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Originally published online as doi:10.1189/jlb.1006610 on February 16, 2007

Published online before print February 16, 2007
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(Journal of Leukocyte Biology. 2007;81:1258-1268.)
© 2007 by Society for Leukocyte Biology

Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17

Susumu Nakae*,1, Yoichiro Iwakura{dagger}, Hajime Suto*,{ddagger} and Stephen J. Galli*,1

* Department of Pathology, Stanford University School of Medicine, Stanford, California, USA;
{ddagger} Atopy Research Center, Juntendo University, Tokyo, Japan; and
{dagger} Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan

1 Correspondence: S.N. and S.G.: Department of Pathology, L-235, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA. E-mail: nakae{at}stanford.edu or snakae{at}nch.go.jn; sgalli{at}stanford.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent evidence from several groups indicates that IL-17-producing Th17 cells, rather than, as once was thought, IFN-{gamma}-producing Th1 cells, can represent the key effector cells in the induction/development of several autoimmune and allergic disorders. Although Th17 cells exhibit certain phenotypic and developmental differences from Th1 cells, the extent of the differences between these two T cell subsets is still not fully understood. We found that the expression profile of cell surface molecules on Th17 cells has more similarities to that of Th1 cells than Th2 cells. However, although certain Th1-lineage markers [i.e., IL-18 receptor {alpha}, CXCR3, and T cell Ig domain, mucin-like domain-3 (TIM-3)], but not Th2-lineage markers (i.e., T1/ST2, TIM-1, and TIM-2), were expressed on Th17 cells, the intensity of expression was different between Th17 and Th1 cells. Moreover, the expression of CTLA-1, ICOS, programmed death ligand 1, CD153, Fas, and TNF-related activation-induced cytokine was greater on Th17 cells than on Th1 cells. We found that IL-23 or IL-17 can suppress Th1 cell differentiation in the presence of exogenous IL-12 in vitro. We also confirmed that IL-12 or IFN-{gamma} can negatively regulate Th17 cell differentiation. However, these cytokines could not modulate such effects on T cell differentiation in the absence of APC.

Key Words: cytokines • T cells • flow cytometry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A disordered Th1/Th2 balance is thought to contribute to the development of several autoimmune and/or allergic diseases. Classically, enhanced Th1 cell activation and IFN-{gamma} production were believed to contribute to the development of both autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), and to certain allergic disorders, such as delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) [1 , 2 ].

However, certain findings have required the revision of this general hypothesis. For example, administration of IFN-{gamma}-neutralizing antibodies can exacerbate MS, and IFN-{gamma} treatment can reduce disease severity [3 ]. Similarly, experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA), which are regarded as a rodent models of MS and RA, respectively, as well as experimental autoimmune uveitis and nephritis, were exacerbated in mice treated with anti-IFN-{gamma}-neutralizing antibodies or in IFN-{gamma}-deficient (IFN-{gamma}–/–) or IFN-{gamma} receptor (IFN-{gamma}R)–/– mice (reviewed in ref. [4 ]). In some studies, DTH or CHS responses were also enhanced or expressed normally in such mice [4 5 6 7 8 ].

Based on the work of several investigators, it is now clear that an additional T cell subset, Th17 cells, which exhibit certain phenotypic and developmental differences from Th1 cells, can have important roles in many processes that formerly were thought to reflect the activities of Th1 cells. Thus, IL-12 can promote the differentiation of Th1 cells, and IL-6 and TGF-ß or IL-23 can promote the differentiation of Th17 cells [9 10 11 12 13 ]. In contrast to IFN-{gamma}–/– mice, IL-17–/– mice exhibited diminished arthritis, EAE, DTH, and CHS [14 15 16 ], suggesting that Th17 cells, not Th1 cells, may represent key pathogenic effector cells in such disorders.

Although much has been learned recently about the biology of Th17 cells, the phenotypic, developmental, and functional characteristics of these cells are still not fully understood. In the present study, we identified additional phenotypic and developmental differences between Th1 cells and Th17 cells in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6 OTII-transgenic [17 ] and C57BL/6J IL-17–/– [14 ] mice and C57BL/6J wild-type, T-bet–/–, IFN-{gamma}–/–, and Rag-1–/– mice (The Jackson Laboratories, Bar Harbor, ME, USA) were housed at the animal care facilities at Stanford University Medical Center (Stanford, CA, USA) under standard temperature, humidity, and timed lighting conditions and were provided mouse chow and water ad libitum and treated in a humane manner in compliance with the "Guide for the Care and Use of Laboratory Animals," prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996), with the permission of the Stanford Institutional Animal Care and Use Committee.

