Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin.
ABSTRACT Naturally occurring CD4(+)CD25(+) regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4(+)CD25(+) non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4(+)CD25(high) T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4(+)CD25(low) or CD4(+)CD25(-) T cells did not. The expanding pure populations of CD4(+)CD25(high) T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4(+)CD25(-) T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4(+)CD25(high) T cell populations retained a broad TCR repertoire and, like CD4(+) CD25(+) T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4(+) and CD8(+) T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.
- SourceAvailable from: uiowa.edu[show abstract] [hide abstract]
ABSTRACT: Several mechanisms control discrimination between self and non-self, including the thymic deletion of autoreactive T cells and the induction of anergy in the periphery. In addition to these passive mechanisms, evidence has accumulated for the active suppression of autoreactivity by a population of regulatory or suppressor T cells that co-express CD4 and CD25 (the interleukin-2 receptor alpha-chain). CD4+ CD25+ T cells are powerful inhibitors of T-cell activation both in vivo and in vitro. The enhancement of suppressor-cell function might prove useful for the treatment of immune-mediated diseases, whereas the downregulation of these cells might be beneficial for the enhancement of the immunogenicity of vaccines that are specific for tumour antigens.Nature reviews. Immunology 07/2002; 2(6):389-400. · 33.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The ultimate goal of any treatment for autoimmune diseases is antigen- and/or site-specific suppression of pathology. Autoaggressive lymphocytes need to be eliminated or controlled to prevent tissue damage and halt the progression of clinical disease. Strong evidence is emerging that the induction of regulatory T (T(Reg)) cells by autoantigens can suppress disease, even if the primary, initiating autoantigens are unknown and if inflammation is progressive. An advantage of these autoreactive T(Reg) cells is their ability to act as bystander suppressors and dampen inflammation in a site-specific manner in response to cognate antigen expressed locally by affected tissues. In this review, we consider the nature and function of such antigen-specific T(Reg) cells, and strategies for their therapeutic induction are discussed.Nature reviews. Immunology 04/2003; 3(3):223-32. · 33.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: There is accumulating evidence that T-cell-mediated dominant control of self-reactive T-cells contributes to the maintenance of immunologic self-tolerance and its alteration can cause autoimmune disease. Efforts to delineate such a regulatory T-cell population have revealed that CD25+ cells in the CD4+ population in normal naive animals bear the ability to prevent autoimmune disease in vivo and, upon antigenic stimulation, suppress the activation/proliferation of other T cells in vitro. The CD25+ CD4+ regulatory T cells, which are naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. They play critical roles not only in preventing autoimmunity but also in controlling tumor immunity and transplantation tolerance.Immunological Reviews 09/2001; 182:18-32. · 12.16 Impact Factor
Selective Survival of Naturally Occurring Human
CD4?CD25?Foxp3?Regulatory T Cells Cultured
Laura Strauss,2*†Theresa L. Whiteside,†Ashley Knights,* Christoph Bergmann,‡
Alexander Knuth,* and Alfred Zippelius2*
Naturally occurring CD4?CD25?regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show
that discrimination of human Treg from effector CD4?CD25?non-nTreg cells and their selective survival and proliferation can
now be achieved using rapamycin (sirolimus). Human purified CD4?CD25highT cell subsets stimulated via TCR and CD28 or by
IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4?CD25lowor CD4?CD25?T cells did
not. The expanding pure populations of CD4?CD25highT cells were resistant to rapamycin-accelerated apoptosis. In contrast,
proliferation of CD4?CD25?T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded
CD4?CD25highT cell populations retained a broad TCR repertoire and, like CD4?CD25?T cells freshly obtained from the
peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein,
CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous
CD4?and CD8?T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo
conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the
availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression
in humans. The Journal of Immunology, 2007, 178: 320–329.
maintenance of immunological self-tolerance, but also in limiting
responses to foreign Ags. Thus, Treg cells have been implicated in
autoimmune diseases, organ transplantation, and infectious dis-
eases (1–3). Importantly, Treg cells can also dampen antitumor T
cell immunity and have been proposed as a potential mechanism of
immune escape (4). Previously, increased proportions of Treg cells
among the total CD4?T cell populations have been described in
the circulation and tumor tissues of patients with different types of
growing body of evidence suggests that regulatory T
(Treg)3cells are capable of inhibiting most types of im-
mune responses. They play key roles not only in the
cancer (5–7). In ovarian cancer, accumulations of Treg cells in the
tumor predicted a significant reduction in survival (8). To date, at
least two types of CD4?Treg cells have been partly characterized
in humans: 1) Treg type 1 and Th3 cells, which are induced in the
periphery upon encountering cognate Ags and secrete IL-10 and
CD4?CD25highFoxp3?T cells (naturally occurring CD4?CD25?
Treg (nTreg) cells) which arise directly in the thymus and have the
CD8?CD25?T cells (11–14). nTreg cells express the transcrip-
tion factor, forkhead box p3 (Foxp3), which has been associated
with their development and suppressive function (15, 16). In ad-
dition, they express glucocorticoid-induced TNFR family related
gene (GITR) and intracellular CTLA-4. More recent studies indi-
cate that CD4?CD25highFoxp3?T cells are a heterogeneous pop-
ulation within CD4?T cells, endowed with different regulatory
functions, including regulation of Ag-specific immune responses
(17, 18). As our knowledge of Treg-mediated immunosuppression
has increased, the potential for therapeutic alterations in immunity
through in vivo and in vitro manipulation of Treg cell numbers
and/or functions in disease holds great promise.
Currently, isolation and expansion of human nTreg cell subsets
into functionally active, disease-specific T cells is, however, dif-
ficult due to the following: 1) the paucity of nTreg cells in the
peripheral blood; 2) the lack of specific identity markers for nTreg
cells; and 3) the absence of protocols for rapid and extensive ex-
pansion of pure nTreg cells with suppressive activity from periph-
eral blood. In humans, CD4?CD25?T cells include suppressor
CD4?CD25highT cells as well as CD4?CD25lowT cells which are
nonsuppressive, activated CD4?T cells. Furthermore, CTLA-4
and GITR are activation Ags and because levels of their expression
vary depending on cell activation, they are not useful for discrim-
inating nTreg from effector T cell populations. Similarly, Foxp3
*Medical Oncology, Department of Internal Medicine, University Hospital, Zurich,
Switzerland;†University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; and
‡Department of Otorhinolaryngology, University Schleswig, Luebeck, Germany
Received for publication June 29, 2006. Accepted for publication October 18, 2006.
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.
1This work was supported in part by a Swiss National Science Foundation special
program, and a grant from the Cancer Research Institute/Ludwig Institute for Cancer
Research Cancer Vaccine Collaborative, the Terry-Fox, Hanne-Liebermann, and
Claudia-von-Schilling Foundation, and UBS Wealth Management. A.Z. was sup-
ported in part by the Emmy-Noether Program (Zi685-2/3) of the Deutsche For-
schungsgemeinschaft. T.L.W. was supported in part by National Institutes of Health
Grant PO-1 DE12321.
