TRAIL is known to play a pivotal role in the inhibition of autoimmune disease. We previously demonstrated that administration of dendritic cells engineered to express TRAIL and myelin-oligodendrocyte glycoprotein reduced the severity of experimental autoimmune encephalomyelitis and suggested that CD4+CD25+ regulatory T cells (Tregs) were involved in mediating this preventive effect. In the current study, we investigated the effect of TRAIL on Tregs, as well as conventional T cells, using TRAIL-deficient mice. Upon induction of experimental autoimmune encephalomyelitis, TRAIL-deficient mice showed more severe clinical symptoms, a greater frequency of IFN-γ–producing CD4+ T (Th1) cells, and a lower frequency of CD4+Foxp3+ Tregs than did wild-type mice. In vitro, conventional T cells stimulated by bone marrow-derived dendritic cells (BM-DCs) from TRAIL-deficient mice showed a greater magnitude of proliferation than did those stimulated by BM-DCs from wild-type mice. In contrast, TRAIL expressed on the stimulator BM-DCs enhanced the proliferative response of CD4+CD25+ Tregs in the culture. The functional TRAILR, mouse death receptor 5 (mDR5), was expressed in conventional T cells and Tregs upon stimulation. In contrast, the decoy receptor, mDc-TRAILR1, was slightly expressed only on CD4+CD25+ Tregs. Therefore, the distinct effects of TRAIL may be due to differences in the mDc-TRAILR1 expression or the signaling pathways downstream of mouse death receptor 5 between the two T cell subsets. Our data suggest that TRAIL suppresses autoimmunity by two mechanisms: the inhibition of Th1 cells and the promotion of Tregs.

TRAIL is a type II membrane protein belonging to the TNF superfamily (1, 2). TRAIL is expressed in a variety of cell types, including lymphocytes, NK cells, NKT cells, dendritic cells (DCs), macrophages, and virus-infected APCs (36). In humans, TRAIL can bind two apoptosis-inducing receptors TRAILR1 (DR4) and TRAILR2 (DR5), as well as two decoy receptors TRAIL-R3 (LIT, DcR1) and TRAIL-R4 (TRUNDD, DcR2). A soluble receptor (osteoprotegerin) is also capable of binding TRAIL (1, 7, 8). In contrast, mice have only one apoptosis-inducing receptor murine TRAILR (MK; mouth death receptor 5 [mDR5]) and two decoy receptors mDc-TRAIL-R1 and mDc-TRAIL-R2 (9).

TRAIL induces apoptosis and activates the transcription factor NF-κB (1, 10). The TRAIL/TRAILR system is implicated in immune regulation and antitumor immunity (11, 12). Indeed, TRAIL-deficient mice are highly susceptible to autoimmune arthritis, diabetes, and experimental autoimmune encephalomyelitis (EAE) (13-15). In addition, TRAIL is involved in the regulation of the Th1/Th2 balance (16). TRAIL on CD4+CD25+ regulatory T cells (Tregs) was reported to induce the death of CD4+CD25 conventional T cells (17).

We previously observed that in vivo administration of embryonic stem cell-derived DCs (ES-DCs) expressing TRAIL, along with myelin-oligodendrocyte glycoprotein (MOG) peptide, decreased the severity of MOG-induced EAE and myelin basic protein-induced EAE (18, 19). In addition, adoptive transfer of CD4+CD25+ Tregs isolated from donor mice treated with the genetically modified ES-DCs protected the recipient mice from subsequently induced EAE (19). Furthermore, TRAIL on ES-DCs enhanced the proliferation of CD4+CD25+ Tregs in vitro (19). With regard to the relationship between TRAIL and Tregs, TRAIL was shown to expand CD4+CD25+ Tregs in a study of experimental autoimmune thyroiditis (EAT) (20). With regard to the ability of TRAIL to induce cell proliferation, it was recently reported that TRAIL promotes the proliferation of vascular smooth muscle cells and carcinoma cells (21, 22).

Therefore, we hypothesized that TRAIL promotes the proliferation of Tregs in certain situations. To evaluate this hypothesis, we investigated the effect of TRAIL on Tregs using TRAIL-deficient mice. Our results suggest that TRAIL induced growth inhibition and apoptosis of IFN-γ–producing CD4+ T cells. In contrast, Tregs were more resistant to TRAIL-induced growth inhibition than were conventional T cells. Furthermore, TRAIL promotes the growth of Tregs.

Six- to eight-week-old wild-type (WT) C57BL/6 mice were purchased from Clea Animal (Tokyo, Japan), and TRAIL-deficient (TRAIL−/−) C57BL/6 mice were kindly provided by Amgen (Thousand Oaks, CA). The two mouse strains were crossed to obtain heterozygotes, which were used as the parental generation. The offspring of the heterozygotes were genotyped, and the WT and TRAIL−/− littermates were used in the experiments. The TRAIL allele was examined by PCR of genomic DNA prepared from tail biopsies, using primer pairs specific to the WT TRAIL allele (5′-GGT TAT CAT CAG CTT CAT GG-3′ and 5′-GAA ATG GTG TCC TGA AAG GTT C-3′) and specific to neomycin phosphotransferase gene (5′-AGC GAG CAC GTA CTC GGA TGG AA-3′ and 5′-CCC ATT CGC CGC CAA GCT CTT CAG CAA TAT-3′). Mice were housed in a specific pathogen-free barrier facility. All experiments were approved by the Animal Research Committee of Kumamoto University.

Mouse MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) and soluble recombinant mouse TRAIL (rTRAIL) were purchased from AnyGen (Gwangju, Korea) and Alexis Biochemicals (San Diego, CA), respectively. Rat anti-mouse CD25 mAb (clone PC61.5.3) was produced, as described previously (19).

The following Abs and secondary reagents were purchased from eBioscience (San Diego, CA), unless otherwise stated: FcR blocking Ab (anti-mouse CD16/CD32, clone 2.4G2; BD Pharmingen, San Diego, CA), PE-conjugated anti-mouse TRAIL (clone N2B2), FITC- and PE-conjugated anti-mouse CD4 (clone RM4-5), PE-conjugated anti-mouse CD8a (clone 53-6.7), FITC-conjugated anti-mouse/human B220 (clone RA3-6B2), FITC-conjugated anti-mouse/rat Foxp3 (clone FJK-16s), FITC-conjugated anti-mouse IL-17A (clone eBio17B7), FITC-conjugated anti-mouse IFN-γ (clone XMG1.2), biotin-conjugated anti-mouse DR5 (clone MD5-1), anti-mouse DcTRAIL-R1 (clone mDcR1-3; BioLegend, San Diego, CA), anti-mouse DcTRAIL-R2 (clone mDcR2-1; BioLegend), FITC-conjugated streptavidin, FITC-conjugated anti-hamster (Armenian) IgG (BioLegend), Armenian Hamster IgG isotype-matched control (BioLegend), FITC- and PE-conjugated rat IgG2a isotype-matched control, FITC-conjugated rat IgG1 isotype-matched control, and biotin-conjugated Armenian Hamster IgG isotype-matched control.

