, as the
level of FOXP3 was found to be equal in T cells having divided
once or several times (Figure 1D).
To exclude the possibility that moDCs could expand the few
contaminating FOXP3+cells rather than convert nonTregs into
Tregs, mature moDCs were cultivated with autologous purified
CD25hiCD127lowTregs (.90% FOXP3). While purified by cell
sorting Tregs completely lost FOXP3 expression when cultivated
alone without any kind of stimulation, expression of FOXP3 was
partially maintained when they were cultured with mature moDCs
(Figure S1). However, they did not proliferate, suggesting that the
contamination by these cells does not contribute significantly to
the pool of cycling FOXP3+cells seen in the DC : nonTregs co-
cultures (Figure S1). We also confirmed that conversion was a
mDC-dependent mechanism, as neither FOXP3 nor CD25 were
induced when non-Tregs were cultured with LPS alone (Figure
S2). SIVmac VLPs have been reported to not affect DC function
and maturation . We nevertheless confirmed that infection of
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DCs with SIVmac VLP alone did not compromise their ability to
induce FOXP3 (Figure S3).
HIV-infected Primary mDCs are Defective in their
Capacity to Induce FOXP3
We next wanted to confirm data obtained with infected
moDCs with primary infected DCs. Purified circulating myeloid
DCs (mDCs) from healthy donors were infected, or not, with
HIV in vitro, and then cultured with autologous nonTregs.
Uninfected mDCs were more efficient at inducing a Treg
phenotypein nonTregs compared
0.39%60.09% in uninfected vs. infected co-cultures, respective-
ly; p=0.018, n=5, Figure 1E), although they were less efficient
than moDCs. However, Treg induction by uninfected, purified
mDCs was comparable to that observed using tissue-derived
Figure 1. Infection of DCs by HIV blocks their induction of CD25 and FOXP3 in autologous nonTregs. (A) moDCs differentiated from
purified CD14+monocytes were stimulated with or without LPS (500 ng/ml). After 24 h, immature or LPS-mature moDCs were co-cultured with
autologous purified nonTregs (CD4+CD25lowCD127hi) for 5 days, at a DC: nonTregs ratio of 1:10. As a control, nonTregs were cultured alone. A
representative flow cytometry data comparing Treg induction by mature or immature moDCs is shown. B) moDCs differentiated as described in A)
were stimulated with LPS (500 ng/ml) for 24 h. They were then infected with HIV YK-JRCSF, in presence or not of SIVmac VLPs (both at a MOI=3). HIV
proviral DNA levels were measured by nested real-time HIV-LTR-Alu PCR 24 h postinfection; HIV DNA levels were normalized based on CD3
quantification (n=5). C) LPS-activated moDC were infected with HIV YK-JRCSF and SIVmac VLP (INF) or left uninfected (UNI), and co-cultured with
autologous purified nonTregs (CD4+CD25lowCD127hi) for 5 days, as described above. Mean 6 SE % of CD25+FOXP3+CD4+T cells (n=18) is shown and
one representative flow cytometry experiment showing CFSE levels in induced FOXP3+cells is shown in D). E) Elutriated fraction of PBMC was stained
with a cocktail of mAbs and mDCs were isolated based on CD142HLA-DR+CD1232CD11c+expression. After overnight stimulation with LPS, these
cells were infected with HIV and cultured with autologous purified CD4+CD25lowCD127hinonTregs for 5 days, as described above. Mean 6 SE % of
CD25+FOXP3+CD4+T cells (n=5) is shown.
