GARP (LRRC32) is essential for the surface expression
of latent TGF-? on platelets and activated FOXP3?
regulatory T cells
Dat Q. Trana,1, John Anderssona, Rui Wangb, Heather Ramseya, Derya Unutmazb, and Ethan M. Shevacha,1
aLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; andbDepartment of
Microbiology, New York University School of Medicine, New York, NY 10016
Edited by Dan R. Littman, New York University Medical Center, New York, NY, and approved June 2, 2009 (received for review February 21, 2009)
TGF-? family members are highly pleiotropic cytokines with di-
verse regulatory functions. TGF-? is normally found in the latent
form associated with latency-associated peptide (LAP). This latent
complex can associate with latent TGF?-binding protein (LTBP) to
produce a large latent form. Latent TGF-? is also found on the
surface of activated FOXP3?regulatory T cells (Tregs), but it is
unclear how it is anchored to the cell membrane. We show that
GARP or LRRC32, a leucine-rich repeat molecule of unknown
function, is critical for tethering TGF-? to the cell surface. We
demonstrate that platelets and activated Tregs co-express latent
TGF-? and GARP on their membranes. The knockdown of GARP
mRNA with siRNA prevented surface latent TGF-? expression on
activated Tregs and recombinant latent TGF-?1 is able to bind
directly with GARP. Confocal microscopy and immunoprecipitation
strongly support their interactions. The role of TGF-? on Tregs
appears to have dual functions, both for Treg-mediated suppres-
sion and infectious tolerance mechanism.
transforming growth factor beta ? Tregs ? latency-associated peptide
differentiation, tissue morphogenesis and modulation of cell
growth, inflammation, matrix synthesis, and apoptosis. Dysregu-
lations in TGF-? function are associated with multiple patho-
logical conditions including tumor cell growth, fibrosis, emphy-
sema, and autoimmunity (1). All 3 TGF-? isoforms are
synthesized as homodimeric proproteins. The proproteins are
cleaved in the Golgi apparatus by a furin-like convertase to
produce the dimeric propeptides called latency-associated pep-
tide (LAP) that noncovalently associates with the dimeric ma-
ture TGF-? to prevent its activity (2). There are multiple
mechanisms of activating TGF-? from its latency by pathways
that include protease plasmin, matrix metalloproteases, throm-
bospondin-1 (TSP1), and certain ?V integrins (3). TGF-? can be
secreted in a small latent form associated with LAP, or this
complex can further associate with latent-TGF-?-binding pro-
tein (LTBP) to produce a large latent form for deposition onto
the extracellular matrix. In addition, small latent TGF-? can be
expressed on the membrane of many cell types, including
megakaryocytes, platelets (4), immature dendritic cells (DCs)
(5), and activated FOXP3?regulatory T cells (Tregs) (6–8), and
However, it is unknown how this membrane small latent TGF-?
is anchored to the cell surface.
It has been recently shown that megakaryocytes and activated
Tregs expressed high levels of mRNA for a member of the
leucine-rich repeat family of proteins that has been termed
GARP or LRRC32 and that platelets express this molecule on
their membrane (9, 10). The GARP or LRRC32 gene consists of
662 aa and encodes an 80-kDa transmembrane protein with an
extracellular region composed primarily of 20 leucine-rich re-
peats (11, 12). As the Garp gene is expressed in multiple cell
types in the mouse during embryogenesis, it has been proposed
GF-? family members (?1, ?2, ?3 isoforms) are highly
pleiotropic cytokines that have critical functions in cell
that Garp plays an important role in development, but its actual
function is unknown (13). Since platelets and activated Tregs
contain both GARP and latent TGF-? on their membranes, we
hypothesized that GARP might bind and anchor latent TGF-?.
Here, we show that GARP is critical for the surface expression
of latent TGF-? by binding to the complex and functioning as its
cell surface receptor.
GARP or LRRC32 Is Selectively Expressed on Activated FOXP3?Tregs.