2Address correspondence and reprint requests to Dr. Laura Strauss, University of
Pittsburgh Cancer Institute, Research Pavilion at the Hillman Cancer Center, 5117
Centre Avenue, Suite 1.27, Pittsburgh, PA 15213-1863; E-mail address: straussl@
upmc.edu or Dr. Alfred Zippelius, Medical Oncology, Department of Internal Med-
icine, University Hospital, Raemistrasse 100, 8091, Zurich, Switzerland; E-mail ad-
3Abbreviations used in this paper: Treg, regulatory T; nTreg, naturally occurring
CD4?CD25?Treg; GITR, glucocorticoid-induced TNFR family related protein; TT,
tetanus toxoid; ANX V, annexin V; LN, lymph node; MFI, mean fluorescence inten-
sity; PI, propidium iodine.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$2.00
The Journal of Immunology
expression—though more specific for Treg—may also be up-reg-
ulated on effector cells following activation (19), and due to its
intracellular localization, Foxp3 cannot be used for separation of
living Treg cells. As a consequence, most current studies using
CD4?CD25?populations for analysis of the phenotype and func-
tions of nTreg cells are not interpretable due to the presence of
contaminating effector CD4?T cells, which reduce or even com-
pletely camouflage suppressive functions of nTreg cells present in
T cell cultures.
It has been shown that the immunosuppressive drug rapamycin
(sirolimus) is a powerful pharmacological agent which blocks in-
tracellular signaling in response to T cell growth factors, e.g., IL-2,
required for the progression of activated T cells from G1into S
phase (20). Though rapamycin has been reported to favor
CD4?CD25?T cell-dependent immunoregulation in vitro and in
vivo (21–25), the exact mechanism of its action on nTreg cells
remains largely unknown. In particular, a direct effect of rapamy-
cin on survival and proliferation of human nTreg cells has not been
To gain insights into the phenotypic and functional characteris-
tics of expanded, phenotypically homogeneous, and highly sup-
pressive populations of human CD25highnTreg cells, we have used
rapamycin to discriminate between CD4?CD25highFoxp3?T cells
with a constitutive expression of CD25 from CD4?CD25?effec-
tor T cells and CD4?CD25?resting T cells. In this study, we
provide new evidence that in the presence of IL-2, rapamycin
alone or rapamycin combined with stimulation signals delivered
via TCR and CD28 is capable of fostering the selective survival
and expansion of CD4?CD25highFoxp3?nTreg cells, which are
endowed with potent suppressive activities and thus may serve as
pure populations of human nTreg cells for future investigations
Materials and Methods
Reagents and Abs
All Abs were purchased from BD Pharmingen except anti-GITR (R&D
Systems) and Foxp3 (eBioscience). Rapamycin, LPS, PHA, PMA, iono-
mycin, and saponin were obtained from Sigma-Aldrich. IL-2 and IL-7
cytokines were obtained from R&D Systems. Monensin was obtained from
BD Pharmingen. T cells were cultured in RPMI 1640 (Invitrogen Life
Technologies) supplemented with human AB serum (8% v/v), L-glutamine
(200 mM), sodium pyruvate (1%), nonessential amino acids (1%), peni-
cillin/streptomycin (1%) (all obtained from Invitrogen Life Technologies),
2-ME (1%) (Sigma-Aldrich), and fluoroquinolon (1%) (ciproxin infusion;
Peptides were synthesized by standard solid-phase chemistry on a mul-
tiple peptide synthesizer (Biosynthesis). The following synthetic peptides
were used: tetanus toxoid947–967(FNNFTVSFWLRVPK) (TT947–967),
MAGE-A3247–258(TQHFV QENYLEY) (MAGE-A3247–258), Melan-A/
(ILKEPVHGV) (HIV476–484). Synthesis of PE-labeled HLA-A2/Melan-
A26–35multimeric complexes was performed as previously described (26).
PBMC were isolated from buffy coat preparations derived from the whole
blood of healthy volunteers (Suisse Blood Center, Zurich, Switzerland and
Central Blood Bank, Pittsburgh, PA) by density sedimentation on Ficoll-
Hypaque gradients. Cells recovered from the gradient interface were
washed twice in RPMI 1640 medium, counted, and immediately used for
MACS and staining. Briefly, CD4?T cells were negatively selected from
the total PBMC using the CD4 isolation kit (Miltenyi Biotec), yielding a
population of CD4?cells with purity of 92–98%. Positive selection on
anti-CD25 magnetic microbeads was then used to separate the negative
fraction containing CD4?CD25?T cells from the CD4?CD25?T cell
fraction, using the CD4?CD25?T Regulatory Cell Isolation kit from
Miltenyi Biotec. Cells were then applied to a second magnetic column,
washed, and eluted again. This procedure led to the complete positive
selection of CD4?CD25?T cells (purity ?96%), and negative depletion of
CD4?CD25?T cells, as measured by flow cytometry. Both cell subsets
were used for in vitro expansion.
In vitro expansion of MACS-purified CD4?T cells
Initially, purified CD4?CD25?or CD4?CD25?T cells were cultured in
Terasaki plates (10 cells/well) with syngeneic irradiated (3000 rad) PBMC
(20,000 cells/well), PHA (1 ?g/ml), and 50 IU IL-2/ml for 7–8 days to
increase their numbers. Next, aliquots of cells (150,000 cells/well) were
transferred to wells of a 96-well plate and cultured with anti-CD3/anti-
CD28 Ab-coated magnetic Dynabeads (CD3/CD28 beads; Dynal Biotech/
Invitrogen Life Technologies) at 4 ? 107beads/ml and 1,000 IU/ml IL-2
for 7 days. Rapamycin at the final concentration of 1 nM was added to half
of the cultures (Fig. 2). After 1 wk of expansion, cells were washed to
remove IL-2 and the beads were removed with a magnetic particle con-
centrator (Miltenyi Biotec). The expanded cells were then transferred to
wells of a 24-well plate and restimulated with CD3/CD28 beads and 1000
IU/ml IL-2. These cells were cultured in the presence or absence of 1 nM
rapamycin for 1 wk (Fig. 2). Cells cultured in the absence of rapamycin are
designated as “R0” and those cultured with 1 nM rapamycin as “R1.” After
every week of expansion, cells sampled for assays were washed twice, the
beads were removed, and the cell number was determined by direct count-
ing to evaluate expansion. Cell aliquots sampled from each group (at least
200,000 cells) were maintained for 48 h in culture medium and low-dose
IL-2 (50 IU/ml) in the absence of CD3/CD28 beads and rapamycin before
To evaluate effects of rapamycin on expansion of fresh MACS-purified
T cells, CD4?CD25?and CD4?CD25?cells were cultured ? rapamycin
in the presence of 50 IU/ml IL-2 or PHA and irradiated syngeneic APC for
7 days and used for assays.
