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Costimulatory effects of IL-1 on the expansion/differentiation of CD4

CD25

Foxp3

and CD4

CD25

Foxp3

T cells

Carine Brinster1and Ethan M. Shevach2

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

Abstract: CD4CD25forkhead box p3 (Foxp3) regulatory T cells (Treg) control peripheral toler- ance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proin- flammatory cytokines such as IL-1 or IL-6 that func- tion as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3 Treg in vitro. When CD4CD25 T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3 T cells was ob- served. The effects of IL-1 were mediated on CD4CD25Foxp3T cells present in the starting population rather than on the DC or on the CD4CD25Foxp3T cells. In contrast, stimula- tion of CD4CD25T cells with plate-bound anti- CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4CD25Foxp3T cell popula- tion composed of NKT cells and non-NKT, IL-17- producing cells. Foxp3Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimu- lated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4CD25 T cells for cellular biotherapy. J. Leukoc. Biol. 84: 000 – 000; 2008.

Key Words: rodentdendritic cellscostimulationcell prolifer- ation

INTRODUCTION

CD4CD25forkhead box p3 (Foxp3) regulatory T cells (Treg) play an important role in modulation of immune re- sponses. Treg were first characterized in mice, where they are generated in the thymus and constitute 5–10% of the CD4T cell pool in the periphery. In humans and mice, they have been shown to suppress CD4and CD8T cell responses in a cell contact-dependent, cytokine-independent manner in vitro and to require activation through the TCR to be functional [1, 2].

Accumulating data have shown that Foxp3Treg play a role in the control of autoimmune disorders, infections, tumors, al- lergy, and transplantation tolerance [3].

Adoptive cellular immunotherapy with CD4CD25Foxp3 T cells has been proposed as a potential therapy for chronic autoimmune diseases and for the induction of tolerance to allografts (graft versus host disease, organ transplantation) [4].

A number of major problems exist with implementation of this approach. First, because of the lack of a cell surface antigen that is specific for Foxp3Treg, their purification requires the use of surrogate markers, such as CD25, which are also ex- pressed on activated, conventional T cells. In general, even highly purified CD4CD25T cells routinely contain 5–10%

or more Foxp3T cells. In addition to the lack of a specific marker, the in vitro expansion of Treg to numbers sufficient for use in cell-transfer studies is hampered by their “anergic”

phenotype and hyporesponsiveness, even with strong TCR- based stimulation. Nevertheless, stimulation of mouse CD4CD25T cells with immobilized anti-CD3 (with or with- out anti-CD28) in the presence of high concentrations of ex- ogenous IL-2 or IL-4 and to a lesser extent, IL-7, leads to their significant expansion over 4 –10 days of culture [5]. However, many of these early studies did not evaluate Foxp3 expression by the expanded cells, and significant outgrowth of CD25Foxp3 T cells was likely. The expansion of human CD4CD25 T cells under similar conditions is complicated further by the observation that under certain stimulatory con- ditions, activated human CD4Foxp3T cells may be induced to express Foxp3 in the absence of Treg functions [6].

There is considerable controversy as to the potential role of accessory cells and their associated costimulatory signals in the expansion of Foxp3 Treg. We [7] and others [8] have previously shown that mature, fully activated bone marrow- derived dendritic cells (BMDC), but not splenic DC, could induce the proliferation of mouse CD4CD25 Treg in the absence of exogenous IL-2; similar results were also reported in humans [9]. Although the mechanism by which activated BMDC stimulate the proliferation of Treg has not been defined, one likely possibility is that they produce significant amounts of proinflammatory cytokines such as IL-1 or IL-6, which function as growth factors for Treg expansion. Indeed, Kubo et al. [10] have recently shown that the addition of anti-IL-1 and

1Current address: Laboratory of Immuno-Hematology, Medical School, Uni- versity of Rennes 1, 2, avenue du Pr. Le´on Bernard, 35 043 Rennes, France.

2Correspondence: LI/NIAID/NIH, Building 10, Room 11N315, 10 Center Drive-MSC 1892, Bethesda, MD 20892, USA. E-mail: eshevach@niaid.nih.gov Received February 5, 2008; revised April 9, 2008; accepted April 16, 2008.

doi: 10.1189/jlb.0208085

0741-5400/08/0084-0001 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 84, August 2008 1

Uncorrected Version. Published on May 13, 2008 as DOI:10.1189/jlb.0208085

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anti-IL-6 antibodies would abrogate the expansion of CD4CD25 Treg cultured with mature BMDC.

