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and IL-10 Induce the Differentiation of Human Type 1 T Regulatory Cells1
*
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; and
DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not TGF-
, induces naive
CD4+ T cells derived from cord blood to differentiate into
Tr1 cells:
IL-10+IFN-
+IL-2-/lowIL-4-.
Naive CD4+ T cells derived from peripheral blood require
both exogenous IL-10 and IFN- for Tr1 cell differentiation. The
proliferative responses of the Tr1-containing lymphocyte populations,
following activation with anti-CD3 and anti-CD28 mAbs, were
reduced. Similarly, cultures containing Tr1 cells displayed reduced
responses to alloantigens via a mechanism that was partially mediated
by IL-10 and TGF-. More importantly, Tr1-containing populations
strongly suppressed responses of naive T cells to alloantigens.
Collectively, these results show that IFN- strongly enhances
IL-10-induced differentiation of functional Tr1 cells, which represents
a first major step in establishing specific culture conditions to
generate T regulatory cells for biological and biochemical analysis,
and for cellular therapy to induce peripheral tolerance in
humans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, promote cell-mediated responses against
bacteria, and Th2 cells which, by producing IL-4, IL-5, and IL-13,
induce Ab synthesis and the anti-parasite mast cell and eosinophil
responses (1). Both subsets originate from a naive T cell
precursor, whose differentiation is influenced by both the mode of
activation and the environment in which it is initially stimulated.
Variables known to influence the development of T cell subsets include
the affinity of the TCR for Ag (2), the duration of the
interaction between the TCR and Ag (3), and differential
costimulation by APCs (4). In addition, it has been shown
that cytokines present upon T cell activation define T cell
differentiation. It is well established that priming of naive T cells
in the presence of IL-12 favors the development of Th1 cells, whereas
IL-4 favors the development of Th2 cells (5, 6).
Activation, in vitro or in vivo, of human or mouse
CD4+ T cells in the presence of IL-10 has been
shown to result in the generation of T cell clones with a cytokine
production profile different from that of Th1 or Th2 cells. These T
cell clones produce significant amounts of IL-10, IFN-, TGF-, and
IL-5, but low amounts of IL-2 and no IL-4 (7, 8).
Functionally, these T cell clones have inhibitory effects on
Ag-specific activation of naive autologous T cells, which are partially
mediated by IL-10 and TGF-. This novel CD4+ T
cell subset was termed T regulatory type 1
(Tr1)3 cells
(7). In addition, in a murine model of inflammatory bowel
disease in SCID mice, cotransfer of Tr1 cell clones together with
pathogenic CD4+ CD45RBhigh
T cells prevented the induction of disease (7). Prevention
of inflammatory bowel disease was only observed in mice that were
administered the Ag recognized by Tr1 cells, demonstrating that Tr1
cells must be activated in vivo via the TCR to exert their regulatory
effects. Donor-derived T cells specific for host alloantigens that
possessed a Tr1 profile of cytokine production were also isolated from
tolerant SCID patients who had been reconstituted with HLA-mismatched
stem cells (8, 9, 10). Together, these data indicate that Tr1
cells can function as regulatory cells in vivo.
CD4+ T cells that, in addition to IL-10, secrete
high levels of TGF- and/or IL-4 and that suppress responses to self
peptides, have been described in several experimental models of
autoimmune diseases (11, 12, 13, 14, 15). Notably, in most cases,
these T regulatory cells appear to arise following repeated Ag
stimulation either in vitro or in vivo. Buer et al. (16)
reported that IL-10-producing T cells generated following repeated
stimulation with influenza hemagglutinin could regulate immune
responses in vivo. Similarly, repetitive in vitro stimulation with
Ag-loaded APCs (17) or in vivo stimulation with
superantigen (18, 19) led to the emergence of
CD4+ T cells that suppressed naive T cell
responses via an IL-10-dependent mechanism. A number of investigators
have also documented the presence of Ag-specific regulatory
CD4+ T cells that, via a TGF--dependent
mechanism, can prevent T cell-mediated diseases (13, 14, 15, 20, 21). These observations support the notion that in addition to
clonal deletion and clonal anergy, clonal suppression mediated by
regulatory T cells that produce suppressive cytokines is an important
mechanism of peripheral tolerance.
Due to their low proliferative capacity, both human and mouse Tr1 cells
are difficult to isolate under standard culture conditions. To better
understand the requirements for Tr1 cell differentiation, we determined
which immunoregulatory cytokines, in addition to IL-10, could drive the
differentiation of this unique T cell subset in vitro. Using culture
conditions in which highly purified naive CD4+ T
cells were stimulated via their TCR in the absence of professional
APCs, it was demonstrated that IFN-, but not TGF-, acts
synergistically with IL-10 to induce the generation of
CD4+ immunosuppressive Tr1 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human blood was obtained from healthy anonymous donors in accordance with local ethical committee approval. The neonatal leukocytes from umbilical cord blood were prepared by centrifugation over Ficoll-Hypaque gradients (Nycomed Amersham, Uppsala, Sweden), and CD4+ T cells were purified by positive or negative selection. For negative selection, purification was performed by the addition of Dynabeads (Dynal, Oxoid, Italy) coupled to mAbs against CD8, CD14, CD19, and CD56. Beads were added at a 4:1 bead-target cell ratio and incubated for 1 h at 4°C. Beads and contaminating cells were removed by magnetic field. For positive selection, cells were purified using the Miltenyi CD4+ T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotech, Bergische Gladbach, Germany). Results obtained with CD4+ T cells isolated by negative or positive selection were identical. Human PBMCs were centrifuged over Ficoll-Hypaque gradients, adherent cells were removed by two rounds of incubation on tissue-culture treated flasks for 1 h at 37°C, and CD4+ CD45RO- cells were purified by negative selection. Nonadherent cells were incubated with anti-CD45RO mAbs (5 µg/106 target cells) (UCHL1; Valter Occhiena, Torino, Italy) for 45 min at 4°C. Following washing, Dynabeads coupled to mAbs directed against CD8, CD14, CD19, CD56, and mouse IgG were added, and CD4+CD45RO- cells were isolated as described above.