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mDCs in macaques . As a similar pattern of Treg induction
was found for mDCs and moDCs, the majority of experiments
were performed with moDCs, due to the paucity of primary
Exposure to HIV is Sufficient to Impair moDCs to Induce
Even low levels of HIV, as low as 0.01 MOI, abolished the
ability of moDCs to induce Tregs (Figure 2A). To determine
what stage of the HIV cycle affects DC function, we treated
DCs with the reverse transcriptase inhibitor AZT before culture
with nonTregs. This treatment did not restore Treg induction,
suggesting that exposure to virus alone was sufficient to block
DC-mediated Treg conversion (Figure 2B). To confirm these
data, we inactivated HIV by treatment with AT-2, a mild
oxidizing reagent that eliminates the infectivity of HIV while
maintaining its structure and ability to be processed for
presentation to T cells . AT-2 YK-JRCSF-exposed moDCs
were unable to induce Tregs, similar to moDCs infected with
the replication-competent YK-JRCSF (Figure 2C). The addition
of SIVmac VLP to AT-2 YK-JRCSF did not change these
results (data not shown).
Figure 2. Exposure to HIV is sufficient to impair DC-mediated conversion by moDCs. A) Different MOIs of infectious YK-JRCSF were used
to infect LPS-stimulated moDCs. Percentage of CD25+FOXP3+T cells was analyzed after 5 days of co-culture. One representative example of 5
independent experiments is shown. B) In some experiments (n=4), infected DC were treated or not with the reverse transcription inhibitor AZT
(1 mM). Flow cytometry data from one representative experiment are shown. C) In some experiments (n=4), LPS-activated moDCs were exposed to
infectious or AT-2-treated YK-JRCSF+SIV VLPs. As a negative control, AT-2-treated microvesicles were used. Flow cytometry data from one
representative experiment are shown.
HIV-Infected DCs Kill Induced Tregs
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moDCs Convert both Naı ¨ve and Memory nonTregs into
Many studies of in vitro Treg conversion have used naı ¨ve T
cells, as murine and human memory T cells could not be
converted to Tregs by in vitro stimulation with TGF-b.
Furthermore, the production of cytokines by memory T cells
can block the conversion of naı ¨ve CD4+T cells into Tregs .
However, other studies suggest that memory human and murine
nonTregs could also be converted into Tregs [17,31–33], and
DC-mediated conversion of human cells does not appear to be
similarly blocked by inhibitory cytokines . We nevertheless
tested whether cytokines produced by memory T cells blocked
Treg conversion by adding anti-IFN-cR1, anti-IL4R or anti-
IL17 Abs to our cultures. FOXP3 induction was not increased
by these antibodies (data not shown). Moreover, the efficiency of
FOXP3 induction was not correlated with either the age of the
donors (r=0.08, p=0.23) or the proportion of memory
CD45RO+T cells in purified nonTregs (r=0.05, p=0.39).
To confirm these data, we purified naı ¨ve and memory nonTregs
by cell sorting, and cultivated them with autologous uninfected
or infected moDCs. DC-mediated conversion could occur in
both nonTreg populations, although conversion was more
efficient for naı ¨ve nonTregs than for memory nonTregs
(Figure 3). Notably, the pattern of Treg induction in unfrac-
tionated nonTregs (containing both naı ¨ve and memory) was
similar to that observed in purified naı ¨ve or memory nonTregs.
Converted Tregs Suppress Proliferation of Autologous
nonTregs, Similar to Circulating Tregs
FOXP3 expression can be transiently upregulated upon T
cell activation, but does not always confer regulatory properties
. It was therefore important to test the functionality of the
CD25+FOXP3+Tregs generated by culture with moDCs. The
intracellular localization of the transcription factor FOXP3
prevents sorting of live converted CD25+FOXP3+T cells. For
this reason, day 5 co-cultures containing both DCs and the
converted FOXP3+cells, were added in bulk to autologous
stimulated nonTreg responder cells at a 10:1 ratio (experimental
scheme is detailed in Figure 4A). This ratio was chosen because
nonTregs cultured with uninfected moDCs contained about
10% FOXP3+cells (Figure 1C), giving an approximate ratio of
1 Treg : 1 responder cell. Importantly, cultures containing
de novo induced FOXP3+cells suppressed the proliferation of
cultivated with infected DCs, which contained less than 1% of
FOXP3+cells, did not suppress the proliferation of responder
nonTregs (Figure 4B). These data strongly suggest that the
FOXP3+cells induced in vitro are suppressive. Furthermore, the
induced Treg were as suppressive as circulating Tregs (Figure 4B
Almost 50% of converted CD25+FOXP3+Tregs expressed
GARP and Cytotoxic T lymphocyte antigen-4 (CTLA-4), markers
associated with Treg function [35,36], whereas CD252FOXP32
cells present in the same cultures express these markers at low
levels (,6%, p=0.006 and ,2%, p,0.001, paired t-tests,
CD25+FOXP3+cells proliferated in vitro, as assessed by their low
levels of CFSE (74.31% 66.60% were CFSElow).