Consistent with a previous publication (10), we found that
GARP mRNA is selectively expressed in fresh human Tregs and
rapidly up-regulated after activation of CD4?CD25hiTregs with
anti-CD3/CD28 and IL-2 (Fig. 1A). Only very low levels of
mRNA were detected in CD4?CD25–T cells after activation for
5 days. While the addition of TGF-?1 resulted in the induction
of FOXP3 mRNA in CD4?CD25–T cells (14), the level of
GARP mRNA was not dramatically increased. Cell surface
expression of either LAP or GARP was not significantly de-
tected on freshly isolated Tregs (CD25hi), but the expression of
both LAP and GARP was rapidly up-regulated after activation
(Fig. 1B). GARP and LAP occasionally could be detected on
?5% of activated CD4?CD127?CD25–T cells (Fig. S1). How-
ever, when we activated the CD4?CD25intpopulation, which
contains mostly CD45RO?FOXP3–T cells and some FOXP3?
Tregs, the vast majority of LAP and GARP could be detected on
FOXP3?Tregs. The rapid appearance and disappearance of
LAP?/GARP?FOXP3–T cells during the 36 h of activation of
CD25hiand CD25intmost likely represent Tregs that have
down-regulated their FOXP3 and could be on their way to cell
death. Platelets also co-expressed LAP and GARP on their
membranes (Fig. 1C). In contrast to previous reports (5), we
were unable to detect any significant surface expression of LAP
or GARP on plasmacytoid or myeloid DCs from peripheral
blood (Fig. 1D). Interestingly, the cell surface expression of both
LAP and GARP requires the Golgi apparatus, since the addition
of monensin or brefeldin A during the activation culture pre-
vented their surface expression (Fig. 1E).
GARP Associates with Latent TGF-? Complex and Is Critical for Its
Surface Expression. Since the kinetics of induction and pattern of
expression of LAP and GARP on activated Tregs appeared to
be similar, we used siRNA technology to knockdown TGF-?1 or
GARP mRNA to assess if the expression of these molecules is
Author contributions: D.Q.T. designed research; D.Q.T., J.A., and H.R. performed research;
R.W. and D.U. contributed new reagents/analytic tools; D.Q.T. and E.M.S. analyzed data;
and D.Q.T. and E.M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
whom correspondencemaybeaddressed. E-mail:email@example.com
This article contains supporting information online at www.pnas.org/cgi/content/full/
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related. Freshly isolated Tregs transfected with TGF-?1 siRNA
and activated for 48 h expressed GARP, but not LAP on their
surface, while Tregs transfected with GARP siRNA failed to
express either molecule. Expression of GARP or LAP was not
affected when Tregs were transfected with a control nonspecific
siRNA (Fig. 2A). This result suggested that GARP was required
for the expression of latent TGF-?1 on the cell surface. To test
whether latent TGF-?1 can associate with GARP, we incubated
the 2 activated Treg populations (TGF-?1 siRNA or GARP
siRNA) with recombinant human (rh) TGF-?1, LAP, LAP plus
TGF-?1, or latent TGF-?1. LAP was only detected on the
TGF-?1 siRNA transfected GARP?LAP–Tregs when they were
incubated with either LAP mixed with TGF-?1 or latent TGF-
?1, but not mature TGF-?1 or LAP alone, indicating that the
TGF-?1/LAP complex was needed for interaction with GARP
(Fig. 2B). We next tested whether the interaction of TGF-?2 or
TGF-?3 with LAP of TGF-?1 results in binding to GARP.
Interestingly, the mixture of TGF-?2 and LAP resulted in
binding to GARP, while the mixture of TGF-?3 and LAP failed
to bind to GARP (Fig. 2C). Although different isoforms of
TGF-? naturally associate with their own distinct LAPs, it has
been reported that LAP of TGF-?1 can bind and inactivate
TGF-?2 and TGF-?3 with apparent Kdvalues of 1.9 and 0.4 nM,
respectively (15). However, our result suggests that either
TGF-?3 does not interact with LAP of TGF-?1 or their inter-
actions produce a conformation that does not bind to GARP.