T cell differentiation
Murine L cell transfectants expressing hCD32 (FCRII), hCD58
(LFA-3), and hCD80 (22) were cultured in RPMI 1640
(BioWhittaker, Bergamo, Italy) supplemented with 10% FCS (Mascia
Brunelli, Milan, Italy), 100 U/ml penicillin/streptomycin
(Bristol-Myers Squibb, Sermoneta, Italy), and 2 mM glutamine (Life
Technologies, Milan, Italy). L cells were detached by incubation with
trypsin-EDTA (Life Technologies) and irradiated (7000 rad) by an x-ray
source. Following washing, cells were plated in 24-well plates
(Corning, Cambridge, MA) at an initial density of 4 x
105 cells/ml in a 500 µl volume of YSSEL medium
(Diaclone, Besançon, France) supplemented with 10% FCS,
1% pooled human serum, and 100 U/ml penicillin/streptomycin (hereafter
referred to as complete medium) and 100 ng/ml of anti-CD3 (OKT3;
Orthoclone, Jansen Cilag, Italy). After the L cells had adhered, 500
µl of CD4+ cord blood or
CD4+CD45RO- peripheral
blood T cells were added at an initial density of 4 x
105 cells/ml in complete medium. For
differentiation by immobilized anti-CD3, the mAb (10 µg/ml) was
immobilized by overnight incubation in 24-well plates in 0.1 M Tris
buffer, pH 9.5. Wells were washed three times with PBS before the
addition of 106 CD4+ cord
blood T cells in complete medium.
All experiments were conducted in the presence of recombinant human
(rh) IL-2 (100 U/ml; Chiron Italia, Milan, Italy) and rhIL-15 (1
ng/ml; R&D Systems, Minneapolis, MN). In addition, the following
polarizing cytokines were added as indicated: rhIL-10 (100 U/ml;
Schering-Plough Research Institute, Kenilworth, NJ), rhIFN- 2b (5
ng/ml; Schering-Plough Research Institute or PeproTech, Rocky Hill,
NJ), rhIL-4 (200 U/ml; Schering-Plough Research Institute), rhIL-12 (5
ng/ml; R&D Systems), TGF-2 (1 ng/ml; R&D
Systems). Anti-human IL-4 mAb (200 ng/ml; PharMingen, San Diego, CA)
and anti-human IL-12 mAb (10 µg/ml; PharMingen) were added to the
Th1 and Th2 polarizing conditions, respectively. T cells were split as
necessary, IL-2 and IL-15 were replenished in all cultures, and IL-4
was replenished only in cultures of Th2 cells. At day 7, T cells were
collected, washed, counted, and restimulated under identical conditions
for an additional 7 days. At day 14 of in vitro culture, cells
were collected, washed, counted, and analyzed for their profile of
cytokine production and proliferative capacity. All cells were cultured
in humidified incubators at 37°C with 5%
CO2.
Analysis of cytokine production
Intracellular cytokines were detected by flow cytometry as
described in Sornasse et al. (23), with slight
modifications. T cells (1 x 106/ml)
were stimulated with immobilized anti-CD3 (10 µg/ml) and soluble
anti-CD28 (1 µg/ml) mAbs (PharMingen) in complete medium. Upon
initiation of the culture, the plates were centrifuged for 5 min at
800 x g. After 3 h of activation, brefeldin A (10
µg/ml; Sigma, Milan, Italy) was added. After a total of 6 h of
activation, T cells were collected, washed in PBS, and fixed with 2%
formaldehyde. After fixation, T cells were permeabilized by incubation
in PBS supplemented with 2% FCS and 0.5% saponin (Sigma).
Permeabilized T cells were incubated with PE-labeled anti-hIL-4,
anti-hIL-2, or anti-hIL-10, and FITC-coupled anti-hIFN-
or anti-hIL-4 mAbs. All mAbs were obtained from PharMingen. After
washing, cells were analyzed using a FACScan flow cytometer (BD
Biosciences, Mountain View, CA), and data were analyzed with
CellQuest software (BD Biosciences). Quadrant markers were positioned
to include 95% of stained, unstimulated cells in the lower left
square.
For detection of IL-5 and TGF-, capture ELISAs were performed on
supernatants of cells that had been stimulated (1 x
106 T cells/ml) with immobilized anti-CD3 (10
µg/ml) and anti-CD28 (1 µg/ml) for 72 h. ELISAs were
performed as described (24), and capture and detection
mAbs were purchased from PharMingen.
Proliferation of polarized T cells
Polarized T cell subsets were compared for their proliferative
capacity following polyclonal or Ag-specific activation. To analyze
proliferation in response to polyclonal activation, 96-well flat-bottom
plates (Costar) were coated overnight with anti-CD3 mAbs (10
µg/ml) and washed three times with PBS. T cells were plated at an
initial density of 2.5 x 105 cells/ml in a
final volume of 200 µl of complete medium and soluble anti-CD28
mAbs (1 µg/ml). Control cultures consisted of T cells cultured in the
absence of anti-CD3 and anti-CD28 mAbs. To determine
proliferation induced by Ag-dependent activation, MLRs were performed.