4B). In contrast,nonTregs
Absence of Treg Conversion by Infected DCs is not Due
to a Soluble Factor
To investigate the mechanisms responsible for the lack of Treg
induction in infected co-cultures, we examined whether this
inhibition involved soluble factors. We first focused on IL-10 and
TGF-b because these cytokines play a critical role in Treg
Figure 3. Naı ¨ve and memory nonTregs are converted into Tregs by uninfected moDCs. Uninfected or infected moDCs were cultured with
purified naı ¨ve (CD4+CD25lowCD127hiCD45RApos, upper panel) and memory (CD4+CD25lowCD127hiCD45RAneg, lower panel) nonTregs. The numbers in
the quadrants represent the percentages of CD25+FOXP3+induced Tregs. One representative example of 2 independent experiments is shown.
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Figure 4. Induced Tregs suppress proliferation of responder T cells similarly to natural Tregs. Ability of induced CD4+FOXP3+CD25+to
suppress the proliferation of autologous responder nonTregs was assessed. A) Scheme showing the experiment design. NonTregs were cultured for 5
days with autologous mature moDCs, infected (INF) or not (UNI). After 5 days, these T cells (‘‘bulk’’) were mixed with CFSE-labelled autologous
nonTregs (resp T), and stimulated by immobilized anti-CD3 Ab (1 mg/ml) and soluble anti-CD28 Ab (0.5 mg/ml) for 4 days. As the frequency of FOXP3+
cells within co-cultures containing uninfected DCs is close to 10%, we used a ‘‘bulk’’: nonTregs ratio of 1:10. At day 4, proliferation of responder
nonTregs was determined by flow cytometric analysis of CFSE levels. Responder nonTregs were also cultured alone or were stimulated alone. As a
control, circulating purified Tregs were added to responder nonTregs at a 2:1 ratio. B) One representative example of 3 independent experiments is
shown. Numbers in each panel represent the percentage of responder cells undergoing proliferation (CFSE low). The small inserts show the
proportion of induced Tregs in the same experiment. C) Graph shows the mean 6 SE % of suppression mediated by converted Tregs or circulating
Tregs compared to proliferation of stimulated nonTregs alone (n=3, paired t-test).
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induction. Treatment with anti-IL-10 completely blocked Treg
conversion in uninfected cultures, whereas addition of rIL-10
increased Treg conversion in these cultures. However, addition of
rIL-10 did not restore Treg conversion in infected co-cultures
(Figure 5A), ruling out that low IL-10 production could be an
underlying mechanism. rIL-10 enhanced the expression of CD25
in FOXP32cells in infected co-cultures (Figure 5A). These
CD25+FOXP32cells did not divide (data not shown), in line with
previous reports showing that exogenous IL-10 induces a
population of anergized T cells [37,38], which fail to suppress
normal CD4+T cell proliferation in vitro .
Similarly to IL-10, levels of TGF-b did not appear to be
limiting, as addition of rTGF-b did not affect the proportion of
converted Tregs in either uninfected or infected co-cultures
(Figure 5B). Moreover, HIV infection did not change TGF-b
mRNA levels in DCs (mean 6 SE: 1.0160.18 arbitrary units vs.