Semiquantitative RT-PCR for Foxp3
For RNA extraction, the RNase easy kit (Qiagen) was used according to the
manufacturer’s recommendation. Extracted RNA was reverse transcribed
using the SuperScript III First-Strand Synthesis System (Invitrogen Life
Technologies). We performed quantitative PCR using the TaqMan ABI
Prism 7000 Sequence Detection System (Applied Biosystems) on cDNA
samples for Foxp3 expression normalized to the housekeeping gene 18s
rRNA. The following primers for PCR were purchased from Applied Bio-
systems: forkhead/winged helix transcription factor (Foxp3), 5?-GCACCT
GAAACC-3? (antisense) in a 20-?l volume. Levels of 18s rRNA were
quantified by using TaqMan PDAR Eukaryotic 18s Endogenous Controls
(Applied Biosystems). The following cycling conditions were used: 10 min
at 95°C, followed by 30 cycles of 15 s at 95°C, and 1 min at 60°C.
Surface and intracellular staining
All CD4?T cell samples were stained with anti-CD4, monocyte-discrim-
inating Abs (anti-CD14, anti-CD32, and anti-CD116). Fresh as well as in
vitro-expanded CD4?CD25?(R0 vs R1) and CD4?CD25?T cells (at least
200,000 cells/tube) were stained with the following mAbs for 15 min at
4°C to determine the “nTreg” surface marker profile: PE-conjugated
anti-CD25, PerCP-labeled anti-CD4, allophycocyanin-conjugated anti-
CD45RO, CCR7, and HLA-DR, FITC-conjugated anti- CD62L, Fas,
GITR, CD45RA, and CD27. Criteria used for designating T cells as
CD4?CD25highwere based on mean fluorescence intensity (MFI) as fol-
lows: with fresh PBMC when MFI was ?20, and with isolated/cultured
CD4?CD25?T cells, which were highly enriched populations, when MFI
was ?500, as illustrated in Fig. 1, A and B. Appropriate isotype controls
were included in all experiments.
Intracellular levels of IL-10 in fresh or in vitro-expanded R0 and R1 T
cells were measured after a 16-h incubation with 25 ng/ml LPS (Sigma-
Aldrich) in the presence of 1 ?g/ml monensin (GolgiStop; BD Pharmin-
gen). Intracellular staining was performed as previously described (27).
Briefly, for detection of intracellular Foxp3, CTLA-4, and IL-10, samples
were first incubated with mAbs against surface markers CD4 and CD25.
After subsequent washes, cells were fixed with PBS containing 4% form-
aldehyde for 20 min at room temperature, washed once with PBS contain-
ing 0.5% BSA and 2 nM EDTA, permeabilized with PBS containing 0.5%
(w/v) BSA and 0.2% (v/v) saponin, and stained with anti-CTLA-4-
allophycocyanin, anti-Foxp3-FITC-labeled or anti-IL-10-PE-labeled mAb
for 30 min at room temperature. Cells were further washed twice with PBS
containing 0.5% BSA and 0.2% saponin, resuspended in FACS flow so-
lution, and immediately analyzed by flow cytometry. Appropriate isotype
controls were used in all experiments.
CD4?CD25?and CD4?CD25?T cells obtained by MACS isolation
and R0 as well as R1 cells were assessed for annexin V (ANX V) binding.
Samples were stained with FITC-conjugated ANX V (Molecular Probes/
321 The Journal of Immunology
Invitrogen Life Technologies) and propidium iodide (PI; Molecular
Probes), according to the instructions provided by the distributor.
Flow cytometry was performed using a FACScan flow cytometer (BD
Biosciences) equipped with CellQuest software (BD Biosciences). The ac-
quisition and analysis gates were restricted to the lymphocyte gate, as
determined by characteristic forward and side scatter properties of lym-
phocytes. Analysis gates were restricted to the CD3?CD4?, CD4?CD25?,
and CD4?CD25highT cell subsets. Usually, 1 ? 105lymphocytes were
collected for analysis. For ANX V-stained samples the acquisition gates
were restricted to the live (PI?) as well as the dead cell (PI?) populations
present in each sample. Six parameters (forward scatter, side scatter, and
four fluorescence channels) were used for list mode data analysis.
T cell repertoire diversity
TCRV ?-chain repertoire of fresh as well as in vitro-expanded R0 and R1
cells was determined by staining with a panel of V? chain-specific Abs
(BD Pharmingen), followed by flow cytometry analysis.
Generation of Ag-specific CD4?25?and CD8?25?T cells
CD4?and CD8?T cells enriched from PBMC using a miniMACS device
were stimulated with HLA-A*0201-restricted Melan-A/MART-126–35- and
HLA-DP*0401-restricted TT947–967peptides. To generate peptide-specific
CD25 on freshly isolated PBMC. B, Expression of CD4 and CD25 on MACS-purified human T lymphocytes. C, MACS-purified T cells after 1 wk of culture
in the presence of 50 IU/ml IL-2. D, MACS-purified T cells after a 1-wk expansion in the presence of PHA (1 ?g/ml) and irradiated APC in the presence
of 50 IU/ml IL-2. E and F, Expression of CD4 and CD25 on MACS-purified T cells after a further 1-wk expansion (in the presence of 50 IU/ml IL-2 or
PHA and irradiated feeders, respectively, in the absence (R0) or presence of 1 nM rapamycin (R1)). In R0 cultures, the mean ? SD values were as follows:
2.5 ? 7% CD25highcells, 23 ? 9% CD25lowcells, and 60 ? 7.1% CD25?cells (E and F, left). In contrast, in R1 cultures, mainly CD25highT cells (95 ?
9.6%), (E and F, right) were present. G and H, Expansion rates and ANX V binding to MACS-purified CD4?CD25?and CD4?CD25?T cells cultured
in the presence (R1) or absence (R0) of rapamycin. The expansion rates of CD4?CD25?(u), CD4?CD25?(?), and CD4?CD25high(f) cells in R0 and
R1 T cells after a 1-wk expansion in 50 IU/ml IL-2 or in the presence of PHA and irradiated feeders are shown in G and H, left. Expansion rates are
calculated based on the ratio of absolute cell counts after expansion vs absolute counts after MACS purification; percentages of CD4?CD25?ANX V?
cells in R0 (u) and R1 (o) cultures after 1 wk of expansion in 50 IU/ml IL-2 or in the presence of PHA and irradiated feeders are shown in G and H, right.
A–H, The results of experiments performed with cells obtained from five healthy individuals. The data are representative flow cytometry dot plots (A–F)
or means ? SD of five experiments.