T cells (5 x 105 cells/ml) were stimulated
with irradiated (6000 rad) allogenic PBMCs (5 x
105 cells/ml) that had been depleted of
CD3+ cells by negative selection. Cells were
cocultured in a final volume of 200 µl of complete medium in 96-well
round-bottom plates (Costar). Neutralizing anti-IL-10R (20 µg/ml)
(3F9, a gift of Kevin Moore, DNAX Research Institute) and/or
anti-TGF-1,2,3 (20 µg/ml) (R&D Systems)
mAbs were added as indicated. After 48 h, wells were pulsed for
16 h with 1 µCi/well [3H]thymidine
(Amersham, Uppsala, Sweden). Cells were harvested, and counted in a
scintillation counter.
To asses the effects of IFN- on proliferation, cord blood T cells
were activated with L cells as described above in the presence of IL-4,
IL-12, or IL-10, with or without IFN-, for the primary stimulation.
After 7 days, the cells were washed and restimulated in the presence of
IL-4, IL-12, or IL-10, with or without IFN-, and incorporation of
[3H]thymidine was measured 3 days after
initiation of the secondary stimulation.
Suppression of naive T cells
Polarized T cell subsets were tested for their ability to suppress the proliferation of naive T cells to alloantigens. Naive autologous CD4+ cord blood T cells were purified as described above and cocultured (2.5 x 105 cells/ml) together with irradiated, allogenic CD3-depleted PBMCs (5 x 105 cells/ml), in the absence or presence of polarized T cells (2.5 x 105 or 5 x 105 cells/ml) in a final volume of 200 µl in complete medium. Control cultures consisted of naive and polarized T cells in the absence of allogenic PBMCs, and polarized T cells plus allogenic PBMCs in the absence of naive T cells. After 4 days, wells were pulsed for 16 h with 1 µCi/well [3H]thymidine.
Effects of IFN- and IL-10 on TCR-mediated signal transduction
CD4+ T cells were purified from PBMCs by
positive selection. Immediately after purification, cells were
resuspended at 8 x 106 cells/ml in
serum-free medium (X-vivo 15; BioWhittaker) and incubated at 37°C for
16 h in the absence or presence of IL-10 (1000 U/ml) and/or
IFN- (50 ng/ml). After 16 h, the cells were stimulated for 10
min at 37°C with 5-µm latex beads (Interfacial Dynamics, Portland,
OR) coated with 1 µg/ml anti-CD3 and 10 µg/ml anti-CD28
mAbs or with 10 µg/ml mouse IgG (Sigma) as a control, at a ratio of
2.5 beads per cell. For samples incubated for 16 h in medium,
IL-10 and/or IFN- were added together with the latex beads for 10
min. Cells were lysed, and whole cell lysates were subjected to
SDS-PAGE and immunoblotting (25). Membranes were
immunoblotted with 4G10 (Upstate Biotechnology, Lake Placid, NY) to
assess whole-cell tyrosine phosphorylation,
anti-phospho-extracellular signal-related kinase 1/2 (Cell
Signaling Technology, Beverly, MA), anti-phospho-p38 (Cell
Signaling Technology), or anti-phospho-c-Jun N-terminal kinase
(Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis
All analyses for statistically significant differences were performed with Student’s paired t test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to induce the differentiation
of Tr1 cells
We have shown that IL-10 is a critical factor for the generation
of Tr1 cells in primary MLRs and in OVA-stimulated cultures of naive T
cells and autologous APCs (7). To determine whether IL-10
alone could induce the differentiation of naive
CD4+ T cells into Tr1 cells, we established in
vitro culture conditions in the absence of professional APCs. In these
cultures, highly purified naive CD4+ cord blood
lymphocytes were stimulated with anti-CD3 mAbs cross-linked onto
CD32+ mouse L cells in the presence or absence of
exogenous IL-10. Similar cultures performed in the presence of IL-12 or
IL-4 resulted in highly polarized populations of Th1 and Th2 cells,
respectively (23). Although activation in the presence of
IL-10 reproducibly increased the total number of IL-10-producing cells
(Fig. 1
, 8.4% compared with 1.6% in the
absence of IL-10), only a small population (on average 1.0%, range
0.4–2.3%, n = 12) of cells displayed the typical Tr1
phenotype, as judged by their characteristic cytokine production
profile:
IL-10+IFN-+IL-2-/lowIL-4-
(Fig. 1).
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, and TGF- has previously been
reported to positively regulate its own production (26).
In addition, both exogenous IL-10 and TGF- are required for
induction of Ag-specific anergy of murine T cells in vitro
(27). Therefore, we analyzed the effect of TGF- on
IL-10-induced Tr1 cell differentiation. Cord blood T cells activated in
the presence of TGF-, with or without IL-10, did not differentiate
into a population of Tr1-polarized cells. Rather, addition of TGF-
resulted in a general suppression of cytokine production, including
that of IL-10. These results are consistent with previous reports
indicating that TGF- strongly inhibits the production of IL-4 and to
a lesser extent inhibits that of IL-2 and IFN- (28, 29).
