Anti-TNF-alpha therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-beta.
ABSTRACT The induction of regulatory T (T reg) cells holds considerable potential as a treatment for autoimmune diseases. We have previously shown that CD4+CD25hi T reg cells isolated from patients with active rheumatoid arthritis (RA) have a defect in their ability to suppress proinflammatory cytokine production by CD4+CD25- [corrected] T cells. This defect, however, was overcome after anti-tumor necrosis factor (TNF)-alpha antibody (infliximab) therapy. Here, we demonstrate that infliximab therapy gives rise to a CD4+CD25hiFoxP3+ T reg cell population, which mediates suppression via transforming growth factor (TGF)-beta and interleukin 10, and lacks CD62L expression, thereby distinguishing this T reg cell subset from natural T reg cells present in healthy individuals and patients with active RA. In vitro, infliximab induced the differentiation of CD62L- T reg cells from CD4+CD25- T cells isolated from active RA patients, a process dependent on TGF-beta. In spite of the potent suppressor capacity displayed by this CD62L- T reg cell population, the natural CD62L+ T reg cells remained defective in infliximab-treated patients. These results suggest that anti-TNF-alpha therapy in RA patients generates a newly differentiated population of T reg cells, which compensates for the defective natural T reg cells. Therefore, manipulation of a proinflammatory environment could represent a therapeutic strategy for the induction of T reg cells and the restoration of tolerance.
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ABSTRACT: Current rheumatoid arthritis (RA) therapies such as biologics inhibiting pathogenic cytokines substantially delay RA progression. However, patient responses to these agents are not always complete and long lasting. This study explored whether substance P (SP), an 11 amino acids long endogenous neuropeptide with the novel ability to mobilize mesenchymal stem cells (MSC) and modulate injury-mediated inflammation, can inhibit RA progression. SP efficacy was evaluated by paw swelling, clinical arthritis scoring, radiological analysis, histological analysis of cartilage destruction, and blood levels of tumor necrosis factor-alpha (TNF-α||, interleukin (IL)-10, and IL-17 in vivo. SP treatment significantly reduced local inflammatory signs, mean arthritis scores, degradation of joint cartilage, and invasion of inflammatory cells into the synovial tissues. Moreover, the SP treatment markedly reduced the size of spleens enlarged by excessive inflammation in CIA, increased IL-10 levels, and decreased TNF-αand IL-17 levels. Mobilization of stem cells and induction of Treg and M2 type macrophages in the circulation were also increased by the SP treatment. These effect of SP might be associated with the suppression of inflammatory responses in RA and, furthermore, blockade of RA progression. Our results propose SP as a potential therapeutic for autoimmune-related inflammatory diseases.Biochemical and Biophysical Research Communications 09/2014; · 2.28 Impact Factor
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ABSTRACT: TNFα plays an important role in autoimmune pathogenesis and is the main therapeutic target of rheumatoid arthritis. However, its underlying mechanism is not completely understood. In this study, we described that Th17 cells were accumulated in synovial fluid, which was attributable to TNFα aberrantly produced in rheumatoid synovium. Interestingly, TNFα cannot induce IL-17 production of CD4(+) T cells directly, but through the monocytes high levels of IL-1β and IL-6 in a TNFRI and TNFRII dependent manner from the active RA patients are produced. TNFα was shown to enhance the phosphorylation level of STAT3 and the expression level of transcription factor RORC of CD4(+) T cells when cultured with CD14(+) monocytes. Treatment with an approved TNFα blocking antibody showed marked reduction in the levels of IL-6, IL-1β, and IL-17 and the expression level of STAT3 phosphorylation in relation to Th17 cell differentiation in patients with rheumatoid arthritis. The study provides new evidence supporting the critical role of TNFα in the pathogenic Th17 cell differentiation in rheumatoid arthritis.Research Journal of Immunology 01/2014; 2014:385352.
The Journal of Experimental Medicine
BRIEF DEFINITIVE REPORT
JEM © The Rockefeller University Press $15.00
Vol. 204, No. 1, January 22, 2007 33–39 www.jem.org/cgi/doi/10.1084/jem.20061531
The numerous descriptions of regulatory T
(T reg) cells in both animal models and, more
recently, in humans have revealed a wide diver-
sity of populations and mechanisms of action.
Thymically derived CD4+CD25+ T reg cells
play a crucial role in the maintenance of self-
tolerance and the prevention of autoimmune
disease. This “natural” subset expresses the tran-
scription factor Foxp3, which has emerged as
an important functional marker of T reg cell
activity, supported by the fact that ectopic
Foxp3 expression is suffi cient to empower naive
T cells with a regulatory function (1). CD4+ T
reg cells can also be generated in the periphery
(adaptive), and many studies have identifi ed
diff erent subpopulations of CD4+ T cells based
on phenotypic and functional properties (2, 3).