Expression of cell surface molecules
Spleens were harvested from OTII mice, and then single cell suspensions of spleen cells were prepared. After lysing RBC, OTII spleen cells (2x106 cells/ml) were cultured for 3 days in the presence of 1 µM OVA peptide, 1 ng/ml recombinant murine (rm)IL-12 (R&D Systems, Minneapolis, MN, USA) for Th1 cells, or with or without 50 ng/ml recombinant humant (rh)IL-23 (R&D Systems) or 10 ng/ml rhTGF-ß (R&D Systems) plus 20 ng/ml rmIL-6 (PeproTech, Rocky Hill, NJ, USA) for Th17 cells, which were then stimulated with 1 µg/ml ionomycin plus 0.1 µg/ml PMA in the presence of 1 µM monensin for 6 h and then incubated with antimouse CD16/CD32 (2.4G2; BD PharMingen, San Jose, CA, USA) in a staining buffer (HBSS containing 2% FCS and 0.1% sodium azide) on ice for 15 min. After FcR blocking, cells were incubated on ice for 40 min with APC antimouse CD4 (RM4-5; BD PharMingen) and PE-Cy7 antimouse CD49b (DX5; eBioscience, San Diego, CA, USA) with isotype-matched, control IgG (BD PharMingen) or specific antibodies for cell surface markers: PE- or Alexa647-conjugated, antimouse CD27 (LG.3A10), CD40 (3/23), CD70 (FR70), CD80 (16-10A1), CD86 (GL1), CD95/Fas (Jo2), CD120b/TNF receptor 2 (TNFR2; TR75-89), CD121a/IL-1 receptor 1 (IL-1R1; 35F5), CD121b/IL-1R2 (4E2), CD153 (RM153), CD184/CXCR4 (2B11), CD193/CCR3 (83103), CD195/CCR5 (C34-3448), and CXCR5 (2G8) antibodies were obtained from BD PharMingen. FITC- or PE-conjugated, antimouse 4-1BB ligand (4-1BBL; TKS-1), B7-H3 (M3.2D7), B7x/B7-H4 (Clone 9), CCR7 (4B12), CD28 (37.51), CD30 (mCD30.1), CD134/OX40 (OX86), CD137/4-1BB (17B5), CD152/CTLA-4 (UC10-4B9), CD154 (MR1), CD178/Fas ligand (FasL; MFL3), ICOS (C398.4A), ICOSL/B7-H2 (HK5.3), programmed death-1 (PD-1; J43), PD ligand 1 (PD-L1)/B7-H1 (MIH6), PD-L2/B7-dendritic cell (DC; TY25), OX40L (RM134L), T cell Ig domain, mucin-like domain-1 (TIM-1; RMT1-17), TIM-2 (RMT2-1, RMT-2-14), TIM-3 (RMT3-23), TLR2 (6C2), TLR4 (MTS510), and TNF-related activation-induced cytokine [TRANCE; also called receptor activator of NF-{kappa}B ligand (RANKL); IK22/5] antibodies were purchased from eBiosciences. PE-antimouse CD120a/TNFR1 (55R-170; Santa Cruz Biotechnology, Santa Cruz, CA, USA), PE-antimouse IL-8RB/CXCR2 (K-19; Santa Cruz Biotechnology), PE-antimouse glucocorticoid-induced TNFR (GITR; 108619; R&D Systems), PE-antimouse CXCR3 (220803; R&D Systems), PE-antimouse CCR6 (140706; R&D Systems), biotin antimouse IL-18R{alpha}/IL-1R5 (BAF856; R&D Systems), or FITC-antimouse T1/ST2/IL-1R4 (DJ8; MD Biosciences, St. Paul, MN, USA) were obtained as indicated. After washing, the cells were fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature. After washing with a permeabilization buffer [0.1% saponin (Sigma Chemical Co., St. Louis, MO) in the staining buffer], cells then were incubated with nonlabeled, FITC- or PE-labeled rat IgG1 (BD PharMingen), FITC- or PE-antimouse IFN-{gamma} (XMG1.2; BD PharMingen), or nonlabeled or PE-antimouse IL-17 (TC11-18H10.1; BD PharMingen) and FITC-antirat IgG1 (RG11/39.4; BD PharMingen) as the second antibody. Expression of cell surface markers on DX5–CD4+IFN-{gamma}+ or DX5–CD4+IL-17+ T cells was analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) using CellQuest software (Becton Dickinson).

Cytokine profiles
To generate Th1 cells or Th17 cells, IL-17–/– OTII or IFN-{gamma}–/– OTII spleen cells were cultured in the presence of 1 µM OVA peptide with 50 ng/ml rmIL-12 or rhIL-23 for 3 days. Cells then were stimulated with 1 µg/ml ionomycin plus 0.1 µg/ml PMA in the presence of 1 µM monensin for 6 h and harvested. Cells were incubated with anti-CD16/CD32, CD4, and DX5 mAb and fixed as described above and then were incubated with antimouse IFN-{gamma} or IL-17 plus anticytokine antibodies or isotype-matched, control IgG. PE-antimouse IL-1{alpha} (ALF-161) and IL-10 (JES5-16E3) and nonlabeled, antimouse lymphotoxin {alpha} (LT{alpha}; AF.B3) were obtained from BD PharMingen. FITC-antimouse IL-4 (BVD6-24G2), IL-6 (MP5-20F3), and TNF (MP6-XT22) were purchased from eBioscience. Biotin-conjugated IL-1Ra (AF-480), IL-13 (BAF413), and IL-17F (BAF2057) were obtained from R&D Systems, and staining for IL-1Ra and IL-13 was performed as described elsewhere [18 , 19 ]. PE-antimouse IL-16 (17.1) was obtained from Caltag (Burlingham, CA, USA). For the second staining, we used FITC-antigoat IgG (705-095-147; Jackson ImmunoReseach, West Grove, PA, USA), FITC-antirat IgG1 (RG11/39.4; BD PharMingen), FITC-antirat IgG2b (RG7/1.30; BD PharMingen), FITC-antihamster IgG (G70-204, G94-56; BD PharMingen), and/or PE-streptavidin.