CD4+ T cells primed in the presence of IL-10 and
IFN- differentiate into Tr1 cells
It has been shown previously that CD4+ T
cells activated by anti-CD3 cross-linked to
CD32+ L cells or in vitro-differentiated
dendritic cells, in the presence of IFN-, produced high levels of
IL-10 (29, 30). Therefore, we investigated the effects of
IL-10 + IFN- on the differentiation of CD4+
cord blood T cells. As shown in Fig. 2A, activation of
CD4+ cord blood T cells in the presence of IL-10
+ IFN- resulted in the differentiation of a significant population
of T cells with a Tr1 cytokine production profile. Under these culture
conditions a significant subset of T cells produced IL-10 (average:
11.5%, range: 7.5–18.8%, n = 12), whereas a
significantly lower proportion of T cells produced IL-2 (average:
4.0%, range: 2.2–11%) or IL-4 (average: 2.1%, range: 0.9–4.2%) in
comparison to the Th1 and Th2 cultures, respectively (Fig. 2C). Cultures differentiated in IL-10 + IFN- contained
more IFN--producing cells (average 15%, range: 7.3–28%) than Th2
cultures, but the proportion of IFN--producing cells was lower than
in Th1 cultures. As expected, addition of IL-4 or IL-12 resulted in the
differentiation of Th2 (IL-4+
IL-10+) and Th1 cells
(IFN-+ IL-2+),
respectively. It is important to note that although in all three
culture conditions a similar proportion of IL-10+
T cells was induced, only in cultures with IL-10 + IFN- did 
50%
of IL-10+ T cells display the expected Tr1
phenotype and were positive for IFN- (average 4.3%, range:
1.5–7.7%) but negative or low for IL-4 and IL-2. Addition of
neutralizing anti-IL-4 and/or anti-IL-12 mAbs (which inhibited
differentiation of Th1 or Th2 cells, respectively) during priming in
the presence of IL-10 + IFN- did not significantly alter this
cytokine production profile (data not shown). T cells primed with IL-10
+ IFN- also produced IL-5 and TGF- (average TGF- levels
detected in supernatants 1.7 ng/ml, range: 0.8–2.8 ng/ml, and
average IL-5 levels 0.6 ng/ml, range: 0.4–0.9 ng/ml, n
= 3) as previously described for Tr1 cells (7).
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could promote the
differentiation of Tr1 cells from CD4+ cord blood
T cells activated by anti-CD3 mAbs immobilized on plastic.
Following two rounds of stimulation with immobilized anti-CD3 mAbs,
polarized T cell populations, which displayed profiles of cytokine
production similar to those obtained with the cells activated by
anti-CD3 cross-linked to
CD32+CD80+CD58+
L cells, were generated (data not shown). However, the overall
percentages of Tr1 cells were lower. These data demonstrate that IL-10
and IFN- act directly on T cells to induce differentiation of Tr1
cells.
We next determined whether priming of naive CD4+
T cells from peripheral blood in the presence of IL-10 + IFN- also
led to the differentiation of Tr1 cells. Similar to cord blood cells,
activation of peripheral blood T cells in the presence of IL-10 +
IFN- reproducibly resulted in a population that contained
IL-10+ T cells (average 7.5%, range 3.8–11.5%)
and a small but statistically significant percentage of
IL-10+IFN-+ cells
(average 4.0%, range 1.4–7.2%, n = 9; Fig. 2
B). Peripheral blood T cells activated in the presence of
IL-10 + IFN- produced less IL-4 (average 3.1%, range: 0.6–4.8%)
and IL-2 (average 7.4%, range 1.2–14%) than cultures differentiated
in the presence of IL-4 or IL-12, respectively (Fig. 2C).
Significant levels of TGF- (average 3.5 ng/ml, range: 0.8–7.6
ng/ml, n = 3) and IL-5 (average 1.5 ng/ml, range:
1.2–1.6 ng/ml, n = 3) were detected in the culture
supernatants of cells primed with IL-10 + IFN- and restimulated for
72 h with anti-CD3 and anti-CD28 mAbs (data not shown).
Thus, priming of CD4+ T cells from cord blood or
peripheral blood in the presence of IL-10 + IFN- results in a
population of cells that display a Tr1 phenotype of cytokine
production:
IL-10+IFN-+IL-2-/lowIL-4-.
Comparison of the effects of IL-10 and IFN- on cord blood and
peripheral blood CD4+ T cells
Analysis of the relative roles of exogenous IL-10 and IFN- in
the differentiation of IL-10-producing T cells from cord blood revealed
that addition of IFN- alone was sufficient to induce the
differentiation of a population of cells with a Tr1 profile of cytokine
production (Fig. 3A). Addition
of either IL-10 or IFN- resulted in the presence of
IL-10+ IL-4- T cells, and
addition of both IL-10 and IFN- induced a variable enhancement of
IL-10+ IL-4- T cells
compared with the numbers induced by IFN- alone. However, IFN-
alone reproducibly induced a higher proportion of
IL-10+ IFN-+ T cells
compared with IL-10 alone. Although the IL-10+
IFN-+ cells were clearly
IL-4-, it cannot be excluded that they produced
low levels of IL-2. Addition of IL-10 to IFN- resulted in a variable
increase in the proportion of
IL-10+IFN-+ cells, but
upon comparison of data from 12 donors, this increase was not
statistically significant (Fig. 3C). As demonstrated
previously, cord blood T cells have an intrinsic ability to produce
high levels of endogenous IL-10 in comparison to peripheral blood T
cells (31, 32). Unpolarized cord blood T cells produced 7-
to 13-fold higher levels of IL-10 (range: 40–130 ng/ml) in comparison
to unpolarized peripheral blood T cells (range: 6–10 ng/ml; data not
shown). Therefore, the variable effects of exogenous IL-10 in enhancing
the proportion of
IL-10+IFN-+ T cells may
be due to the variable production of endogenous IL-10.
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in inducing the
differentiation of Tr1 cells, we tested naive T cells derived from
peripheral blood. In Fig. 3B it is shown that, in contrast
to cord blood T cells, naive CD4+ peripheral
blood T cells reproducibly required both exogenous IL-10 and IFN- to
differentiate into cells with a Tr1 cytokine production pattern.
