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IMMUNOBIOLOGY
From the Departments of Immunology and Paediatrics,
Hammersmith Campus, Imperial College School of Medicine, London, United
Kingdom; and Department of Rheumatology and Molecular Medicine,
University of Leeds, United Kingdom.
Despite thymic deletion of cells with specificity for
self-antigens, autoreactive T cells are readily detectable in the
normal T-cell repertoire. In recent years, a population of
CD4+ T cells that constitutively express the interleukin-2
receptor- Immune tolerance to self is essential to prevent
autoimmunity. Clonal elimination of autoreactive T cells in the thymus
is effective but incomplete and, consequently, peripheral mechanisms of
tolerance such as anergy, immune deviation, deletion, and
regulation/suppression are important.
A role for immunoregulatory T cells in the maintenance of
self-tolerance was first suggested in the 1970s, but a succession of in
vitro artifacts cast doubts upon the experimental data. However, more
recent evidence from rodent models has implicated regulatory T cells in
transplantation tolerance1-4 and the prevention of
autoimmune disease.5-11 The phenotype and mechanisms of
action of such cells are only partially defined. Two major cell types have been described: cytokine-secreting Th2, Th3, and Tr1 cells, which
predominantly release interleukin-4 (IL-4), IL-10, and transforming growth factor Spontaneously occurring populations of T cells that regulate
autoimmune inflammation have been described in animals. One such population is the CD4+CD25+ subset found in
normal mice.8,10 These cells resemble anergic cells in
vitro and can suppress the responses of
CD4+CD25 The existence of immunoregulatory cells in human self-tolerance,
particularly in the pathogenesis of autoimmune diseases, is poorly
understood. This study examined the possibility that a spontaneously
arising population of T cells with regulatory properties existed in
man. Such a population was found in both adults and newborn infants,
and the phenotype, function, and mechanism of action of these cells
were explored.
Subjects
Culture media, reagents, and antibodies
CTLL-2 cells CTLL-2 cells are a murine cell line that responds to murine IL-2 and IL-4 but only to human IL-2. Cells were maintained in culture in supplemented RPMI medium with 10% fetal calf serum (Biowhittaker, Wokingham, United Kingdom) and human recombinant (rh)-IL-2 (10 U/mL, Boehringer Mannheim, Mannheim, Germany). They were subcultured every 2 to 3 days. Cells were rested in medium without IL-2 overnight prior to use in assays.CD4+CD25+ and
CD4+CD25 
cells were obtained by negative selection (flow through), and CD4+CD25+ cells were positively selected
(eluted column-retained cells). In some experiments, CD4+
cells were purified by flow cytometry using biotinylated anti-CD25 (7G7) antibodies followed by streptavidin-PE and FITC-conjugated anti-CD4 monoclonal antibodies (catalog No. 340133, Becton Dickinson) on a FACStar cell sorter (Becton Dickinson). The purity of the cell
population was measured by flow cytometry and was between 90% to 95%
for CD4+CD25 cells and between 80% to 90%
for CD4+CD25+ cells. Cord blood mononuclear
cells were isolated by density gradient centrifugation over Lymphoprep.
Red blood cells and reticulocytes were removed by incubating cord blood
mononuclear cells with mouse anti-human glycophorin A followed by
incubation with goat anti-mouse immunoglobulin G magnetic particles
(BioMag, Polysciences, Warrington, United Kingdom). Cord blood
CD4+ T cells and CD4+CD25 cells
were isolated as described above.
Accessory cells Irradiated (30 Gy) T-cell-depleted PBMCs were used as accessory cells (ACs). PBMCs were incubated with anti-CD3 antibodies (OKT3) followed by magnetic bead separation (Dynal) to remove T cells.Dendritic cells Human monocyte-derived dendritic cells were generated by culturing adherent cells from PBMCs in the presence of GM-CSF and IL-4. After 7 days, dendritic cells were harvested and were used in coculture experiments with CD4+CD25+ or CD4+CD25 cells. After coculture, dendritic
cells were isolated by removing CD4+ cells using magnetic
beads (Dynal).
Mixed lymphocyte reactions About 1 × 104 responder cells were cocultured with 5 × 104 stimulator cells per well in 96-well plates (final volume 200 µL) in the presence or absence of 30 units rh-IL-2, for 5 days. 3H-thymidine was added in the last 12 hours of the culture. The cells were harvested at the end of the culture, and 3H-thymidine incorporation was assessed by liquid scintillation spectrometry. All experiments were performed in triplicate.Proliferation assays About 1 × 104 to 2 × 104 responder cells (CD4+CD25 or
CD4+CD25 cells) were cocultured with
1 × 104 (or specified otherwise) ACs per well in 96-well
plates (final volume 200 µL/well). PHA, at a final concentration of 2 µg/mL, was added with or without 10 U/mL rh-IL-2. After 60 hours of
culture, 3H-thymidine was added and the cells were cultured
for a further 12 hours before 3H-thymidine incorporation
was assessed.
Transwell assays Transwells of pore size 0.4 µm were used (Costar, High Wycombe, United Kingdom); 1.5 × 105 CD4+CD25 cells per well were cultured in
24-well plates in the presence of ACs (1.5 × 105
cells/well) with 2 µg/mL PHA (referred to as lower wells). Equal numbers of CD4+CD25+ or
CD4+CD25 cells were added into either the
Transwells (referred to as upper wells) or the lower wells directly.