Moreover, while TGF-?2 could interact with LAP to bind to
GARP, it appears to be less efficient than with TGF-?1 based on
the lower mean fluorescence intensity of LAP detection. The
interaction of various molecules including ?V integrins and
TSP1 with the RGD sequence in LAP has been implicated in
releasing and activating TGF-? (4, 16, 17). Using Jurkat cells
expressing surface GARP, we tested whether TSP1, RGD, or
RGDS peptides can block the binding of latent TGF-?1 to
GARP. Preincubation of rhTSP1 or RGD/RGDS peptides did
not block the binding of latent TGF-?1 to GARP (Fig. 2D). This
result suggested that it is unlikely that the binding of GARP to
latent TGF-?1 occurs via the RGD site on LAP or that latent
TGF-?1 binds via the RGD site to another cell surface molecule
that then interacts with GARP. To further support the associ-
ation of GARP with LAP, we performed immunoprecipitation
and colocalization confocal imaging studies. When LAP was
immunoprecipitated from the surface of 48 h activated Tregs,
GARP could be detected by immunoblot (Fig. 2E). Likewise,
confocal imaging of surface-stained LAP and GARP on 48 h
activated Tregs strongly demonstrated their colocalization (Fig.
3). Finally, to determine whether GARP and latent TGF-?1 can
directly bind to each other, we performed a flow cytometric
protein-protein interaction assay. Dynabeads were conjugated to
either anti-LAP or anti-GARP mAbs and then incubated with a
mixture of latent TGF-?1 and GARP-Fc fusion proteins, LAP
and GARP-Fc, or TGF-?1 and GARP-Fc followed by flow
cytometric detection of the anti-LAP Dynabead complex with
fluorochrome conjugated anti-hFc or anti-GARP Dynabead
complex with anti-LAP Abs. Beads conjugated with GARP
bound latent TGF-?1, but not LAP and beads conjugated with
latent TGF-?1, but not LAP, bound GARP (Fig. 2F). This result
indicates that GARP can directly bind to latent TGF-?1.
Regulation of GARP Is Independent of FOXP3.SinceGARPandLAP
expression was observed in FOXP3?Tregs, we next evaluated
whether the de novo induction of FOXP3 in CD4?FOXP3–T
cells was sufficient to induce GARP and LAP expression on the
cell surface. Naïve CD45RA?and memory CD45RO?T cells
activated with anti-CD3/CD28 in the presence of TGF-?1 can be
induced to express FOXP3 (Fig. 4A) although such cells lack
regulatory function (18). In contrast to Tregs, activation of naïve
or memory T cells in the presence or absence of TGF-?1 failed
to express significant levels of surface LAP or GARP on primary
stimulation (Fig. S1A) or following multiple rounds of restimu-
CD4?CD25–T cells and Tregs (CD25hi) at 12, 24, 120 h, or 120 h with TGF-?1. Tregs were rested until day 14 (0 h) and restimulated for 18 h. (B) Flow cytometric
analysis of surface LAP, GARP and intracellular FOXP3 on fresh (0 h) and activated Tregs (CD25hi) and CD4?CD25intT cells. (C) Surface staining of LAP and GARP
on platelets based on FSC/SSC and CD61 expression. (D) LAP and GARP surface staining of plasmacytoid and myeloid DCs from PBMCs by gating on CD303?Lin-1–
and CD1c?, respectively. (E) Surface LAP and GARP expression on Tregs after 12 h activation in the absence (none) or presence of monensin or brefeldin A for
the last 8 h. Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant for B–E.
GARP and LAP are selectively expressed on activated FOXP3?Tregs and platelets. (A) Level of FOXP3 and GARP mRNA on fresh (0 h) and activated
www.pnas.org?cgi?doi?10.1073?pnas.0901944106Tran et al.
lation without TGF-?1 (Fig. 4A and Fig. S1B). It has been shown
that high level and prolonged expression of FOXP3 following
lentiviral-mediated transfection can result in the complete ac-
quisition of the Treg phenotype in CD4?FOXP3–T cells (19).