Cultures with IFN- alone did not contain statistically significant
higher proportions of
IL-10+IFN-+ cells than
cultures differentiated in the presence of IL-10 alone (average 0.5%
with IL-10 and 1.0% with IFN-, n = 9). In contrast,
addition of both IL-10 and IFN- resulted in a statistically
significant increase in both the total proportion of IL-10-producing T
cells (average 7.5, range 3.8–11.5%) and the
IL-10+IFN-+ cells
(average 4.0%, range 1.4–7.2%) in comparison to IFN- alone (Fig. 3C).
CD4+ T cells activated in the presence of IL-10 and
IFN- are impaired in their responses to polyclonal activation and
alloantigens
In addition to their cytokine production profile, Tr1 cells are functionally characterized by their intrinsic low proliferative capacity (7, 8). Therefore, we investigated whether the presence of a population of cells with a Tr1 cytokine production profile correlated with an impaired ability of the cultures to proliferate in response to polyclonal activation or alloantigens in primary MLRs.
In comparison to Th1 and Th2 control cultures, cultures of cord blood
containing Tr1 cells had a reduced proliferative responses to
activation by anti-CD3 and anti-CD28 mAbs (Fig. 4A). In addition, the cells
differentiated in IL-10 + IFN- reproducibly failed to proliferate
significantly in response to alloantigens in comparison to Th2 and Th1
cultures (Fig. 4B). Importantly, addition of neutralizing
anti-IL-10R and anti-TGF- mAbs resulted in partial (average
5-fold increase) restoration of the proliferation in response to
alloantigens (Fig. 4B), indicating that the reduced
proliferative responses were due, in part, to the production of IL-10
and TGF- in the cultures. Thus, IL-10 + IFN- induced the
differentiation of a population of T cells that not only displayed a
Tr1-like profile of cytokine production, but also had the intrinsically
low proliferative potential of regulatory T cells.
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alone was sufficient to differentiate cord blood T cells into
cells with a reduced capacity to proliferate following polyclonal
activation (Fig. 5A) or
stimulation with alloantigens (Fig. 5B). In contrast,
addition of IFN- alone had a minimal effect on the proliferative
responses of peripheral blood T cells, and only T cells cultured in
both IL-10 and IFN- showed the characteristic reduction in
proliferation to polyclonal activation (Fig. 5C) and a
strongly reduced proliferative response to alloantigens (Fig. 5D).
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suppress the proliferative response of naive
autologous T cells to alloantigens
To establish the functional capacity of in vitro-differentiated
Tr1 cells to act as regulatory/suppressor cells (7), we
tested whether
IL-10+IFN-+IL-2-/lowIL-4-
T cells differentiated in the presence of IFN- and/or IL-10 could
suppress the response of autologous CD4+ T cells.
As shown in Fig. 6, addition of T cells
primed in the absence or presence of IL-10 had a minimal effect on the
proliferation of naive autologous cells. In contrast, T cells cultured
in the presence of IFN-, or IL-10 + IFN-, strongly suppressed the
proliferation of naive autologous T cells. On average, T cells from
cultures with IFN- + IL-10 inhibited the proliferation of naive
cells by 54.8 ± 11.4% (n = 3) when cocultured at
a ratio of 1:1 (naive:polarized) and by 58.1 ± 12.6% at a ratio
of 1:2.
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do not affect TCR-mediated intracellular
signaling or proliferation of CD4+ T cells
It has been described that IL-10 prevents T cell activation
(33) and that IFN- may mediate anti-proliferative
effects by interfering with TCR signaling (34). Therefore,
we determined whether IL-10 and/or IFN- act by altering TCR-mediated
intracellular signals in our system. As shown in Fig. 7A, addition of IFN- and/or
IL-10 either 16 h before, or at the time of, TCR activation
affected neither whole-cell tyrosine phosphorylation nor activation of
the mitogen-activated protein (MAP) family kinases, extracellular
signal-related kinase 1/2, c-Jun N-terminal kinase 1/2 or p38. However,
both IL-10 and IFN- were biologically active as judged by their
ability to induce tyrosine phosphorylation of STAT-3 and STAT-1,
respectively (data not shown).
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did not have significant anti-proliferative
effects when we activated cord blood T cells at day 0 and/or
at day 7 in the presence of IFN- and the indicated polarizing
cytokines (IL-4, IL-12, or IL-10) and measured the proliferative
responses after 3 days. Addition of IFN- during the primary,
secondary, or both the primary and secondary stimulation periods had
only a modest inhibitory effect (30% reduction) on proliferation of
cells cultured with IL-12 or IL-10, whereas no significant effect was
observed in cells cultured with IL-4 (Fig. 7B). Moreover,
results in Fig. 7C show that T cells cultured in IL-4,
IL-12, or IL-10 + IFN- had similar increases in numbers during the
primary (7-day) stimulation period. In contrast, at the end of the
secondary stimulation, during which significant differentiation of Tr1,
Th1, and Th2 cells was observed, the recovery of T cells
cultured in the presence of IL-10 + IFN- was significantly lower
compared with that of cultures with IL-4 or IL-12.
Together, these data indicate that IFN- does not act as a general
anti-proliferative agent, but that it drives the differentiation of
Tr1 cells which, upon reactivation, suppress the proliferation of
bystander non-Tr1 cells. It should be noted that despite the low cell
recovery at day 14 in cultures with IL-10 + IFN-, a significant
number of Tr1 cells could be recovered. Based on the average percentage
of IL-10+IFN-+ cells
(Fig. 3C) and the average fold increases, culture of
106 naive CD4+ T cells in
IL-10 + IFN- would result in 2 x 106
Tr1 cells at day 14 of culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have additive
effects on the differentiation of a population of cells that displays a
Tr1 cytokine production profile:
IL-10+IFN-+IL-2-/lowIL-4-.