CD25 expression on fresh, activated, and cultured CD4?CD25?T cells before and after rapamycin exposure. A, Expression of CD4 and
322 PHENOTYPE AND FUNCTION OF HUMAN RAPAMYCIN-INDUCED nTregs
T cells, peptide-pulsed irradiated syngeneic APCs were cocultured with
CD4?or CD8?T cells in the presence of IL-2 (25 IU/ml) and IL-7 (10
ng/ml) as described (27). To select for peptide-specific T cells in these
cultures, flow cytometry was used to sort T cells stained by multimers or
T cell-secreting cytokines in response to peptide stimulation. The isolated
T cells were cloned and cultured as previously described (27).
Suppression of proliferation and cytokine secretion
Initially, suppression of proliferation mediated by MACS-purified
CD4?CD25?and CD4?CD25?T cells before and after exposure to rapa-
mycin was analyzed using an allogeneic system. Suppressive functions of
these cells immediately after isolation, after 1 wk expansion (in the pres-
ence of 50 IU IL-2/ml or PHA and irradiated APC) or after 3 wk expansion
(in the presence of CD3/CD28 beads and 1000 IU IL-2/ml) cocultured with
fresh allogeneic responder CD4?CD25?or CD8?CD25?T cells was de-
termined. For this purpose, the responder T cells were labeled with 0.5 ?M
CFSE (Molecular Probes) and stimulated with OKT3 (1 ?g/ml) (American
Type Culture Collection) and soluble anti-CD28 Ab (1 ?g/ml; Miltenyi
Biotec). Suppressor cells, either CD4?CD25?or CD4?CD25?cell frac-
tions, were added at the suppressor:responder ratio of 1:1. Cells were in-
cubated for 5 days in a CO2incubator at 37°C.
Next, Ag-specific suppression mediated by CD4?CD25?R0 or R1 T
cells that were expanded for 3 wk was analyzed in an autologous in vitro
system. CD4?CD25?TT947–967peptide-specific T cell line ZH-P1 and
CD8?CD25?Melan-A/MART127–35peptide-specific T cell clone ZH-
P7/2 were labeled with 0.5 ?M CFSE (Molecular Probes) and incubated
with either R0 or R1 T cells at the 1:1 ratio. Peptide-specific responder T
cells were stimulated with the relevant peptide-pulsed (2 ?g/ml) APC (au-
tologous PBMCs) or with PHA (1 ?g/ml).
All CFSE data were analyzed using the ModFit software provided by
Verity Software House. The percentages of suppression were calculated
based on the proliferation index of responder cells alone compared with the
proliferation index of cultures containing responders and Tregs. The pro-
gram determines the percent of cells within each peak and the sum of all
peaks in the control culture is taken as 100% of proliferation and 0% of
For IFN-? ELISPOT analysis, the CD4?CD25?TT947–967peptide-
specific T cell line ZH-P1 and CD8?CD25?Melan-A27–35peptide-specific
T cell clone ZH-P7/2 were stimulated either with the relevant peptide
(TT947–967, Melan-A/MART126–35, respectively) or irrelevant peptides
(MAGE-A3247–258, HIV-POL476–484, respectively) using HLA-DP*0401?
and HLA-A*0201?target cells, respectively. Twenty-four-hour ELISPOT
assays were performed as previously described (27). Maximum IFN-?
release was determined by stimulation of responder cells with PMA/
Differences between groups were assessed using the Student t test. Values
of p were two tailed. Values of p ? 0.05 were considered significant.
Purification of CD4?CD25?T cells obtained from PBMC of
We first determined that CD4?CD25?T cells comprised 2–5% of
CD4?T cells in the peripheral blood of 10 healthy individuals
analyzed. The CD25highT cells defined as described in Materials
and Methods represented 0.5–1% of the total CD4?T cell popu-
lation, while the CD25lowT cells accounted for 2.5% of CD4?T
cells (Fig. 1A). Next, CD4?CD25?T cells were isolated using
MACS on anti-CD4/anti-CD25 beads. The purity of positively se-
lected CD4?CD25?T cells was ?96%. After MACS isolation,
the CD4?CD25?cell fraction contained both CD4?CD25highand
CD4?CD25lowcells (Fig. 1B).
Culture system for expansion of CD4?CD25?T cells
Fig. 2 is a schema designed for expansion of isolated CD4?
CD25?human T cells. It is based on our initial finding that
these cells plated at the usual concentrations of ?105cells/well
in 96-well plates failed to proliferate even when IL-2, PHA, and
APCs were added. We determined that plating CD4?CD25?T
cells at 10 or fewer cells per well in Terasaki plates allowed for
their initial expansion to 15–20 ? 103cells/well and their transfer
to wells of 96-well plates 7 days later. To obtain sufficient cell
numbers for subsequent culture ? rapamycin, CD4?CD25?T
cells remained in wells of a 96-well plate for 1 wk, yielding
?100–150 ? 103cells. At this point, the cells were divided into
parallel R0 and R1 cultures. Viable cell counts performed at each
transfer showed that the fold expansion was 50- to 100-fold for R0
as well as for R1 cultures at the end of the entire 3-wk expansion
Phenotypic changes in cultured CD4?CD25?T cells
The MACS-isolated cells were plated in the presence of 50 IU of
IL-2 or in PHA and autologous feeder cells and assessed for phe-
notype after 1 wk of culture. In the presence of IL-2 alone, CD4?
T cells down-regulated surface expression of CD25 (Fig. 1C). In
contrast, most CD4?T cells stimulated with PHA and autologous
feeder cells retained CD25 expression (Fig. 1D). These cultures
were next incubated in the presence (R1) or absence (R0) of 1 nM
rapamycin for 7 days. In IL-2 or PHA-containing R0 cultures, few
CD4?T cells were CD4?CD25highand most were CD25?(Fig. 1,
E and F, left). In contrast, the parallel R1 cultures were enriched in
CD4?CD25highT cells (Fig. 1, E and F, right). The viable cell
counts and ANX V-binding assays performed on day 7 indicated
that most CD4?CD25?T cells, which rapidly outgrow in the R0
cultures, are not sensitive to apoptosis (Fig. 1, G and H). In con-
trast, the R1 cultures largely contained proliferating CD4?CD25?
T cells and CD4?CD25?T cells, representing a minority popu-
lation, were highly sensitive to apoptosis (Fig. 1, G and H). These
data are consistent with the preferential expansion of CD4?
CD25?T cells accompanied by the significant inhibition of CD4?
CD25?T cell expansion due to a high frequency of ANX V-
binding cells in the R1 cultures (Fig. 1, G and H).
CD4?CD25?T cells exposed to rapamycin. The numbers of R0 or R1 cells
per well on week 1, 2, or 3 of expansion are given in the respective boxes.
A schemafortheexpansion protocol ofpurified
323The Journal of Immunology
These initial experiments indicated that rapamycin-treated
CD4?CD25?cells could expand up to 1- to 2.5-fold in cultures
supplemented with IL-2 or PHA and autologous APC. The optimal
rapamycin concentration for expansion of nTreg cells was next
determined. In contrast to experiments performed previously in
mice (25), doses of rapamycin ?10 nM considerably decreased
proliferation of MACS-purified CD4?CD25?T cells (data not
shown). Conversely, lower doses of rapamycin (?1 nM) together
with CD3/CD28 beads and 1000 IU/ml IL-2 showed no difference
in expansion rates for MACS-purified CD4?CD25?and CD4?