Examples include IL-10–secreting Tr1 cells,
which can inhibit colitis in mice (4, 5), and
Th3 cells, which play a role in mediating toler-
ance via the production of TGF-β (6). TGF-β
has also been shown to mediate suppression by
CD4+CD25+ T reg cells in murine models of
autoimmunity (7). In addition, TGF-β can also
confer a suppressive phenotype on polyclonally
activated naive T cells in vitro, leading to an
expansion of FoxP3+ T reg cells (8–10).
The manipulation of the peripheral pool of
T reg cells has been a particular focus for the
treatment of autoimmune diseases and trans-
plantation. Our previous data showing that T
reg cells from patients with rheumatoid arthri-
tis (RA) are functionally defective, and that
after infl iximab therapy this defect is reversed,
have emphasized the potential of biological
therapy (11). An intriguing observation was
the increased number of peripheral blood
CD4+CD25hi T reg cells seen only in RA pa-
tients responding to infl iximab. Thus, TNF-α
blockade might, as a consequence of the re-
duced infl ammation, either “recall” T reg cells
from previously infl amed joints or lead to the
diff erentiation of “new” T reg cells. Here, we
demonstrate that infl iximab treatment induced
the diff erentiation of a population of T reg cells
Anti–TNF-α therapy induces a distinct
regulatory T cell population in patients
with rheumatoid arthritis via TGF-β
Suchita Nadkarni, Claudia Mauri, and Michael R. Ehrenstein
Centre For Rheumatology, Department of Medicine, Windeyer Institute, University College London, London W1T 4JF,
The induction of regulatory T (T reg) cells holds considerable potential as a treatment for
autoimmune diseases. We have previously shown that CD4+CD25hi T reg cells isolated from
patients with active rheumatoid arthritis (RA) have a defect in their ability to suppress
proinfl ammatory cytokine production by CD4+CD25− T cells. This defect, however, was
overcome after anti–tumor necrosis factor (TNF)-훂 antibody (infl iximab) therapy. Here, we
demonstrate that infl iximab therapy gives rise to a CD4+CD25hiFoxP3+ T reg cell population,
which mediates suppression via transforming growth factor (TGF)-훃 and interleukin 10,
and lacks CD62L expression, thereby distinguishing this T reg cell subset from natural
T reg cells present in healthy individuals and patients with active RA. In vitro, infl iximab
induced the differentiation of CD62L− T reg cells from CD4+CD25− T cells isolated from
active RA patients, a process dependent on TGF-훃. In spite of the potent suppressor capacity
displayed by this CD62L− T reg cell population, the natural CD62L+ T reg cells remained
defective in infl iximab-treated patients. These results suggest that anti–TNF-훂 therapy in RA
patients generates a newly differentiated population of T reg cells, which compensates for
the defective natural T reg cells. Therefore, manipulation of a proinfl ammatory environment
could represent a therapeutic strategy for the induction of T reg cells and the restoration
C. Mauri and M.R. Ehrenstein contributed equally to this work.
The online version of this article contains supplemental material.
34 DIFFERENTIATION OF REGULATORY T CELLS BY ANTI–TNF-α THERAPY | Nadkarni et al.
expressing Foxp3 and low levels of CD62L through conver-
sion of CD4+CD25− T cells.
RESULTS AND DISCUSSION
The expanded population of T reg cells from infl iximab-
treated patients is Foxp3+ and CD62L−
First, we addressed whether the increased number of
CD4+CD25hi T cells observed in RA patients responding to
infl iximab therapy corresponded to an increase of T reg cells
or simply refl ected an increase in the percentage of activated
T cells. Peripheral CD4+ T cells isolated from healthy, active
RA patients and those treated with infl iximab were analyzed
for the expression of the transcription factor Foxp3 by intra-
cellular staining. A two- to threefold increase in the percentage
of CD4+Foxp3+ cells was observed in the PBMCs of post-
infl iximab patients compared with active RA patients or
healthy individuals, suggesting that the increase in CD25 ex-
pression in post-anti–TNF-α–treated patients refl ects in-
creased numbers of T reg cells (Fig. 1 A). There was no
signifi cant diff erence in FoxP3 expression in CD4+ T cells
from active RA patients compared with healthy controls. To
better characterize the phenotype of the T reg cells after
infl iximab, we measured surface markers representative of
activation, memory, and regulation. A signifi cant increase in
the percentage of CD4+CD25hiCD62L− was observed in
PMBCs from infl iximab-treated patients compared with RA
patients with active disease before infl iximab and healthy
individuals (Fig. 1 B). As previous data has indicated that the
Figure 1. Increased numbers of Foxp3+CD62L−CD4+ T cells found
in infl iximab-treated RA patients compared with patients with
active disease and healthy controls. (A) Representative FACS plots gated
on the CD4+ population depicting PBMCs from healthy controls, active
RA patients, and infl iximab-treated RA patients stained with anti-CD4 and
anti-Foxp3. The percentages of Foxp3+ cells in the CD4+ gate for individual
RA patients, before and after infl iximab, and healthy controls are shown
in the chart. (B) Histograms were gated on CD4+CD25high cells (as indicated),
and the expression of CD62L is shown. (C) PBMCs from the same
groups as in A were stained with anti-CD4, anti-Foxp3, and anti-CD62L.