Cytokines of the IL-1 family have been shown to display pleiotropic effects and to be involved in host defense and the neuroimmune-endocrine network regulation. Polymorphism or absence of the IL-1R antagonist gene belonging to the IL-1 family cluster has thus been associated with the severity/

susceptibility of inflammatory diseases such as ankylosing spondylitis or chronic inflammatory polyarthropathy [11, 12].

Similarly, IL-1␤ and -␣, the most characterized members of this family, were shown with TNF-␣ to play a role in the pathogenesis and development of rheumatoid arthritis [13, 14].

The direct effects of the IL-1 family of cytokines on the survival, expansion, or function of Foxp3 Treg have not yet been investigated. Here, we have analyzed in-depth the co- stimulatory effects of IL-1 on the expansion of Foxp3Treg in vitro. We demonstrate that in the presence of splenic DC and soluble anti-CD3, the addition of IL-1␣,␤ results in the ex- pansion of Foxp3T cells. However, the costimulatory effects of IL-1 on the expansion of Foxp3Treg were not mediated by acting on the splenic DC or on the Foxp3T cells themselves but on the few CD4CD25Foxp3 T cells that routinely contaminate cell sorter-purified CD4CD25T cell prepara- tions. Surprisingly, when CD4CD25T cells were stimulated by immobilized anti-CD3 and IL-1␣,␤ in the absence of DC, few Foxp3 could be isolated, and a marked expansion of IL-17-producing NKT cells and conventional CD4 T cells was observed. We conclude from these studies that IL-1 has pleiotropic effects on CD4CD25Foxp3T cells. In the pres- ence of DC and IL-1, CD4CD25Foxp3T cells facilitate the selective expansion of Foxp3 Treg. However, under DC-free conditions, IL-1 promotes the expansion of NKT and non-NKT, IL-17-producing T cells that may actually be pathogenic T cells. The implication of these findings for the design of pro- tocols for the preparation of Foxp3Treg for therapeutic use will be discussed. In addition, these results suggest that a complex, fine-tuning mechanism might exist for the regulation of the expansion of Foxp3 Treg in vivo, depending on the nature of the surrounding cytokine environment.

MATERIALS AND METHODS Mice

BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD, USA). Type I IL-1R-deficient (⫺/⫺) mice (backcrossed on a C57BL/6 background) [15] were obtained from Taconic Farms (Germantown, MD, USA) and CD1(d)⫺/⫺mice (backcrossed 11 times on a BALB/cJ back- ground) [16] from The Jackson Laboratories (Bar Harbor, ME, USA). Yasmine Belkaid [Mucosal Immunology Unit and Immunobiology Section, Labora- tory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA] kindly provided enhanced GFP (eGFP) Foxp3 knock-in mice. All mice were bred at the NIH under specific pathogen-free conditions and used at 6 – 8 weeks old.

Splenic DC purification and in vitro activation

After isolation, spleens were fragmented and digested in the presence of liberase blendzyme II (Roche Molecular Biochemicals, Indianapolis, IN, USA) and DNase (2␮g/ml; Roche Applied Science, Indianapolis, IN, USA) for 30 min at 37°C in complete medium (modified RPMI 1640, supplemented by 10%

FBS, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 10 mM Hepes, 4⫻10⫺7M␤-ME, 1 mM essential amino acids, 1 mM sodium pyruvate;

all from Biofluids, Rockville, MD, USA). RBCs were removed using an ammonium chloride-potassium lysis buffer (Biosource, Camarillo, CA, USA).

T and NK cells were depleted after addition of PE-conjugated anti-NK (DX5 clone) and anti-CD3 (2C-11 clone) antibodies (BD PharMingen, San Diego, CA, USA) and anti-PE magnetic beads on an autoMACS (Miltenyi Biotech, Auburn, CA, USA). CD11ccells were then isolated by positive selection on mass spectrometry magnetic columns (Miltenyi Biotech). The purity was 90 – 95%. For their activation in vitro, the CD11ccells were cultured overnight in complete medium supplemented with LPS (Escherichia coli strain 0111:B4, Sigma Chemical Co., St. Louis, MO, USA) at a concentration of 100 ng/ml.