Spleen and inguinal lymph node (ILN) cells were incubated with FcR-blocking Ab on ice for 15 min and subsequently labeled with specific Abs at 4°C before analysis using a FACScan flow cytometer (BD Biosciences, San Jose, CA). In some experiments, intracellular cytokine analysis was performed, as described previously (23, 24). Briefly, spleen or ILN cells were resuspended at 5 × 106 cells/ml in RPMI 1640 supplemented with 10% FBS and 2-ME. PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (10 μg/ml; all from Sigma-Aldrich, St. Louis, MO) were added to the cells. After incubation for 5 h, cells were washed and stained with PE-conjugated anti-mouse CD4. The fixation and permeabilization of cells were performed using IntraPrep reagent (Beckman Coulter, Fullerton, CA), according to the manufacturer’s instructions, and intracellular cytokines were stained with FITC-conjugated anti-mouse IFN-γ or IL-17A. To demonstrate the specificity of staining, isotype-matched control mAbs were also used. The stained cells were analyzed by flow cytometry.

EAE was induced in 6–8-wk-old female mice by s.c. immunization with 200 μg MOG 35–55 peptide emulsified in CFA containing 2 mg/ml heat-killed Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI) at the base of the tail on day 0. Additionally, mice received 350 ng Bordetella pertussis toxin (Calbiochem, San Diego, CA) i.p. in 0.5 ml PBS on days 0 and 2. In some experiments, to deplete CD25+ T cells, anti-mouse CD25 mAb (clone PC61.5.3, 400 μg/mouse) or rat IgG (Chemicon International, Temecula, CA) as a control was injected i.p. at 14 d before the immunization. Residual CD4+Foxp3+ T cells in spleen cells were examined by flow cytometry after 12 d (2 d before the immunization). Clinical signs of EAE were assessed with a 0–6-point scoring system: 0, normal; 1, weakness of the tail and/or paralysis of the distal half of the tail; 2, loss of tail tonicity and abnormal gait; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, forelimb paralysis or moribundity; and 6, death.

The generation of bone marrow-derived DCs (BM-DCs) was carried out, as described previously (25). In brief, bone marrow cells from WT or TRAIL−/− mice were cultured in RPMI 1640 supplemented with 10% FBS, GM-CSF (500 U/ml), and 2-ME for 9 d. To induce maturation of DCs, immature BM-DCs were stimulated with 1 μg/ml LPS during the final 48 h.

Twenty-one days after immunization with MOG peptide, spleen or ILN cells were isolated and cultured (2.5 × 105 cells/well) with MOG peptide (0, 1, or 3.2 μM) or Con A (5 μg/ml; Sigma-Aldrich) as a positive control in 96-well flat-bottom plates. The cells were cultured for 3 d, and [3H]thymidine (248 GBq/mM) was added to the culture (37 kBq/well) for the final 16 h. At the end of culture, cells were harvested onto glass fiber filters (Wallac, Vernon Hills, IL), and the incorporation of [3H]thymidine was measured by scintillation counting.

CD4+CD25 conventional T cells and CD4+CD25+ Tregs from the spleens of unprimed WT or TRAIL−/− mice were purified using the MACS cell-sorting system (Miltenyi Biotec, Auburn, CA), as described previously (19). Cell purity was confirmed to be >95% by FACS analysis. The proliferation of CD4+CD25 conventional T cells and CD4+CD25+ Tregs was analyzed, as described previously (26), with some modifications. In brief, CD4+CD25 conventional T cells or CD4+CD25+ Tregs (4.0 × 104 cells) were cocultured with LPS-stimulated and X-ray irradiated (45 Gy) BM-DCs (2.0 × 104 cells) in the presence of 1 μg/ml anti-CD3 mAb (clone 145-2C11; BD Biosciences) and 10 U/ml human IL-2 in 96-well round-bottom plates for 3 d. In some experiments, CD4+CD25 conventional T cells or CD4+CD25+ Tregs (1.0 × 105 cells/well) from TRAIL−/− mice were stimulated with 3 μg/ml plate-bound anti-CD3 and anti-CD28 mAb (clone 37.51; BD Biosciences) and 20 U/ml human IL-2 in the presence or absence of soluble rTRAIL for 3 d. [3H]thymidine was added to the culture for the final 12 h, and the proliferation of T cells was quantified by measuring [3H]thymidine incorporation.

CD4+CD25 conventional T cells or CD4+CD25+ Tregs (1.0 × 105 cells) isolated from the spleens of TRAIL−/− mice were stimulated with 3.0 μg/ml plate-bound anti-CD3 and anti-CD28 mAb and 20 U/ml human IL-2 for 72 h. These T cells were stained with biotin-conjugated anti-mouse DR5 and subsequently stained with FITC-conjugated streptavidin. They were also stained with anti-mouse DcTRAIL-R1 or anti-mouse DcTRAIL-R2, stained with FITC-conjugated anti-hamster (Armenian) IgG, and analyzed on a flow cytometer.

Seventeen days after immunization with MOG peptide, animals were sacrificed, and the spinal cords were flushed out with PBS. The spinal cords were fixed in 4% paraformaldehyde for 72 h, embedded in paraffin, and cross-sectioned using standard protocols. Sections were stained with H&E.

The two-tailed Student t test was used to determine any statistically significant differences. A p value <0.05 was considered statistically significant.

It was reported that TRAIL−/− mice have enlarged lymph nodes, spleens, and thymuses (15). As shown in Fig. 1A–C, the frequencies of CD4+, CD8+, and CD4+Foxp3+ T cells and B cells in TRAIL−/− mice were similar to those of WT mice. In contrast, the frequency of IFN-γ–producing CD4+ T (Th1) cells in the spleen and ILNs in TRAIL−/− mice was significantly greater than in WT mice (Fig. 1D). The frequency of IL-17–producing CD4+ T (Th17) cells was similar in WT and TRAIL−/− mice (Fig. 1E). Spleen and ILNs of 6–8-wk-old TRAIL−/− mice were slightly larger than those of WT mice, and the total number of cells of these tissues in TRAIL−/− mice were slightly greater than in WT mice (Table I). Thus, the difference in the absolute number of Th1 cells between WT and TRAIL−/− mice was significant. These results suggest that TRAIL is involved in the regulation of the number of Th1 cells in the steady state.

FIGURE 1.

Increased frequency of Th1 cells in TRAIL−/− mice. The frequencies of specific lymphocytes in the thymus (A), spleen (B), and ILNs (C) of WT or TRAIL−/− mice were assessed by flow cytometry. A, The frequencies of double-negative (DN), double-positive (DP), CD8+ single-positive (SP CD8), and CD4+ single-positive (SP CD4) cells among thymocytes are shown. The frequencies of B220+ B cells, CD8+ T cells, and CD4+ T cells in the spleen (B) and ILNs (C) are shown. The frequency of CD4+ Foxp3+ T cells detected by intracellular staining with anti-Foxp3 mAb is also shown. D and E, Whole spleen and ILN cells were stimulated with PMA plus ionomycin in the presence of brefeldin A for 5 h, followed by intracellular cytokine staining of IFN-γ and IL-17A, and analysis by flow cytometry. Dot plots (left panels) and bar graphs (right panels) represent the frequencies of IFN-γ+ (D) and IL-17A+ (E) CD4+ T cells within the total CD4+ T cells in the whole spleen or ILNs. Bar graphs indicate the mean frequency ± SD of five mice per group. **p < 0.01.

FIGURE 1.