The Transwells were inserted onto the 24-well plate. ACs
(1.5 × 105 cells/well) were present in all upper wells.
After 60 hours of culture, the cells in the lower and upper wells were
harvested separately and transferred to 96-well plates.
3H-thymidine was added, and the cells were cultured for a
further 12 hours before the incorporation of 3H-thymidine
was measured.
Limiting dilution analyses Serial dilutions of responder cells in 24 replicates were cocultured with 5 × 104 irradiated stimulator cells per well in 96-well plates for 5 days. The exact number of responder cells depended on the number of responder cells obtained from each individual, with top dilutions ranging from 2 × 104 to 8 × 104 cells per well. Wells were scored positive if the counts were above 3 SD of the average count of the control (wells containing only irradiated stimulator cells). The frequency, confidence interval, and 2 value for each assay were calculated by
the maximum likelihood method using GLIM software (NAG, Oxford, United
Kingdom). For all data, a probability estimate of the data conforming
to single-hit kinetics was calculated.
Cytokine ELISA Supernatants were taken from cultures at 72 hours. Antibodies from clones 9D7, 9016.2, and 43-11 (Immunokontact, Witney, United Kingdom) were used as capture antibodies for detection of IL-10, TGF- 1, and interferon  (IFN-), respectively. The corresponding detection antibodies used in the assays are 12G8 (Pharmingen), anti-TGF- chicken immunoglobulin (R & D Systems), and 45-15 (Immunokontact).
Real-time polymerase chain reaction quantitation of mRNA CD4+CD25+ and CD4+CD25 cells were purified as mentioned
above. Cells were solubilized in a guanidinium buffer (6 M guanidinium thiocyanate, 0.1 mM citrate, 1% [vol/vol] sarcosyl, 0.4%
[vol/vol] -mercaptoethanol, 0.1 mM sodium acetate). Lysates were
subjected to a protein extraction using phenol, and RNA was recovered
by precipitation with isopropanol. Total RNA (1 µg) extracted from control or activated T cells was incubated with 1.5 µg
oligo-d(T)15 primer at 95°C for 15 minutes and then
cooled on ice for 5 minutes. A first-strand complementary DNA synthesis
was performed by adding 400 units Superscript II reverse transcriptase
(Gibco) containing RT buffer (25 mM Tris-HCl, pH 8.3; 37.5 mM KCl; 1.5 mM MgCl2), 10 mM dithiothreitol, and deoxyribonucleoside
triphosphate mixture (50 µM each) and incubating at 42°C for 90 minutes. The reaction was stopped, and secondary structures denatured,
by incubation at 75°C for 10 minutes. Real-time polymerase chain
reaction (PCR) was performed using an ABI7700 sequence detection system
(PE Applied Biosystems, London, United Kingdom) in the presence of
Sybro-green. This fluorochrome incorporates stoichiometrically
into the amplification product, providing real-time quantification of
the double-stranded DNA PCR product. Primers for each gene of interest
were designed for use under real-time PCR conditions, to amplify an
80- to 100-base pair fragment with 59°C annealing temperature
(Primer Express, PE Applied Biosystems). Because of the high homology
between members of each family, sequences targeted for primer design
were restricted to the lowest homolog regions. The optimization of the
real-time PCR reaction was then performed according to the
manufacturer's instructions. For each analysis, transcription of the
gene of interest was compared with transcription of the housekeeping
gene GAPDH, which was amplified in parallel.
A subset of CD4+ T cells constitutively expressing IL-2
receptor- T cells and 80% to
90% for CD4+CD25+ T cells (Figure
1A). In this study, we used peripheral
samples from adult healthy volunteers and routine venesection samples from primary polycythemic individuals who were screened to exclude white cell disorders.
CD4+CD25+ T cells are phenotypically distinct from recently activated CD4+ T cells that express CD25 Because CD25 is up-regulated during T-cell activation, it was important to determine whether the CD4+CD25+ cells isolated from peripheral blood of healthy individuals simply represented recently activated cells. We compared the surface expression of activation makers on CD4+CD25+ cells from peripheral blood with CD4+CD25+ cells obtained from PHA-stimulated PBMCs. We found that most CD4+CD25+ cells from peripheral blood expressed CD45RO, CD62 ligand (CD64L), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) but not CD69, CD40 ligand (CD40L) (Figure 1B). In contrast, CD4+ cells in which CD25 expression was induced by PHA stimulation exhibited high levels of CD69, and a much higher proportion of these cells were CD45RO, whereas the level of CD62L expression was similar
(Figure 1C).
CD4+CD25+ T cells are hyporesponsive to polyclonal T-cell stimuli in vitro Despite the constitutive expression of CD25, the proliferative responses of CD4+CD25+ T cells to PHA or soluble anti-CD3 in the presence of autologous ACs and to HLA-mismatched stimulator cells were considerably lower than those of CD4+CD25 T cells (Figure
2A-C). The
CD4+CD25+ cells also produced less IL-2 and
IFN- in response to these stimuli (Figure 2E,F). However, when IL-2
was added into the culture, the responsiveness of
CD4+CD25+ cells was restored (Figure
2A,D).
CD4+CD25+ T cells suppress the responses of
CD4+CD25 cells, the responses to PHA or
soluble anti-CD3 in the presence of autologous ACs were significantly
suppressed (Figure 3A,B) and the
production of IL-2 and IFN- was profoundly inhibited (Figure 3C,D).