CD25?T cells compared with expansion cultures without rapamycin
to be optimal for expansion of CD4?CD25?T cells and used for all
Fig. 3A illustrates expansion of CD4?CD25?, CD4?CD25?, and
CD4?CD25highT cell populations in parallel R0 and R1 cultures
during a 3-wk expansion period. The cultures were monitored weekly
for cell numbers and the percentage of CD4?CD25highT cells.
MACS-purified CD4?CD25?T cells proliferated readily in the ab-
sence of rapamycin (R0), reaching considerable fold expansion by
week 3. However, CD4?CD25?as well as CD4?CD25highT cells
were a minor component of R0 cultures. In contrast, while expansion
of CD4?CD25?and CD4?CD25?T cells was delayed in the pres-
ence of rapamycin (R1), by week 3, vigorous proliferation of
CD4?CD25?and CD4?CD25highT cells occurred, leading to their
50- to 100-fold expansion. CD4?CD25?T cells were not proliferat-
ing in R1 cultures (Fig. 3A), while a significant enrichment in
CD4?CD25highT cells was routinely observed (Fig. 3B).
ANX V binding to MACS-purified CD4?CD25?T cells exposed
Previous experiments showed that rapamycin blocked proliferation of
CD4?CD25?T cells (25) and our initial data were in agreement with
this possibility. To analyze whether rapamycin selectively induces
apoptosis of CD4?CD25?T cells, we examined ANX V binding to
fresh and cultured CD4?CD25?and CD4?CD25?T cells after their
exposure to rapamycin. As shown in Fig. 4A, in normal fresh PBMCs
exposed to rapamycin for 48 h, the CD4?CD25?T cells bind little
ANX V (0.1 ? 1.2%). In contrast, the CD4?CD25?T cell subset
showed significantly higher (p ? 0.00001) percentages of ANX V?
CD4?CD25?T cells are sensitive to rapamycin-induced apoptosis.
Also, a significantly higher percentage of ANX V?cells was ob-
served in week 3 R1 cultures of CD4?CD25?T cells (80 ? 13 vs
as shown in Fig. 4B for a representative culture. In contrast, ANX V
binding to CD4?CD25?and CD4?CD25?T cell subset was similar
when these cells were cultured without rapamycin (23 ? 14 vs 20 ?
15%; p ? 0.44; data not shown). Our results are consistent with the
conclusion that rapamycin blocks proliferation of CD4?CD25?T
cells by inducing their apoptosis, while CD4?CD25?Treg are resis-
tant to apoptosis and continue to proliferate in the presence of 1 nM
and CD4?CD25?T cells in the presence or absence of rapamycin.
A, Expansion rate of CD4?CD25?(u), CD4?CD25?(?), and CD4?
CD25high(f) cells in the presence (R1) or absence (R0) of rapamycin with
CD3/CD28 beads and 1000 IU/ml IL-2 throughout a 3-wk expansion pe-
riod was assessed at weekly intervals. The expansion rate reflects the ratio
of absolute cell counts after expansion vs that after MACS purification.
B, The percentage of CD4?CD25highT cells in R0 and R1 cultures after a
3-wk culture. The data are means ? SD of experiments performed with
cells of five healthy individuals.
Expansion rates of in vitro-expanded human CD4?CD25?
rapamycin. A, Percentages of ANX V?CD4?CD25?and CD4?CD25?
cells in fresh PBMC after exposure of the cells to rapamycin for (48 h).
B, R1 and R0 T cells were stained with ANX V and assessed by FACS
analysis. In all cases, gates were set on CD4?CD3?T cells. The data are
representative dot plots from experiments performed with cells of five nor-
ANX V binding to fresh or cultured CD4?T cell subsets ?
324PHENOTYPE AND FUNCTION OF HUMAN RAPAMYCIN-INDUCED nTregs
Phenotypic characteristics of MACS-purified CD4?CD25?
T cells expanded by rapamycin
Foxp3 has been considered to be a major phenotypic marker of
Treg. Therefore, its expression was determined in fresh CD4?
CD25?and CD4?CD25?T cells as well as after 3 wk of expan-
sion in R0 and R1 cultures. Initially, mRNA for Foxp3 was mea-
sured, using Foxp3 specific real-time quantitative PCR (Fig. 5A).
Later, intracellular flow cytometry analysis of cells stained with
Foxp3-specific Abs was used (Fig. 5B). Neither fresh CD4?
CD25?R0 T cells nor fresh CD4?CD25?R1 T cells expressed
Foxp3 mRNA. In contrast, CD4?CD25?T cells before and after
exposure to rapamycin expressed Foxp3 mRNA (Fig. 5A), and
CD4?CD25?R1 T cells expressed significantly higher levels of
Foxp3 mRNA (p ? 0.044) compared with CD4?CD25?R0 T
cells (Fig. 5A). We have also studied Foxp3 protein expression by
fresh and rapamycin-expanded CD4?CD25?T cells by flow cy-
tometry. As shown in Fig. 5B, CD4?CD25?T cells in 3-wk
R0 cultures contained 10 ? 6.5% of Foxp3?cells. In contrast,
CD4?CD25?T cells in 3-wk R1 cultures were all Foxp3 positive
(98 ? 2.5%).
To establish the phenotype of T cells expanded in the presence
of rapamycin relative to that of fresh CD4?CD25highT cells, mul-
ticolor flow cytometry was performed. First, the phenotype of
fresh PBMC before exposure to rapamycin was determined. The
CD4?CD25highT cell subset in fresh PBMC expressed higher lev-
els of CD62L, GITR, CTLA-4, CD45RO, Fas, CCR7, and
HLA-DR markers compared with the CD4?CD25lowsubset or to-
tal CD4?CD25?T cells (Fig. 5C). R1 cells after 1 as well as 3 wk
of expansion expressed significantly higher levels of the same
markers present on the CD4?CD25highsubset in fresh PBMC (Fig.
5C; p ? 0.05). In R1 cultures, CD4?CD25highcells, which rep-
resent the proliferating and expanded subset, are CD45RO?
(?40%), CCR7?(?80%), and CD27?(?10%), a phenotype re-
sembling the central memory phenotype (28). The expression of all
“nTreg” markers was higher on CD4?CD25?T cells after 3 wk of
expansion as compared with nTreg after 1 wk of expansion; how-
ever, the difference was not statistically significant (p ? 0.65)
(Fig. 5C). No significant difference in expression of the above-
listed markers was observed in R1 cells expanded with ?1 nM
rapamycin vs R0 cells (data not shown).