Expression of transcription factors
OTII spleen cells (2x106 cells/ml) were cultured for 3 days + 1 µM OVA peptide + 1 ng/ml rmIL-12 (R&D Systems) for Th1 cells or + 50 ng/ml rhIL-23 (R&D Systems) for Th17 cells. Cells stimulated with 1 µg/ml ionomycin + 0.1 µg/ml PMA + 1 µM monensin for 6 h were incubated with antimouse CD16/CD32 (2.4G2; BD PharMingen) in a staining buffer (HBSS containing 2% FCS and 0.1% sodium azide) on ice for 15 min. After FcR blocking, cells were incubated on ice for 40 min with APC antimouse CD4 (RM4-5; BD PharMingen) + PE-Cy7 antimouse CD49b (DX5; eBioscience). Cells were fixed and stained with FITC-antimouse IFN-{gamma} or nonlabeled, antimouse IL-17 mAb + FITC-antirat IgG1 (RG11/39.4; BD PharMingen) as the second antibody. After washing, cells were incubated with PE-anti-T-bet antibody (4B10; Santa Cruz Biotechnology), PE-antimouse forkhead box p3 (Foxp3; FJK16 s; eBioscience), or anti-GATA-3 mAb (HG3-31) + PE-antimouse Ig (BD PharMingen) as the second antibody.

Effects of IL-17 or IFN-{gamma} on T cell differentiation
CD4+ or CD8+ splenic T cells (2x105 cells/well in 96-well plates) were purified by magnetic cell sorting [14 ] and stimulated with plate-coated, anti-CD3 mAb (0.3 or 1.0 µg/ml) ± various concentrations of cytokines for 48 h. rmIFN-{gamma}, rmIL-1ß, and rmTNF or rmIL-18 and rmIL-17 were from PeproTech or R&D Systems, respectively. IFN-{gamma} and IL-17 levels in culture supernatants were measured by ELISA [20 ]. Splenic CD4+ T cells from wild-type OTII, IFN-{gamma}–/–OTII, or IL-17–/–OTII mice were cultured in the presence of T cell-depleted, wild-type OTII, IFN-{gamma}–/–OTII, or IL-17–/–OTII spleen cells (T:APC=1:5) plus 1 µM OVA peptides with 10 ng/ml rmIL-12, rhIL-23, rmIL-1ß, rmIL-18, and/or rmTNF for 72 h. Cells were then stimulated with 1 µg/ml ionomycin + 0.1 µg/ml PMA + 1 µM monensin for 6 h and harvested. IFN-{gamma} and IL-17 expression in CD4+TCRV{alpha}2+ cells was determined by FACS as described above. FITC-anti-TCRV{alpha}2 mAb (B20.1; BD PharMingen) was used for detection of OVA-specific, TCR-expressing T cells [17 ].

Statistics
The unpaired Student’s t test, two-tailed, was used for statistical evaluation of the results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell surface phenotype
IL-23 can induce IL-17 production by memory T cells but not by naïve T cells [21 22 23 24 ], and IL-23 is required for Th17 cell survival and function [11 ]. IL-12-derived Th1 cells and IL-23-derived Th17 cells expressed certain CD28 family molecules (CD28, CTLA-4, ICOS, and PD-1) and B7 family molecules (CD80, CD86, and PD-L1; Fig. 1 ). However, the intensity of ICOS, CTLA-4, and PD-L1 expression on Th17 cells was greater than that on Th1 cells. By contrast, the expression of ICOSL, PD-L2, B7x/B7-H4, and B7-H3 was hardly detectable on Th1 or Th17 cells (Fig. 1) .


Figure 1
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  		Figure 1. Expression of members of the CD28 and B7 family of molecules on Th1 cells and Th17 cells. OTII spleen cells were cultured with OVA peptides in the presence of rmIL-12 (for Th1 cells) or rhIL-23 (for Th17 cells) for 72 h. Cells were then stimulated with ionomycin and PMA with monensin for 6 h. The expression of cell surface molecules on DX5–CD4+IFN-{gamma}+ Th1 cells or DX5–CD4+IL-17+ Th17 cells was determined by flow cytometry. Shaded areas show staining with isotype-matched, control antibody; bold lines show specific antibody (Ab) staining. Figures show representative results from at least three independent experiments. The first and second numbers in the panels are the mean fluorescence intensity (MFI) for staining for the surface structure noted and for the control antibody, respectively.