The suppression occurred in a dose-dependent manner, and 50%
inhibition was observed at a ratio of 1:1
CD4+CD25+:CD4+CD25
cells (Figure 3E). Addition of IL-2 to the coculture abrogated this
suppression (Figure 3A).
The suppressive function of CD4+CD25+ cells cannot be explained by passive IL-2 consumption by these cells One mundane explanation for the suppressive property of CD4+CD25+ cells was that these cells competed with CD4+CD25 cells for growth factors such
as IL-2. To investigate this possibility, we measured the proliferation
of the IL-2-dependent murine CTLL-2 cells to a titration of
recombinant human IL-2 in the presence or absence of either
CD4+CD25+ or CD4+CD25
cells. Because neither CD4+CD25+ nor
CD4+CD25 cells proliferated in response to
rh-IL-2 alone, the proliferation measured in the assay can be
considered as solely contributed by the proliferation of CTLL-2 cells.
The proliferation of the CTLL-2 cells was unaffected by the presence of
CD4+CD25+ or CD4+CD25
cells (Figure 4A), suggesting that
neither cell population consumed a significant amount of IL-2. The
suppressive property of CD4+CD25+ cells is thus
unlikely to be the result of passive consumption of IL-2 by these
cells.
The suppressive function of CD4+CD25+ cells does not appear to be mediated by soluble factors To determine whether the suppressive function of CD4+CD25+ cells is mediated by secretion of soluble factors, a Transwell system was used. As shown in Figure 4C, if CD4+CD25+ cells were mixed with CD4+CD25 cells in the lower well, inhibition
of proliferation was observed. However, if the
CD4+CD25+ cells were placed in the upper well,
which also contained ACs, and were thus physically separated from the
CD4+CD25 cells by a semipermeable membrane,
no inhibition was observed, although the
CD4+CD25+ cells remained hyporesponsive (Figure
4D). These observations suggest that soluble factors do not play an
essential role in mediating the suppressive function of
CD4+CD25+ cells and that the suppression
requires cell-to-cell contact. Moreover, these data argue further
against IL-2 consumption being the mechanism of suppression.
The suppressive function of CD4+CD25+ cells
is not mediated by IL-10 or TGF- , were responsible for the suppressive effects of this T-cell population, the levels of these cytokines were measured in the supernatants of stimulated cultures. In only 1 of 3 experiments were
these cytokines detected and then only at very low concentrations (Figure 4B). To exclude the possibility that the ELISA assay was insufficiently sensitive to detect biologically active concentrations of TGF-, purified TGF- was titrated into cultures of
mitogen-stimulated CD4+CD25+ T cells to
determine the concentration required to reproduce the inhibition caused
by the CD25+ cells. This revealed that a concentration in
excess of 80 pg/mL was needed to see significant inhibition of T-cell
proliferation; the ELISA assay was able to detect concentrations as low
as 1.0 pg/mL (data not shown). Thus, it is unlikely that functionally significant amounts of TGF- were secreted by the CD25+
population. Furthermore, addition of neutralizing antibodies against
these 2 cytokines failed to reverse the suppression caused by the
CD25+ cells (Figure 4E) or reverse the hyporesponsiveness
(data not shown).
CD4+CD25+ T cells do not inhibit APC function One possible target of the suppressive effects of the CD4+CD25+ cells is the antigen-presenting cell (APC), as we have shown previously in the regulation mediated by anergic T cells.19 To address this possibility, we cultured premature monocyte-derived dendritic cells with either CD4+CD25+ cells or CD4+CD25 cells in the presence of PHA for 48 hours. Although dendritic cells that had been cocultured with
CD4+CD25+ cells expressed a lower level of CD80
and CD86 than those cocultured with CD4+CD25
cells, the levels of these molecules were similar to dendritic cells
that had been cocultured with medium only (Figure
5A). The expression of CD40 and HLA-DR
was comparable. Furthermore, dendritic cells that had been precultured
with CD4+CD25+ T cells were fully competent in
functional assays (data not shown). By exclusion, these findings
suggest that these regulatory cells act directly on neighboring T
cells.
A member of the Notch family and one of its ligands are overexpressed on CD4+CD25+ cells One candidate set of molecules in T-cell-mediated regulation is the Notch family of receptors and its ligands, which belong to the Jagged and Delta molecular families. Thus, overexpression of Jagged-1 in antigen-pulsed APCs induced tolerance in naive T cells, which could then be adoptively transferred.21 Additionally, certain family members were differentially transcribed when a human T-cell clone was anergized or activated (F.P. and J.I., unpublished observations, 2000). There are no staining reagents that reliably detect or block the various family members on living cells, and we therefore used real-time PCR quantitation of the corresponding transcripts in CD4+CD25+ and CD4+CD25 cells. This revealed that deltex, a
positive regulator of the Notch signaling pathway,22,23
was highly up-regulated in CD4+CD25+ cells
compared with CD4+CD25 cells. Furthermore,
upon stimulation, the transcription of Notch-4 and Delta-1 dramatically
increased only in CD4+CD25+ cells, concurrent
with a rise in Hes-1 transcript (Figure 5B). The expression of Notch-1,
Jagged-1, and Jagged-2 was similar among
CD4+CD25+ and
CD4+CD25 cells (data not shown). Hes-1 is a
downstream mediator of Notch pathway signaling,24 and
these data suggest that stimulation of
CD4+CD25+ cells effects a change in Notch
signaling, possibly via modulation of Notch-4. Additionally, the rise
in Delta-1 transcription raises the possibility that stimulated
CD4+CD25+ T cells become capable of
Notch-mediated T cell-to-T cell communication. Thus, when
antigen-specific T-cells were transfected with Delta-1, they were able
to inhibit the responses of antigen-experienced cells and became
capable of linked suppression.25 These observations suggest that Notch signaling contributes to the regulatory effects of
these cells.