Although the TGF?-induced FOXP3?cells expressed similar
level of FOXP3 as the Tregs upon restimulation, they fail to
express surface latent TGF-? or GARP (Fig. S1B) and continue
to lack Treg phenotype. Therefore, it does not appear that
expression of FOXP3 in non-Tregs is sufficient to drive the
surface expression of GARP and LAP.
To evaluate whether FOXP3 was essential for the expression
of GARP and LAP on Tregs, we first knocked down the level of
FOXP3 on CD25hicells with siRNA for 5 days and determined
the induction of GARP and LAP after 48 h of restimulation.
Although FOXP3 expression was completely suppressed with
the siRNA compared to the nonspecific control, the expression
of GARP and LAP was not affected (Fig. 4B). This result
indicates that the rapid expression of GARP and LAP was not
controlled by an immediate downstream effect of FOXP3.
However, it remains possible that FOXP3 might have an indirect
effect on GARP expression after a more prolonged period of
suppression of FOXP3 expression. Since virtually all T cells
express TGF-?1 mRNA and are capable of secreting TGF-?1,
we evaluated whether forced expression of GARP in
CD4?FOXP3?T cells was sufficient to permit surface expres-
sion of latent TGF-?. Indeed, transduction of GARP into
CD4?FOXP3?T cells allows for the surface expression of LAP,
but not an increase in the percentage of FOXP3?cells (Fig. 4C).
Therefore it appears that GARP associates with latent TGF-?
intracellularly and transports it to the cell surface via the Golgi
apparatus. Furthermore, this result demonstrates that the failure
to detect latent TGF-? on activated non-Tregs was due to the
lack of GARP expression in these cells.
Dual Role of TGF-? on Tregs for Mediating Suppression and Infectious
Tolerance. As the kinetics for induction of cell surface expression
of GARP and LAP closely resembled the kinetics for activation
and rested in IL-2 culture medium for 24 h before stimulation for 48 h to assess surface expression of GARP and LAP. (B) Tregs transfected with TGF-?1 or GARP siRNA
were activated for 48 h then incubated for 30 min at 37 °C with 5 ?g/mL mature rhTGF-?1, LAP, LAP ? TGF-?1, or latent TGF-?1 and then surface stained for LAP. (C)
GARP transfected Jurkat cells were incubated for 30 min at 37 °C without (NONE) or with 5 ?g/mL LAP, TGF-?1, TGF-?2, or TGF-?3 alone or mixed with LAP and then
(LTGF?1) or preincubated for 20 min at 37 °C with a 10-fold excess of TSP1, RGD, or RGDS peptides before latent TGF-?1 incubation and then stained for surface LAP.
(E) Tregs were expanded in vitro for 14 days and then restimulated for 48 h before immunoprecipitation with anti-LAP (left lane) or isotype (right lane) followed by
immunoblot with anti-GARP. (F) Dynabeads conjugated to ?GARP (Left) or ?LAP (Right) were incubated with a mixture of GARP-Fc ? TGF-?1 (shaded histogram),
GARP-Fc ? LAP (dashed histogram), or GARP-Fc ? latent TGF-?1 (solid histogram) followed by flow cytometric analysis with PE-labeled anti-hLAP (Left) or anti-hFc
(Right). Data are representative of 3 independent experiments for A, B, C, D, and F and 2 for E. Numbers indicate percentage in each quadrant for A–D.
Surface expression of latent TGF-?1 requires GARP association. (A) Tregs were transfected with nonspecific (siNS), TGF-?1 (siTGF?1), or GARP (siGARP) siRNA
Tregs. Tregs activated for 48 h from 3 different donors (D1, D2, D3) were
surface-stained with anti-GARP and anti-LAP or isotype controls. The cells
were imaged with a Leica SP2-AOBS confocal microscope.