Suppressor functions of MACS-purified CD4?CD25?and
CD4?CD25?T cells expanded by rapamycin
To evaluate suppressive activity of R0 and R1 T cells after MACS
isolation and 1 wk of expansion, cocultures (in the presence of
IL-2 or PHA and irradiated APC) were set up with CFSE-labeled
Foxp3 before and after rapamycin exposure were determined by real-time quantitative RT-PCR in fresh MACS-purified CD4?CD25?, fresh MACS-
purified CD4?CD25?T cells, and in MACS purified cells in R0 and R1 cultures after 3 wk of expansion. ?, Significant differences (p ? 0.044) between
R0- and R1-treated cells or cultures. B, Flow cytometry of CD4?CD25highT cells in R1 and R0 cultures after 3 wk of expansion T cells were stained with
Foxp3 mAb and assessed by FACS analysis. C, Flow cytometry for expression of various markers on fresh CD4?CD25?T cells and R1 T and R0 T cells
after 1 and 3 wk of culture. Cells were stained with mAb against CD62L, CD27, GITR, CTLA-4, CD45RO, Fas, CCR7, and HLA-DR, and evaluated by
multiparameter FACS analysis. Gates were set on CD4?CD25highcells (f) or CD4?CD25lowcells (u). The data are mean percentages of positive cells ?
SD. Experiments were performed with fresh and cultured cells obtained from five normal individuals.
Phenotypic characterization of fresh, activated, and in vitro-expanded CD4?CD25?T cells ? rapamycin. A, Relative levels of mRNA
325 The Journal of Immunology
fresh allogeneic CD4?CD25?T cells (ratio 1:1) responding to
OKT3 and anti-CD28 Ab (Fig. 6A). The mean ? SD percentage of
inhibition was 43 ? 2.5% for fresh CD4?CD25?T cells obtained
after MACS isolation from healthy donors. No suppression was
observed with CD4?CD25?T cells after 1 wk of expansion in
IL-2 alone or with CD4?CD25?T cells after 1 wk of expansion
with PHA and irradiated APC (Fig. 6A). These cells tended to lose
CD25 expression (see Fig. 1, C and D) and suppressor functions,
possibly due to a rapid outgrowth of CD4?CD25?populations
(see Fig. 3A). In contrast, as seen in Fig. 6A, the mean ? SD
percentages of inhibition obtained with CD4?CD25?T cells from
R1 cultures were: 62 ? 7.2% (fresh), 71 ? 3.5% (at 1 wk ? IL-2),
and 74 ? 6.5% (at 1 wk plus PHA/APC), respectively.
Furthermore, we analyzed the suppressive activity of R0 and R1
T cell cultures after 3 wk of expansion with CD3/CD28 beads and
1000 IU/ml IL-2 on proliferation of fresh allogeneic CD4?CD25?
and CD8?CD25?T cells (ratio 1:1) responding to OKT3 and anti-
CD28 Ab, using CFSE assays (Fig. 6B). The mean ? SD percent-
age of inhibition obtained with cells of five healthy donors was
82 ? 4.5 and 98 ? 2.35%, respectively (Fig. 6B), when R1 cells
were tittered in at the 1:1 ratio. Much lower levels of suppression
were observed (12 ? 4.5 and 16 ? 6.7%, respectively) when R0
T cells were added to the cocultures (Fig. 6B).
To study Ag-specific suppression in an autologous in vitro sys-
tem, syngeneic R0 and R1 T cells after 3 wk of expansion with
CD3/CD28 beads and 1000 IU/ml IL-2 were added at 1:1 ratios to
CD4?CD25?T cell line ZH-P1 and CD8?CD25?T cell clone
ZH-7/2, which are specific for TT947–967peptide and Melan-A/
MART126–35peptide, respectively. For this purpose, the specific T
cells were stimulated with either 1) PHA or 2) APC pulsed with
cultured human CD4?CD25?T cells ? rapamycin.
A, Freshly MACS-purified, CD4?CD25?T cells were
labeled with CFSE and stimulated with OKT3 (1 ?g/
ml) and anti-CD28 mAb (1 ?g/ml) in the presence of 50
IU/ml IL-2. Fresh allogeneic MACS-purified CD4?
CD25?T cells or the same expanded cells from R0 and
R1 cultures (7 days in 50 IU/ml IL-2 or 7 days in PHA
and irradiated APC) were added at the start of the cul-
ture (ratio 1:1). Cell division represented by the dilution
of CFSE was analyzed using the ModFit software as
described in Materials and Methods. The inset next to
control culture (responder cells alone) shows the per-
centages of cells in each peak after 5 days of prolifer-
ation. Histograms show CFSE intensity (x-axis) and the
number of events (y-axis). The acquisition gates were
restricted to the lymphocyte gate, as determined by
characteristic forward and side scatter properties of
lymphocytes. Analysis gates on the ModFit program
were restricted to the CD3?CD4?and CD4?CFSE?T
cell subsets, as shown on the left. The results of one
representative experiment of five performed are shown.
The percentages of proliferation inhibition are indicated
in each panel. B, Freshly MACS-purified, CD4?
CD25?, or CD8?CD25?T cells were labeled with
CFSE and stimulated with OKT3 (1 ?g/ml) and anti-
CD28 mAb (1 ?g/ml) in the presence of 50 IU/ml IL-2.
Allogeneic T cells from R0 and R1 cultures (after 3 wk
of expansion with CD3/CD28 beads and 1000 IU/ml
IL-2) were added at the start of the culture (ratio 1:1)
and flow cytometry was performed on day 5. Note pro-
found inhibition of proliferation by R1 but not R0 T
cells. A representative experiment of five performed
with PBMC of different normal donor is shown. C, A
suppression of proliferation by R0 or R1 T cells in an
autologous system. CD4?CD25?T cell line (5 ? 104
cells) (left) and CD8?CD25?T cell clone (5 ? 104
cells) (right) were labeled with CFSE and stimulated
with TT947–967or MELANA26–36peptides and irradi-
ated autologous PBMC (2.5 ? 105) in the presence of
IL-2. Autologous R1 and R0 T cells (after 3 wk of ex-
pansion with CD3/CD28 beads and 1000 IU/ml IL-2)
were added at the start of the culture (ratio 1:1) and
results were analyzed on day 5 of culture. One repre-
sentative experiment of three performed with PBMC of
the same (HLA?A2?DR4?) donor is shown.
Suppression of proliferation by fresh or
326PHENOTYPE AND FUNCTION OF HUMAN RAPAMYCIN-INDUCED nTregs
the cognate peptides TT947–967and Melan-A/MART126–35, re-
spectively. After 5 days, specific CD4?and CD8?T cell clones
proliferated vigorously in response to cognate peptides in the ab-
sence of R1-expanded cells (Fig. 6C). However, proliferation of
CD4?and CD8?CD25?T cells after peptide stimulation was
strongly inhibited upon coculture with syngeneic R1 T cells (Fig.