The presence of CD4+CD25+ T cells is responsible for nonlinear kinetics in limiting dilution analyses of alloresponses We used limiting dilution assays to detect subpopulations of cells exhibiting multihit kinetics consistent with suppressive activity in coculture. Figure 6 shows a limiting dilution analysis plot of a mixed lymphocyte reaction using unfractionated CD4+, CD4+CD25, or
CD4+CD25+ cells as responders. A significant
deviation from single-hit kinetics was observed with unfractionated
CD4+ cells, whereas depletion of the
CD4+CD25+ population led to restoration of
single-hit kinetics and a significant increase in frequency. When
enriched CD4+CD25+ cells were used, multihit
kinetics was observed. These results suggest that the
CD4+CD25+ subpopulation contains cells with a
suppressive or regulatory function.
Cells with similar phenotype and function to adult CD4+CD25+ cells are found in umbilical venous blood from healthy newborn infants We observed that a proportion of CD4+ T cells from umbilical venous blood in newborn infants express CD25 (P.J.D. et al, unpublished observation, 2000). We tested whether this subpopulation of CD4+CD25+ cells showed similar phenotype and behavior to their adult counterparts. About 5% to 15% of CD4+ cells expressed CD25, and these had a similar phenotype to adult CD4+CD25+ cells: neonatal CD4+CD25+ cells expressed CD62L and CTLA4 but not CD69, CD40L. However, most neonatal cells expressed CD45RA, with a small percentage expressing CD45RO. Indeed, the CD4+CD45RO+ cells were confined to the CD4+CD25+ population (Figure 7A). Because of the relatively small volume of umbilical venous blood that could be collected, it was not possible to obtain sufficient numbers of CD4+CD25+ cells for detailed functional analysis. However, by examining the kinetics of limiting dilution analysis against allogeneic stimulator cells using either CD4+CD25 cells or unfractionated
CD4+ cells as responders, we again found that the
unfractionated CD4+ population exhibited significant
deviation from single-hit kinetics, and depletion of
CD4+CD25+ cells restored the single-hit
kinetics and led to an increase in frequency (Figure 7B). These
findings suggest that the CD4+CD25+ cells from
cord blood also possess regulatory function.
CD4+CD25+ cells retain their suppressive properties after in vitro expansion As described in Figures 2 and 3, the CD4+CD25+ cells could be driven to divide by stimulation in the presence of exogenous IL-2. Given that cell division can lead to recovery of responsiveness by T cells rendered anergic in vitro, we tested the suppressive activity of the CD4+CD25+ cells after 2 days of stimulation with coimmobilized anti-CD3 and anti-CD28 antibodies, followed by 2 days in rh-IL-2. As shown in Figure 8, the CD25+ cells retained their suppressive properties after in vitro expansion. In contrast, CD4+CD25
cells treated in exactly the same manner were not suppressive. These
data indicate that the CD4+CD25+ cell
population is a stable cell lineage that can be expanded while
retaining its regulatory function.
In this study, CD4+CD25+ T cells were
detected in peripheral blood samples from adult volunteers and in
umbilical venous blood taken from newborn infants. These cells had a
distinctive phenotype, expressing a mixture of markers of memory and
naive T cells, and caused substantial inhibition of proliferation and
cytokine secretion by CD4+CD25 The results cannot be attributed to the recent activation of
CD4+ T cells in vivo inducing expression of CD25. Indeed,
the similarity of results between healthy volunteers, individuals with
primary polycythemia, and newborns argues against an inflammatory
origin in any one of these groups. Individuals with primary
polycythemia were screened to exclude white cell disorders, and
neonatal samples were derived from infants delivered by elective
cesarean section, making it unlikely that parturition was responsible
for inducing this population of cells. Equally, these findings are
unlikely to be the result of an in vitro artifact because of the use of anti-CD25 monoclonal antibody in the positive selection process. The
clone of antibody that we employed has been shown not to inhibit anti-CD3-stimulated proliferation or IL-2 binding of T
cells.26,27 Furthermore, we have confirmed that the
addition of this antibody in concentrations up to 10 µg/mL did not
affect the proliferation of CD4+ T cells in response to PHA
in the presence of autologous ACs (data not shown). In addition, our
observation that PHA-stimulated CD4+ cells (that had
up-regulated CD25 expression) were phenotypically distinct from
naturally occurring CD4+CD25+ cells argues
further against these being recently activated CD4+ cells.