 
Some members of the TNF superfamily of molecules (CD154, CD153, and TRANCE/RANKL) or of the TNFR superfamily (OX40, Fas, CD27, 4-1BB, and GITR) were observed on Th1 and IL-23-induced Th17 cells, and the TNF superfamily molecules OX40L, FasL, CD70, and 4-1BBL and the TNFR superfamily molecules TNFR1, TNFR2, CD40, and CD30 were barely detectable on either T cell subset (Fig. 2 ). IL-23-induced Th17 cells expressed CD153, TRANCE/RANKL, Fas, and CD27 more strongly than did Th1 cells (Fig. 2) . Thus, although Th1 and Th17 cells exhibit a similar pattern of expression of molecules in the B7-CD28 and TNF-TNFR families, the two T cell subsets differ in the intensity of their expression of some of these molecules. The intensity of ICOS, CTLA-4, PD-L1, CD153, TRANCE, Fas, and CD27 expression was similar in Th17 cells maintained without supplemental cytokines (OVA peptide alone), IL-23-stimulated Th17 cells, IL-6- and TGF-ß1-stimulated Th17 cells, and IFN-{gamma}–/– OTII cells maintained in the presence of OVA peptides with rmIL-23 and anti-IL-4 mAb (data not shown).


Figure 2
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  		Figure 2. Expression of members of the TNF and TNFR family of molecules on Th1 cells and Th17 cells. The expression of cell surface molecules on DX5–CD4+IFN-{gamma}+ Th1 cells or DX5–CD4+IL-17+ Th17 cells was determined by flow cytometry as described in Figure 1 . Shaded areas show staining with isotype-matched, control antibody; bold lines show specific antibody staining. Figures show representative results from at least three independent experiments. The first and second numbers in the panels are the MFI for staining for the surface structure noted and for the control antibody, respectively.

 
Certain members of the IL-1/TLR family or TIM family and some chemokine receptors are regarded as T cell subset-specific markers. Thus, IL-18R{alpha}, TIM-3, and CXCR3 are expressed predominantly on Th1 cells [25 26 27 ], and T1/ST2, TIM-1, TIM-2, and CCR3 are expressed predominantly on Th2 cells [26 27 28 29 ]. Like Th1 cells, Th17 cells expressed IL-18R{alpha}, TIM-3, and CXCR3 but hardly expressed T1/ST2, IL-1R1, IL-1R2, TLR2, TLR4, TIM-1, TIM-2, CCR3, CCR5, CXCR1, and CXCR4 (Fig. 3 ). CXCR5, a marker for follicular B helper T cells (TFH cells) [30 ], was hardly detectable on Th17 or Th1 cells (Fig. 3) . Expression of IL-18R{alpha}, TIM-3, and CXCR3, but not T1/ST2 and TIM-1, was also observed in IL-6- and TGF-ß1-induced Th17 cells and in Th17 cells maintained without supplemental cytokines (data not shown), and IL-4+ Th2 cells induced by incubation with rmIL-4, anti-IFN-{gamma} mAb, and anti-IL-12 mAb expressed small levels of T1/ST2 and TIM-1 but not IL-18R{alpha}, TIM-3, or CXCR3 (data not shown).


Figure 3
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  		Figure 3. Expression of members of the IL-1/TLR and TIM families of molecules and chemokine receptors on Th1 cells and Th17 cells. The expression of cell surface molecules on DX5–CD4+IFN-{gamma}+ Th1 cells or DX5–CD4+IL-17+ Th17 cells was determined by flow cytometry as described in Figure 1 . Shaded areas show staining with isotype-matched, control antibody; bold lines show specific antibody staining. Figures show representative results from at least three independent experiments. The first and second numbers in the panels are the MFI for staining for the surface structure noted and for the control antibody, respectively.

 
These results support the conclusion that the Th17 cell lineage resembles the Th1 cell lineage in its expression of cell surface markers but differs significantly from Th2 cells or TFH cells.

Intracellular cytokine and transcription factor profiles
Th1 cells are major producers of IFN-{gamma}, and Th2 cells are major producers of IL-4, IL-5, and IL-13. Regulatory T cells (Treg), Tr1 cells, and/or Th3 cells produce IL-10 and/or TGF-ß1. Th17 cells were identified, in humans and mice, as non-Th1/Th2 cells and as producers of IL-17, TNF, and/or GM-CSF [31 ]. TNF and LT{alpha} or GM-CSF are also expressed by Th1 cells or by Th1 and Th2 cells, respectively [31 32 33 ]. Th17 cells but neither Th1 nor Th2 cells also express mRNA for IL-17, IL-17F [22 ], and IL-6 [23 ]. Certain T cell lineages express IL-1{alpha} [34 ] or IL-1Ra [18 ], and CD8+ T cells are a source of IL-16 [35 ].

By intracellular FACS analysis, Th2-type cytokines (e.g., IL-4 and IL-13) were hardly detectable in Th1 or Th17 cells (Fig. 4A ). Consistent with the results reported by others [22 , 23 , 31 ], we found that Th1 and Th17 cells produced TNF and that IL-17F was produced by Th17 cells but hardly by Th1 cells by FACS (Fig. 4A) . It was reported that Th17 cells expressed IL-6 mRNA [23 ], but we did not detect production of IL-6 or of LT{alpha}, IL-1{alpha}, IL-1Ra, IL-10, or IL-16 by Th1 or Th17 cells (data not shown). When DO11.10 cells were cultured in the presence of OVA peptides plus Borrelia burgdorferi lysates or Mycobacterium bovis bacillus Calmette-Guerin strain Danish, IL-17+ Th17 cells also produced GM-CSF [31 ]. By contrast, in our culture conditions (without these components from pathogens), GM-CSF production was barely detectable in Th1 or Th17 cells (Fig. 4A) . Thus, the pattern of cytokine production by Th17 cells differs from that of Th1, Th2, Th3, and/or Tr1 cells, although the intracellular cytokine profile of each of these T cell subsets clearly may be affected by variation in the culture conditions used.