Furthermore, a direct differentiation from naive T cells toward a Tr1
phenotype was observed, suggesting that Tr1 cells arise directly from a
CD4+ T cell precursor. Analysis of the
proliferative responses of populations containing Tr1 cells revealed
that priming in the presence of IL-10 + IFN- results in a
synergistic reduction in the proliferative capacity following
polyclonal activation or stimulation with alloantigens. Proliferation
to alloantigens was partially restored by addition of neutralizing
anti-IL-10R and anti-TGF- mAbs. These data correlate with
our previous observations that Tr1 cells have an intrinsically low
proliferative capacity (8) and that they have
immunoregulatory functions that are partially mediated by IL-10 and
TGF- (7). Furthermore, our results suggest that, in
addition to IL-10 and TGF-, other soluble or cell-surface proteins
are involved in regulation of immune responses by Tr1 cells.
Importantly, cultures containing Tr1 cells suppressed the proliferation
of naive autologous T cells, demonstrating that in vitro-differentiated
Tr1 possess the key functional property of T regulatory cells and can
mediate active suppression (35). The suppressive effects
of in vitro-differentiated Tr1 cells are remarkably potent considering
that only 1.5–7.7% of the cells in cultures with IL-10 + IFN- are
Tr1 cells (i.e.,
IL-10+IFN-+). Thus fewer
than 4000 Tr1 cells are capable of suppressing the proliferation of
50,000 naive CD4+ T cells by 50%.
Cord blood and peripheral blood CD4+ T cells
differ in their requirements for exogenous cytokines for the
differentiation of Tr1 cells. Addition of IFN- alone to cord blood T
cells was, in some cases, sufficient to induce a population of T cells
with a Tr1-cytokine production profile and immunosuppressive
properties. Cord blood T cells have the intrinsic ability to produce
high levels of IL-10 (31, 32), which could be further
enhanced by the presence of IFN- (29, 30). Therefore,
autocrine IL-10 production by cord blood T cells may alleviate the
requirement for exogenous IL-10. In contrast, peripheral blood T cells,
which produced 7- to 13-fold less endogenous IL-10 than cord blood T
cells, required both exogenous IL-10 and IFN- to induce the
differentiation of functional Tr1 cells.
It has previously been shown that IL-12 can also induce the
differentiation of
IL-10+IFN-+ cells from
PBMC (36, 37). In our system using purified
CD4+ T cells, we observed that differentiation of
cord blood, but not peripheral blood, in the presence of IL-12 resulted
in a significant population of
IL-10+IFN-+ T cells.
However, the presence of these
IL-10+IFN-+ T cells was
also associated with a high percentage of IL-2-positive cells, and no
reduction in proliferation or suppressive activity. These data suggest
that if Tr1 cells were present, their regulatory effects were largely
reversed in the presence of "classical" Th1 cells that clearly
represent the majority of T cells following culture with IL-12.
IL-10 and IFN- have been shown to prevent activation of T cells and
can affect the growth of multiple cell types (33, 34). In
addition, both cytokines can promote cell-cycle arrest in
CD4+ T cells (38, 39). However,
there are also reports documenting enhancing effects of both IL-10
(40) and type I IFNs (41, 42) on survival and
proliferation of T cells. Our experiments showed that neither IFN-
nor IL-10 had an effect on TCR-mediated activation of MAP family
kinases, indicating that these cytokines did not act by preventing
activation of early events in TCR-mediated signal transduction. In
addition, we did not observe any significant anti-proliferative
effects of IFN- and/or IL-10, as measured by thymidine incorporation
3 days after activation, whereas there was a striking decrease in the
total number of cells recovered at the end of the culture in the
presence of these cytokines. These data suggest that IFN- and IL-10
do not act as general anti-proliferative agents, but rather as
factors that induce the differentiation of Tr1 cells, which themselves
have a low proliferative capacity, and in addition inhibit the growth
of non-Tr1 cells present in the culture.
Our data indicate that there is a difference between the cytokines
required for the differentiation and the effector functions of Tr1
cells. Although TGF- in part mediates the immunosuppressive
functions of Tr1 cells, and has immunosuppressive effects on T
cell-mediated pathology (13, 43), we found that TGF-
had no effect on the differentiation of these cells. Rather, a general
suppression of cytokine production (IL-2, IL-4, IL-10, and IFN-) by
T cells primed in the presence of TGF- was observed. These data are
in agreement with recent reports describing that TGF- inhibits
production of IFN- and IL-4, and differentiation of murine Th1 and
Th2 cells (44, 45).
In contrast to TGF-, IL-10 was important for both the
differentiation and effector functions of Tr1 cells. IL-10 was required
for the differentiation of Tr1 cells derived from peripheral blood, and
despite the fact that cord blood cells produced considerable amounts of
endogenous IL-10, in several independent experiments, exogenous IL-10
further increased the number of IL-10+ cord blood
T cells. IL-10 is well characterized as an effector cytokine involved
in regulation of T cell-mediated responses via both direct and indirect
mechanisms (reviewed in Ref. 33), but its role in
differentiation of Th cells has not been extensively addressed. The
present data support previous studies that demonstrated that T cell
priming in the presence of APCs and IL-10 results in cells that produce
IL-10 and TGF- and have immunoregulatory properties
(7).
