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* Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; and
Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
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Abstract |
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. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A variety of cells that display regulatory function in vitro or in vivo have been described. These can be subdivided into different subsets based on the expression of cell surface markers, production of cytokines, and mechanisms of action. Recent studies focused on CD25 as the best marker for regulatory CD4+ T cells in mice and humans (12, 13, 14). CD4+CD25+ T cells, which constitute 
10% of peripheral murine CD4+ T cells, express little CD45RB, and a significant proportion expresses CTLA-4 (15, 16, 17, 18). They show a partially anergic phenotype, in that they proliferate poorly upon TCR stimulation in vitro and their growth is dependent on exogenous IL-2.
However, increasing evidence suggests that the peripheral CD4+CD25- T cell population also possesses some regulatory activity (19, 20, 21, 22, 23, 24), although specific markers for CD4+CD25- regulatory T cells remain unclear. Stephens and Mason (23) have described a population of CD4+CD25- T cells that can prevent autoimmunity in thymectomy/irradiation model in rat, but only if recent CD25- thymic emigrants have been deleted from the population. Similarly, CD4+CD25- T cells appear to be responsible for the resistance of mice expressing a transgenic TCR specific for myelin basic protein to the spontaneous development of autoimmune encephalomyelitis (20). In fact, CD4+CD25+ T cell population is heterogeneous, and although a relatively high proportion of these cells may be regulatory T cells, but it is not likely to be true that the entire population is regulatory or that all regulatory T cells express CD25.
Very recently, it has been demonstrated that glucocorticoid-induced TNFR family-related gene (GITR), a member of the TNF-nerve growth factor receptor gene superfamily, is predominantly expressed on CD4+CD25+ T cells and on CD4+CD25+CD8- thymocytes in normal naive mice (25). In addition, removal of GITR-expressing T cells or administration of a mAb to GITR produced organ-specific autoimmune disease in otherwise normal mice (25).
In the present study, we conducted a series of experiments focusing on GITR as a regulatory T cell marker for intestinal mucosal regulatory T cell to investigate the characteristics of CD4+CD25- regulatory T cells, and to understand how the mucosal immune system is controlled by GITR-associated regulatory T cells.
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Materials and Methods |
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Female BALB/c, C.B-17 SCID, and C57BL/6 mice were purchased from Japan Clear (Tokyo, Japan). Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Tokyo Medical and Dental University. Mice were used at 7–12 wk of age. All experiments were approved by the regional animal study committees.
Antibodies
The following mAbs and reagents were purchased from BD PharMingen (San Diego, CA), except anti-mouse GITR mAb (DTA-1, rat IgG2a), and used for purification of cell populations and flow cytometry analysis: RM4-5, PE-conjugated anti-mouse CD4 (rat IgG2a); 7D4, FITC-conjugated anti-mouse CD25 (rat IgM); PC61, PE-conjugated anti-mouse CD25 (rat IgG1); DTA-1, biotinylated anti-GITR (25); 9H10, PE-conjugated anti-CTLA-4 (hamster IgG); M290, PE-conjugated anti-mouse CD103 (
E integrin) (rat IgG2a); PK136, PE-conjugated anti-mouse NK1.1 (mouse IgG2a); DX5, FITC-conjugated anti-mouse pan-NK cells (rat IgM); H28-710, FITC-conjugated anti-mouse TCR -chain (hamster IgG); GL4, FITC-conjugated anti-mouse TCR 
-chains (hamster IgG); isotype control Abs, biotin-conjugated rat IgG2, FITC-conjugated rat IgM, PE-conjugated rat IgG2a, and PE-conjugated mouse IgG2a; PE-conjugated streptavidin; CyChrome-conjugated streptavidin.
Purification of T cell subsets
CD4+ T cells were isolated from spleen cells from BALB/c mice using the anti-CD4 (L3T4) MACS system (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instruction. Enriched CD4+ T cells (96–97% pure, as estimated by a FACSCalibur (BD Biosciences, Sunnyvale, CA)) were then labeled with PE-conjugated anti-mouse CD4 (RM4-5), FITC-conjugated anti-CD45RB (16A), FITC-conjugated anti-CD25 (7D4), biotinylated anti-GITR mAb (DTA-1), and streptavidin PE. Subpopulations of CD4+ cells were generated by two-color sorting on a FACSVantage (BD Biosciences). All populations were >98.0% pure on reanalysis.