Increased frequency of Th1 cells in TRAIL−/− mice. The frequencies of specific lymphocytes in the thymus (A), spleen (B), and ILNs (C) of WT or TRAIL−/− mice were assessed by flow cytometry. A, The frequencies of double-negative (DN), double-positive (DP), CD8+ single-positive (SP CD8), and CD4+ single-positive (SP CD4) cells among thymocytes are shown. The frequencies of B220+ B cells, CD8+ T cells, and CD4+ T cells in the spleen (B) and ILNs (C) are shown. The frequency of CD4+ Foxp3+ T cells detected by intracellular staining with anti-Foxp3 mAb is also shown. D and E, Whole spleen and ILN cells were stimulated with PMA plus ionomycin in the presence of brefeldin A for 5 h, followed by intracellular cytokine staining of IFN-γ and IL-17A, and analysis by flow cytometry. Dot plots (left panels) and bar graphs (right panels) represent the frequencies of IFN-γ+ (D) and IL-17A+ (E) CD4+ T cells within the total CD4+ T cells in the whole spleen or ILNs. Bar graphs indicate the mean frequency ± SD of five mice per group. **p < 0.01.

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Table I.
Numbers of total, CD4+, CD4+Foxp3+, IFN-γ+CD4+, and IL-17A+CD4+ T cells in the thymus, spleen, and ILNs in naive WT and TRAIL−/− mice
OrganCell SubsetGroup (n = 5 Mice/Group)Total Cell Number
Thymus Total cells WT 9.7 ± 2.9 × 107 
  TRAIL−/− 11.1 ± 1.0 × 107 
 DN cells WT 4.0 ± 1.6 × 106 
  TRAIL−/− 5.2 ± 1.1 × 106 
 DP cells WT 7.8 ± 2.6 × 107 
  TRAIL−/− 8.9 ± 1.1 × 107 
 SP CD8+ T cells WT 4.8 ± 1.2 × 106 
  TRAIL−/− 5.4 ± 0.8 × 106 
 SP CD4+ T cells WT 9.9 ± 2.5 × 106 
  TRAIL−/− 14.7 ± 0.6 × 106* 
Spleen Total cells WT 8.6 ± 1.6 × 107 
  TRAIL−/− 10.2 ± 1.7 × 107 
 B cells WT 4.1 ± 1.1 × 107 
  TRAIL−/− 4.5 ± 0.6 × 107 
 CD8+ T cells WT 1.0 ± 0.3 × 107 
  TRAIL−/− 1.2 ± 0.3 × 107 
 CD4+ T cells WT 1.4 ± 0.4 × 107 
  TRAIL−/− 1.9 ± 0.4 × 107 
 CD4+Foxp3+ T cells WT 1.7 ± 0.6 × 106 
  TRAIL−/− 2.3 ± 0.5 × 106 
 IFN-γ+CD4+ T cells WT 0.6 ± 0.2 × 106 
  TRAIL−/− 1.3 ± 0.2 × 106* 
 IL-17A+CD4+ T cells WT 0.3 ± 0.1 × 106 
  TRAIL−/− 0.5 ± 0.2 × 106 
ILNs Total cells WT 2.3 ± 0.8 × 106 
  TRAIL−/− 3.3 ± 0.5 × 106 
 B cells WT 0.7 ± 0.3 × 106 
  TRAIL−/− 0.8 ± 0.3 × 106 
 CD8+ T cells WT 0.8 ± 0.3 × 106 
  TRAIL−/− 1.1 ± 0.1 × 106** 
 CD4+ T cells WT 0.9 ± 0.2 × 106 
  TRAIL−/− 1.4 ± 0.2 × 106** 
 CD4+Foxp3+ T cells WT 1.0 ± 0.4 × 105 
  TRAIL−/− 1.3 ± 0.2 × 105 
 IFN-γ+CD4+ T cells WT 1.4 ± 0.7 × 104 
  TRAIL−/− 4.3 ± 1.3 × 104* 
 IL-17A+CD4+ T cells WT 2.1 ± 0.9 × 104 
  TRAIL−/− 3.0 ± 1.6 × 104 
OrganCell SubsetGroup (n = 5 Mice/Group)Total Cell Number
Thymus Total cells WT 9.7 ± 2.9 × 107 
  TRAIL−/− 11.1 ± 1.0 × 107 
 DN cells WT 4.0 ± 1.6 × 106 
  TRAIL−/− 5.2 ± 1.1 × 106 
 DP cells WT 7.8 ± 2.6 × 107 
  TRAIL−/− 8.9 ± 1.1 × 107 
 SP CD8+ T cells WT 4.8 ± 1.2 × 106 
  TRAIL−/− 5.4 ± 0.8 × 106 
 SP CD4+ T cells WT 9.9 ± 2.5 × 106 
  TRAIL−/− 14.7 ± 0.6 × 106* 
Spleen Total cells WT 8.6 ± 1.6 × 107 
  TRAIL−/− 10.2 ± 1.7 × 107 
 B cells WT 4.1 ± 1.1 × 107 
  TRAIL−/− 4.5 ± 0.6 × 107 
 CD8+ T cells WT 1.0 ± 0.3 × 107 
  TRAIL−/− 1.2 ± 0.3 × 107 
 CD4+ T cells WT 1.4 ± 0.4 × 107 
  TRAIL−/− 1.9 ± 0.4 × 107 
 CD4+Foxp3+ T cells WT 1.7 ± 0.6 × 106 
  TRAIL−/− 2.3 ± 0.5 × 106 
 IFN-γ+CD4+ T cells WT 0.6 ± 0.2 × 106 
  TRAIL−/− 1.3 ± 0.2 × 106* 
 IL-17A+CD4+ T cells WT 0.3 ± 0.1 × 106 
  TRAIL−/− 0.5 ± 0.2 × 106 
ILNs Total cells WT 2.3 ± 0.8 × 106 
  TRAIL−/− 3.3 ± 0.5 × 106 
 B cells WT 0.7 ± 0.3 × 106 
  TRAIL−/− 0.8 ± 0.3 × 106 
 CD8+ T cells WT 0.8 ± 0.3 × 106 
  TRAIL−/− 1.1 ± 0.1 × 106** 
 CD4+ T cells WT 0.9 ± 0.2 × 106 
  TRAIL−/− 1.4 ± 0.2 × 106** 
 CD4+Foxp3+ T cells WT 1.0 ± 0.4 × 105 
  TRAIL−/− 1.3 ± 0.2 × 105 
 IFN-γ+CD4+ T cells WT 1.4 ± 0.7 × 104 
  TRAIL−/− 4.3 ± 1.3 × 104* 
 IL-17A+CD4+ T cells WT 2.1 ± 0.9 × 104 
  TRAIL−/− 3.0 ± 1.6 × 104 

Mean absolute cell numbers were calculated from total cell numbers for the whole organ; values are rounded to the first decimal place.

*p < 0.01; **p < 0.05, compared with WT mice.

DN, double negative; DP, double positive; SP CD4, CD4+ single positive; SP CD8, CD8+ single positive.

We used a MOG-induced EAE disease model to investigate the influence of TRAIL in vivo. It was reported that TRAIL−/− mice have exacerbated EAE disease (13). In agreement with this report, we observed an earlier onset of EAE and more severe disease in TRAIL−/− mice compared with WT mice (Fig. 2A, Table II).