Figure 4
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  		Figure 4. Cytokine and transcription factor expression and requirements in Th1 cells versus Th17 cells. (A) Expression of cytokines in DX5–CD4+IFN-{gamma}+ Th1 cells or DX5–CD4+IL-17+ Th17 cells was assessed by flow cytometry. (B) Expression of GATA-3 in BALB/c spleen cells stimulated with anti-CD3 and anti-CD28 mAb in the presence of rmIL-4, anti-IFN-{gamma} mAb, and anti-IL-12 mAb for 5 days and that of Foxp3 in naïve BALB/c spleen cells was assessed by flow cytometry. (C) Expression of transcription factors (T-bet, GATA-3, and Foxp3) in DX5–CD4+IFN-{gamma}+ Th1 cells or DX5–CD4+IL-17+ Th17 cells was assessed by flow cytometry. The first and second numbers in the T-bet panels are the MFI for staining for T-bet and for the control antibody, respectively. Figures are representative results from at least three independent experiments.

 
Like cytokine profiles, expression profiles of transcription factors are also different in various T cell subsets. T-bet, GATA-3, or Foxp3 participates in the development of Th1, Th2, or Tr1 cells, respectively [36 , 37 ]. GATA-3-positive CD4+ T cells and Foxp3-positive CD4+ T cells could be detected by FACS in Th2-conditioned BALB/cJ spleen cells [38 ] and in naïve BALB/cJ spleen cells, respectively (Fig. 4B) . However, neither Th1 nor Th17 cells expressed GATA-3 or Foxp3 by FACS (Fig. 4C) . Consistent with other reports [10 , 39 40 41 ], we found that whereas Th1 cells expressed T-bet, T-bet expression was barely detectable in Th17 cells (Fig. 4B) .

Effects of IL-1, IL-18, and TNF on T cell differentiation
IFN-{gamma} production is markedly impaired yet detectable in IL-12–/– mice [42 ], and IL-17 production is reduced but not absent in IL-23–/– mice [43 ]. It is known that IL-18, IL-1ß, or TNF can promote IFN-{gamma} production synergistically with IL-12 [44 , 45 ]. Similarly, factor(s) besides IL-23 can contribute to IL-17 production and/or Th17 cell differentiation. For example, it was shown that IL-18 and IL-6, but not TNF and IL-1ß, can promote IL-17 production by CD4+ T cells [31 ]. Moreover, TGF-ß1 plus IL-6, TNF, and IL-1, in the presence of microbial components, can promote Th17 cell differentiation [10 , 12 , 13 ].

We found that production of IFN-{gamma} versus IL-17 by splenic CD4+OTII T cells exhibited distinct patterns of cytokine responsiveness. IFN-{gamma} production by CD4+OTII T cells stimulated with plate-coated, anti-CD3 mAb was promoted by IL-18, IL-12, or IL-1ß but not by TNF (Fig. 5A ). When T cells were stimulated with a relatively high dose of anti-CD3 mAb (1, 3, or 10 µg/ml), we detected no effects of rIL-1 or rTNF on OX40, ICOS, GITR, PD-1, 4-1BB, or CD154 surface expression on T cells [18 , 20 , 46 ]. However, we obtained results similar to those shown in Figure 5A when CD4+OTII T cells were stimulated with a low dose of plate-coated, anti-CD3 mAb (0.1 or 0.3 µg/ml; data not shown). We also observed that IL-12, IL-18, IL-1ß, or TNF each promoted IFN-{gamma} production strongly by CD4+OTII T cells stimulated with 0.3 µg/ml plate-coated, anti-CD3 mAb but induced little or no IL-17 production (Fig. 5B and 5E) .


Figure 5
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  		Figure 5. IL-1, IL-18, or TNF can promote Th17 cell differentiation. CD4+T cells from OTII mice were stimulated for 48 h with 1.0 µg/ml (A and D) or 0.3 µg/ml (B–F) of plate-coated, anti-CD3 mAb in the presence of various concentrations of cytokines: rmIL-18, rmIL-1ß, rmTNF, rhIL-23, or rmIL-12 (A and D), rmIL-18, rmIL-1ß, or rmTNF + 1 ng/ml rmIL-12 (B and E), and rmIL-18, rmIL-1ß, or rmTNF plus 1 ng/ml rhIL-23 (C and F). (A–F) Data are shown as the average ± SD. *, P < 0.05, versus corresponding values for cells incubated without that cytokine (0 ng/ml). (G) OTII (n=6) or IFN-{gamma}–/– OTII (n=6) cells were cultured with T cell-depleted spleen cells as APC (T:APC=1:5) in the presence of 1 µM OVA peptides ± 10 ng/ml rhIL-23 ±10 ng/ml rmIL-1ß, 10 ng/ml rmIL-18, and 10 ng/ml rmTNF for 72 h. Cells were then stimulated with 0.1 µg/ml PMA, 1 µg/ml ionomycin, and 1 µM monensin for 6 h. Intracellular cytokine profiles were assessed by flow cytometry. (G) Data are shown as the average + SD. *, P < 0.005, versus corresponding values for OVA peptides alone; {dagger}, P < 0.005, versus corresponding values for wild-type OTII cell culture; {ddagger}, P < 0.005, versus corresponding values for OVA peptides plus rhIL-23 alone in OTII or IFN-{gamma}–/–OTII cell culture; ¶, P < 0.005, versus corresponding values for OVA peptides alone in IFN-{gamma}–/– OTII cell culture.