Indeed, in vitro stimulation and expansion of
CD4+CD25+ cells did not abolish their
suppressive phenotype, while similar treatment to
CD4+CD25 In the mouse, regulatory CD4+CD25+ cells appear
to arise in the thymus in that CD4+CD25+
thymocytes behave similarly to mature peripheral
CD4+CD25+ T cells.28 In this
respect, it is interesting that 5% to 15% of the CD4+ T
cells from umbilical venous blood samples of normal-term infants express CD25. Neonatal CD4+CD25+ T cells have a
similar phenotype to their adult counterparts except that they more
frequently express CD45RA. However, the functional implication of this
differential expression of CD45 isoforms is unclear. In mice, both
CD45RBlo ("memory") and CD45RBhi
("naive") subsets of CD4+CD25+ cells
inhibit the proliferation of CD4+CD25 T-cell anergy has been implicated in the maintenance of peripheral
tolerance. Although anergic human T-cell clones have been generated by
a variety of methods in vitro, most of the in vivo evidence has come
from rodent models, and naturally occurring anergic T cells have not
previously been described in man. In this study, the behavior of
CD4+CD25+ cells isolated from peripheral blood
of healthy individuals was characteristic of anergic cells. To our
knowledge, this is the first set of data indicative of the existence of
naturally occurring anergic T cells in vivo. We have previously
demonstrated that human and murine anergic T-cell clones could inhibit
antigen-specific and allospecific T-cell proliferation17,19
and prolong skin allograft survival in vivo.31 Similarly,
CD4+CD25+ cells were also capable of inhibiting
the responses of CD4+CD25 The mechanism(s) of regulation effected by these cells remains to be
elucidated fully. From the results obtained here, it does not involve
known regulatory cytokines or passive consumption of IL-2 and appears
to require cell-to-cell contact. We have demonstrated previously that
anergic murine T-cell clones inhibit the responses of responsive T-cell
clones via inhibition of the APCs in a cognate manner.19
More recently, Cederbom and colleagues32 demonstrated that
murine CD4+CD25+ T cells down-regulate the
expression of CD80 and CD86 on dendritic cells. However, in this study,
although CD4+CD25+ cells were less efficient in
inducing the up-regulation of costimulation molecules CD80 and CD86 on
monocyte-derived dendritic cells compared with
CD4+CD25 It has been suggested recently that Notch and its ligands may be
important in the induction and maintenance of tolerance. Delta-1
expression is increased on peripheral T cells during the induction of
tolerance with high-dose peptide delivered intranasally. Furthermore,
antigen-specific CD4+ T cells transfected with Delta-1
inhibited the response of antigen-primed T cells and induced linked
suppression.25 Additionally, overexpression of human
Jagged-1, a Notch ligand, on murine APCs induces naive peripheral
CD4+ T cells to become regulatory cells and transfer
antigen-specific tolerance to recipient mice.21 We have
also observed the up-regulation of these family member genes in an
anergic human T-cell clone (F.P. and J.I, unpublished observations,
2000). In this respect, it is interesting that we found
differential transcription of deltex, which facilitates Notch signaling
in CD4+CD25+ T cells. Furthermore, following
stimulation with anti-CD3 and anti-CD28, Notch-4 and Delta-1
transcriptions were up-regulated, along with Hes-1, a downstream
mediator of Notch signaling. These data imply a role of Notch and its
ligands in the development or maintenance of the regulatory function of
CD4+CD25+ cells. Currently, blocking studies
are not possible in that no antibodies are available to detect or
inhibit the interactions between Notch:Notch ligand family members,
possibly reflecting the high degree of conservation of these protein
between species. Another molecule that is expressed on
CD4+CD25+ cells, but not
CD4+CD25 Some of the most persuasive evidence for the existence of
"suppressor" cells has come from limiting dilution
analysis.33-35 A zigzag appearance of the semilogarithmic
plot has been attributed to the presence of more than one responder
cell type, one of which has regulatory effects. More recently, Dozmorov
and colleagues36,37 proposed a 2-cell subtype model to
explain these observations. The first subtype (LPC1) exhibited
single-hit kinetics and was responsive; the second subset (LPC2)
exhibited multihit kinetics and had the ability to inhibit the
responses of the first subset. In their studies using murine
CD4+ T cells, they found that the "suppressor" subset
was contained within the "memory" CD4+ T-cell
population and addition of IL-2 or neutralizing antibodies to IL-10
restored single-hit kinetics.38 In keeping with these findings, we showed that the human CD4+CD25+
cells exhibited multihit kinetics in response to alloantigens. In
contrast, the CD4+CD25 Taken together, our results demonstrate that a proportion of CD4+ T cells from adult peripheral blood and neonatal umbilical venous blood constitutively express CD25 and are distinct from recently activated CD4+ T cells. This subset of CD4+ T cells resembles anergic cells and possesses suppressive function in vitro. The suppression appears to be cell-to-cell contact dependent and may involve the Notch signaling between neighboring T cells. This subpopulation of CD4+ T cells has similar characteristics to CD4+CD25+ in mice, which are critical in the maintenance of self-tolerance and the prevention of autoimmune disease. Human CD4+CD25+ cells may have an equally important role in the regulation of autoimmunity and transplant tolerance.
We thank Drs Andrew George and Hans Stauss for critical review of the manuscript, Drs Ragnar Lindstedt, Silvia Vendetti, and Juo-Guan Chai for helpful discussions, Sue Douglas and Gary Warns for technical assistance. We also thank the nursing staff on the Haematology Day Unit of Hammersmith Hospital for arranging the collection of blood samples. We are grateful for the financial support of the Wellcome Trust, Garfield Weston Foundation, and Action Research and Wellbeing.
Submitted March 27, 2001; accepted April 24, 2001.
Supported by the Wellcome Trust, Garfield Weston Foundation, and Action Research and Wellbeing. W.F.N. is a recipient of a Wellcome Trust training fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert I. Lechler, Dept of Immunology, Hammersmith Campus, Imperial College School of Medicine, Du Cane Rd, London W12 0NN, United Kingdom; e-mail: r.lechler{at}ic.ac.uk.