After activation, DC were collected and analyzed for their phenotype by flow cytometry. They were stained with the following antibodies (all from BD PharMingen): anti-CD11c-biotinylated (HL3 clone) and streptavidin-allophy- cocyanin, PE-conjugated anti-CD86 (GL1 clone), anti-CD80 (16-10A1 clone), anti-CD40 (3/23 clone), or anti-I-A/I-E (M5/114.15.2 clone).

CD4CD25T cell purification

CD4CD25T cells were purified using an autoMACS as described previ- ously [2]. For most of the experiments, CD4CD25T cells were sorted after staining with FITC-conjugated anti-CD4 (RM4-5 clone) and PE-conjugated anti-CD25 (PC61 clone) after FcR blocking (2.4G2 clone; all purchased from BD PharMingen). Foxp3 CD4 CD25 T cells were sorted according to eGFP expression from the eGFP knock-in mice, Tricolor-conjugated anti-CD4 (Caltag, Burlingame, CA, USA), and PE-conjugated anti-CD25.

CFSE labeling

CD4CD25 T cells were labeled with 1 ␮M CFSE for 8 min at room temperature and washed three times in FBS and complete medium before being seeded.

Analysis of the CD4CD25T cell population

CD4 T cells were purified by autoMACS from peripheral and mesenteric lymph node suspensions and stained with the following mAb: PE-conjugated anti-CD25 (PC61 clone, BD PharMingen) and FITC-conjugated anti-Foxp3 (eBiosciences, San Diego, CA, USA). NKT cells were detected using the allophycocyanin-conjugated CD1 tetramer provided by the NIH tetramer fa- cility (Emory University, Atlanta, GA, USA). The cells were stained 1 h at 4°C with the CD1d tetramer.

CD4CD25T lymphocyte cultures and proliferation assays

After activation, splenic DC were collected and washed twice to remove any cytokines. Viable cells were counted after exclusion of dead cells by trypan blue. eGFP CD4CD25 T cells and CFSE-labeled CD4CD25 were seeded at 3⫻ 104cells/well in triplicates in 96-well plates at a DC:T cell ratio of 1:1. The cultures were grown for 72 h in the presence of soluble anti-CD3 (1␮g/ml, 2C-11 clone, BD PharMingen) at 37°C. IL-1␣ and IL-1␤ were added at a final concentration of 5 ng/ml (2.5 ng/ml each), IL-4 at 5 ng/ml, and IL-2 at 50 U/ml. In some experiments, the anti-IL-2 antibody (clone S4B6, BD PharMingen) was added at a concentration of 20␮g/ml. eGFPCD4CD25 T cells and CD4CD25were also seeded at 3⫻ 104cells/well in triplicates in 96-well plates with plate-bound anti-CD3 (5 ␮g/ml) for 5 days, with or without IL-1␣,␤ or IL-2.

For the3H-TdR-based proliferation assays, the cells were pulsed for the last 18 h with 1␮Ci3H-TdR (Amersham Biosciences, Piscataway, NJ, USA) before being collected and assessed for radioactivity. All results were expressed as the mean cpmSDof triplicate cultures.

Intracellular cytokine and Foxp3 staining

For intracellular cytokine staining, the cells were harvested, pooled, and seeded in 24-well plates for 6 h in the presence of plate-bound anti-CD3 and anti-CD28 antibodies (3␮g/ml each). Monensin (2 ␮M; Calbiochem, La Jolla, CA, USA) was added in the last 2 h. The cells were then fixed in 4%

paraformaldehyde, washed, permeabilized in PBS containing 0.1% saponin and 0.1% BSA, and stained with allophycocyanin-coupled anti-IL-2 (JES6-

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5H4 clone), allophycocyanin-anti-IL-13 (kindly provided by W. E. Paul), allophycocyanin-anti-IFN-␥ (XMG1.2 clone), PE-anti-IL-17 (TC11-18H10 clone), and PE-anti-IL-4 (11B11 clone). Foxp3 staining was performed accord- ing the manufacturer’s instructions (Foxp3 staining kit, eBiosciences).

Flow cytometry analysis

Flow cytometry acquisition of the samples was performed on a FACSCalibur (BD Immunocytometry Systems, Mountain View, CA, USA) and analyzed using CellQuest威 and FlowJo威 softwares.