 
IL-17 production by CD4+OTII T cells stimulated with plate-coated, anti-CD3 mAb was strongly promoted by IL-23, more weakly promoted by IL-1ß or IL-18, but not detectably affected by IL-12 or TNF (Fig. 5D) . In the presence of IL-23, IL-17 production was enhanced by IL-18, IL-1ß, or TNF, but only IL-18 enhanced IFN-{gamma} production (Fig. 5C and 5F) .

In tests of the ability of cytokines to enhance development of Th17 cells in vitro, IL-23 plus IL-18, IL-1ß, or TNF increased IL-17+ Th17 cells slightly in populations of CD4+OTII cells or in IFN-{gamma}–/–OTII cells, which had been stimulated with OVA peptides compared with IL-23 alone (Fig. 5G) . Moreover, the combination of IL-23 plus IL-18, IL-1ß, and TNF also increased Th17 cells in these populations more than did IL-23 alone or IL-23 plus any single cytokine (IL-18, IL-1ß, or TNF; Fig. 5G and data not shown). IL-18, IL-1ß, or TNF also promoted IFN-{gamma} production in the presence of IL-12 and IL-17 production in the presence of IL-23 in wild-type CD4+ T cells and CD8+ T cells (data not shown).

Effects of IFN-{gamma} and IL-17 in Th17 and Th1 cell differentiation
IL-12, IFN-{gamma}, and IL-4 suppress Th17 cell differentiation [22 , 23 , 39 ]. However, when purified CD4+ T cells were stimulated with plate-coated, anti-CD3 mAb in the presence of various concentrations of rmIFN-{gamma} ± rhIL-23, exogenous IFN-{gamma} did not affect IL-17 production by CD4+ T cells or Th17 cell differentiation, as assessed by FACS (Fig. 6A and data not shown).


Figure 6
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  		Figure 6. Regulation of Th1 versus Th17 cell differentiation by IL-12/IFN-{gamma} and IL-23/IL-17. (A) CD4+T spleen cells from wild-type mice were stimulated with 1.0 µg/ml plate-coated, anti-CD3 mAb without (upper and lower panels: {blacksquare}, n=3) or with 1 ng/ml rhIL-23 (upper panel: •, n=3) or 1 ng/ml rmIL-12 (lower panel: •, n=3) plus various concentrations of rmIFN-{gamma} (upper panel) or rmIL-17 (lower panel) for 48 h. (B) IL-17 or IFN-{gamma} levels in supernatants of CD4+T spleen cells from wild-type (n=4), IFN-{gamma}–/– (n=4), or IL-17–/– (n=4) mice were stimulated for 48 h with 1.0 µg/ml plate-coated, anti-CD3 mAb ± 10 ng/ml rmIL-12 or 10 ng/ml rhIL-23. Data are average ± (A) or + (B) SD and are representative of results from two independent experiments. *, P < 0.05, versus corresponding values for cultures without cytokines (–). ND, Not detected. (C) CD4+TCRV{alpha}2+ wild-type OTII (OTII), IFN-{gamma}–/– OTII, or IL-17–/–OTII spleen cells cocultured with T cell-depleted, wild-type OTII (OTII), IFN-{gamma}–/– OTII, or IL-17–/–OTII spleen cells (T:APC=1:5) in the presence of 1 µM OVA peptides ± 10 ng/ml rmIL-12 or 10 ng/ml rhIL-23 for 72 h were stimulated with 0.1 µg/ml PMA and 1 µg/ml ionomycin plus 1 µM monensin for 6 h. Intracellular cytokine profiles were assessed by flow cytometry. These are representative results of FACS analyses obtained in the three independent experiments performed, each of which gave similar results.

 
We next confirmed recently reported findings [47 ] in showing that exogenous IL-17 had no detectable effect on IFN-{gamma} production by CD4+ T cells or Th1 cell differentiation, as assessed by FACS, when wild-type CD4+ T cells were stimulated with plate-coated, anti-CD3 mAb in the presence of various concentrations of rmIL-17 ± rmIL-12 (Fig. 6A and data not shown). Extending such observations, we found that IFN-{gamma}–/–CD4+ T cells stimulated with plate-coated, anti-CD3, with or without rhIL-23, exhibited wild-type levels of IL-17 production and that IL-17–/–CD4+ T cells stimulated with plate-coated, anti-CD3, with or without rmIL-12, exhibited wild-type levels of IFN-{gamma} production (Fig. 6B) . Thus, under the conditions tested, neither exogenous nor endogenous IFN-{gamma} or IL-17 significantly influenced IL-23-dependent IL-17 production or IL-12-dependent IFN-{gamma} production in anti-CD3-stimulated, purified CD4+ T cells. We also obtained similar findings using CD8+ T cells (data not shown).