The histograms indicate the expression of CD62L in the CD4+Foxp3+
population. Data from individual patients are shown in the chart.
(D) CCR7 and CD45RO expression on the CD4+Foxp3+ T cell population
in the different patient groups. (E) CCR7 and CD45RO expression on
the CD4+FoxP3+CD62L− population from an RA patient treated with
infl iximab. Results are representative of six patients or healthy individuals
for each group. The dashed line in the FACS plots represents the isotype
control for the specifi c marker examined.
JEM VOL. 204, January 22, 2007
BRIEF DEFINITIVE REPORT
most potent CD4+CD25hi T reg cell subset expressed CD62L
(12), we next assayed the expression of CD62L in the
CD4+Foxp3+ population. Although most CD4+Foxp3+
cells from healthy individuals and patients with active RA
expressed CD62L, the profi le of expression was remarkably
diff erent after infl iximab treatment, where the majority of
CD4+Foxp3+ cells expressed low levels of CD62L (Fig.
1 C). There was no change in the percentage of Foxp3+ cells
or shift in CD62L expression in patients responding to metho-
trexate (not depicted). Compared with the T reg cells found
in healthy individuals and patients with active disease, RA T
reg cells post-infl iximab expressed CD45RO but had a re-
duced expression of CCR7 (Fig. 1 D). Further examination
of the CD4+Foxp3+CD62L− RA T reg cells post-infl iximab
revealed that these cells lacked CCR7 expression and remained
CD45RO+ (Fig. 1 E).
CD62L− T reg cells from infl iximab-treated RA patients
mediate their suppressive action through TGF-훃 and IL-10
We next tested whether the CD4+CD25hiCD62L− T reg
cells isolated from infl iximab-treated patients were function-
ally suppressive. CD4+CD25hi were FACS sorted according
to their expression of CD62L (identifi ed as CD62L+ and
CD62L− T reg cells; Fig. S1, available at http://www.jem
.org/cgi/content/full/jem.20061531/DC1), and their regula-
tory capacity was assessed with respect to inhibition of pro-
liferation and TNF-α and IFN-γ production by autologous
CD4+CD25− T cells. In agreement with previous studies
(12), CD62L+ T reg cells isolated from healthy individuals
were more potent at suppressing CD4+CD25− T cell pro-
liferation than CD62L− T reg cells (Fig. 2 A). Conversely,
CD62L− T reg cells isolated from RA patients after infl ix-
imab exhibited a more potent suppression of T cell prolifera-
tion than their CD62L+ T reg cell counterparts. Similarly,
we observed a “switch” in the suppressor population from
CD62L+ T reg cells in healthy individuals to CD62L− T reg
cells in patients after infl iximab, with respect to inhibition of
IFN-γ and TNF-α production (Fig. 2 B). There was a sub-
stantial reduction in the suppressor potency of CD62L+ T
reg cells isolated from patients with active RA compared
with healthy individuals, confi rming that CD4+CD25hi T
reg cells are defective in RA patients (11). Of importance,
the potency of the CD62L+ T reg cells after anti–TNF-α
therapy was not restored to levels found in healthy controls
(Fig. 2 B). Collectively, these results raise the intriguing
possibility that the natural T reg cells (CD62L+ T reg cells)
remain defective in patients even after infl iximab therapy,
and that the previously reported restoration of function of
CD4+CD25hi T reg cells after infl iximab therapy could be
due to the diff erentiation of a new population of CD62L−
T reg cells.
To explore the eff ector mechanisms of the CD62L− T
reg cells derived from post-infl iximab RA patients, we exam-
ined the cytokine dependency of their suppressive eff ect.
T reg cells were cocultured with autologous CD4+CD25− T
cells in the presence of neutralizing mAbs to TGF-β and
IL-10, previously recognized as regulatory cytokines involved
in the suppressive mechanism of adaptive T reg cells (2). The
data shown in Fig. 2 C demonstrate that in healthy individu-
als, the suppressive eff ect of CD62L− T reg cells was unal-
tered by the neutralization of TGF-β or IL-10. In contrast,
Figure 2. CD62L− T reg cells from infl iximab-treated RA patients
are more potent suppressors than their CD62L+ counterparts and
mediate their suppressive effects through IL-10 and TGF-훃.