6C) but not syngeneic R0 T cells. The mean ? SD percentage of
inhibition obtained with cells of the same donor in three experi-
ments was 85 ? 1.5% for CD4?TT peptide-specific T cell line
ZH-P1 (Fig. 6C) and 58 ? 6% for CD8?CD25?MART1-peptide
specific T cell clone ZH-7/2 (Fig. 6C). The mean percentages of
inhibition of CD4?CD25?- and CD8?CD25?-specific T cells by
syngeneic R0 or R1 T cells after stimulation with PHA peptide
were similar to those obtained after peptide stimulation (data not
To determine whether rapamycin induces suppressor T cells in
CD4?CD25?T cells, we analyzed inhibition of fresh allogeneic
CD4?CD25?T cell or CD8?CD25?T cell proliferation in re-
sponse to OKT3, anti-CD28 mAb, and IL-2 by CD4?CD25?T
cells after 7 days or 3 wk of expansion, respectively, in the pres-
ence or absence of rapamycin. CFSE-labeled responders stimu-
lated with OKT and anti-CD28 Ab were cultured alone or cocul-
ture with CD4?CD25?R0 or R1 cells at the ratio of 1:1 (Fig. 7).
No evidence was obtained for induction of suppressor T cells in
these cultures as indicated by low levels of suppressor activity in
R1 cocultures (R0 cocultures not shown). The data indicated that
rapamycin does not induce suppressor T cells in CD4?CD25?
populations during a 7-day or 3-wk incubation.
Next, we determined the suppressive activities of R1 T cells on
cytokine secretion using IFN-? ELISPOT assays. In contrast to R0
T cells, both syngeneic (Fig. 8) and allogeneic (data not shown) R1
T cells strongly inhibited IFN-? secretion of TT947–967peptide-
specific CD4?T cells (99.1 ? 0.5 and 97.3 ? 1%, respectively;
Fig. 8, top) and Melan-A/MART126–35peptide-specific CD8?T
cells (98.7 ? 3 and 95.8 ? 0.6%, respectively; Fig. 8, bottom)
upon peptide stimulation. Similar results were obtained upon stim-
ulation with PHA (data not shown).
IL-10 expression by MACS-purified CD4?CD25?T cells
expanded by rapamycin
To distinguish nTreg cells from IL-10-secreting Treg cells (9),
intracellular IL-10 expression was determined in both fresh
CD4?CD25?and in vitro-expanded R0 and R1 T cells after stim-
ulation with LPS. Neither fresh CD4?CD25?T cells nor R0 and
R1 T cells after in vitro expansion expressed IL-10 as measured by
flow cytometry (data not shown).
TCRV? repertoire of MACS-purified CD4?CD25?T cells
expanded by rapamycin
TCRV? chain repertoire of R0 and R1 T cells was determined
using V? chain-specific Abs in a flow cytometry-based assay. We
or absence of rapamycin does not induce Treg. Freshly MACS-purified
CD4?CD25?or CD8?CD25?T cells were labeled with CFSE and cul-
tured with OKT (1 ?g/ml) and anti-CD28 mAb (1 ?g/ml) in the presence
of IL-2 for 5 days. Allogeneic CD4?CD25?T cells were first cultured in
the presence of PHA and irradiated APC for 7 days or CD3/CD28 beads
and 1000 IU/ml IL-2 for 3 wk ? rapamycin. Next, these cells were added
at the 1:1 ratio to the CFSE-labeled responder cells and the cocultures
incubated for 5 days. The observed percentage of suppression in these
cocultures was very low (5–12%). One representative experiment (for ra-
pamycin-expanded CD4?CD25?cells) of five performed is shown.
Incubation of purified CD4?CD25?T cells in the presence
CD4?CD25?T cells cultured ? rapamycin. Top, Autologous R1 and R0
T cells (after 3 wk of expansion with CD3/CD28 beads and 1000 IU/ml
IL-2) were coincubated at the 1:1 ratio with CD4?CD25?TT947–967pep-
tide-specific polyclonal T cell line ZH-P1 for 16 h. IFN-? release was
analyzed in an IFN-? ELISPOT assay using HLA-DP*0401-positive B-
LCL cell lines pulsed with a relevant (TT947–967) peptide or an irrelevant
(Mage-A3247–258) peptide as stimulators (top). Bottom, Autologous R1 and
R0 T cells (after 3 wk of expansion with CD3/CD28 beads and 1000 IU/ml
IL-2) were coincubated at the 1:1 ratio with CD8?CD25?Melan-A26–35-
specific T cell clone ZH-P7/2 for 16 h. IFN-? release was analyzed in an
IFN-? ELISPOT assay using HLA-A*0201-positive B-LCL cell lines
pulsed with a relevant peptide (Melan-A26–35) or an irrelevant (HIV-
POL476–484) peptide as stimulators (bottom). The data are numbers of
spots/5 ? 103cells/well in 24-h ELISPOT assays. The percentages of
inhibition in the number of IFN-?-secreting positive cells by the R0 and R1
T cells are indicated in top and bottom. The data are from one represen-
tative experiment of three performed with PBMC of one HLA-DP*0401?
Suppression of IFN-y secretion by purified human
327The Journal of Immunology
found that R1 T cells after a 3-wk expansion displayed a poly-
clonal TCRV? chain repertoire, comparable to CD4?CD25?T
cells of fresh PBMC and to 3 wk-expanded R0 T cells of the same
donor (data not shown).
We demonstrate here that human CD4?CD25highFoxp3?nTreg
cells upon stimulation via TCR and CD28 or by IL-2 undergo
rapid and robust expansion when rapamycin is added. Purified
CD4?CD25?T cells exposed to rapamycin: 1) exhibit a strong
proliferative capacity; 2) show a Foxp3?GITR?CTLA?4?phe-
notype; 3) have a potential for homing to lymph node (LN) as
indicated by CD62L and CCR7 expression; 4) mediate potent sup-
pressive activities against syngeneic or allogeneic CD4?and
CD8?T cells, T cell clones or populations with defined antigenic
specificity as well as polyclonal T cells with various antigenic
specificities. In contrast, purified CD4?CD25?T cells cultured in
the absence of rapamycin (R0) are mainly composed of nonsup-
pressor T cells with a CD25lowphenotype. The ex vivo use of
rapamycin as outlined here should open the avenue for the in vitro
generation of highly efficient nTreg cell populations in humans.
In the last few years, several strategies have been developed for
long-term culture and polyclonal expansion of human nTreg cells
(29–31). It has been demonstrated that the combined stimulation
via TCR, CD28, and IL-2R is required to transiently induce human
Treg proliferation and this strategy is especially effective with anti-
CD3/CD28 beads or artificial APC (29–31). MACS-based purifi-
cation has previously been used to obtain enriched CD4?CD25?