However, different results were obtained when CD4+ T cells from OVA-specific, TCR-expressing OTII transgenic mice were cultured with APC in the presence of OVA peptides. Under these conditions, IFN-{gamma}+IL-17– Th1 cells and IL-17+IFN-{gamma}– Th17 cells were observed (Fig. 6C) . A small population of IFN-{gamma}+IL-17+ Th cells was also detected (Fig. 6C) . In this setting, we found that IL-12 promoted Th1 cell differentiation but suppressed Th17 cell differentiation, whereas IL-23 suppressed Th1 cell differentiation but promoted Th17 cell differentiation (Fig. 6C and Table 1 ). Th17 cell differentiation was also increased significantly in IFN-{gamma}–/– versus IFN-{gamma}+/+ OTII cells, whether or not rhIL-23 was present, and was suppressed significantly by rmIL-12 in IFN-{gamma}–/–OTII and IFN-{gamma}+/+OTII cells (Fig. 6C and Table 1 ). Results similar to those shown in Figure 6C also were obtained when IFN-{gamma}–/– OTII cells were maintained in the presence of OVA peptides with rmIL-23 and anti-IL-4 mAb (data not shown).


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  		Table 1. Regulation of Th1 Versus Th17 Cell Differentiation by IL-12/IFN-{gamma} and IL-23/IL-17

 
By contrast, under these conditions (i.e., in the presence of APC and OVA peptides), levels of Th1 cell differentiation in IL-17–/–OTII cells were significantly lower than those in OTII cells (Fig. 6C and Table 1 ), perhaps as IL-17 is required for optimal antigen-specific T cell priming [14 , 15 ]. However, rmIL-12 enhanced the percentage of Th1 cells to a greater extent in IL-17–/–OTII cells than in wild-type OTII cells, whereas rhIL-23 had little or no effect on the percentage of Th1 cells in IL-17–/–OTII cells (Fig. 6C and Table 1 ). In these culture conditions, expression of CD44, CD45RB, and CD62L was similar among Th1 cells and Th17 cells from wild-type OTII, IFN-{gamma}–/–OTII, and IL-17–/–OTII mice (data not shown).

Thus, we now report the new finding that IL-23 or IL-17 can suppress Th1 cell differentiation in the presence of exogenous IL-12. We also confirmed that IL-12 or IFN-{gamma} can negatively regulate Th17 cell differentiation in vitro [22 , 39 ]. As neither IFN-{gamma} nor IL-17 appears to influence Th17 or Th1 cell differentiation directly (Fig. 6A and 6B) , the suppressive effects of these cytokines in this setting may reflect actions on T cell–APC interactions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified previously unknown differences in the cell surface molecule expression and development of Th17 cells and Th1 cells. We found that Th17 cells expressed the Th1-lineage marker, CXCR3, but not certain Th2-lineage markers such as T1/ST2, TIM-1, and TIM-2. We also confirmed that Th17 cells, like Th1 cells, expressed IL-18R{alpha} [41 ] and TIM-3 [48 ]. Our findings thus extend and complement those from other groups in indicating that the expression pattern of cell surface molecules by Th17 cells is closer to that of Th1 cells than that of Th2 cells.

We also detected quantitative differences in the levels of expression of certain cell surface molecules of Th1 and Th17 cells. It is known that some costimulatory molecules, including CD28, ICOS, and OX40, can promote IL-17 production [15 , 40 ]. It is reasonable to surmise that the function of Th17 cells may also be influenced by their levels of surface expression of other molecules in the CD28 or B7 families or the TNF superfamily.

We found that the expression intensity of CTLA-1, ICOS, PD-L1, CD153, Fas, and TRANCE/RANKL was greater on Th17 cells than on Th1 cells. TRANCE/RANKL (or osteoclast differentiation factor), which is also expressed on osteoblasts, can promote osteoclast differentiation and is critical for osteoclast-mediated bone resorption [49 ]. IFN-{gamma} can suppress TRANCE/RANKL-mediated osteoclastogenesis by inducing the rapid degradation of TNFR-associated factor 6, which is required for TRANCE/RANKL and IL-17 signal transduction [9 , 50 ]. Our findings are consistent with the possibility that this pathway can contribute to the ability of IFN-{gamma} to suppress the development of arthritis [4 ].

It is notable that IL-17 can promote TRANCE/RANKL-mediated osteoclast differentiation by inducing PGE2-mediated TRANCE/RANKL expression on osteoblasts [51 ]. Our findings therefore also raise the possibility that TRANCE/RANKL-expressing Th17 cells, rather than (or in addition to) Th1 cells, may contribute to osteoclast-mediated bone resorption by participating in direct TRANCE/RANKL-RANK interactions between Th17 cells and osteoclast progenitor cells. Supporting this notion, it has been reported that TRANCE/RANKL+Th17 cells are detectable in the lesions of arthritis patients [52 ].