CD4+CD25−, CD4+CD25hiCD62L+, and CD4+CD25hiCD62L− were FACS
(MoFlo) sorted from the PBMCs of healthy controls, active RA patients, and
infl iximab-treated RA patients. In all experiments, cells were stimulated
with 2 μg/ml of soluble anti-CD3/CD28. (A) CD4+CD25− T cells were
cocultured with either CD62L+ or CD62L− T reg cells (2:1 ratio shown) for
5 d, with [3H]thymidine added in the last 18 h of culture. Mean triplicate
values shown from six patients. (B) CD4+CD25− T cells were cultured
alone or cocultured at a 2:1 ratio with either CD62L+ or CD62L− T reg
cells for 48 h. Cells were intracellularly stained for TNF-α and IFN-γ. Data
shown expressed as mean ± SE of six patients and six healthy controls,
and are represented as the percentage inhibition of cytokine production
compared with CD4+CD25− T cells alone. The means for percent IFN-γ+/
TNF-α+ cells are as follows: healthy CD4+CD25− 3.2/3.4, CD4+CD25−/
CD62L+ T reg cells 0.7/0.9, CD4+CD25−/CD62L− T reg cells 1.9/2.1; active
RA CD4+CD25− 5.2/13.2, CD4+CD25−/CD62L+ T reg cells 3.6/9.0,
CD4+CD25−/CD62L− T reg cells 3.1/7.8; post-infl iximab CD4+CD25−
4.2/8.6, CD4+CD25−/CD62L+ T reg cells 3.8/6.3; and CD4+CD25−/CD62L−
T reg cells 1.1/2.2. (C) CD4+CD25− T cells from healthy and infl iximab-
treated RA patients were cultured alone or with CD62L− T reg cells
(2:1 ratio) for 48 h with anti-CD3/CD28 alone or in the presence of 2 μg/ml
anti–TGF-β1, 0.5 μg/ml anti–IL-10, or anti–TGF-β1 and anti–IL-10 together,
and stained for TNF-α and IFN-γ. Data depicted represent mean ± SE
of six patients and healthy controls. (D) CD4+CD25– T cells were
cocultured, either directly or separated by a transwell membrane, with
CD62L− T reg cells from RA patients after infl iximab, and stimulated with
2 μg/ml anti-CD3/CD28 for 48 h. Cells were intracellularly stained for
TNF-α and IFN-γ, and the results are depicted as the percentage inhibition
of cytokine production compared with CD4+CD25– T cells alone.
Data represent mean ± SE of four patients.
36 DIFFERENTIATION OF REGULATORY T CELLS BY ANTI–TNF-α THERAPY | Nadkarni et al.
the neutralization of TGF-β, and to a lesser extent IL-10,
signifi cantly impaired the suppressive capacity of the CD62L−
T reg cells from infl iximab-treated patients. When the action
of both TGF-β and IL-10 was blocked, the suppressive activ-
ity of these CD62L− T reg cells was almost abolished. The
potency of CD62L+ T reg cells from healthy individuals was
unaltered by blockade of IL-10 or TGF-β (not depicted).
The results reported here appear at odds with our previous
fi ndings showing that suppression required contact between
T reg cells isolated from infl iximab patients and their auto-
logous CD4+CD25− T cells (11). However, the two results
may not be directly comparable because in the previous
work, suppression was assayed without separating the
CD62L+ and CD62L− T reg cell subsets. Therefore, we as-
sayed the functional properties of purifi ed CD62L− T reg
cells isolated from RA patients after infl iximab therapy to
dissect out their mode of action. The data shown in Fig.
2 D demonstrate that cell contact is required to eff ect
maximal suppression of TNF-α and IFN-γ production by
CD4+CD25− T cells, although some cytokine suppression is
still observed when cell contact is prevented. The fact that
the T reg cells isolated from patients after infl iximab required
both cell contact and cytokines for their suppressive eff ect
could be explained by data indicating that TGF-β can medi-
ate suppression through cell contact, when the predominant
form of TGF-β is membrane bound (13). In addition, in
some experimental systems, IL-10 production also depends on
cell contact between T reg cells and CD4+CD25− T cells (14).
Collectively, these results suggest that infl iximab therapy
gives rise to, or recruits from the periphery, a population of
T reg cells that phenotypically and functionally diff ers from
natural T reg cells present in healthy individuals or in patients
with active RA.
In vitro infl iximab stimulation of CD4+CD25− T cells
from RA patients induced a CD62L− T reg cell population
To investigate the possible provenance of CD62L− T reg
cells, infl iximab was added in vitro to CD4+CD25− T cells
isolated from active RA or healthy individuals, and the ex-
pression of Foxp3 was measured by intracellular staining. The
addition of infl iximab to purifi ed active RA CD4+CD25−
T cells resulted in a substantial increase in the percentage of
CD4+Foxp3+ cells (Fig. 3 A), whereas no such eff ect was
seen when CD4+CD25− T cells were isolated from healthy
individuals. Neutralization of TGF-β in this culture system
completely prevented the diff erentiation of the CD4+Foxp3+
T cell population from the CD4+CD25− T cells (Fig. 3 A).