FIGURE 2.

Increased severity and protracted course of MOG-induced EAE in TRAIL−/− mice. A, WT and TRAIL−/− mice (10 mice/group) were immunized with MOG 35–55 peptide on day 0, and pertussis toxin on day 0 and 2, as described in 1Materials and Methods. Values represent the mean clinical score for each group. The data are summarized in Table II. B, Isolated spleen cells (2.5 × 105) (left panel) or ILN cells (right panel) from WT or TRAIL−/− mice 21 d after immunization were cultured with MOG peptide (0, 1, or 3.2 μM) for 3 d. As a positive control, respective cells were also stimulated with Con A (5 μg/ml). The proliferative response was quantified by measuring [3H]thymidine incorporation in the final 12 h of the culture. *p < 0.05; **p < 0.01. The results are expressed as the mean of a triplicate assay ± SD. The data are representative of three independent and reproducible experiments with similar results. C and D, Spinal cords isolated at day 17 after immunization from WT or TRAIL−/− mice were examined histologically. Cross-sections of paraffin-embedded spinal cord samples were stained with H&E and are representative of sections taken from three mice per group. Scale bars, 200 μm. Original magnification ×100 (C) and ×250 (D).

FIGURE 2.

Increased severity and protracted course of MOG-induced EAE in TRAIL−/− mice. A, WT and TRAIL−/− mice (10 mice/group) were immunized with MOG 35–55 peptide on day 0, and pertussis toxin on day 0 and 2, as described in 1Materials and Methods. Values represent the mean clinical score for each group. The data are summarized in Table II. B, Isolated spleen cells (2.5 × 105) (left panel) or ILN cells (right panel) from WT or TRAIL−/− mice 21 d after immunization were cultured with MOG peptide (0, 1, or 3.2 μM) for 3 d. As a positive control, respective cells were also stimulated with Con A (5 μg/ml). The proliferative response was quantified by measuring [3H]thymidine incorporation in the final 12 h of the culture. *p < 0.05; **p < 0.01. The results are expressed as the mean of a triplicate assay ± SD. The data are representative of three independent and reproducible experiments with similar results. C and D, Spinal cords isolated at day 17 after immunization from WT or TRAIL−/− mice were examined histologically. Cross-sections of paraffin-embedded spinal cord samples were stained with H&E and are representative of sections taken from three mice per group. Scale bars, 200 μm. Original magnification ×100 (C) and ×250 (D).

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Table II.
Exacerbation of MOG-induced EAE in TRAIL−/− mice
GroupDisease Incidence (n)Death (n)Day of Onset (Mean ± SD)Peak Clinical Score (Mean ± SD)
WT mice 10/10 0/10 9.6 ± 0.6 2.2 ± 0.4 
TRAIL−/− mice 10/10 0/10 7.3 ± 0.8 3.4 ± 0.3 
GroupDisease Incidence (n)Death (n)Day of Onset (Mean ± SD)Peak Clinical Score (Mean ± SD)
WT mice 10/10 0/10 9.6 ± 0.6 2.2 ± 0.4 
TRAIL−/− mice 10/10 0/10 7.3 ± 0.8 3.4 ± 0.3 

The data are combined from two experiments, including those shown in Fig. 2A. The values for onset day and mean peak clinical score are rounded to the first decimal place.

We also analyzed the response of T cells to MOG isolated from MOG-immunized mice. Twenty-one days after immunization, spleen and ILN cells isolated from the mice were cultured with MOG peptide. As shown in Fig. 2B, the proliferative response of spleen and ILN cells isolated from TRAIL−/− mice to MOG peptide was much greater compared with WT mice. In contrast, there was no significant difference in the response to Con A between WT and TRAIL−/− mice. Histological examination of the spinal cords was performed to determine whether the difference in clinical symptoms observed between WT and TRAIL−/− mice correlated with the degree of inflammatory infiltration in the CNS. Consistent with the data shown in Fig. 2B, more intensive infiltration of inflammatory cells into the spinal cord was observed in TRAIL−/− mice than in WT mice (Fig. 2C, 2D).

Because the frequency of Th1 cells in TRAIL−/− mice was greater than that in WT mice in an unimmunized state (Fig. 1D), we considered the possibility that the more severe EAE disease in TRAIL−/− mice was caused by the greater frequency of Th1 cells. To assess this possibility, we analyzed the frequency of CD4+ T, Th1, and Th17 cells and Tregs in whole spleen and ILN cells on days 7, 13, 17, and 21 in EAE-induced WT and TRAIL−/− mice. An increase in the frequency of total CD4+ T cells in the spleen was observed on days 7 and 13 in TRAIL−/− mice but not in WT mice (Fig. 3A, left panel). The frequency of Tregs in TRAIL−/− mice was less than in WT mice at all assay points in spleen and ILNs (Fig. 3B). In contrast, the frequency of Th1 cells in TRAIL−/− mice was greater than that in WT mice (Fig. 3C). In contrast, no significant difference in the frequency of Th17 cells was observed between TRAIL−/− mice and WT mice (Fig. 3D). We also calculated the absolute cell numbers of the respective cells in the spleen (Table III) and ILNs (Table IV). The difference in the absolute numbers of Tregs between TRAIL−/− mice and WT mice was significant on day 7 for the spleen (Table III). These results indicate that TRAIL functions to increase Tregs and decrease Th1 cells upon induction of EAE. The increased proliferative response of the TRAIL−/− mice-derived cells to MOG (Fig. 2B) may be attributed to the greater population of Th1 cells and smaller population of Tregs compared with WT mice.

FIGURE 3.

Increased Th1 cells and decreased Tregs in TRAIL−/− mice upon EAE induction. Spleen (left panels) and ILN (right panels) cells from WT or TRAIL−/− mice were analyzed by flow cytometry at days 7, 13, 17, and 21 after immunization. A, Frequency of whole CD4+ T cells. B, Frequency of CD4+Foxp3+ T cells in whole cells. C and D, Cells were stimulated with PMA plus ionomycin in the presence of brefeldin A for 5 h and subsequently analyzed for the production of IFN-γ and IL-17A by intracellular staining and flow cytometry. Frequencies of IFN-γ+CD4+ T cells (C) and IL-17A+CD4+ T cells (D) in total CD4+ T cells are shown. Bar graphs represent the mean frequency ± SD of four or five mice per group. *p < 0.05; **p < 0.01.

FIGURE 3.

Increased Th1 cells and decreased Tregs in TRAIL−/− mice upon EAE induction. Spleen (left panels) and ILN (right panels) cells from WT or TRAIL−/− mice were analyzed by flow cytometry at days 7, 13, 17, and 21 after immunization. A, Frequency of whole CD4+ T cells. B, Frequency of CD4+Foxp3+ T cells in whole cells. C and D, Cells were stimulated with PMA plus ionomycin in the presence of brefeldin A for 5 h and subsequently analyzed for the production of IFN-γ and IL-17A by intracellular staining and flow cytometry. Frequencies of IFN-γ+CD4+ T cells (C) and IL-17A+CD4+ T cells (D) in total CD4+ T cells are shown. Bar graphs represent the mean frequency ± SD of four or five mice per group. *p < 0.05; **p < 0.01.