T cells for in vitro expansion. However, though straightforward,
this method results in a considerable contamination with conven-
tional CD4?CD25lowT cells. Consequently, the addition of high
doses of IL-2 may promote an overgrowth of cells with modest or
no suppressor activity. Thus, Battaglia et al. (25) showed that cul-
tured murine CD4?CD25?T cells not exposed to rapamycin lose
their suppressive function. Others, using sorting for selection of
only CD4?CD25highcells, were able to generate suppressor cell
lines from adult blood (32). However, as previously noted, even
sorted populations may contain a mix of conventional and Treg
cells (32), and relatively few CD25highT cells are recovered by
this technique.In contrast,
CD4?CD25?T cells followed by a large-scale expansion in the
presence of rapamycin yields a population of cells with a defined
phenotype and strong suppressive function.
Our data suggest that in the presence of rapamycin (1 nM),
human Treg subsets present in the peripheral blood survive and
expand, while the other T cells (i.e., CD4?CD25low) are inhibited
from proliferation. We demonstrated that fresh or activated
CD4?CD25?T cells exposed to rapamycin (1 nM) in the presence
of CD3/CD28 beads and 1000 IU/ml IL-2 do not proliferate, com-
pared with CD4?CD25?T cells cultured under the same condi-
tions but in the absence of rapamycin. In contrast, rapamycin-
exposed human CD4?CD25highT cells vigorously expand for at
least 3 wk in culture. Rapamycin also enhances PHA-stimulated
expansion of Treg cells. We hypothesize that this expansion of
CD4?CD25highT cells results from rapamycin-driven selection of
T cells at a unique state of activation, with rapamycin-induced
apoptosis of CD4?CD25?T cells. Sensitivity of these T cells to
apoptosis, as indicated by ANX V binding, and resistance to ap-
optosis of fresh as well as rapamycin-expanded CD4?CD25highT
cells, suggest that the mechanism of rapamycin-based selection
involves the death pathway. It is also noteworthy that rapamycin
concentrations higher than 10 nM significantly decreased expan-
sion rates of CD4?CD25highT cells, while doses lower than 1 nM
were not effective in inducing their expansion in our hands (data
MACS separationof human
not shown). This indicates the existence of a threshold for rapa-
mycin-mediated responses that could be related to the activation
state of CD4?CD25?T cells. Importantly, Treg cultured in the
presence of IL-2 without rapamycin do not mediate suppression,
while RT cells are highly suppressive (Fig. 6). In contrast to the
findings reported by Valmori et al. (33), we demonstrate that the
suppressive function of R1 T cells is maintained after rapamycin
withdrawal (data not shown).
Phenotypic characteristics of RT cells are qualitatively similar
to those of freshly isolated CD4?CD25highT cells. We show here
that rapamycin-expanded nTreg, like fresh nTreg, show high ex-
pression levels of CD25, Foxp3, CD62L, CD45RO, GITR,
CTLA-4, CCR-7, Fas, and HLA-DR. In contrast, CD4?CD25?T
cells expanded in the absence of rapamycin express significantly
lower levels of these markers. Furthermore, CD4?CD25?and
CD4?CD25lowT cells expanded in the presence of rapamycin
showed low marker expression (Fig. 5C). This observation sug-
gests that rapamycinpromotes
CD4?CD25highT cells that constitutively express high levels of
the markers characterizing the suppressor phenotype. Furthermore,
RT cells displayed a broad TCRV? usage similar to that of
CD4?CD25?from fresh PBMC. As this TCRV? chain repertoire
remained virtually unchanged after in vitro expansion in the pres-
ence of rapamycin, it appears that RT cells represent a polyclonal
expansion of T cells originating from precursors present in the
RT cells expressed significantly higher levels of CD95 (Apo-1/
Fas) molecules on the surface as compared with R0 T cells. Upon
TCR stimulation of CD4?CD25highcells or in RT cells, we ob-
served no significant activation-induced cell death in agreement
with recent findings (34), suggesting that these T cells were resis-
tant to apoptosis despite high levels of CD95 expression. In con-
CD4?CD25?T cell cultures, as shown by ANX V binding, while
expanded RT cells remained alive. Similar apoptosis-mediated de-
pletion of activated T cells and, consequently, an increase in
CD4?CD25?T cells upon rapamycin treatment has been de-
scribed in allotransplant and autoimmune animal models (21, 35).
It is believed that successful expansion of Treg is a prerequisite
for future adoptive transfers of these cells in human diseases. In
transplantation or autoimmunity, expansion and transfers of Treg
might be beneficial, while in cancer, their attenuation could allow
for recovery of antitumor immune responses. After 1–3 wk of ex-
pansion, T cells exposed to rapamycin (RT cells) represented a
pure population of CD4?CD25highFoxp3?T cells (?98%). Fur-
ther phenotypic analysis showed that a considerable fraction ex-
pressed CD62L and CCR7, two receptors also present on T cells
and some subsets of memory T cells and considered to be respon-
sible for lymphocyte homing to secondary lymphoid organs (36).
Indeed, accumulating data in animal models systems of autoim-
munity or allotransplantation suggest that the LN is a critical site
for tolerance induction (36, 37). The phenotype of human RT cells
suggests that these cells could migrate to secondary lymphoid or-
gans and to exert suppressive function in a site-specific manner.
Although our results indicate that rapamycin can be used for the
expansion of Treg cells with a defined phenotype and suppressive
functions from the peripheral blood of normal volunteers, in vitro
expansion of Treg from blood of subjects with disease may not be
equally feasible. In patients with autoimmune diseases, these cells
might be less responsive or present in fewer numbers (38, 39). We
are in the process of developing a protocol for a large-scale ex-
pansion of human Treg with rapamycin, and our preliminary data
indicate that a 105expansion index after 3 wk of culture is achiev-
able with peripheral blood-derived CD4?CD25?T cells of normal
survivalofa subset of
apoptosis in the
328 PHENOTYPE AND FUNCTION OF HUMAN RAPAMYCIN-INDUCED nTregs
donors. Also, it will be of interest to expand nTreg cell subsets
isolated from cancer patients, especially patients with leukemia
treated with hemopoietic stem cell transplantation. A possibility of
therapy with expanded Treg to ameliorate graft-vs-host disease has
been considered (40). In addition to therapeutic use, the availabil-
ity of purified Treg in numbers sufficient for detailed phenotypic
and functional analysis of these cells and understanding of their
mode of action or Ag specificity is scientifically important.
In summary, we have shown that human polyclonal CD4?
CD25?nTreg cells capable of mediating potent suppression sur-
vive and readily proliferate upon exposure to rapamycin. Ex-
panded CD4?CD25highnTreg cells obtained from the peripheral
circulation of normal donors show a “characteristic” suppressor
phenotype and maintain expression of LN homing receptors. The
data presented here may considerably accelerate the development
of immunotherapeutic approaches for the treatment of autoimmune
diseases or posttransplant alloreactions by the adoptive transfer of
We thank Dr. Immanuel Luescher for multimer synthesis and Conny
Schneider and Heidi Mattlin for technical assistance.
The authors have no financial conflict of interest.
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329The Journal of Immunology