RESULTS

IL-1␣ and IL-1␤ drive the proliferation of CD4CD25T cells when cultured in the presence of mature splenic DC

We [7] and others [8] have previously shown that TCR stimu- lation in the presence of mature BMDC, but not splenic DC, would drive the expansion of CD4CD25 T cells in the absence of IL-2. One study [10] suggested that the production of proinflammatory cytokines by activated BMDC played an important role in driving this response, as the addition of anti-IL-6 and anti-IL-1␣,␤ antibodies to these cultures inhib- ited proliferation. However, in preliminary studies, we failed to see any effects of anti-IL-1 and/or anti-IL-6 on the proliferation of CD4CD25T cells in the presence of mature BMDC. To directly examine the role of IL-1 in the expansion of CD4CD25 Treg, we cultured mature splenic DC with cell sorter-purified CD4CD25T cells in the presence or absence of exogenous IL-1␣ and -␤. As previously reported [7], no

proliferation was observed in the presence of soluble anti-CD3 and mature splenic DC, but a substantial response was seen when IL-1␣ and -␤ were added to the cultures; the addition of exogenous IL-2 resulted in a somewhat greater proliferative response (Fig. 1A). Although the starting population was

⬃96% Foxp3(Fig. 1B), analysis of Foxp3 expression after 3 days of culture indicated that⬃80% of the cells expanded in the presence of IL-1 still expressed high levels of Foxp3 but that some Foxp3T cells (⬃20%) were also present (Fig. 1B).

Foxp3and Foxp3subsets expanded, with an average of six rounds of division for the Foxp3 subset (Fig. 1B, upper panels), and the Foxp3 subset proliferated faster and com- pletely diluted CFSE (Fig. 1B, lower panels).

To determine if the effect of IL-1 was mediated directly on the responder CD4CD25Treg or indirectly on the splenic DC via the induction of soluble factors or costimulatory mol- ecules, we purified splenic DC and CD4CD25T cells from IL-1R⫺/⫺mice. Although we observed a reduced percentage and number of CD4 and CD8 T cells in the lymph nodes from IL-1R⫺/⫺mice, the percentage of CD25Foxp3cells in the CD4population was similar to that seen in wild-type mice (data not shown). CD4CD25T cells isolated from wild-type mice demonstrated a significant, proliferative response when cultured with splenic DC from IL-1R⫺/⫺mice. CD4CD25 T cells isolated from IL-1R⫺/⫺mice failed to proliferate when cultured in the presence of IL-1 and splenic DC from wild-type mice (Fig. 1C). These results indicate that in the presence of mature splenic DC, IL-1 exerts its costimulatory effects by acting directly on CD4CD25 T cells and not on the DC.

Fig. 1.IL-1␣,␤ drive the proliferation of CD4CD25T cells in the presence of ma- ture splenic DC. (A) FACS-purified CD4CD25T cells were stimulated with soluble anti-CD3 (1␮g/ml), mature splenic DC, in the presence or absence of IL-1␣,␤

(5 ng/ml) or IL-2 (50 U/ml).3H-TdR incor- poration was measured after 72 h of culture, and values are represented as the mean cpm of triplicate culture wells SD. (B) FACS-purified CD4CD25 T cells were stained for the Foxp3 expression. The sorted cells were CFSE-labeled and stimu- lated with soluble anti-CD3 and mature splenic DC in the presence of IL-1␣,␤ for 3 days. The cells were then analyzed for their CFSE dilution profile and Foxp3 expres- sion. The histograms represent the CFSE divisions of the gated Foxp3and Foxp3 populations. (C) FACS-purified CD4CD25 T cells sorted from wild-type (wT) or IL- 1R⫺/⫺ mice were cultured with mature splenic DC (sp.DC) isolated from wild-type or IL-1R⫺/⫺mice and anti-CD3 (1␮g/ml) in the presence or absence of IL-1␣,␤ (5 ng/ml). 3H-TdR incorporation was mea- sured after 72 h of culture. These data are representative of four separate experiments.

ns, Not significant.