1.5460.32 in uninfected vs. infected moDCs, respectively;
To get a broader picture of the involvement of soluble
factors, we next tested whether an inhibitory soluble factor was
present in DC supernatants from infected cultures. Ruling that
mechanism out, our data show that supernatants from infected
DCs did not compromise the induction of Tregs by uninfected
Figure 5. Soluble factors are not responsible for the lack of Treg induction by infected DCs. A) Flow cytometric analysis of the percentage
of induced CD25+FOXP3+T cells by DC, in the presence of anti-IL-10 Ab (10 mg/ml) or rIL-10 (10 ng/ml). One representative example of 3
independent experiments is shown. B) Flow cytometry analysis of the percentage of induced CD25+FOXP3+T cells by DCs, in the presence of rTGF-
b(10 ng/ml). One representative example of 4 independent experiments is shown. C) Flow cytometry analysis of the percentage of induced Tregs
after culture with uninfected (UNI) DCs for 5 days, in presence of DC supernatants collected from cultures containing either uninfected or infected
DCs (6 hours post infection). D) In the same experiment, nonTregs were either cultured at 10:1 ratio with uninfected or infected moDCs or they were
mixed at the 10:0.5:0.5 ratio (nonTregs : infected moDCs : uninfected moDCs). One representative example of 3 independent experiments is shown.
HIV-Infected DCs Kill Induced Tregs
PLOS ONE | www.plosone.org8 August 2012 | Volume 7 | Issue 8 | e42802
moDCs (Figure 5C); conversely, supernatants from uninfected
DCs did not restore Treg conversion in infected co-cultures
(data not shown). Furthermore, in agreement with previous
studies , separation of DCs and nonTregs by a membrane
completely abolished Treg conversion in uninfected co-cultures
(data not shown), confirming the necessity of physical contact.
Notably, the percentage of FOXP3+cells decreased dramatically
when uninfected moDCs were mixed with in vitro infected
moDCs at a 1:1 ratio (Figure 5D).
In vitro HIV Infection Alters moDC Phenotype
Impaired Treg conversion could be due to the altered
expression of maturation and costimulatory molecules by
infected moDCs  or primary myeloid DCs [41–43]. In
agreement with these previous data, in vitro HIV-infected and
exposed moDCs expressed lower levels of costimulatory and
maturation molecules than uninfected mature moDCs (Figure 6).
The expression of these molecules was consistently higher on in
vitro HIV-infected and exposed moDCs than on unstimulated
uninfected DCs, while their capacity to induce FOXP3+cells
was consistently lower than that of unstimulated uninfected
DCs, suggesting that the levels of costimulatory molecules did
not affect the capacity to induce Tregs. The inhibitory
costimulatory molecule programmed death ligand-1 (PDL-1)
has been shown in some models to induce Tregs . As
infected DCs expressed lower levels of PD-L1 than uninfected
DCs, we directly tested this mechanism, but the addition of
rPDL-1 did not restore Treg conversion in infected cultures
(mean 6 SE % of converted CD25+FOXP3+
0.25%60.09% in infected co-cultures and 0.07%60.01% after
addition of rPDL-1; p=0.29, n=3).
Infected moDCs and Primary mDCs Induce Death of
HIV infection did not increase DC death after 3 days of co-
cultures (p=0.49, Figure 7A), although, by that time, Treg
induction by infected DCs was already severely decreased
compared to that induced by uninfected DC (Figures 7A). In
contrast, in our experimental system using moDCs, T cell death
was significantly higher in infected co-cultures than in uninfected
ones (p,0.001, Figure 7B). The percentage of induced Tregs in
uninfected co-cultures was inversely correlated with the percentage
of T cell death (Figure 7C), suggesting that death of induced Treg
could be a mechanism for their low percentage. Confirming this
hypothesis, treatment of the DC:non-Treg cultures with pan-
caspase inhibitor Z-VAD increased Treg conversion (Figure 7D) as
well as T cell survival (Figure 7E) in both infected and uninfected
co-cultures, thus directly linking Treg death to the decreased
conversion observed in HIV-exposed DC cultures. In contrast,
addition of exogenous IL-2 in infected co-cultures neither rescued
cell viability nor restored Treg conversion (not shown). Of note,
these results were confirmed using primary DCs. Z-VAD
treatment significantly increased Treg conversion (Figure 7D)
and decreased T cell death (Figure 7E), although the effect was
partial as the percentage of Treg conversion in Z-VAD treated
cultures remained significantly lower than that in uninfected
DC-T cell contact is crucial for the regulation of immune
responses and any perturbation in DC function would result in
significant changes in the tolerance/immunity balance. Due to
their location at mucosal surfaces and their expression of HIV
Figure 6. HIV impairs the maturation and activation of moDCs. Expression of maturation, activation and HLA-DR molecules was analyzed by
flow cytometry in uninfected immature (dotted line), in vitro infected over night mature (solid black line) and uninfected mature moDCs (dashed line).