In this study, addition of IFN- resulted not only in enhanced IL-10
and IFN- production compared with cultures in IL-10 alone, but also
in down-regulation of IL-4 and IL-2 synthesis, consistent with previous
reports (29, 30, 46, 47, 48). Through its ability to inhibit
production of IL-4, IFN- has previously been implicated in the
differentiation of human Th1 cells (49). However, our data
show that IFN- is not involved in the differentiation of Th1 cells,
but rather, together with IL-10, is a key factor in the differentiation
of Tr1 cells.
The receptors for IL-10 and IFN- are structurally similar: both are
members of the class II cytokine receptor family (50) and
they activate similar pathways of intracellular signal transduction.
However, the molecular mechanism(s) by which IL-10 and IFN- induce
the differentiation of Tr1 cells are unknown. It is well documented
that the Jak/STAT pathway is the key intracellular pathway involved in
differentiation of Th1 and Th2 cells (51). IL-10 activates
STAT-1 and STAT-3 (52, 53), whereas IFN- activates
STAT-1, -2, and -3 in most cells (54), and in human
lymphocytes can also activate STAT-4 and -5 (42, 49). It
remains to be determined which members of the Jak/STAT pathway are
required for the differentiation of Tr1 cells.
In the human, priming of naive T cells with type 2 dendritic cells
(DC2) cultured in the presence of IL-3 and activated by CD40 ligand
results in polarization toward Th2 cells (55). However,
viral-infected DC2 cells produce high levels of IFN- (56, 57); therefore, it is tempting to speculate that DC2 cells may
also be involved in the differentiation of Tr1 cells.
The hypothesis that type I IFNs may not only be involved in innate
immunity but also in the generation of immunoregulatory T cells in vivo
is supported by the observation that in the absence of functional IFN
responses, viral infection results in a profound Th2-mediated
inflammation (58). Furthermore, treatment with IFN- of
patients suffering from relapsing-remitting multiple sclerosis results
in increased levels of IL-10 production by mononuclear cells and
favorably alters the disease course (59, 60, 61).
A number of T regulatory cell subsets have been identified, and there
is growing evidence that these cells are essential for controlling
immune responses (reviewed in Refs. 35, 62, 63). The
relationship between Tr1 cells and other types of
CD4+ T regulatory cells, such as those that are
CD25+, CD45RBlow, or
TGF-+ (Th3) is unclear. Although we have much
to learn about the biology of T regulatory cells, it is clear that via
expression of inhibitory cell-surface molecules and production of
cytokines such as IL-10 and TGF-, T regulatory cells maintain
peripheral tolerance to self and foreign Ags, and that in their absence
the host may succumb to a variety of autoimmune and chronic
inflammatory diseases (11, 13, 35, 63, 64). We have shown
that IL-10 and IFN- promote the in vitro differentiation of type 1 T
regulatory cells. The ability to isolate and culture Tr1 cells in vitro
is a crucial step toward learning more about their basic biology and
exploring the use of these cells as a "cellular therapy" to
regulate immune responses to self- or allo-Ags in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria-Grazia Roncarolo, San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, Milan, Italy 20132.
3 Abbreviations used in this paper: Tr1, T regulatory type 1; MAP, mitogen-activated protein; DC2, type 2 dendritic cell; rh, recombinant human.
Received for publication December 5, 2000. Accepted for publication March 1, 2001.
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B. Bohle, A. Breitwieser, B. Zwolfer, B. Jahn-Schmid, M. Sara, U. B. Sleytr, and C. Ebner A Novel Approach to Specific Allergy Treatment: The Recombinant Fusion Protein of a Bacterial Cell Surface (S-Layer) Protein and the Major Birch Pollen Allergen Bet v 1 (rSbsC-Bet v 1) Combines Reduced Allergenicity with Immunomodulating Capacity J. Immunol., June 1, 2004; 172(11): 6642 - 6648. [Abstract] [Full Text] [PDF]
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 S. M. Blois, C. D. Alba Soto, M. Tometten, B. F. Klapp, R. A. Margni, and P. C. Arck Lineage, Maturity, and Phenotype of Uterine Murine Dendritic Cells Throughout Gestation Indicate a Protective Role in Maintaining Pregnancy Biol Reprod, April 1, 2004; 70(4): 1018 - 1023. [Abstract] [Full Text] [PDF]
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S. G. Zheng, J. H. Wang, M. N. Koss, F. Quismorio Jr., J. D. Gray, and D. A. Horwitz CD4+ and CD8+ Regulatory T Cells Generated Ex Vivo with IL-2 and TGF-{beta} Suppress a Stimulatory Graft-versus-Host Disease with a Lupus-Like Syndrome J. Immunol., February 1, 2004; 172(3): 1531 - 1539. [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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 M.-T. Lin, B. Storer, P. J. Martin, L.-H. Tseng, T. Gooley, P.-J. Chen, and J. A. Hansen Relation of an Interleukin-10 Promoter Polymorphism to Graft-versus-Host Disease and Survival after Hematopoietic-Cell Transplantation N. Engl. J. Med., December 4, 2003; 349(23): 2201 - 2210. [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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T. Nagai, O. Devergne, T. F. Mueller, D. L. Perkins, J. M. van Seventer, and G. A. van Seventer Timing of IFN-{beta} Exposure during Human Dendritic Cell Maturation and Naive Th Cell Stimulation Has Contrasting Effects on Th1 Subset Generation: A Role for IFN-{beta}-Mediated Regulation of IL-12 Family Cytokines and IL-18 in Naive Th Cell Differentiation J. Immunol., November 15, 2003; 171(10): 5233 - 5243. [Abstract] [Full Text] [PDF]