Moreover, infl iximab induced a signifi cant increase in TGF-β
production from CD4+CD25− T cells from patients with
active RA, but not healthy individuals (Fig. 3 B). To de-
termine whether infl iximab modulates natural T reg cell
Foxp3 expression, CD4+CD25hi T cells were isolated from
healthy individuals or patients with active RA and cultured
with infl iximab. The results in Fig. 3 C show that infl iximab
Figure 3. The addition of infl iximab in vitro to activated RA
CD4+CD25− T cells induced a population of FoxP3+ T cells that
were functionally suppressive. (A) Purifi ed CD4+CD25− T cells from
both healthy controls and active RA patients were stimulated with 2 μg/ml
anti-CD3/CD28, ±10 μg/ml infl iximab. In some wells, 2 μg/ml anti–
TGF-β was added. Cells were cultured for 24 h and then intracellularly
stained for FoxP3. Representative FACS plots are shown as well as the
pooled data (n = 8 patients and 8 healthy individuals). Percentages shown
are of the purifi ed CD4+CD25− fraction. (B) Supernatants were collected
from cells cultured in A before staining and were tested for TGF-β
production by ELISA. (C) Purifi ed CD4+CD25high T cells from healthy
individuals and patients with active RA were incubated with 2 μg/ml
anti-CD3/CD28, ±10 μg/ml infl iximab, and the percentages of Foxp3+
cells are shown. (D) Histograms depicting CD62L expression in the T cells
after culture as in A. Black line, isotype control; shaded area, CD62L.
(E) CD4+CD25+ from the cultures in A were isolated, recultured at a 1:2 ratio
with freshly isolated autologous CD4+CD25− T cells for an additional 2 d,
and stained for TNF-α and IFN-γ. Data are depicted as the percentage
of cytokine inhibition (n = 8).
JEM VOL. 204, January 22, 2007
BRIEF DEFINITIVE REPORT
did not aff ect the expression of FoxP3 on CD4+CD25hi
T reg cells, supporting the evidence that this agent targets
CD4+CD25− T cells rather than modulating preexisting
T reg cells. We next measured CD62L expression after culture
of CD4+CD25− T cells with infl iximab. The addition of in-
fl iximab to CD4+CD25− T cells from active RA patients, but
not from healthy individuals, led to a reduction in CD62L
expression (Fig. 3 D). Thus, these in vitro infl iximab-generated
T cells share reduced CD62L expression as a common feature
with the T reg cells present in the PBMCs of RA patients
that had received infl iximab therapy.
We sought to confi rm that the Foxp3+ T cells diff eren-
tiated upon infl iximab stimulation possess suppressor activity.
Because the majority of purifi ed CD4+CD25− T cells had
acquired CD25 expression when stimulated with anti-CD3/
CD28, with or without infl iximab, the CD4+CD25+ T cells
were purifi ed and cultured with freshly isolated autolo gous
CD4+CD25− T cells. Only the CD4+CD25+ T cells de rived
from active RA patients, but not healthy individuals, which
had been cocultured with infl iximab, were able to suppress
production of IFN-γ and TNF-α from freshly isolated
CD4+CD25− T cells (Fig. 3 E).
The potential of T reg cells to modulate immune re-
sponses has led to considerable interest in their use for the
treatment of autoimmune disease. Two broad therapeutic ap-
proaches have been considered; fi rst, to expand T reg cells in
vitro with the intention of infusing these cells into patients,
and second, to manipulate the immune system in vivo result-
ing in an increase in T reg cells. Infl iximab, a monoclonal
anti–TNF-α antibody, appears to be an example of the latter
and demonstrates that inhibition of a proinfl ammatory cyto-
kine can result in an expanded T reg cell population. Infl ix-
imab led to the diff erentiation of a population of T reg cells
identifi ed as FoxP3+CD25hiCD62L−CCR7− in patients
with RA. These T reg cells are phenotypically distinct from
natural T reg cells, which express CD62L and CCR7, but
also diff er functionally as their suppressor capacity depends
on the production of TGF-β and IL-10. Murine studies have
demonstrated the importance of IL-10 and/or TGF-β
in providing increased regulatory potency to CD4+CD25+
T reg cells in the face of an infl ammatory response (7, 15, 16)
or in the prevention of graft rejection (17). In addition, IL-
10–mediated suppression is used by CD4+CD25hiHLA-DR−
T reg cells found in healthy individuals (18). Of relevance to
this work, CD4+CD25+CD62L− T reg cells, producing
both IL-10 and TGF-β, have been identifi ed in diabetic mice
treated with a combination of anti-CD3 and insulin (19). In
patients with diabetes, anti-CD3 therapy expanded a popula-
tion of CD4+CD25+CD62L− T cells (20). Thus, anti-CD3
treatment in the context of diabetes and anti–TNF-α therapy
for RA both induce a population of T reg cells with a mem-
ory phenotype (CCR7−CD62L−) (21), perhaps suggesting
a similar provenance.
The T reg cells found in post-infl iximab RA patients
may have arisen either from CD4+CD25− T cells that had
already begun to diff erentiate along a Foxp3+ pathway in
vivo, or from CD4+CD25− responder T cells. The purifi ed
CD4+CD25− T cells still contain a small fraction of Foxp3+
cells, which could be targeted by infl iximab. CD127, which
negatively correlates with Foxp3 expression, could be used
to further exclude Foxp3+ cells in the CD4+CD25− T cells
(22). However, TGF-β appeared to mediate the infl iximab-
driven increase in Foxp3+ T reg cells, which is known to
target CD4+CD25− T cells rather than natural T reg cells
(8–10). Moreover, the data shown in Fig. 3 C demonstrate
that infl iximab has no eff ect on CD4+CD25hiFoxp3+ T cells.