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The costimulatory effects of IL-1 on the expansion of CD4CD25Foxp3 cells are mediated indirectly via CD4CD25Foxp3 T cells

Although the results above strongly suggested that IL-1 was acting directly on the CD4CD25 population, it remained possible that the target for the action of IL-1 was the minor population of CD4CD25Foxp3T cells that always con- taminate the CD4CD25Foxp3 cells (3–10% present in sorted CD4CD25T cells). To address this issue, we made use of the recently engineered mice expressing eGFP in the Foxp3 locus, sorted the CD4CD25eGFP cells [99.3%

eGFP(Foxp3); data not shown], and cultured them with mature splenic DC and IL-1 (Fig. 2A). In the presence of IL-1, the proliferative responses of the CD4CD25eGFPT cells were three- to fourfold lower than the CD4CD25 T cells from wild-type mice but similar in the presence of exogenous IL-2. In addition, the recovery of the CD4CD25eGFPcells (less than or equal to onefold) was much lower than that of CD4CD25T cells (three- to fourfold increase) after 3 days of culture (data not shown).

One possible explanation for the costimulatory effects of the CD4CD25Foxp3 subset on the proliferation of the CD4CD25Foxp3 population is that the former produces IL-2 that drives the proliferation of the Foxp3 T cells. To assess this possibility, we cultured CD4CD25T cells with mature splenic DC and IL-1 in the presence or absence of anti-IL-2. The proliferation of CD4CD25 T cells was re- duced by ⬃50% (Fig. 2B), indicating that the CD4CD25Foxp3T cells were capable of producing IL-2.

Taken together, these results suggest that IL-1 is primarily mediating its costimulatory effects on the expansion of CD4CD25Foxp3 T cells by acting indirectly on the CD4CD25Foxp3 population to induce the production of IL-2, as well as potentially other stimulatory cytokines, or by inducing the expression of costimulatory molecules on the surface of the Foxp3cells that augment the proliferation of the Foxp3cells.

IL-1 induces the expansion of CD4CD25Foxp3 T cells in the absence of allophycocyanin

To more directly analyze the effects of IL-1 in the activation of CD4CD25 T cells, we stimulated highly purified

CD4CD25 T cells from peripheral lymph nodes of C57BL/6 mice (90.5–96.8% Foxp3; data not shown) with plate-bound anti-CD3 in the presence or absence of IL-1.

Surprisingly, CD4CD25 T cells proliferated as well or better in the presence of IL-1 as the CD4CD25 T cells stimulated with exogenous IL-4 (Fig. 3, left panel; or IL-2;

data not shown). The extent of cellular expansion on Day 5 was comparable in cultures stimulated with IL-1 or IL-4 (mean of 3.3⫾1.0 in IL-1 and 4.2⫾1.3 in IL-4). Consistent with the data presented in Figure 2, sorted CD4CD25eGFP cells failed to proliferate when cultured in the presence of IL-1 but readily proliferated in the presence of exogenous IL-2 (Fig. 3, right panel).

IL-1 selectively induces the expansion of Foxp3 NKT cells and IL-17-producing cells

As IL-1 appeared to exert a potent, costimulatory effect on the expansion of CD4CD25Foxp3 T cells rather than the Foxp3cells, it was important to analyze in detail the pheno- type of the expanded cells. Seventy-five percent of the recov- ered cells were Foxp3, expressed low levels of CD4, and were enriched in V␤8, CD1d-tetramerNKT cells (⬃25% CD1d-

Fig. 2. CD4CD25Foxp3T cells modu- late the proliferation of CD4CD25Foxp3 T cells. (A) FACS-sorted CD4CD25and CD4CD25eGFPT cells were stimulated with soluble anti-CD3 (1 ␮g/ml), mature splenic DC in the presence or absence of IL-1␣,␤ (5 ng/ml) or IL-2 (50 U/ml).3H-TdR incorporation was measured after 72 h of culture. (B) FACS-purified CD4CD25T cells were stimulated with soluble anti-CD3 (1 ␮g/ml) and mature splenic DC in the presence of IL-1␣,␤ (5 ng/ml). The anti- IL-2 antibody (clone S4B6) was added at 20

␮g/ml.

Fig. 3. IL-1␣,␤ costimulate the expansion of CD4CD25Foxp3T cells when CD4CD25T cells are stimulated with plate-bound anti-CD3. FACS- sorted CD4CD25 T cells were stimulated with plate-bound anti-CD3 (5

␮g/ml) in the presence of medium alone, IL-1␣,␤ (5 ng/ml), or IL-4 (5 ng/ml).