Colocalization of GARP and latent TGF-?1 on the surface of activated
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of Treg suppressor function (20), we next evaluated the role of
these molecules in Treg-mediated suppression. Tregs that lacked
the surface expression of LAP following treatment with TGF-?1
siRNA or the expression of GARP and LAP following treatment
with GARP siRNA were significantly less suppressive than Tregs
treated with the control siRNA in an in vitro suppression assay
(Fig. 5A and B). Although the role of cell surface or secreted
has remained controversial (6, 21), these results indicate that
TGF-? contributes moderately to Treg-mediated suppression in
vitro, but TGF-? does not appear to be the dominant mechanism
since suppression was not completely abrogated. We have shown
previously that activated mouse Tregs can induce Foxp3 expres-
sion in CD4?FOXP3?T cells during a 4-day co-culture (7).
Similarly, co-culture of activated human Tregs treated with
nonspecific, but not TGF-?1 or GARP siRNA, induced FOXP3
in CD4?FOXP3?T cells (Fig. 5C). We have not tested whether
these induced FOXP3?T cells have regulatory functions, as we
are unable to separate them from the large population of
FOXP3?T cells in the culture.
Our results clearly demonstrate that GARP or LRRC32 func-
tions as a carrier and cell surface receptor for latent TGF-?1.
Using siRNA technology, we have shown in vitro that
GARP?LAP?cells can bind latent TGF-?1, but we have not
been able to determine whether in vivo the binding of latent
TGF-?1 to GARP occurs exclusively intracellularly or whether
GARP can also bind secreted latent TGF-?1 or latent TGF-?
associated with LTBP. Although the GARP/LAP complex is
expressed by platelets, within the immune system GARP/LAP
expression is mostly observed on activated functional FOXP3?
Tregs (9). We did not observe significant surface expression of
GARP or LAP in CD19?/CD20?B cells, CD14?monocytes,
CD8?T cells, natural killer (NK) cells, NK T cells, and imma-
ture/mature monocyte-derived DCs. However, it is possible that
under certain conditions and with activation, these cells might
express GARP. Thus far, the predominant function of GARP/
LAP on human Tregs remains elusive. Our functional studies
FACS-sorted Tregs (CD25hi), naïve (CD45RA?), and memory (CD45RO?) T cells
were activated for 5 days in the absence or presence of TGF-?1 then rested for 7
days and restimulated for 48 h without TGF-?1 before analysis of FOXP3 with
surface GARP and LAP expressions. (B) Tregs were transfected with nonspecific
and analysis of FOXP3 with surface GARP and LAP expressions. (C) Naïve T cells
expressions among RFP?and RFP?cells (Right). Data are representative of 3
independent experiments. Numbers indicate percentage in each quadrant.
GARP is required for the surface expression of latent TGF-?1. (A)
and preactivated for 24 h before assessing their suppression of the proliferation of CD4?CD25–T cells stimulated with HLA-DR?APCs and soluble anti-CD3 for
3 days and pulsed with3H-TdR. The top panel is the FOXP3 expression, and the middle panel is the surface GARP and LAP expression of the 3 Treg populations
were activated with HLA-DR?APCs and soluble anti-CD3 for 3 days alone (top row) or in the presence of Tregs transfected with siNS, siGARP, or siTGF?1 at a
ratio of 2:1, 4:1, and 8:1 responder:Treg (bottom rows). (C) CFSE-labeled CD4?CD25?CD127?CD45RA?T cells were stimulated with anti-CD3/CD28 Dynabeads
Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant.