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Table III.
Cell numbers of total, CD4+, CD4+Foxp3+, IFN-γ+CD4+, and IL-17A+CD4+ T cells in the spleen in EAE-induced WT and TRAIL−/− mice
  Days After Immunization
Cell SubsetGroup (n = 4–5 Mice/Group)7131721
Total cells WT 1.7 ± 0.1 × 108 2.1 ± 0.6 × 108 1.8 ± 0.2 × 108 1.8 ± 0.4 × 108 
 TRAIL−/− 1.8 ± 0.3 × 108 2.0 ± 0.3 × 108 2.7 ± 0.2 × 108* 2.2 ± 0.3 × 108 
CD4+ T cells WT 1.5 ± 0.2 × 107 1.9 ± 0.5 × 107 1.3 ± 0.3 × 107 1.5 ± 0.5 × 107 
 TRAIL−/− 2.0 ± 0.3 × 107** 2.4 ± 0.3 × 107 2.3 ± 0.2 × 107* 1.5 ± 0.7 × 107 
CD4+Foxp3+ T cells WT 1.9 ± 0.2 × 106 3.3 ± 0.7 × 106 2.8 ± 0.1 × 106 2.4 ± 0.7 × 106 
 TRAIL−/− 1.3 ± 0.2 × 106** 2.0 ± 0.3 × 106** 2.8 ± 0.3 × 106 2.0 ± 0.6 × 106 
IFN-γ+CD4+ T cells WT 2.4 ± 0.2 × 106 3.8 ± 1.3 × 106 1.8 ± 0.4 × 106 2.0 ± 1.1 × 106 
 TRAIL−/− 4.4 ± 0.6 × 106* 6.5 ± 1.5 × 106 5.1 ± 1.5 × 106** 3.4 ± 1.4 × 106 
IL-17A+CD4+ T cells WT 1.3 ± 0.2 × 106 2.1 ± 0.9 × 106 1.7 ± 0.8 × 106 1.2 ± 0.6 × 106 
 TRAIL−/− 2.1 ± 0.3 × 106* 3.0 ± 0.6 × 106 3.3 ± 1.2 × 106 1.7 ± 1.0 × 106 
  Days After Immunization
Cell SubsetGroup (n = 4–5 Mice/Group)7131721
Total cells WT 1.7 ± 0.1 × 108 2.1 ± 0.6 × 108 1.8 ± 0.2 × 108 1.8 ± 0.4 × 108 
 TRAIL−/− 1.8 ± 0.3 × 108 2.0 ± 0.3 × 108 2.7 ± 0.2 × 108* 2.2 ± 0.3 × 108 
CD4+ T cells WT 1.5 ± 0.2 × 107 1.9 ± 0.5 × 107 1.3 ± 0.3 × 107 1.5 ± 0.5 × 107 
 TRAIL−/− 2.0 ± 0.3 × 107** 2.4 ± 0.3 × 107 2.3 ± 0.2 × 107* 1.5 ± 0.7 × 107 
CD4+Foxp3+ T cells WT 1.9 ± 0.2 × 106 3.3 ± 0.7 × 106 2.8 ± 0.1 × 106 2.4 ± 0.7 × 106 
 TRAIL−/− 1.3 ± 0.2 × 106** 2.0 ± 0.3 × 106** 2.8 ± 0.3 × 106 2.0 ± 0.6 × 106 
IFN-γ+CD4+ T cells WT 2.4 ± 0.2 × 106 3.8 ± 1.3 × 106 1.8 ± 0.4 × 106 2.0 ± 1.1 × 106 
 TRAIL−/− 4.4 ± 0.6 × 106* 6.5 ± 1.5 × 106 5.1 ± 1.5 × 106** 3.4 ± 1.4 × 106 
IL-17A+CD4+ T cells WT 1.3 ± 0.2 × 106 2.1 ± 0.9 × 106 1.7 ± 0.8 × 106 1.2 ± 0.6 × 106 
 TRAIL−/− 2.1 ± 0.3 × 106* 3.0 ± 0.6 × 106 3.3 ± 1.2 × 106 1.7 ± 1.0 × 106 

Data are mean ± SD. Mean absolute cell numbers were calculated from total cell numbers for the whole spleen. The values are rounded to the first decimal place.

*p < 0.01; **p < 0.05, compared with WT mice.

Table IV.
Cell numbers of total, CD4+, CD4+Foxp3+, IFN-γ+CD4+, IL-17A+CD4+ T cells in the ILNs in EAE-induced WT and TRAIL−/− mice
  Days After Immunization
Cell SubsetsGroup (n = 4–5 Mice/Group)7131721
Total cells WT 2.4 ± 1.1 × 106 12.9 ± 3.5 × 106 7.7 ± 4.1 × 106 7.5 ± 3.7 × 106 
 TRAIL−/− 4.5 ± 0.7 × 106* 11.7 ± 4.6 × 106 10.8 ± 3.8 × 106 9.4 ± 3.5 × 106 
CD4+ T cells WT 0.5 ± 0.2 × 106 2.6 ± 0.5 × 106 1.7 ± 0.9 × 106 1.9 ± 1.2 × 106 
 TRAIL−/− 0.9 ± 0.2 × 106 2.6 ± 1.2 × 106 2.1 ± 0.6 × 106 2.1 ± 1.0 × 106 
CD4+FoxP3+ T cells WT 1.0 ± 0.5 × 105 3.8 ± 1.2 × 105 2.5 ± 1.1 × 105 2.5 ± 1.1 × 105 
 TRAIL−/− 1.5 ± 0.2 × 105 2.6 ± 1.1 × 105 2.4 ± 1.0 × 105 2.4 ± 0.9 × 105 
IFN-γ+CD4+ T cells WT 0.7 ± 0.5 × 105 1.4 ± 0.2 × 105 0.9 ± 0.6 × 105 0.5 ± 0.2 × 105 
 TRAIL−/− 2.8 ± 0.6 × 105** 2.4 ± 0.9 × 105 1.8 ± 0.8 × 105 1.2 ± 0.3 × 105* 
IL-17A+CD4+ T cells WT 0.5 ± 0.2 × 105 1.3 ± 0.2 × 105 0.5 ± 0.4 × 105 0.4 ± 0.2 × 105 
 TRAIL−/− 1.2 ± 0.2 × 105** 1.6 ± 0.7 × 105 1.2 ± 1.0 × 105 0.6 ± 0.2 × 105 
  Days After Immunization
Cell SubsetsGroup (n = 4–5 Mice/Group)7131721
Total cells WT 2.4 ± 1.1 × 106 12.9 ± 3.5 × 106 7.7 ± 4.1 × 106 7.5 ± 3.7 × 106 
 TRAIL−/− 4.5 ± 0.7 × 106* 11.7 ± 4.6 × 106 10.8 ± 3.8 × 106 9.4 ± 3.5 × 106 
CD4+ T cells WT 0.5 ± 0.2 × 106 2.6 ± 0.5 × 106 1.7 ± 0.9 × 106 1.9 ± 1.2 × 106 
 TRAIL−/− 0.9 ± 0.2 × 106 2.6 ± 1.2 × 106 2.1 ± 0.6 × 106 2.1 ± 1.0 × 106 
CD4+FoxP3+ T cells WT 1.0 ± 0.5 × 105 3.8 ± 1.2 × 105 2.5 ± 1.1 × 105 2.5 ± 1.1 × 105 
 TRAIL−/− 1.5 ± 0.2 × 105 2.6 ± 1.1 × 105 2.4 ± 1.0 × 105 2.4 ± 0.9 × 105 
IFN-γ+CD4+ T cells WT 0.7 ± 0.5 × 105 1.4 ± 0.2 × 105 0.9 ± 0.6 × 105 0.5 ± 0.2 × 105 
 TRAIL−/− 2.8 ± 0.6 × 105** 2.4 ± 0.9 × 105 1.8 ± 0.8 × 105 1.2 ± 0.3 × 105* 
IL-17A+CD4+ T cells WT 0.5 ± 0.2 × 105 1.3 ± 0.2 × 105 0.5 ± 0.4 × 105 0.4 ± 0.2 × 105 
 TRAIL−/− 1.2 ± 0.2 × 105** 1.6 ± 0.7 × 105 1.2 ± 1.0 × 105 0.6 ± 0.2 × 105 

Data are mean ± SD. Mean absolute cell numbers were calculated from total cell numbers for the whole spleen. The values are rounded to the first decimal place.