Filled histograms represent the level of expression of each marker by unstained mature moDCs. One representative example of 19 experiments is
HIV-Infected DCs Kill Induced Tregs
PLOS ONE | www.plosone.org9 August 2012 | Volume 7 | Issue 8 | e42802
Figure 7. DC-mediated Treg conversion is partially restored in infected co-cultures by inhibition of apoptosis. (A) Percentage of dead
DCs was analyzed by flow cytometry using an amine reactive dye (LIVE/DEAD kit, Invitrogen). Graph shows the mean % 6 SE of dead CFSE2CD32
cells in co-cultures after 3 days and representative dot plots of converted Tregs after 3 days of both uninfected and infected co-cultures are shown (3
independent experiments, paired t-test). B) Percentage of dead T cells was analyzed by flow cytometry using an amine reactive dye (LIVE/DEAD kit,
Invitrogen). Graph shows the mean % 6 SE of dead CD3+CD4+T cells in moDCs-nonTregs co-cultures (19 experiments, paired t-test). C) Percentage of
induced Tregs and percentage of dead T cells were correlated in each experiment, using linear regression analysis. D) Mature moDCs or purified
mDCs were co-cultured with autologous purified nonTregs in presence or not of the pan-caspase inhibitor Z-VAD (50 mM). The inhibitor was replaced
twice a day for 5 days. The graph represents the mean % 6 SE of converted FOXP3+cells in uninfected untreated co-cultures or infected Z-VAD-
treated co-cultures for both moDCs (n=4, paired t-test) and mDCs (n=3, paired t test). E) Mean % 6 SE of dead CD3+CD4+T cells in infected
untreated or Z-VAD treated co-cultures for both moDCs (n=4, paired t-test) and mDCs (n=3, paired t test) is shown.
HIV-Infected DCs Kill Induced Tregs
PLOS ONE | www.plosone.org10 August 2012 | Volume 7 | Issue 8 | e42802
receptors, DCs are important targets of HIV infection. HIV-
infected myeloid DCs exhibit defective maturation even upon
strong microbial stimulation [45,46], a result confirmed by our
study, although opposite results were obtained if HIV-1 Bal
infected DCs were stimulated by CD40L . How these defects
affect the ability of circulating DCs to induce Tregs is poorly
understood, and clarifying this issue was the goal of our study.
We show that uninfected LPS-matured moDCs induce a
higher proportion of Tregs than immature DCs. LPS is a
classical DC stimulus, which we used as a model to examine
whether mature DCs could induce Tregs in an autologous
system. Of note, similar defects were obtained with DCs
activated with gram-positive
phenomenon.The finding that
induce Tregs more efficiently than immature moDCs is in
agreement with previous reports [19,40,48], although it appears
paradoxical because immature DCs classically induce tolerance
through the production of regulatory cytokines and induction of
Tregs both in vivo and in vitro [49,50]. However, an important
characteristic of our model is that we used an autologous system
without T cell-specific stimulation. In this system, mature DCs
could trigger Treg induction as a mechanism of protection
against excessive immune responses. Of note, our experiments
demonstrated the crucial role of DC-T cell contact in Treg
conversion as thisprocess
separating these two cell populations, in line with previous
studies [40,51], suggesting the involvement of TCR-mediated
signals in this pathway. It has been shown that costimulatory
molecules on mature DCs are required to maintain self
tolerance . Immature DCs may be better at inducing Tr1
cells, IL-10 producing cells that do not express FOXP3 .