Dual functions of TGF-? in Treg-mediated suppression and infectious tolerance mechanism. (A) Tregs were transfected with siNS, siGARP, or siTGF?1
www.pnas.org?cgi?doi?10.1073?pnas.0901944106Tran et al.
indicate that the GARP/LAP complex does contribute to Treg-
mediated suppression in vitro but whether this result also holds
true in vivo is unknown and remains controversial. We have
recently proposed (7) that a major role of latent TGF-? on the
surface of murine Tregs is to convert responder T cells into
FOXP3?Tregs through a mechanism of infectious tolerance
when both populations are activated in concert via their TCRs.
also be involved in human Tregs. The regulation of expression of
GARP at the mRNA level (10) in the mouse and the regulation
of the expression of cell surface LAP are similar to that seen on
has not been evaluated due to the lack of anti-murine GARP
antibodies. As GARP and LAP are only expressed on TCR-
activated FOXP3?Tregs, 1 major function of this complex is to
target delivery of TGF-?1 to sites of ongoing immune responses
where Tregs can be activated by recognition of their cognate
antigens on APCs. Following release of active TGF-?1, it might
act locally on FOXP3–T cells to convert them to FOXP3?Tregs
(7), to generate Th17 effectors if an inflammatory milieu is
present (22), to act directly on the DCs to modulate their
functions, or to signal in an autocrine manner to maintain Treg
functions. A major question that remains to be addressed is the
mechanism by which active TGF-?1 is generated from the latent
GARP/LAP complex either by cell-associated molecules such as
the ?V integrins or by soluble factors.
Mutations in both GARP and LAP have been reported and
result in complex clinical conditions. A base substitution from
arginine to tryptophan in the coding region of the GARP gene
has been identified in a large Samaritan kindred with Usher
syndrome type 1, an autosomal recessive disease characterized
by profound congenital sensorineural deafness, vestibular dys-
function, and progressive visual loss (23). It is unclear whether
this mutation would result in a defective GARP protein that
would fail to associate with LAP or potentially lead to enhanced
release of active TGF-?. Mutations in the TGF-?1 signal peptide
or the LAP coding region result in enhanced TGF-? activity as
seen in Camurati-Engelmann disease, a rare, autosomal domi-
nant condition characterized by sclerosing bone dysplasia and
neurological deficiencies (24). Detailed studies of lymphocyte
function, platelet function, or studies of tissue healing, remod-
eling, and fibrosis have not been performed in these patients.
Mutations or deletions in GARP would only affect membrane
bound TGF-?, and not the secreted pool, and therefore offer an
TGF-? to the regulation of immunity and inflammation. A
recent study using antisense morpholino oligonucleotide to
knockdown GARP in zebrafish demonstrated that GARP might
appears to be associated with infertility since up-regulation of
GARP transcripts were observed in infertile human endome-
trium compared with fertile controls (26). Lastly, GARP mRNA
is highly amplified in different tumors (27–34), but surface
expression of GARP and its association with TGF-? in tumors
has not been studied. Tumor cells may use GARP to express
TGF-? or to capture TGF-? from their surroundings resulting in
local suppression of anti-tumor immune responses or the induc-
tion of Tregs. Further studies of the regulation of GARP
expression may lead to the development of drugs that can
enhance or suppress the expression of GARP and membrane
TGF-? and might be useful to treat autoimmunity or cancers and
fibrotic diseases, respectively. Therefore, our discovery of the
critical role of GARP in controlling surface expression of latent
TGF-? will provide insights into another importance pathway of
TGF-? regulation of morphogenesis, immune homeostasis, in-
flammation, and tissue remodeling.
Materials and Methods
Cell Purification. Peripheral blood was obtained from healthy adult donors
Health and approved by the NIAID institutional review board. The study was
conducted in accordance with the Declaration of Helsinki. PBMCs were pre-
pared over Ficoll-Paque Plus gradients (GE Healthcare). The CD4?cells were
enriched over the AutoMACS with CD4 microbead (Miltenyi). The cells were
CD4?CD25int (intermediate), and CD4?CD127–CD25hi (high). In some experiments,
the cells were FACS-sorted for CD4?CD127?CD25–CD45RA?or CD45RO?.
Human APCs for in vitro suppression assay were obtained by depleting T cells
from PBMCs with CD3 microbead (Miltenyi) using the AutoMACS followed by
positive selection with HLA-DR microbead.