Recent fi ndings have revealed that TGF-β can also have a
proinfl ammatory role dependent on the immunological en-
vironment (23). Thus, in the presence of proinfl ammatory
cytokines, TGF-β contributes to the generation of immuno-
pathogenic IL-17–secreting T cells (24–26). Consequently,
the prerequisite for the generation of T reg cells, rather than
IL-17–secreting T cells, by TGF-β is the removal of proin-
fl ammatory cytokines, specifi cally IL-6, but also TNF-α and
IL-1. Infl iximab can serve this function well as it is known
to rapidly reduce levels of IL-6 (27), the critical partner for
TGF-β in the production IL-17–producing T cells.
Although Foxp3 expression has been clearly linked to
T reg cell activity in murine studies, this association has been
less well established in humans. This is particularly true in the
context of in vitro activation studies where transient expres-
sion of Foxp3 can occur (28). The ability of infl iximab to
confer a suppressive capacity on CD4+CD25− T cells could
be explained by the presence of activated T cells producing
the immunoregulatory cytokines IL-10 and TGF-β. These
T reg cells could be similar to activated Tr1 or Th3 cells identi-
fi ed by others (4–6).
We have previously shown that CD4+CD25hi T reg cell
function in RA patients was defective and that this defect
was reversed after infl iximab treatment (11). However, the
data presented here lead to an important reinterpretation of
our previous fi nding, specifi cally that the naturally derived
T reg cells, defi ned as CD4+CD25hiCD62L+, appear to remain
defective after treatment with anti–TNF-α therapy. The res-
toration of function of the CD4+CD25hi population after in-
fl iximab therapy is most likely to be due to the suppressive
eff ects of the newly diff erentiated CD62L− T reg cells. In
patients with RA receiving infl iximab, the diff erentiation of
adaptive T reg cells could be further amplifi ed by their abil-
ity to confer a further suppressive activity on CD4+CD25−
T cells (29). One could envisage a feedback mechanism
in which infl iximab drives the production of TGF-β by
T cells, which would then induce their diff erentiation into
FoxP3+CD62L− T reg cells. Why natural T reg cells remain
defective after the neutralization of the proinfl ammatory en-
vironment by infl iximab is unclear. It is possible that the re-
versal of the proinfl ammatory milieu by TNF-α blockade
may only be partial, suffi cient to allow the generation of
T reg cells from CD4+CD25− T cells but insuffi cient to restore
function to the natural CD62L+ T reg cells. It is tempting to
speculate that the induction of T reg cells after infl iximab
therapy could lead to restoration of tolerance and might partly
38 DIFFERENTIATION OF REGULATORY T CELLS BY ANTI–TNF-α THERAPY | Nadkarni et al.
explain the exciting observation that infl iximab can induce
remission in some patients with early RA after cessation of
therapy (30). In conclusion, our results indicate that T reg
cells can be induced, and tolerance restored, by targeting specifi c
proinfl ammatory cytokines such as TNF-α.
MATERIALS AND METHODS
Patient population. 31 patients with active RA, fulfi lling the revised clas-
sifi cation criteria of the American College of Rheumatology for RA, were
evaluated before and 4–6 mo after anti–TNF-α therapy (see reference 11 for
infl iximab regime and further details for patient selection and assessment of
disease activity, all of which remained the same for this study). 20 healthy
individuals were used as controls. This study was approved by the University
College London Hospital (UCLH) Ethics Committee.
Antibodies. The following antibodies were used: FITC–anti-CD4 (RPA-
T4), PE-Cy5–anti-CD25 (M-A251), PE–anti-CD62L (Dreg-56), PE–CY7-
anti–TNF-α (Mab11), PE–Cy7-anti–IFN-γ (45.B3), PE-FoxP3 (PCH-101),
APC–anti-CCR7 (3D12), and PE–anti-CD45RO (UCLH1). T cells were
activated with soluble anti-CD3 (HIT-3a) and anti-CD28 (CD28.2) as indi-
cated. All antibodies were from BD Biosciences, except FoxP3, which was
from eBioscience. For neutralization experiments, anti–TGF-β1 (9016.2) and
anti–human IL-10 (25209) were used (R&D Systems). Infl iximab, a chimeric
IgG1 anti–TNF-α mAb, was donated by Schering-Plough.
Cell isolation. Human blood mononuclear cells (PBMCs) were isolated by
Ficoll-Paque (GE Healthcare) and cultured in RPMI 1640 with 100
U/μg/ml penicillin/streptomycin (Life Technologies) and 10% FCS (Sera
Laboratories International, Ltd.). Magnetic bead separation was performed
as described previously (11), with all magnetic columns and magnetically la-
beled beads purchased from Miltenyi Biotec. To purify cells using FACS
sorting (MoFlo), cells were stained with the above conjugated antibodies
(CD4, CD25, and CD62L). Cells were sorted according to gates as indi-
cated in Fig. S1.