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H. J. P. M. Koenen, E. Fasse, and I. Joosten CD27/CFSE-Based Ex Vivo Selection of Highly Suppressive Alloantigen-Specific Human Regulatory T Cells J. Immunol., June 15, 2005; 174(12): 7573 - 7583. [Abstract] [Full Text] [PDF]
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G. Darrasse-Jeze, G. Marodon, B. L. Salomon, M. Catala, and D. Klatzmann Ontogeny of CD4+CD25+ regulatory/suppressor T cells in human fetuses Blood, June 15, 2005; 105(12): 4715 - 4721. [Abstract] [Full Text] [PDF]
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T. M. Brusko, C. H. Wasserfall, A. Agarwal, M. H. Kapturczak, and M. A. Atkinson An Integral Role for Heme Oxygenase-1 and Carbon Monoxide in Maintaining Peripheral Tolerance by CD4+CD25+ Regulatory T Cells J. Immunol., May 1, 2005; 174(9): 5181 - 5186. [Abstract] [Full Text] [PDF]
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T. Bonnefoix, P. Bonnefoix, P. Perron, J.-Q. Mi, W. F. Ng, R. Lechler, J.-C. Bensa, J.-Y. Cahn, and D. Leroux Quantitating Effector and Regulatory T Lymphocytes in Immune Responses by Limiting Dilution Analysis Modeling J. Immunol., March 15, 2005; 174(6): 3421 - 3431. [Abstract] [Full Text] [PDF]
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C. Y. Liu, M. Battaglia, S. H. Lee, Q.-H. Sun, R. H. Aster, and G. P. Visentin Platelet Factor 4 Differentially Modulates CD4+CD25+ (Regulatory) versus CD4+CD25- (Nonregulatory) T Cells J. Immunol., March 1, 2005; 174(5): 2680 - 2686. [Abstract] [Full Text] [PDF]
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A. Balandina, S. Lecart, P. Dartevelle, A. Saoudi, and S. Berrih-Aknin Functional defect of regulatory CD4+CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis Blood, January 15, 2005; 105(2): 735 - 741. [Abstract] [Full Text] [PDF]
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W. R. Godfrey, D. J. Spoden, Y. G. Ge, S. R. Baker, B. Liu, B. L. Levine, C. H. June, B. R. Blazar, and S. B. Porter Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function Blood, January 15, 2005; 105(2): 750 - 758. [Abstract] [Full Text] [PDF]
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S. Lindley, C. M. Dayan, A. Bishop, B. O. Roep, M. Peakman, and T. I.M. Tree Defective Suppressor Function in CD4+CD25+ T-Cells From Patients With Type 1 Diabetes Diabetes, January 1, 2005; 54(1): 92 - 99. [Abstract] [Full Text] [PDF]
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E. A. Moseman, X. Liang, A. J. Dawson, A. Panoskaltsis-Mortari, A. M. Krieg, Y.-J. Liu, B. R. Blazar, and W. Chen Human Plasmacytoid Dendritic Cells Activated by CpG Oligodeoxynucleotides Induce the Generation of CD4+CD25+ Regulatory T Cells J. Immunol., October 1, 2004; 173(7): 4433 - 4442. [Abstract] [Full Text] [PDF]
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C. A. Thornton, J. W. Upham, M. E. Wikstrom, B. J. Holt, G. P. White, M. J. Sharp, P. D. Sly, and P. G. Holt Functional Maturation of CD4+CD25+CTLA4+CD45RA+ T Regulatory Cells in Human Neonatal T Cell Responses to Environmental Antigens/Allergens J. Immunol., September 1, 2004; 173(5): 3084 - 3092. [Abstract] [Full Text] [PDF]
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W. R. Godfrey, Y. G. Ge, D. J. Spoden, B. L. Levine, C. H. June, B. R. Blazar, and S. B. Porter In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures Blood, July 15, 2004; 104(2): 453 - 461. [Abstract] [Full Text] [PDF]
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O. Boyer, D. Saadoun, J. Abriol, M. Dodille, J.-C. Piette, P. Cacoub, and D. Klatzmann CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis Blood, May 1, 2004; 103(9): 3428 - 3430. [Abstract] [Full Text] [PDF]
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N. Misra, J. Bayry, S. Lacroix-Desmazes, M. D. Kazatchkine, and S. V. Kaveri Cutting Edge: Human CD4+CD25+ T Cells Restrain the Maturation and Antigen-Presenting Function of Dendritic Cells J. Immunol., April 15, 2004; 172(8): 4676 - 4680. [Abstract] [Full Text] [PDF]
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F. J. Clark, R. Gregg, K. Piper, D. Dunnion, L. Freeman, M. Griffiths, G. Begum, P. Mahendra, C. Craddock, P. Moss, et al. Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4+CD25high regulatory T cells Blood, March 15, 2004; 103(6): 2410 - 2416. [Abstract] [Full Text] [PDF]
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E. M. Aandahl, J. Michaelsson, W. J. Moretto, F. M. Hecht, and D. F. Nixon Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens J. Virol., March 1, 2004; 78(5): 2454 - 2459. [Abstract] [Full Text] [PDF]
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L. A. Stephens, A. N. Barclay, and D. Mason Phenotypic characterization of regulatory CD4+CD25+ T cells in rats Int. Immunol., February 1, 2004; 16(2): 365 - 375. [Abstract] [Full Text] [PDF]
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M. Stanzani, S. L. R. Martins, R. M. Saliba, L. S. St. John, S. Bryan, D. Couriel, J. McMannis, R. E. Champlin, J. J. Molldrem, and K. V. Komanduri CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans Blood, February 1, 2004; 103(3): 1140 - 1146. [Abstract] [Full Text] [PDF]