Flow Cytometric Analysis. FOXP3 expression was detected with anti-FOXP3
mAbs after fixing and permeabilizing the cells with a Fixation/Permeabiliza-
tion kit (eBioscience). LAP and GARP expression was surface stained with
anti-LAP and anti-GARP mAbs before fixation/permeabilizing for FOXP3 de-
tection. For GARP, a secondary detection with fluorochrome conjugated
anti-mouse IgG2b was needed.
Antibodies and Reagents. CD4, CD25, CD45RA, CD45RO, anti-mouse IgG1,
anti-mouse IgG2b, goat (Fab?)2 anti-hFc, and carboxyfluorescein succinimidyl
ester (CFSE) were from Invitrogen. Linage-1 mixture, CD127, streptavidin,
monensin, and brefeldin A were from BD. CD1c and CD303 were from Milte-
nyi. Anti-LAP unconjugated and PE-conjugated IgG1 mAbs (clone 27232),
thrombospondin-1 were from R&D Systems. Unconjugated anti-GARP IgG2b
of human IgG1 were from Alexis Biochemicals. rhTGF-?1, -?2, -?3, and rhIL-2
were from Peprotech. RGD and RGDS peptides were from Sigma-Aldrich.
Anti-CD3 (UCHT1), -CD28, and -FOXP3 (clone 236A/E7) mAbs were from
eBioscience. FACSCalibur was used for data acquisition and the data were
analyzed with FlowJo software (Tree Star).
Cell Culture. For cell stimulation, 24-well culture plates (Corning) were coated
in complete RPMI 1640 supplemented with 2% heat-inactivated autologous
human serum and 100 U/mL rhIL-2 (Peprotech). For induction of FOXP3, naïve
CD4?CD127?CD25–CD45RA?and memory CD45RO?T cells were stimulated
for 5 d with plate-bound anti-CD3/CD28 (5 ?g/mL) ? 5 ng/mL rhTGF-?1
(Peprotech). For expansion, Tregs were stimulated with anti-CD3/CD28 Treg
Dynabead (Invitrogen) and 100 U/mL IL-2. For in vitro suppression assay,
50,000 FACS-sorted allogeneic CD4?CD25–T cells were unlabeled or labeled
with 2 ?M CFSE and stimulated with 25,000 nonirradiated autologous CD3-
depleted HLA-DR?APCs and 0.25 ?g/mL OKT3 (Ortho Biotech) alone or with
the last 6–8 h or FACS analysis for CFSE labeled experiment. For the infectious
tolerance experiments, 50,000 CFSE-labeled CD4?CD25–CD127?CD45RA?T
cells were stimulated with anti-CD3/CD28 Dynabeads at 2:1 cell-to-bead ratio
for 5 days alone (top panel) or with 25,000 preactivated (24 h) Tregs trans-
fected with siNS, siGARP, or siTGF?1 and analyzed for FOXP3 induction in the
CFSE-labeled cells. The cells were placed in 96-well flat bottom plates and
the suppression and infectious tolerance experiments, the Tregs were first
transfected by electroporation then rested for 24 h in 100 U/mL IL-2 before
preactivation with plate-bound anti-CD3/CD28 for 24 h before testing their
Quantitative Real-Time PCR Analysis. Total RNA was extracted from cells with
an RNeasy Plus Kit (Qiagen). RT-PCR was performed with ?1 ?g of isolated
RNA for cDNA synthesis using SuperScript II RNase H- Reverse Transcriptase
(Invitrogen). Real-time PCR was performed in triplicate according to the
Taqman Universal 2? master mix and run on the ABI/PRISM 7900 Sequence
expression was normalized to the 18S rRNA and calculated according to the
comparative Ct method as described by Applied Biosystems. The TagMan
Gene Expression Assays from FOXP3 (Hs01085834_m1) and GARP
(Hs00194136_m1) were from Applied Biosystems.