Flow cytometric analysis and cytokine detection. Cells were surface
stained as described previously (11) using the above mentioned conjugated
antibodies (CD4, CD25, and CD62L). For intracellular analysis of TNF-α
and IFN-γ production, cells were cultured at 2 × 105 for 48 h with PMA,
ionomycin, and Golgi Plug added in the fi nal 5 h of culture. TGF-β1 was
quantifi ed using an ELISA kit (R&D Systems). In some experiments, 24-
well transwell plates (Costar) with 0.4-μm membrane supports were used.
Proliferation assays. In all experiments, T reg cells were cultured at 2 ×
105 cells/ml, with CD4+CD25− T cell numbers adjusted accordingly for
ratio experiments. Cells were cultured in 96-well U-bottomed plates (Nunc)
for 5 d with [3H]thymidine added in last 18 h of culture. Proliferation was
measured using a liquid scintillation counter.
Statistical analysis. Statistical signifi cance was determined using Student’s
t test, with p-values <0.05 regarded as statistically signifi cant.
We thank Ayad Eddaoudi for providing technical assistance with MoFlo FACS sorting.
S. Nadkarni and C. Mauri have been supported by a Medical Research Council
PhD studentship and the Wellcome Trust (grant no. 068629), respectively. This work
was supported by UCLH Charity Trust and Schering-Plough Corporation.
The authors have no confl icting fi nancial interests.
Submitted: 20 July 2006
Accepted: 5 December 2006
R E F E R E N C E S
1. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regula-
tory T cell development by the transcription factor Foxp3. Science.
2. Levings, M.K., and M.G. Roncarolo. 2005. Phenotypic and functional
diff erences between human CD4+CD25+ and type 1 regulatory T cells.
Curr. Top. Microbiol. Immunol. 293:303–326.
3. Bluestone, J.A., and A.K. Abbas. 2003. Natural versus adaptive regula-
tory T cells. Nat. Rev. Immunol. 3:253–257.
4. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J.E.
de Vries, and M.G. Roncarolo. 1997. A CD4+ T-cell subset inhib-
its antigen-specifi c T-cell responses and prevents colitis. Nature. 389:
5. Asseman, C., S. Mauze, M.W. Leach, R.L. Coff man, and F. Powrie.
1999. An essential role for interleukin 10 in the function of regulatory
T cells that inhibit intestinal infl ammation. J. Exp. Med. 190:995–1004.
6. Fukaura, H., S.C. Kent, M.J. Pietrusewicz, S.J. Khoury, H.L. Weiner, and
D.A. Hafl er. 1996. Induction of circulating myelin basic protein and
proteolipid protein-specifi c transforming growth factor-beta1-secreting
Th3 T cells by oral administration of myelin in multiple sclerosis
patients. J. Clin. Invest. 98:70–77.
7. Green, E.A., L. Gorelik, C.M. McGregor, E.H. Tran, and R.A. Flavell.
2003. CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells
through TGF-beta-TGF-beta receptor interactions in type 1 diabetes.
Proc. Natl. Acad. Sci. USA. 100:10878–10883.
8. Fantini, M.C., C. Becker, G. Monteleone, F. Pallone, P.R. Galle, and
M.F. Neurath. 2004. Cutting edge: TGF-beta induces a regulatory phe-
notype in CD4+CD25− T cells through Foxp3 induction and down-
regulation of Smad7. J. Immunol. 172:5149–5153.
9. Chen, W., W. Jin, N. Hardegen, K.J. Lei, L. Li, N. Marinos, G.
McGrady, and S.M. Wahl. 2003. Conversion of peripheral CD4+CD25−
naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of
transcription factor Foxp3. J. Exp. Med. 198:1875–1886.
10. Zheng, S.G., J.D. Gray, K. Ohtsuka, S. Yamagiwa, and D.A. Horwitz.
2002. Generation ex vivo of TGF-beta-producing regulatory T cells
from CD4+CD25− precursors. J. Immunol. 169:4183–4189.
11. Ehrenstein, M.R., J.G. Evans, A. Singh, S. Moore, G. Warnes, D.A.
Isenberg, and C. Mauri. 2004. Compromised function of regulatory
T cells in rheumatoid arthritis and reversal by anti-TNFα therapy. J. Exp.
12. Fu, S., A.C. Yopp, X. Mao, D. Chen, N. Zhang, M. Mao, Y. Ding, and
J.S. Bromberg. 2004. CD4+ CD25+ CD62+ T-regulatory cell subset
has optimal suppressive and proliferative potential. Am. J. Transplant.
13. Nakamura, K., A. Kitani, and W. Strober. 2001. Cell contact–
dependent immunosuppression by CD4+CD25+ regulatory T cells is
mediated by cell surface–bound transforming growth factor β. J. Exp.
14. Dieckmann, D., C.H. Bruett, H. Ploettner, M.B. Lutz, and G. Schuler.
2002. Human CD4+CD25+ regulatory, contact-dependent T cells in-
duce interleukin 10–producing, contact-independent type 1–like regu-
latory T cells. J. Exp. Med. 196:247–253.