3H-TdR incorporation was determined after 5 days of culture.

CD4CD25eGFP T cells were stimulated with plate-bound anti-CD3 (5

␮g/ml) in the presence of medium, IL-1␣,␤, or IL-2 (50 U/ml). 3H-TdR incorporation was measured after 5 days of culture. This experiment was performed three times with similar results.

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tetramer;⬃25% V␤8; Fig. 4A). In contrast, the Foxp3 cells were uniformly CD4high and were ⬃20% V␤8. The Foxp3CD1d tetramer cells also expressed CD94 (25.2% of the CD1d tetramer cells) and NKG2A,C,E (43.8% of the CD1d tetramer cells) receptors but were negative for the NK1.1 marker (data not shown). In addition to their unique pattern of cell surface antigen expression, intracellular cyto- kine staining of the Foxp3cells, expanded in the presence of IL-1 (Fig. 4B), revealed that they were markedly enriched in IL-17 (56.2% of total cells representing⬃75% of the Foxp3 cells) and IL-13 producers (22.4% of total cells representing

⬃30% of the Foxp3cells). Approximately 20% of the Th17 cells were CD1d tetramer cells. The capacity of IL-1 to expand the CD4CD25Foxp3 cells was unique, as CD4CD25 T cells expanded in IL-2 or IL-4 (Fig. 4C) remained predominantly Foxp3 and contained few CD1d tetramer cells (⬍2%) or IL-17 producers (⬍1%). Taken together, these results indicate that the polyclonal activation of the CD4CD25population in the absence of DC but in the presence of IL-1 results in the outgrowth of several distinct Foxp3T cells subsets including NKT cells capable of pro- ducing IL-17 and IL-13, as well as a subset of non-NKT, IL-17-producing cells.

CD4CD25Foxp3 NKT cells can be detected in normal lymph nodes

CD4CD25NKT cells have not been described previously in the mouse, although present in human peripheral blood [17]. We next determined whether small numbers of CD4CD25Foxp3 CD1d tetramer cells were present in the starting populations of CD4CD25T cells used in our studies. We purified CD4 T cells from peripheral lymph nodes of BALB/c, C57BL/6, and CD1d⫺/⫺ mice and ana- lyzed them for expression of CD25, invariant V␣14V␤8 TCR (with the CD1d tetramer), and Foxp3 (Fig. 5). Eight to 11% of the CD4population in all strains expressed CD25 (Fig. 5A). When the CD4CD25subset was analyzed (Fig.

5B), 1.2% of the cells from C57BL/6 mice bound the CD1d tetramer (Fig. 5B, middle panel). Few CD4CD25Foxp3 CD1d tetramercells were detected in BALB/c (Fig. 5B, top panel), and none was present in CD1d⫺/⫺ mice (Fig. 5B, bottom panel) or ␤2-microglobulin⫺/⫺ mice (data not shown). Although NKT cells represent, at the most, only 1%

of the starting CD4CD25 population, they appear to expand selectively during TCR stimulation in the presence of IL-1.

Fig. 4. CD4CD25T cells expanded in the presence of IL-1␣,␤ are enriched in Foxp3CD1d tetramerNKT cells and IL-17-producing T cells. (A) CFSE profile, phenotype (CD4, Foxp3, CD1d tetramer, V␤8 expression), and (B) cytokine intracellular staining of cells recovered 5 days after culture of the CD4CD25 T cells with plate-bound anti-CD3 (5␮g/ml) and IL-1␣,␤. (C) CFSE profile, phenotype (Foxp3, CD1d tetramer), and cytokine intracellular staining of cells recovered 5 days after culture of CD4CD25T cells with plate-bound anti-CD3 (5␮g/ml) and IL-2 (50 U/ml) or IL-4 (5 ng/ml). The results are shown as a representative experiment from three separate experiments.