*p < 0.05; **p < 0.01, versus WT mice.

Recently, it was reported that activated CD4+CD25+ Tregs displayed TRAIL-dependent cytotoxicity against CD4+CD25 conventional T cells in vivo (17). To investigate the effect of TRAIL on CD4+CD25+ Tregs in EAE disease, we depleted CD4+CD25+ Tregs in WT and TRAIL−/− mice by administering anti-CD25 mAb before induction of EAE.

We first analyzed the degree of reduction in Foxp3+ Tregs after injection of anti-CD25 mAb. Consistent with previous reports (27), the injection of anti-CD25 mAb reduced Foxp3+ Tregs in the spleen to less than half of the control levels in both types of mice (Fig. 4A, 4B).

FIGURE 4.

Depletion of CD4+CD25+ Tregs exacerbates MOG-induced EAE in WT and TRAIL−/− mice. A and B, Depletion of CD4+Foxp3+ T cells was verified by flow-cytometric analysis of spleen cells on day 12 after anti-CD25 mAb injection or isotype-matched control mAb (400 μg/mouse). A, Dot plots represent CD4 and Foxp3 expression in spleen cells from WT (left panels) and TRAIL−/− (right panels) mice, and the frequency of CD4+Foxp3+ T cells is shown. B, Numbers of CD4+Foxp3+ T cells in the spleen from WT and TRAIL−/− mice. Bar graphs indicate the mean cell numbers ± SD of three mice per group. C, WT or TRAIL−/− mice (n = 5 mice/group) were injected i.p. with anti-mouse CD25 mAb or isotype-matched control mAb (400 μg/mouse) 14 d before immunization with MOG 35–55 peptide and pertussis toxin, as described in Fig. 2A. Values represent the mean clinical score for each group. The data are summarized in Table V.

FIGURE 4.

Depletion of CD4+CD25+ Tregs exacerbates MOG-induced EAE in WT and TRAIL−/− mice. A and B, Depletion of CD4+Foxp3+ T cells was verified by flow-cytometric analysis of spleen cells on day 12 after anti-CD25 mAb injection or isotype-matched control mAb (400 μg/mouse). A, Dot plots represent CD4 and Foxp3 expression in spleen cells from WT (left panels) and TRAIL−/− (right panels) mice, and the frequency of CD4+Foxp3+ T cells is shown. B, Numbers of CD4+Foxp3+ T cells in the spleen from WT and TRAIL−/− mice. Bar graphs indicate the mean cell numbers ± SD of three mice per group. C, WT or TRAIL−/− mice (n = 5 mice/group) were injected i.p. with anti-mouse CD25 mAb or isotype-matched control mAb (400 μg/mouse) 14 d before immunization with MOG 35–55 peptide and pertussis toxin, as described in Fig. 2A. Values represent the mean clinical score for each group. The data are summarized in Table V.

Close modal
Table V.
Statistical parameters for MOG-induced EAE in mice depleted of CD4+CD25+ Tregs
GroupDisease IncidenceDeathDay of Onset (Mean ± SD)Peak Clinical Score (Mean ± SD)
WT mice control 5/5 0/5 9.8 ± 0.4 2.9 ± 0.6 
WT mice CD25 depleteda 5/5 0/5 7.2 ± 1.2 3.7 ± 0.1 
TRAIL−/− mice control 5/5 0/5 7.2 ± 0.4 3.7 ± 0.1 
TRAIL−/− mice CD25 depleteda 5/5 0/5 6.4 ± 0.5 4.2 ± 0.3 
GroupDisease IncidenceDeathDay of Onset (Mean ± SD)Peak Clinical Score (Mean ± SD)
WT mice control 5/5 0/5 9.8 ± 0.4 2.9 ± 0.6 
WT mice CD25 depleteda 5/5 0/5 7.2 ± 1.2 3.7 ± 0.1 
TRAIL−/− mice control 5/5 0/5 7.2 ± 0.4 3.7 ± 0.1 
TRAIL−/− mice CD25 depleteda 5/5 0/5 6.4 ± 0.5 4.2 ± 0.3 

The data are combined from two separate experiments, including those shown in Fig. 4. The values for onset day and peak clinical score are rounded to the first decimal place.

a

CD25+ T cells were depleted by treatment with anti-CD25 mAb.

Based on the above results, we induced EAE in WT and TRAIL−/− mice after administration of control mAb (rat IgG) or anti-CD25 mAb and investigated clinical features. Consistent with the data shown in Fig. 2A, TRAIL−/− mice treated with control mAb developed more severe EAE than did control mAb-treated WT mice (Fig. 4C, Table V). In WT and TRAIL−/− mice, the depletion of Tregs before EAE induction resulted in more severe disease. Therefore, Tregs inhibit the disease, regardless of the presence of TRAIL. The exacerbation caused by Treg depletion was more drastic in WT mice than in TRAIL−/− mice, suggesting that the disease-inhibiting effect of Tregs may be mediated, in part, by TRAIL.

We observed that there is a greater frequency of Th1 cells and lower frequency of Tregs in EAE-induced TRAIL−/− mice than in WT mice. These observations in vivo suggest that TRAIL negatively regulates Th1 cells and positively regulates Tregs. We next examined the effect of TRAIL on cell proliferation in vitro.

TRAIL was expressed by LPS-stimulated BM-DCs from WT mice (Fig. 5A). Naive CD4+CD25 conventional T cells and CD4+CD25+ Tregs were isolated from the spleen of WT or TRAIL−/− mice and cocultured with X-ray–irradiated BM-DCs derived from WT or TRAIL−/− mice in the presence of anti-CD3 mAb and IL-2. The culture was continued for 3 d, and the proliferation of T cells was measured at the end of the culture. As shown in Fig. 5B, the proliferation of CD4+CD25 conventional T cells cocultured with BM-DCs from TRAIL−/− mice was significantly greater than those cocultured with BM-DCs from WT mice. The greatest magnitude of proliferation was observed when the CD4+CD25 conventional T cells and BM-DCs were derived from TRAIL−/− mice. Thus, the proliferation of CD4+CD25 conventional T cells was greatest in the absence of TRAIL. These results suggest that TRAIL downmodulates the proliferation of CD4+CD25 conventional T cells and that TRAIL on BM-DCs and T cells exerts this effect.