ICOS signaling can enhance IL-4 production by Th2 cells [53 , 54 ] and IL-17 production by Th17 cells [40 ]. ICOS is also required for Treg cell function [55 ]. However, the fact that ICOS signaling can enhance aspects of Th2 or Th17 cell-associated responses does not make it possible to predict how the presence of absence of ICOS signaling might influence particular Th2- or Th17-associated responses in vivo. For example, like IL-17–/– mice [16 ], ICOS–/– mice exhibited attenuated development of collagen-induced arthritis [56 ]. Conversely, in contrast to IL-17–/– mice [47 ], ICOS–/– mice showed increased susceptibility to EAE [56 ]. Similarly, mice treated with anti-ICOS-neutralizing antibody exhibited reduced airway hypersensitivity induced by OVA with alum [57 ], whereas IL-17–/– mice exhibited no abnormality in airway hypersensitivity induced by OVA with alum as well as increased Th2 cytokine levels in bronchoalveolar lavage fluids [14 ]. Thus, although ICOS appears to play important roles in the pathogenesis of various disorders through its ability to activate/regulate Th2, Th17, and/or Treg cells, one cannot use the intensity of expression of this molecule solely or other costimulatory molecules on in vitro-derived Th cells to draw conclusions about all of the potential functions of such costimulatory molecules in vivo.

Th1 and Th17 cells express CXCR3 and IL-18R. CXCL10, a ligand for CXCR3, and IL-18, the ligand for IL-18R, were identified originally as IFN-{gamma}-inducible protein 10 kDa and as IFN-{gamma}-inducing factor [44 ], respectively. There is evidence that depending on the setting, the CXCL10–CXCR3 and/or IL-18–IL-18R pathways may enhance or suppress the development or magnitude of Th1 cell- and Th17 cell-mediated disorders. For example, CXCR3–/– or CXCL10–/– mice exhibited exacerbated development of EAE [58 , 59 ], as did IFN-{gamma}–/– mice [4 ], whereas CXCL10–/– mice or mice that had received a neutralizing antibody to CXCL10 exhibited attenuated CHS responses [8 , 60 ], as did IL-17–/– mice [14 ]. In contrast to IFN-{gamma}–/– or IFN-{gamma}R–/– mice, IL-18–/– mice or mice that had received a neutralizing antibody to IL-18 exhibited attenuated development of EAE, CIA, and CHS [61 62 63 ]. Consistent with the possibility that IL-18 can influence the expression of Th17 cell-associated responses, we found that IL-17 production by CD4+OTII T cells stimulated in vitro with plate-coated, anti-CD3 mAb was significantly, albeit rather modestly, promoted by IL-18 (Fig. 5D) . The idea that IL-18 can promote Th17 cell-associated biological responses is also supported by the results of our recent in vivo studies conducted with OVA TCR-transgenic OTII mice. We showed that OVA-induced airway neutrophilia in OTII mice, which is promoted by Th17 cells and suppressed by IFN-{gamma}, was attenuated by IL-18 deficiency, indicating that IL-18 enhances the expression of these Th17 cell-mediated immune responses in vivo [64 ].

In the context of in vivo models of disease, cytokines can influence T cell subsets directly or indirectly, e.g., via interactions with other cell types that respond to these cytokines. We demonstrated that neither IFN-{gamma} nor IL-17 appeared to influence Th17 or Th1 cell differentiation directly (Fig. 6A and 6B) , whereas in the presence of APC, IFN-{gamma} and IL-17 were able to negatively regulate IL-23-mediated Th17 cell differentiation and IL-12-mediated Th1 cell differentiation, respectively. Others have also reported observations consistent with indirect effects of IFN-{gamma} on Th17 cell differentiation or of IL-23 on Th1 cell differentiation. Thus, Harrington et al. [39 ] reported that Th17 cell differentiation was inhibited by exogenous IFN-{gamma} when IFN-{gamma}–/– CD4+ T cells cocultured with IFN-{gamma}–/– APC were stimulated with anti-CD3 mAb in the presence of rIL-23 and rIFN-{gamma}. Also, it has been reported that IL-23 partially inhibited IFN-{gamma} production in splenocytes that lacked IL-12Rß2 [22 ] but not when they included APC, which lacked IL-12 [22 , 39 ]. Finally, Langrish et al. [23 ] reported that the administration of rIL-23 in vivo resulted in reduced numbers of Th1 cells in a model of EAE. The latter study provides evidence that IL-23 can influence Th1 cell biology in vivo, although additional work will be required to know the extent to which this might occur by direct versus indirect mechanisms.

The effects of IFN-{gamma} and IL-17 on Th cell development in our in vitro coculture system may reflect actions of these cytokines on T cell–APC interactions. Supporting this possibility, it has been reported that cross-linking between CD154 on T cells and CD40 on APC such as DC can result in the production of IL-12 by APC and that IFN-{gamma} can potentiate IL-12 production by APC synergistically with CD40 signals [65 , 66 ]. Perhaps Th1 cell-derived IFN-{gamma} induces IL-12 production by APC, leading to IL-12-mediated suppression of Th17 cell differentiation. Taken together, our observations provide further support for the importance of T cell–APC interactions in regulating Th17 cell differentiation.


   ACKNOWLEDGEMENTS
 
This work was supported by United States Public Health Service grants (to S. J. G.) AI-23990, AI-070813, CA-72074, and HL-67674. We thank Hideo Yamane (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) for technical suggestions regarding GATA-3 staining and the members of the Galli lab for helpful discussions.

Received October 3, 2006; revised November 29, 2006; accepted January 10, 2007.


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