Importantly, the FOXP3+T cells generated by uninfected
moDCs not only expressed markers associated with natural Tregs
such as CD25 or CTLA-4 [36,53] but appear to be functional in
suppressing the proliferation of conventional T cells, a result in
agreement with previous reports [19,40]. As FOXP3 expression
cannot be used for live cell sorting, we did not formally prove that
these CD25+FOXP3+cells were suppressive. It is entirely possible
that a suppressive factor was present in the bulk cultures; however,
this putative factor is closely associated with the presence of
CD25+FOXP3+cells, as bulk cultures with ,1% FOXP3+cells
did not suppress the proliferation of responder cells.
In the current study, we found that peripheral blood mature
moDCs as well as mDCs induced a lower proportion of Tregs than
uninfected DCs. Importantly, similar data were obtained with
primary mDCs and moDC, confirming that moDCs constitute a
good model to study this particular aspect of myeloid DC biology.
However, these new results contrast with our recent data, which
showed that tissue isolated mDCs from SIV-chronic infected
macaques induced higher levels of Tregs compared to uninfected
macaques . A potential explanation for the differences
between our two studies could be that mDC-mediated conversion
is more or less efficient at different stages of infection. Our in vitro
system mimics the early phase of the infection, when DCs are
exposed to high doses of virus. In line with these observations,
extensive Treg death occurs in the highly pathogenic model of
acute SIV-infection of pigtailed macaques , whereas increased
proportions of Tregs are found during chronic HIV/SIV infection
[10,11,13,22], although the mechanisms underlying these differ-
ences have not been studied in these reports. Thus, it is possible
that Treg conversion can occur when viral loads are relatively low,
such as in the chronic phase, but is severely impaired during the
acute phase . Further studies conducted with in vivo
differentiated DCs, purified from different tissues of SIV-infected
bacteria, suggestinga broad
macaques at different stages of infection, will be needed to clarify
this important question.
As several pathways are known to be involved in DC-
mediated conversion, we studied which pathway(s) could be
defective in HIV-infected moDCs. Our results show that the
low Treg induction by both HIV-exposed moDCs and primary
mDCs, is mainly due to the selective death of induced Tregs in
contact with these DCs. Supporting this hypothesis, Treg
induction was inversely correlated with T cell death. Moreover,
a significant improvement in Treg induction was achieved by
blocking caspases. Killing of T cells by HIV-infected DCs has
already been reported, but pDCs have been more extensively
studied than mDCs . In particular, HIV infection of pDCs
activates TRAIL, and these DCs induce apoptosis of CD4+T
cells thatexpress thedeath
Interestingly, Lichtner et al. showed that HIV-pulsed moDCs
induce apoptosis in approximately 40% of autologous uninfected
T cells, via multiple caspase-dependent mechanisms, including
FasL, TRAIL, TNF-a and TWEAK . Similarly, Hardy et
al. showed that HIV induced expression of TRAIL on pDCs
and turned them into killer pDCs . The fact that we could
not completely rescue Treg induction by the pan-caspase
inhibitor could have several explanations. One could be that
the dose we used was not sufficient to achieve full blockade.
Alternatively, other mechanisms, such as decreased expression of
costimulatory molecules or PDL-1 by infected DCs, could play
a minor, but synergistic role with their increased killing of
Tregs. One intriguing result in our model is that Tregs appear
more susceptible to apoptotic death than nonTregs. Baatar et
al. showed that Tregs express high levels of FasL , which
could constitute a potential mechanism.
Several important factors must also be noted. In the experiment
where infected DCs were mixed with uninfected DCs, Treg
conversion was not restored. A likely explanation is that there was
a sufficient number of infected DCs able to kill converted Tregs.