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H. Jonuleit and E. Schmitt The Regulatory T Cell Family: Distinct Subsets and their Interrelations J. Immunol., December 15, 2003; 171(12): 6323 - 6327. [Full Text] [PDF]
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H. J. P. M. Koenen, E. Fasse, and I. Joosten IL-15 and Cognate Antigen Successfully Expand De Novo-Induced Human Antigen-Specific Regulatory CD4+ T Cells That Require Antigen-Specific Activation for Suppression J. Immunol., December 15, 2003; 171(12): 6431 - 6441. [Abstract] [Full Text] [PDF]
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G. F. Hoyne Notch signaling in the immune system J. Leukoc. Biol., December 1, 2003; 74(6): 971 - 981. [Abstract] [Full Text]
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T. Keler, E. Halk, L. Vitale, T. O'Neill, D. Blanset, S. Lee, M. Srinivasan, R. F. Graziano, T. Davis, N. Lonberg, et al. Activity and Safety of CTLA-4 Blockade Combined with Vaccines in Cynomolgus Macaques J. Immunol., December 1, 2003; 171(11): 6251 - 6259. [Abstract] [Full Text] [PDF]
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A. Foussat, F. Cottrez, V. Brun, N. Fournier, J.-P. Breittmayer, and H. Groux A Comparative Study between T Regulatory Type 1 and CD4+CD25+ T Cells in the Control of Inflammation J. Immunol., November 15, 2003; 171(10): 5018 - 5026. [Abstract] [Full Text] [PDF]
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E. S. Yvon, S. Vigouroux, R. F. Rousseau, E. Biagi, P. Amrolia, G. Dotti, H.-J. Wagner, and M. K. Brenner Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells Blood, November 15, 2003; 102(10): 3815 - 3821. [Abstract] [Full Text] [PDF]
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E. Anastasi, A. F. Campese, D. Bellavia, A. Bulotta, A. Balestri, M. Pascucci, S. Checquolo, R. Gradini, U. Lendahl, L. Frati, et al. Expression of Activated Notch3 in Transgenic Mice Enhances Generation of T Regulatory Cells and Protects against Experimental Autoimmune Diabetes J. Immunol., November 1, 2003; 171(9): 4504 - 4511. [Abstract] [Full Text] [PDF]
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S. Vigouroux, E. Yvon, H.-J. Wagner, E. Biagi, G. Dotti, U. Sili, C. Lira, C. M. Rooney, and M. K. Brenner Induction of Antigen-Specific Regulatory T Cells following Overexpression of a Notch Ligand by Human B Lymphocytes J. Virol., October 15, 2003; 77(20): 10872 - 10880. [Abstract] [Full Text] [PDF]
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S. Jiang, N. Camara, G. Lombardi, and R. I. Lechler Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo Blood, September 15, 2003; 102(6): 2180 - 2186. [Abstract] [Full Text] [PDF]
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 N. Ardjomand, J. C. McAlister, N. J. Rogers, P. H. Tan, A. J. T. George, and D. F. P. Larkin Modulation of Costimulation by CD28 and CD154 Alters the Kinetics and Cellular Characteristics of Corneal Allograft Rejection Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3899 - 3905. [Abstract] [Full Text] [PDF]
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 T. Azuma, T. Takahashi, A. Kunisato, T. Kitamura, and H. Hirai Human CD4+ CD25+ Regulatory T Cells Suppress NKT Cell Functions Cancer Res., August 1, 2003; 63(15): 4516 - 4520. [Abstract] [Full Text] [PDF]
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A. Hug, M. Korporal, I. Schroder, J. Haas, K. Glatz, B. Storch-Hagenlocher, and B. Wildemann Thymic Export Function and T Cell Homeostasis in Patients with Relapsing Remitting Multiple Sclerosis J. Immunol., July 1, 2003; 171(1): 432 - 437. [Abstract] [Full Text] [PDF]
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C. P. Gray, P. Arosio, and P. Hersey Association of Increased Levels of Heavy-Chain Ferritin with Increased CD4+ CD25+ Regulatory T-Cell Levels in Patients with Melanoma Clin. Cancer Res., July 1, 2003; 9(7): 2551 - 2559. [Abstract] [Full Text] [PDF]
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C. Pridgeon, G. P. Lennon, L. Pazmany, R. N. Thompson, S. E. Christmas, and R. J. Moots Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright,CD94bright,CD158negative phenotype Rheumatology, July 1, 2003; 42(7): 870 - 878. [Abstract] [Full Text] [PDF]
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 A. D. Salama, N. Najafian, M. R. Clarkson, W. E. Harmon, and M. H. Sayegh Regulatory CD25+ T Cells in Human Kidney Transplant Recipients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1643 - 1651. [Abstract] [Full Text] [PDF]
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D. S. Game, M. P. Hernandez-Fuentes, A. N. Chaudhry, and R. I. Lechler CD4+CD25+ Regulatory T Cells Do Not Significantly Contribute to Direct Pathway Hyporesponsiveness in Stable Renal Transplant Patients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1652 - 1661. [Abstract] [Full Text] [PDF]