15. Liu, H., B. Hu, D. Xu, and F.Y. Liew. 2003. CD4+CD25+ regulatory
T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4.
J. Immunol. 171:5012–5017.
16. Uhlig, H.H., J. Coombes, C. Mottet, A. Izcue, C. Thompson, A.
Fanger, A. Tannapfel, J.D. Fontenot, F. Ramsdell, and F. Powrie.
2006. Characterization of Foxp3+CD4+CD25+ and IL-10-
secreting CD4+CD25+ T cells during cure of colitis. J. Immunol.
17. Kingsley, C.I., M. Karim, A.R. Bushell, and K.J. Wood. 2002.
CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4-
and IL-10-dependent immunoregulation of alloresponses. J. Immunol.
18. Baecher-Allan, C., E. Wolf, and D.A. Hafl er. 2006. MHC class II
expression identifi es functionally distinct human regulatory T cells.
J. Immunol. 176:4622–4631.
19. Bresson, D., L. Togher, E. Rodrigo, Y. Chen, J.A. Bluestone, K.C.
Herold, and M. von Herrath. 2006. Anti-CD3 and nasal proinsulin
combination therapy enhances remission from recent-onset autoimmune
diabetes by inducing Tregs. J. Clin. Invest. 116:1371–1381.
20. Herold, K.C., J.B. Burton, F. Francois, E. Poumian-Ruiz, M. Glandt,
and J.A. Bluestone. 2003. Activation of human T cells by FcR
JEM VOL. 204, January 22, 2007
BRIEF DEFINITIVE REPORT
nonbinding anti-CD3 mAb, hOKT3gamma1(Ala-Ala). J. Clin. Invest.
21. Huehn, J., K. Siegmund, J.C. Lehmann, C. Siewert, U. Haubold,
M. Feuerer, G.F. Debes, J. Lauber, O. Frey, G.K. Przybylski, et al.
2004. Developmental stage, phenotype, and migration distinguish
naive- and eff ector/memory-like CD4+ regulatory T cells. J. Exp. Med.
22. Liu, W., A.L. Putnam, Z. Xu-Yu, G.L. Szot, M.R. Lee, S. Zhu, P.A.
Gottlieb, P. Kapranov, T.R. Gingeras, B.F. de St Groth, et al. 2006.
CD127 expression inversely correlates with FoxP3 and suppressive
function of human CD4+ T reg cells. J. Exp. Med. 203:1701–1711.
23. Weaver, C.T., L.E. Harrington, P.R. Mangan, M. Gavrieli, and K.M.
Murphy. 2006. Th17: an eff ector CD4 T cell lineage with regulatory
T cell ties. Immunity. 24:677–688.
24. Veldhoen, M., R.J. Hocking, C.J. Atkins, R.M. Locksley, and B.
Stockinger. 2006. TGFbeta in the context of an infl ammatory cyto-
kine milieu supports de novo diff erentiation of IL-17-producing T cells.
25. Mangan, P.R., L.E. Harrington, D.B. O’Quinn, W.S. Helms, D.C.
Bullard, C.O. Elson, R.D. Hatton, S.M. Wahl, T.R. Schoeb, and C.T.
Weaver. 2006. Transforming growth factor-beta induces development
of the T(H)17 lineage. Nature. 441:231–234.
26. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T.B. Strom, M. Oukka, H.L.
Weiner, and V.K. Kuchroo. 2006. Reciprocal developmental pathways
for the generation of pathogenic eff ector TH17 and regulatory T cells.
27. Feldmann, M., F.M. Brennan, and R.N. Maini. 1996. Role of cyto-
kines in rheumatoid arthritis. Annu. Rev. Immunol. 14:397–440.
28. Gavin, M.A., T.R. Torgerson, E. Houston, P. DeRoos, W.Y. Ho, A.
Stray-Pedersen, E.L. Ocheltree, P.D. Greenberg, H.D. Ochs, and A.Y.
Rudensky. 2006. Single-cell analysis of normal and FOXP3-mutant
human T cells: FOXP3 expression without regulatory T cell development.
Proc. Natl. Acad. Sci. USA. 103:6659–6664.
29. Zheng, S.G., J.H. Wang, J.D. Gray, H. Soucier, and D.A. Horwitz.
2004. Natural and induced CD4+CD25+ cells educate CD4+CD25−
cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10.
J. Immunol. 172:5213–5221.
30. Quinn, M.A., P.G. Conaghan, P.J. O’Connor, Z. Karim, A. Greenstein,
A. Brown, C. Brown, A. Fraser, S. Jarret, and P. Emery. 2005. Very
early treatment with infl iximab in addition to methotrexate in early,
poor-prognosis rheumatoid arthritis reduces magnetic resonance im-
aging evidence of synovitis and damage, with sustained benefi t after
infl iximab withdrawal: results from a twelve-month randomized, double-
blind, placebo-controlled trial. Arthritis Rheum. 52:27–35.