siRNA Experiments. GARP, TGF-?1, FOXP3, and nonsilencing control siRNAs
were from Invitrogen (Stealth Select RNAi). To transfect the Tregs, 200 pmols
siRNA were mixed with 100 ?L human T cell Nucleofector solution (Amaxa
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Biosystems), and 5 ? 106cells were resuspended in this mixture. The cell Download full-text
(Amaxa Biosystems) and placed in 37 °C prewarmed 100 IU/mL IL-2 culture
medium. For the GARP and TGF-?1 knockdown experiments, the transfected
Tregs were rested in 100 U/mL IL-2 for 24 h before stimulation with plate-
bound anti-CD3/CD28. For the FOXP3 knockdown experiments, the trans-
fected Tregs were rested for 5 d before stimulation with anti-CD3/CD28.
48 h with anti-CD3/CD28, then incubated with anti-LAP or IgG (Jackson
Immunoresearch) for 1 h at 4 °C and then washed to remove unbound Abs.
The cells were detergent solubilized in 1% Brij 96 lysis buffer containing
protease inhibitors. LAP complexes were precipitated with anti-mouse IgG
Dynabeads. Immunoprecipitates were resolved on 10%–14% acrylamide gels
branes (Amersham). Blots were incubated with the anti-GARP and then
horseradish peroxidase-conjugated anti-IgG2b. Reactivity was revealed by
enhanced chemiluminescence. For flow cytometric protein-protein interac-
tion experiment, 4.5 ?m (1 ? 106) anti-mouse IgG Dynabeads (Invitrogen)
were incubated in PBS with either 20 ?g/mL anti-LAP or anti-GARP mAbs at
room temperature (RT) for 20 min and placed on magnet to wash away the
supernatant. The anti-LAP or anti-GARP conjugated Dynabeads were incu-
bated with a mixture of 20 ?g/mL GARP-Fc ? 20 ?g/mL LAP, GARP-Fc ? latent
then placed on magnet to wash away the supernatant. Finally the anti-LAP
Dynabead samples were stained with goat anti-hFc PE and the anti-GARP
streptavidin PE and then analyzed by flow cytometry.
Confocal Microscopy. Tregs were activated for 48 h then surface-stained with
anti-GARP IgG2b and anti-LAP IgG1 mAbs followed by anti-mouse IgG2b AF
IgG2b with anti-IgG1 AF 647 or anti-LAP IgG1 with anti-IgG2b AF 568 were
used. Tregs were then fixed with 4% paraformaldehyde in PBS for 30 min at
4 °C and cytospun onto slides, permeabilized with 0.05% Triton X-100 for 5
min at RT. After 3 PBS washes, the nuclei were stained with 40 ng/mL Hoechst
33342 (Invitrogen) for 5 min. Slides were rinsed and mounted with a coverslip
using Fluoromount-G (Southern Biotechnology). Images were collected on a
SP2-AOBS confocal microscope (Leica Microsystems) by the NIAID Biological
GARP Transduction. CD4?T cells were purified using magnetic Dynabeads.
activated by anti-CD3/CD28-coated Dynabeads and transduced with control
HIV-derived HDV expressing red fluorescent protein (RFP) lentiviral vectors
(Clontech Laboratories) or encoding GARP gene as previously described (10)
and expanded for 7 days in 200 U/mL IL-2 culture medium. The cells were then
restimulated for 48 h with plate-bound anti-CD3/CD28 and analyzed for
same vectors and maintained in regular RPMI media with 10% FCS.
ACKNOWLEDGMENTS. We thank Carol Henry, Tom Moyer, and Calvin Eigsti
in the National Institute of Allergy and Infectious Diseases Flow Cytometry
Section for sorting our cells; Cynthia Matthews in the Department of Trans-
Allergy and Infectious Diseases Biological Imaging Facility for performing the
confocal microscopy. This work was supported by the National Institute of
Allergy and Infectious Diseases Intramural Research Program and National
Institutes of Health Grant R01 AI065303 (to D.U.).
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