FIGURE 5.

Effects of TRAIL on the proliferation of CD4+CD25 conventional Th cells and CD4+CD25+ Tregs. A, BM-DCs derived from WT or TRAIL−/− mice were stimulated with 1 μg/ml LPS for 48 h before the expression of TRAIL was analyzed by flow cytometry. Staining patterns of TRAIL (black line) and isotype-matched control (gray line) are shown. CD4+CD25 conventional T cells (4.0 × 104) (B) or CD4+CD25+ Tregs (4.0 × 104) (C) isolated from the spleens of unimmunized WT or TRAIL−/− mice were cocultured with irradiated LPS-stimulated BM-DCs (2.0 × 104) derived from WT or TRAIL−/− mice for 3 d in the presence of soluble anti-CD3 mAb (1 μg/ml) and human IL-2 (10 U/ml). CD4+CD25 conventional T cells (1.0 × 105) (D) or CD4+CD25+ Tregs (1.0 × 105) (E) isolated from the spleens of unimmunized TRAIL−/− mice were stimulated with plate-bound anti-CD3 mAb (3 μg/ml), anti-CD28 mAb (3 μg/ml), and human IL-2 (20 U/ml) in the presence or absence of soluble rTRAIL for 3 d. T cell proliferation was quantified by measuring [3H]thymidine incorporation in the final 12 h of culture. The results are expressed as the mean ± SD of a triplicate assay. The data are representative of more than three independent experiments with similar results. *p < 0.05; **p < 0.01. KO, TRAIL−/−.

FIGURE 5.

Effects of TRAIL on the proliferation of CD4+CD25 conventional Th cells and CD4+CD25+ Tregs. A, BM-DCs derived from WT or TRAIL−/− mice were stimulated with 1 μg/ml LPS for 48 h before the expression of TRAIL was analyzed by flow cytometry. Staining patterns of TRAIL (black line) and isotype-matched control (gray line) are shown. CD4+CD25 conventional T cells (4.0 × 104) (B) or CD4+CD25+ Tregs (4.0 × 104) (C) isolated from the spleens of unimmunized WT or TRAIL−/− mice were cocultured with irradiated LPS-stimulated BM-DCs (2.0 × 104) derived from WT or TRAIL−/− mice for 3 d in the presence of soluble anti-CD3 mAb (1 μg/ml) and human IL-2 (10 U/ml). CD4+CD25 conventional T cells (1.0 × 105) (D) or CD4+CD25+ Tregs (1.0 × 105) (E) isolated from the spleens of unimmunized TRAIL−/− mice were stimulated with plate-bound anti-CD3 mAb (3 μg/ml), anti-CD28 mAb (3 μg/ml), and human IL-2 (20 U/ml) in the presence or absence of soluble rTRAIL for 3 d. T cell proliferation was quantified by measuring [3H]thymidine incorporation in the final 12 h of culture. The results are expressed as the mean ± SD of a triplicate assay. The data are representative of more than three independent experiments with similar results. *p < 0.05; **p < 0.01. KO, TRAIL−/−.

Close modal

To investigate the proliferation of CD4+CD25+ Tregs, the experiments were repeated using BM-DCs and CD4+CD25+ Tregs derived from WT or TRAIL−/− mice (Fig. 5C). As a result, the proliferation of CD4+CD25+ Tregs in the absolute absence of TRAIL was less than that observed in the other three combinations, suggesting that TRAIL upregulates the proliferation of CD4+CD25+ Tregs. Collectively, TRAIL may inhibit the growth of CD4+CD25 conventional T cells and enhance the growth of CD4+CD25+ Tregs.

To further analyze the effect of TRAIL on CD4+CD25 conventional T cells and CD4+CD25+ Tregs, both T cells derived from TRAIL−/− mice were stimulated with plate-bound anti-CD3, anti-CD28 mAb, and IL-2 in the presence or absence of soluble rTRAIL (0–100 ng/ml). The proliferation of T cells was measured at the end of the 3-d culture. As shown in Fig. 5D, the proliferation of CD4+CD25 conventional T cells was inhibited by the addition of soluble rTRAIL in a dose-dependent manner. In contrast, the proliferation of CD4+CD25+ Tregs was not affected by soluble rTRAIL, even when 100 ng/ml was added (Fig. 5E).

We examined the level of expression of the TRAILR mDR5 and decoy receptors mDc-TRAIL-R1 and mDc-TRAIL-R2 on CD4+CD25 conventional T cells and CD4+CD25+ Tregs derived from TRAIL−/− mice. After stimulation with anti-CD3 and CD28 mAb and IL-2, mDR5 expression was upregulated in both T cell subsets (Fig. 6). With regard to decoy receptors, mDc-TRAIL-R2 was not expressed in either T cell subset. In contrast, mDc-TRAIL-R1 was slightly expressed in CD4+CD25+ Tregs but not in CD4+CD25 conventional T cells. The difference in the expression of mDc-TRAIL-R1 between the two T cell subsets may affect their responsiveness to TRAIL.

FIGURE 6.

The expression of the functional TRAILR and two decoy receptors on CD4+CD25 conventional T cells and CD4+CD25+ Tregs. CD4+CD25 conventional T cells (1.0 × 105) (A) or CD4+CD25+ Tregs (1.0 × 105) (B) isolated from the spleens of unimmunized TRAIL−/− mice were stimulated with plate-bound anti-CD3 (3 μg/ml) and anti-CD28 mAb (3 μg/ml) and human IL-2 (20 U/ml) for 72 h. Then, mDR5, mDc-TRAIL-R1, and mDcTRAIL-R2 on each T cell subset were analyzed by flow cytometry. Staining patterns of mDR5, mDc-TRAIL-R1, and mDcTRAIL-R2 (black lines) and isotype-matched control (gray lines) are shown.

FIGURE 6.

The expression of the functional TRAILR and two decoy receptors on CD4+CD25 conventional T cells and CD4+CD25+ Tregs. CD4+CD25 conventional T cells (1.0 × 105) (A) or CD4+CD25+ Tregs (1.0 × 105) (B) isolated from the spleens of unimmunized TRAIL−/− mice were stimulated with plate-bound anti-CD3 (3 μg/ml) and anti-CD28 mAb (3 μg/ml) and human IL-2 (20 U/ml) for 72 h. Then, mDR5, mDc-TRAIL-R1, and mDcTRAIL-R2 on each T cell subset were analyzed by flow cytometry. Staining patterns of mDR5, mDc-TRAIL-R1, and mDcTRAIL-R2 (black lines) and isotype-matched control (gray lines) are shown.

Close modal

Under normal conditions, the frequency of Tregs in TRAIL−/− mice was similar to that in WT mice (Fig. 1B, 1C). However, upon induction of EAE, the frequency of Tregs in TRAIL−/− mice was significantly lower than that in WT mice (Fig. 3B). Wang et al. (20) reported that the administration of rTRAIL induced CD4+CD25+ Tregs in mice that were immunized with thyroglobulin to develop EAT. These in vivo observations collectively suggest that the proliferative effect of TRAIL on Tregs becomes apparent when exposed to the risk for autoimmunity.