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W. Janssens, V. Carlier, B. Wu, L. VanderElst, M. G. Jacquemin, and J.-M. R. Saint-Remy CD4+CD25+ T Cells Lyse Antigen-Presenting B Cells by Fas-Fas Ligand Interaction in an Epitope-Specific Manner J. Immunol., November 1, 2003; 171(9): 4604 - 4612. [Abstract] [Full Text] [PDF]
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M. Lohning, A. Richter, T. Stamm, J. Hu-Li, M. Assenmacher, W. E. Paul, and A. Radbruch Establishment of memory for IL-10 expression in developing T helper 2 cells requires repetitive IL-4 costimulation and does not impair proliferation PNAS, October 14, 2003; 100(21): 12307 - 12312. [Abstract] [Full Text] [PDF]
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 Z. Mallat, A. Gojova, V. Brun, B. Esposito, N. Fournier, F. Cottrez, A. Tedgui, and H. Groux Induction of a Regulatory T Cell Type 1 Response Reduces the Development of Atherosclerosis in Apolipoprotein E-Knockout Mice Circulation, September 9, 2003; 108(10): 1232 - 1237. [Abstract] [Full Text] [PDF]
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K. S. Goudy, B. R. Burkhardt, C. Wasserfall, S. Song, M. L. Campbell-Thompson, T. Brusko, M. A. Powers, M. J. Clare-Salzler, E. S. Sobel, T. M. Ellis, et al. Systemic Overexpression of IL-10 Induces CD4+CD25+ Cell Populations In Vivo and Ameliorates Type 1 Diabetes in Nonobese Diabetic Mice in a Dose-Dependent Fashion J. Immunol., September 1, 2003; 171(5): 2270 - 2278. [Abstract] [Full Text] [PDF]
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K. A. Papadakis, C. Landers, J. Prehn, E. A. Kouroumalis, S. T. Moreno, J.-C. Gutierrez-Ramos, M. R. Hodge, and S. R. Targan CC Chemokine Receptor 9 Expression Defines a Subset of Peripheral Blood Lymphocytes with Mucosal T Cell Phenotype and Th1 or T-Regulatory 1 Cytokine Profile J. Immunol., July 1, 2003; 171(1): 159 - 165. [Abstract] [Full Text] [PDF]
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Z.-m. Chen, M. J. O'Shaughnessy, I. Gramaglia, A. Panoskaltsis-Mortari, W. J. Murphy, S. Narula, M. G. Roncarolo, and B. R. Blazar IL-10 and TGF-{beta} induce alloreactive CD4+CD25- T cells to acquire regulatory cell function Blood, June 15, 2003; 101(12): 5076 - 5083. [Abstract] [Full Text] [PDF]
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 K. Asadullah, W. Sterry, and H. D. Volk Interleukin-10 Therapy--Review of a New Approach Pharmacol. Rev., June 1, 2003; 55(2): 241 - 269. [Abstract] [Full Text] [PDF]
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D. Trabattoni, M. Saresella, M. Biasin, A. Boasso, L. Piacentini, P. Ferrante, H. Dong, R. Maserati, G. M. Shearer, L. Chen, et al. B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression Blood, April 1, 2003; 101(7): 2514 - 2520. [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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S. Rutella, L. Pierelli, G. Bonanno, S. Sica, F. Ameglio, E. Capoluongo, A. Mariotti, G. Scambia, G. d'Onofrio, and G. Leone Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells Blood, September 18, 2002; 100(7): 2562 - 2571. [Abstract] [Full Text] [PDF]
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 K. Asadullah, W. Sterry, and H.-D. Volk Analysis of Cytokine Expression in Dermatology Arch Dermatol, September 1, 2002; 138(9): 1189 - 1196. [Abstract] [Full Text] [PDF]
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P. A. Taylor, T. M. Friedman, R. Korngold, R. J. Noelle, and B. R. Blazar Tolerance induction of alloreactive T cells via ex vivo blockade of the CD40:CD40L costimulatory pathway results in the generation of a potent immune regulatory cell Blood, May 29, 2002; 99(12): 4601 - 4609. [Abstract] [Full Text] [PDF]
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C. P. Gray, P. Arosio, and P. Hersey Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells Blood, May 1, 2002; 99(9): 3326 - 3334. [Abstract] [Full Text] [PDF]
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F. J. Barrat, D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, and A. O'Garra In Vitro Generation of Interleukin 10-producing Regulatory CD4+ T Cells Is Induced by Immunosuppressive Drugs and Inhibited by T Helper Type 1 (Th1)- and Th2-inducing Cytokines J. Exp. Med., March 4, 2002; 195(5): 603 - 616. [Abstract] [Full Text] [PDF]
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P. McGuirk, C. McCann, and K. H.G. Mills Pathogen-specific T Regulatory 1 Cells Induced in the Respiratory Tract by a Bacterial Molecule that Stimulates Interleukin 10 Production by Dendritic Cells: A Novel Strategy for Evasion of Protective T Helper Type 1 Responses by Bordetella pertussis J. Exp. Med., January 22, 2002; 195(2): 221 - 231. [Abstract] [Full Text] [PDF]
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R. M. Steinman and M. C. Nussenzweig Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance PNAS, January 1, 2002; (2002) 231606698. [Abstract] [Full Text] [PDF]
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J. M. Phillips, N. M. Parish, M. Drage, and A. Cooke Cutting Edge: Interactions Through the IL-10 Receptor Regulate Autoimmune Diabetes J. Immunol., December 1, 2001; 167(11): 6087 - 6091. [Abstract] [Full Text] [PDF]
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G. Trinchieri Regulatory Role of T Cells Producing both Interferon {gamma} and Interleukin 10 in Persistent Infection J. Exp. Med., November 19, 2001; 194(10): F53 - F57. [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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