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 A. Lundgren, E. Suri-Payer, K. Enarsson, A.-M. Svennerholm, and B. S. Lundin Helicobacterpylori-Specific CD4+ CD25high Regulatory T Cells Suppress Memory T-Cell Responses to H. pylori in Infected Individuals Infect. Immun., April 1, 2003; 71(4): 1755 - 1762. [Abstract] [Full Text]
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A. M. Wolf, D. Wolf, M. Steurer, G. Gastl, E. Gunsilius, and B. Grubeck-Loebenstein Increase of Regulatory T Cells in the Peripheral Blood of Cancer Patients Clin. Cancer Res., February 1, 2003; 9(2): 606 - 612. [Abstract] [Full Text] [PDF]
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W. F. Ng, M. Hernandez-Fuentes, R. Baker, A. Chaudhry, and R. I. Lechler Reversibility with Interleukin-2 Suggests that T Cell Anergy Contributes to Donor-Specific Hyporesponsiveness in Renal Transplant Patients J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2983 - 2989. [Abstract] [Full Text] [PDF]
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M. K. Levings, R. Sangregorio, C. Sartirana, A. L. Moschin, M. Battaglia, P. C. Orban, and M.-G. Roncarolo Human CD25+CD4+ T Suppressor Cell Clones Produce Transforming Growth Factor {beta}, but not Interleukin 10, and Are Distinct from Type 1 T Regulatory Cells J. Exp. Med., November 18, 2002; 196(10): 1335 - 1346. [Abstract] [Full Text] [PDF]
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K. Namba, N. Kitaichi, T. Nishida, and A. W. Taylor Induction of regulatory T cells by the immunomodulating cytokines {alpha}-melanocyte-stimulating hormone and transforming growth factor-{beta}2 J. Leukoc. Biol., November 1, 2002; 72(5): 946 - 952. [Abstract] [Full Text] [PDF]
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F. Annunziato, L. Cosmi, F. Liotta, E. Lazzeri, R. Manetti, V. Vanini, P. Romagnani, E. Maggi, and S. Romagnani Phenotype, Localization, and Mechanism of Suppression of CD4+CD25+ Human Thymocytes J. Exp. Med., August 5, 2002; 196(3): 379 - 387. [Abstract] [Full Text] [PDF]
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P. Hoffmann, J. Ermann, M. Edinger, C. G. Fathman, and S. Strober Donor-type CD4+CD25+ Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation J. Exp. Med., August 5, 2002; 196(3): 389 - 399. [Abstract] [Full Text] [PDF]
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C. A. Piccirillo, J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura, H. Mizuhara, and E. M. Shevach CD4+CD25+ Regulatory T Cells Can Mediate Suppressor Function in the Absence of Transforming Growth Factor {beta}1 Production and Responsiveness J. Exp. Med., July 15, 2002; 196(2): 237 - 246. [Abstract] [Full Text] [PDF]
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D. Dieckmann, C. H. Bruett, H. Ploettner, M. B. Lutz, and G. Schuler Human CD4+CD25+ Regulatory, Contact-dependent T Cells Induce Interleukin 10-producing, Contact-independent Type 1-like Regulatory T Cells J. Exp. Med., July 15, 2002; 196(2): 247 - 253. [Abstract] [Full Text] [PDF]
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H. Jonuleit, E. Schmitt, H. Kakirman, M. Stassen, J. Knop, and A. H. Enk Infectious Tolerance: Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells J. Exp. Med., July 15, 2002; 196(2): 255 - 260. [Abstract] [Full Text] [PDF]
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M. Guimond, A. Balassy, M. Barrette, S. Brochu, C. Perreault, and D. C. Roy P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells Blood, June 28, 2002; 100(2): 375 - 382. [Abstract] [Full Text] [PDF]
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R. M. Steinman and M. C. Nussenzweig Inaugural Article: Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance PNAS, January 8, 2002; 99(1): 351 - 358. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
chain, CD25, has been shown to play a pivotal role in the
maintenance of self-tolerance in rodent models. This study investigated
whether such a regulatory population exists in humans. A
population of CD4+CD25+ T cells, taken from the
peripheral blood of healthy individuals and phenotypically
distinct from recently activated CD4+ T cells, was
characterized. These cells were hyporesponsive to conventional T-cell stimuli and capable of suppressing the responses of CD4+CD25
) and CD4+CD25
) cells to various
stimuli are shown. In each experiment, 1 × 104 to
2 × 104 responder cells were cultured with PHA (2 µg/mL) plus 1 × 104 irradiated autologous ACs with or
without IL-2 (10 U/mL, A); irradiated allogeneic PBMCs (B); soluble
anti-CD3 with ACs (C); and soluble anti-CD3 with IL-2 (10 U/mL) in the
presence of ACs (D). Conc indicates concentration. (E-F) Production of
IL-2 (E) and IFN-
) (total
2 × 104 cells/well) were stimulated in the presence of
irradiated autologous ACs (1 × 104 cells/well) with PHA
(2 µg/mL, A) in the absence or presence of IL-2 (10 U/mL); soluble
anti-CD3 for 3 days (B). Proliferation was measured by
3H-thymidine incorporation.(C,D) Production of IL-2 (C)
and IFN-
) or CD4+CD25+ (1 × 104)
cells (
) or
CD4+CD25
) cells (2 × 104
cells/well) for 36 hours with 3H-thymidine added during the
last 12 hours of culture. The proliferation of
CD4+CD25+ or CD4+CD25
) or
CD4+CD25
cells and CD4(+)CD8(