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Mature splenic DC facilitate the growth of CD4CD25Foxp3T cells

Although stimulation of CD4CD25 T cells by plate-bound anti-CD3 in the presence of IL-1 promotes the expansion of NKT cells and Th17 cells rather than Foxp3Treg, stimulation of the same population with soluble anti-CD3 in the presence of IL-1 and mature splenic DC facilitated the expansion of CD4CD25Foxp3 Treg (Fig. 1, A and B). To resolve this paradox, we stimulated CD4CD25 T (90 –95% Foxp3) cells with plate-bound anti-CD3 in the presence of IL-1 and mature splenic DC for 5 days (Fig. 6). Under these stimulatory conditions, ⬃60% of the recovered cells were still Foxp3, and⬃40% were Foxp3. About 25% of the Foxp3cells were

CD1d tetramerNKT cells, and⬃50% were IL-17-producers.

Thus, mature splenic DC favor the growth of Foxp3Treg over Foxp3T cells, but the latter remains necessary for the expan- sion of Foxp3cells.

DISCUSSION

The initial goal of this work was to determine the mechanism by which highly activated BMDC were capable of inducing the expansion of Foxp3Treg by TCR stimulation in the absence of exogenous cytokines. As the study of Kubo et al. [10] raised the possibility that IL-1 and/or IL-6 production by the acti- vated DC played a role in the stimulatory activity of the DC, we tested whether the addition of IL-1 to cultures of nonstimula- tory splenic DC and CD4CD25 T cells would lead to ex- pansion of the Treg. We observed marked stimulation of the Foxp3cells, but some outgrowth of Foxp3T cells was also observed. To determine the cellular target of the IL-1, we used criss-cross combinations of IL-1R⫺/⫺DC and CD4CD25T cells. These studies directly demonstrated that the activity of the IL-1 was mediated on the CD4CD25 population. Sur- prisingly, when we purified Treg from mice that were trans- genic for the eGFP reporter gene in the Foxp3 locus rather than on the basis of CD25 expression, much less proliferation of the Foxp3population was observed. This strongly suggested that CD25Foxp3T cells were the cellular targets for IL-1. Sim- ilar results were observed when we stimulated eGFP Treg cells with plate-bound anti-CD3 and IL-1 in the absence of splenic DC.

These results are in agreement with a recent study in which it was shown that IL-1␤ would activate CD4CD25Foxp3T cells in the presence of splenic DC by directly acting on the T cells and not the DC [18]. This study also suggested that Foxp3T cells were not able to suppress IFN-␥ production by CD4CD25Foxp3T cells in the presence of IL-1␤ and that IL-1␤ rendered the CD25Foxp3cells resistant to suppres- sion. However, when we measured IL-2 production by the CD4CD25Foxp3cells using the cytokine capture assay and by intracellular staining, we observed suppression in the pres- ence of Foxp3 T cells (data not shown). Moreover, no IFN-␥ production in the CD4CD25Foxp3subset was detected by intracellular staining after 3 days of culture (data not shown).

Fig. 5. CD4CD25NKT cells can be detected in mouse peripheral lymph nodes, which (A) from BALB/c, C57BL/6, or CD1(d)⫺/⫺mice (on a BALB/c background) were stained with anti-CD25 and CD1d tetramers. (B) Foxp3 expression and CD1d tetramer binding on gated CD25 T cells. Data are representative of three different experiments with similar results.

Fig. 6. The expansion of Foxp3T cells stimulated with plate-bound anti-CD3 and IL-1 is dependent on the presence of mature DC and CD4CD25Foxp3 T cells. FACS-purified CD4CD25T cells were cultured with mature splenic DC in the presence of plate-bound anti-CD3 (5␮g/ml) and IL-1␣,␤ (5 ng/ml). The cells were harvested after 5 days of culture and stimulated for 6 h with anti-CD3 and anti-CD28 (3␮g/ml each). Their phenotype, Foxp3 expression, and intracellular cytokine production were analyzed.

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The discrepancy between the two studies is most likely sec- ondary to the percent of Foxp3 T cells in the CD4CD25 population at the beginning of the cultures (95–97% in our study versus 74% in the study by O’Sullivan et al. [19]). It is possible that in the presence of a large number of CD4CD25 effector cells or different subsets of effector cells, Foxp3Treg may only inefficiently control cytokine production. We have not yet completely analyzed the mechanisms by which CD4CD25Foxp3effector T cells costimulate the prolifer- ation/survival of CD4CD25Foxp3Treg in the presence of mature splenic DC and IL-1. As anti-IL-2 inhibited⬃50% of the proliferative response, it is likely that IL-1-induced IL-2 production by the CD4CD25Foxp3T cells plays an impor- tant role. The induction of other cytokines or costimulatory signals that act directly on the Foxp3 cells or function indi- rectly by acting via the DC is also possible.