Alternatively, the exposure of uninfected DCs to the infected ones
may have altered their physiology and render them unable to
Of note, selective Treg death was not due to a deficit in
cytokines important for Treg survival, as addition of exogenous
TGF-b or IL-2 to HIV-infected cultures did not decrease Treg
death nor did they restore Treg conversion. Second, Treg death
was not due to a virus transfer from DCs to T cells, and their
subsequent death due to a direct cytopathic effect of infection.
Indeed, similar death and low Treg induction were found when
inactivated virus, unable to infect DCs or T cells, were used or
when AZT was added to the cultures. Last, our data do not
support the hypothesis that increased, and early death of HIV-
exposed DC, which would also be controlled by addition of a pan-
caspase inhibitor, plays a major role in their defective induction of
Treg. Indeed, DC death was similar in uninfected and infected co-
cultures at an early stage of co-culture (3 days), when defective
induction of Treg was already notable (Figure 7). Taken together,
our results suggest that Treg conversion is an early and dynamic
event, triggered by contact with mature DC, followed by
expansion in vitro of these induced Tregs an exposure of DC to
HIV profoundly affects this pathway early on.
In summary, our data suggest that death of induced Tregs may
play a role during the acute phase of pathogenic infection. As
Tregs control HIV replication in several cellular targets such as T
cells and macrophages [59,60], killing of Tregs by DCs exposed to
high titers of virus could hamper their capacity to restrain HIV
replication in the early phase of infection, and thus could be an
important factor in HIV pathogenesis.
receptor TRAILR2 .
HIV-Infected DCs Kill Induced Tregs
PLOS ONE | www.plosone.org11 August 2012 | Volume 7 | Issue 8 | e42802
Tregs. Circulating Tregs (CD4+CD25hiCD127low) or nonTregs
(CD4+CD25lowCD127hi) were purified by cell sorting, CFSE-
labelled, and cultured with autologous LPS-activated uninfected
(UNI mature) moDCs for 5 days. One representative flow
cytometric analysis (out of 3 independent experiments) of
expression of CD25, FOXP3 and CFSE is shown. Numbers
represent the percentage of positive cells.
Uninfected moDCs do not expand circulating
FOXP3 expression. Bead-purified nonTregs from PBMCs were
left unstimulated (grey lines), or cultured with LPS overnight
(dotted line) or for 5 days (black line). FOXP3 expression is shown
for one representative example for each group (n=3/group).
LPS stimulation of nonTregs does not induce
mediated Treg conversion. In 4 independent experiments,
LPS-stimulated moDCs were infected with VLP SIVmac alone
(MOI=3), or left uninfected, before co-culture with autologous
nonTregs. Percentage of CD25+FOXP3+T cells was analyzed 5
days later. One representative experiment is shown.
VLP SIVmac alone does not block DC-
CTLA-4. Mean 6 SE % of GARP+or CTLA-4+cells in
converted CD25+FOXP3+Tregs and CD252FOXP32T cells is
shown in A (n=5) and B (n=6), respectively. P values correspond
to paired t-tests.
Converted Tregs express GARP as well as
We thank the NIH AIDS Research and Reference Reagent Program for
the IL-2, cell lines and HIV lab strains and Dr. Larry Wahl to kindly
provide elutriated CD14+monocytes and CD142cells counterpart. We
thank Dr. Cimarelli for kindly providing SIV-macVLP plasmid, Eva
Moore for recruiting patients, Drs. Barbara Shacklett, Julia Shaw, Celine
Silva-Lages and Mr/Mrs Casey Wells and Kris Orsborn for expert
assistance and review of this manuscript.
Conceived and designed the experiments: PP CF CAC. Performed the
experiments: PP MEMF LKR. Analyzed the data: PP MEMF LKR. Wrote
the paper: PP CF CAC.
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HIV-Infected DCs Kill Induced Tregs
PLOS ONE | www.plosone.org 13August 2012 | Volume 7 | Issue 8 | e42802