To investigate the effect of TRAIL in Tregs, CD4+CD25+ Tregs isolated from WT or TRAIL−/− mice were cocultured with BM-DCs derived from both mice in the presence of anti-CD3 mAb and IL-2. When CD4+CD25+ Tregs isolated from TRAIL−/− mice were stimulated by TRAIL−/−-derived BM-DCs, the proliferation was less than when stimulated by WT-derived BM-DCs. This result suggests that TRAIL positively regulates the proliferation of Tregs. In contrast, when CD4+CD25+ Tregs isolated from WT mice were stimulated by WT-derived BM-DCs, the proliferation was similar to when they were stimulated by TRAIL−/−-derived BM-DCs (Fig. 5C). This indicates that the proliferative response of CD4+CD25+ Tregs from WT mice was not affected by the expression of TRAIL on the stimulator cells. Ren et al. reported (17) that CD4+CD25+ Tregs stimulated with anti-CD3/CD28 mAb and IL-2 were mostly TRAIL+. Thus, in our experiments, BM-DCs, as well as CD4+CD25+ Tregs, from WT mice should have expressed TRAIL (Fig. 5C). Therefore, TRAIL was not present in the culture only when BM-DCs and CD4+CD25+ Tregs were from TRAIL−/− mice. This may explain why decreased proliferation of Tregs was only observed when CD4+CD25+ Tregs and BM-DCs were TRAIL deficient.

The frequency of Th1 cells was greater in TRAIL−/− mice than in WT mice, in the steady state (Fig. 1D) and upon induction of EAE (Fig. 3C). The difference in the frequency of Th1 cells between WT and TRAIL−/− mice indicates that TRAIL negatively regulates Th1 cells. A previous study revealed that Th1 cells were sensitive to TRAIL-induced cell death (16, 28). It was also reported that TRAIL functionally inactivated Th1 cells. A recent study revealed that the administration of rTRAIL decreased the production of IFN-γ in mice with EAT (29). Elevated production of IFN-γ was observed in murine CMV-infected TRAILR-deficient mice in comparison with WT mice (30). Our in vitro experiments also showed an inhibitory effect of TRAIL on the proliferation of CD4+CD25 conventional T cells (Fig. 5B, 5D). Collectively, these results suggest that CD4+CD25 conventional T cells, especially Th1 cells, may be downregulated qualitatively and quantitatively by TRAIL through multiple pathways, including the induction of cell death, functional inactivation, and inhibition of growth.

In contrast to Th1 cells, there was no difference in the frequency of Th17 cells between WT and TRAIL−/− mice in steady-state or disease conditions (Figs. 1E, 3D).

The addition of soluble TRAIL did not elicit positive proliferative responses in CD4+CD25+ Tregs stimulated with plate-coated anti-CD3 mAb and anti-CD28 mAb (Fig. 5E). This may be because TRAIL needs to be in a transmembrane form to exert the proliferative effect on CD4+CD25+ Tregs. In contrast, downmodulation of the proliferation of CD4+CD25+ Tregs was never observed, even in the presence of 100 ng/ml of rTRAIL, the dose that affected the proliferation of CD4+CD25 conventional T cells (Fig. 5D, 5E). Other groups reported that Th2 cells, distinct from Th1 cells, were resistant to TRAIL-induced apoptosis (7, 16). Collectively, TRAIL exerts distinct effects on the different T cell subsets.

The functional TRAILR mDR5 was upregulated in CD4+CD25 conventional T cells and CD4+CD25+ Tregs (Fig. 6). In contrast, one of the decoy receptors, mDc-TRAIL-R1, was expressed in CD4+CD25+ Tregs but not in CD4+CD25 conventional T cells (Fig. 6). mDc-TRAIL-R1 expression might function in a similar manner as the two human decoy receptors TRAIL-R3 and TRAIL-R4 (31, 32) to affect the responsiveness of Tregs to TRAIL.

However, the level of the expression of mDc-TRAIL-R1 on CD4+CD25+ Tregs was low. Therefore, the differences in the responsiveness to TRAIL may not be accounted for only by the difference in the expression of mDc-TRAIL-R1. There may be some differences in the signaling pathways downstream of mDR5 between conventional T cells and Tregs. With regard to the cell type-specific signaling pathways downstream of mDR5, upregulation of cellular FLIP in Th2 cells was reported (16, 28). Further investigations are required to explore the distinct functions of TRAIL in different T cell subsets.

Consistent with a previous study by Cretney et al. (13), we observed that TRAIL−/− mice developed more severe EAE than did WT mice (Fig. 2A). Moreover, removal of TRAIL by systemic application of TRAILR led to a more severe course of EAE (33). These observations indicate that TRAIL suppresses autoimmunity in EAE. TRAIL−/− mice showed a lower frequency of Foxp3+ Tregs in the spleen and ILNs after EAE induction compared with WT mice (Fig. 3B). In vitro, TRAIL on stimulator cells enhanced the growth of CD4+CD25+ Tregs (Fig. 5C). These results indicate that TRAIL positively regulates Tregs. The ability of Tregs to suppress severity of EAE was demonstrated in studies by several groups (34, 35), and this is supported by the data shown in Fig. 4C. Collectively, it is suggested that the ability of TRAIL to ameliorate EAE is mediated, at least in part, by the increase in Tregs.

A method to negatively manipulate immune responses is needed for the treatment of autoimmune, allergic, and inflammatory diseases. Control of alloreactive immune responses is the key to treating graft rejection and graft-versus-host disease in transplantation medicine. For these purposes, administration of genetically modified DCs with enhanced immune-regulatory functions may be a promising strategy. Using directed differentiation of embryonic stem cells or induced pluripotent stem cells into DCs, we have developed efficient methods for genetically engineering mouse and human DCs (25, 36, 37). Among the molecules implicated in immune-regulatory functions, we found that TRAIL was the most effective in controlling alloreactive immunity (S. Hirata et al., submitted for publication). The exploration of TRAIL function may be of considerable significance for understanding the physiological mechanisms of immune regulation, as well as for the development of novel medical technology to control immunity.

Disclosures The authors have no financial conflicts of interest.

This work was supported in part by Grants-in-Aid Nos. 18014023, 19591172, and 19059012 from the Ministry of Education, Culture, Sports, Science and Technology, Japan; the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, launched as a project commissioned by the Ministry of Education, Culture, Sports, Science and Technology, Japan; a Research Grant for Intractable Diseases from the Ministry of Health and Welfare, Japan; and grants from the Japan Science and Technology Agency, the Uehara Memorial Foundation, and the Takeda Science Foundation, Advanced Education Program for Integrated Clinical, Basic and Social Medicine, Graduate School of Medical Sciences, Kumamoto University (Program for Enhancing Systematic Education in Graduate Schools, Ministry of Education, Culture, Sports, Science and Technology, Japan).

Abbreviations used in this paper:

BM-DC

bone marrow-derived dendritic cell

DC

dendritic cell

DN

double negative

DP

double positive

EAE

experimental autoimmune encephalomyelitis

EAT

experimental autoimmune thyroiditis

ES-DC

embryonic stem cell-derived dendritic cell

ILN

inguinal lymph node

KO

TRAIL−/−

mDR5

mouse death receptor 5

MOG

myelin-oligodendrocyte glycoprotein

rTRAIL

recombinant mouse TRAIL

SP CD4

CD4+ single positive

SP CD8

CD8+ single positive

Treg

regulatory T cell

WT

wild-type.

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