We observed different results when we stimulated CD4CD25 T cells with plate-bound anti-CD3 and IL-1 in the absence of DC. We used plate-bound anti-CD3 to mimic the conditions that are used in the clinic with anti-CD3/anti- CD28 beads to expand human Treg. In short-term cultures, stimulation of cell sorter-purified CD4CD25 T cells in the presence of IL-4 and to a lesser extent, IL-2 led to a prefer- ential outgrowth of Foxp3 T cells. However, it should be noted that significant growth of contaminating Foxp3cells was seen. Although several papers have claimed marked expansion of human or mouse CD4CD25 T cells in the presence of high concentration of IL-2, few of these studies have carefully examined Foxp3 expression in the expanded populations [20, 21]. Some studies have used a mAb to human Foxp3 that has now been shown to stain activated human T cells nonspecifi- cally [6]. Although cell expansion was seen in our studies in the presence of IL-1, the expanded population consisted pri- marily of Foxp3 cells. Detailed phenotypic studies of these expanded cells demonstrated that they were composed of a mixture of NKT cells and conventional T cells, and both populations contained a high percentage of IL-17-producing cells. It appears that the expanded NKT cells were generated from a small number of CD4CD25 NKT cells that we identified in normal lymph node populations of C57BL/6 mice and that have also been described in man [22]. An IL-17- producing NK1.1NKT cell population similar to the one we have expanded in the presence of IL-1 has recently been shown to be present in the lung and the liver of normal mice and to potentially play a role in neutrophil recruitment [23].

Taken together, our studies confirm and extend the findings of O’Sullivan et al. [19] that IL-1 is a selective, costimulatory factor for CD4CD25Foxp3T cells. The function of this population of activated effector T cells in normal lymphoid tissue is unknown. It has been hypothesized that IL-2 pro- duced by this population is required for the maintenance of Foxp3 Treg under homeostatic conditions in normal mice [15]. In cultures containing splenic DC, IL-1 stimulation of CD4CD25Foxp3 T cells resulted in expansion and/or survival of Foxp3 T cells. One signal provided by the splenic DC was the induction of IL-2 production by the CD4CD25Foxp3 T cells. Previous studies [7, 8] with BMDC also demonstrated that CD86 expression on the DC played a major role in the stimulation of the growth of

CD4CD25T cells, and this may have been secondary to costimulation of IL-2 production by the CD4CD25Foxp3 T cells or to a direct effect of CD86 on the CD4CD25Foxp3 T cells. In the absence of splenic DC, the major effects of IL-1 appear to be expansion/differentiation of IL-17-producing NKT and conventional T cells. It is difficult to extrapolate these findings from in vitro studies to the in vivo situation. During an inflammatory response in vivo, one might expect to have acti- vated DC and CD4CD25Foxp3 and CD4CD25Foxp3 T cells in the same microenvironment (e.g., an active rheuma- toid joint) that might contain high levels of proinflammatory cytokines such as IL-1. Depending on the ratios of the different cell populations and cytokines, the end result might be expansion of Treg or expansion of pathogenic, IL-17-pro- ducing T cells [15]. Lastly, a large number of protocols have been proposed for the expansion of Foxp3 Treg for use in cellular biotherapy. Our results strongly suggest that appro- priate caution must be exercised when these protocols in- volve the use of DC that produce IL-1 or complex mixtures of growth factors that might contain IL-1, resulting in activation of CD4CD25Foxp3effector cells leading to expansion of pathogenic, autoreactive T cells rather than tolerogenic Treg.

Careful analysis of cytokine production, particularly IL-17, by the expanded “Treg populations” should be included in every study. Such measures should ensure the production of bona fide Treg cells for use in cellular immunotherapy.

ACKNOWLEDGMENTS

These studies were supported by funds from the Intramural Program of NIAID. The authors thank Mohammed Oukka and Yasmine Belkaid for providing eGFP Foxp3 knock-in mice.

We also thank Sarah Tanskley, Thomas Moyer, and Carol Henry for cell sorting and Ste´phane Caucheteux for helpful discussions.

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