In vivo experimental design
We performed a series of in vivo experiments below to investigate the role of CD4+GITR+T cells on the regulation of murine chronic colitis. Experiment 1: To assess regulatory T cell activity in CD4+GITR+ T cells, we used the classical SCID-transfer colitis model (26). C.B-17 SCID mice were injected i.p. with one or two subpopulations of sorted CD4+ T cell in PBS: 1) CD4+CD45RBhigh alone (3 x 105 per body, as a positive control); 2) CD4+CD45RBhigh + CD4+CD45RBlow (each 3 x 105 per body, as a negative control); 3) CD4+CD45RBhigh (3 x 105 per body) + CD4+GITR+ (1 x 105 per body); or 4) CD4+GITR- alone (3 x 105 per body). Mice were sacrificed at 4 wk after T cell transfer, because percentage of decrease of original body weight in one of experimental group reached 20%. Experiment 2: To assess functional role of GITR, we used purified anti-murine GITR mAb (rat IgG2a, DTA-1). C.B-17 SCID mice were injected i.p. with CD4+CD45RBhigh + CD4+CD45RBlow T cells (each 3 x 105 per body, as a negative control), and also were given 1 mg control rat IgG or anti-GITR mAb by i.p. injection weekly from the day of T cell transfer over a period of 6 wk. As a positive control, C.B-17 SCID mice were injected i.p. with CD4+CD45RBhigh T cells alone (3 x 105 per body). Experiment 3: To address the possibility that CD4+CD25- T cells in CD4+GITR+ T cell population function as regulatory T cells, we divided CD4+GITR+ T cells into CD4+CD25-GITR+ and CD4+CD25+GITR+ cells. C.B-17 SCID mice were then injected i.p. with one or two subpopulations of sorted CD4+ T cell in PBS: 1) CD4+CD45RBhigh + CD4+CD45RBlow (each 3 x 105 per body, as a negative control); 2) CD4+CD45RBhigh alone (3 x 105 per body, as a postive control); 3) CD4+CD45RBhigh (3 x 105 per body) + CD4+CD25+GITR+ cells (1 x 105 per body); or 4) CD4+CD45RBhigh (3 x 105 per body) + CD4+CD25-GITR+ cells (1 x 105 per body). Mice were sacrificed and analyzed 7 wk after T cell transfer.
Disease monitoring and clinical scoring
The recipient SCID mice after T cell transfer were weighed initially, then three times per week thereafter. They were observed for clinical signs of illness: hunched over appearance, piloerection of the coat, diarrhea, and blood in the stool. Mice were sacrificed and assessed for a clinical score that is the sum of four parameters, as follows: hunching and wasting, 0 or 1; colon thickening, 0–3 (0, no colon thickening; 1, mild thickening; 2, moderate thickening; 3, extensive thickening); and stool consistency, 0–3 (0, normal beaded stool; 1, soft stool; 2, diarrhea; and an additional point was added if gross blood was noted) (27).
Histological examination
Tissue samples were fixed in PBS containing 6% neutral-buffered Formalin. Paraffin-embedded sections (5 µm) were stained with H&E. Three tissue samples from the proximal, middle, and distal parts of the colon were prepared. The sections were analyzed without prior knowledge of the type of T cell reconstitution or treatment. The area most affected was graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of a previously described scoring system (28), as follows: mucosa damage, 0; normal, 1; 3–10 intraepithelial cells (IEL)/high power field (HPF) and focal damage, 2; >10 IEL/HPF and rare crypt abscesses, 3; >10 IEL/HPF, multiple crypt abscesses and erosion ulceration, submucosa damage, 0; normal or widely scattered leukocytes, 1; focal aggregates of leukocytes, 2; diffuse leukocyte infiltration with expansion of submucosa, 3; diffuse leukocyte infiltration, muscularis damage, 0; normal or widely scattered leukocytes, 1; widely scattered leukocyte aggregates between muscle layers, 2; leukocyte infiltration with focal effacement of the muscularis, 3; extensive leukocyte infiltration with transmural effacement of the muscularis.
Preparation of mucosal lamina propria mononuclear cells
Colonic lamina propria mononuclear cells (LPMCs) were isolated using a method, as described previously (29). In brief, the entire length of intestine was opened longitudinally, washed with PBS, and cut into small (5-mm) pieces. To remove epithelium including IEL, the dissected mucosa was incubated twice with Ca2+ Mg2+-free HBSS containing 1 mM DTT (Sigma-Aldrich, St. Louis, MO) for 30 min, and then serially incubated twice in medium containing 0.75 mM EDTA (Sigma-Aldrich) for 60 min at 37°C under gentle shaking. The supernatants from these incubations, which included the epithelium and IELs, were deserted, and the residual fragments were pooled and treated with 2 mg/ml collagenase A (Worthington Biomedical, Freehold, NJ) and 0.01% DNase (Worthington) in 5% CO2 humidified air at 37°C for 2 h. The cells were then pelleted twice through a 40% isotonic Percoll solution, after which they were further purified by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation (40/75%). Enriched lamina propria (LP) CD4+ T cells were obtained by positive selection using an anti-CD4 (L3T4) MACS magnetic separation system. The resultant cells when analyzed by FACSCalibur contained >96% CD4+ cells.
Flow cytometry
To detect the surface expression of a variety of molecules, isolated splenocytes or LPMCs were preincubated with an FcR-blocking mAb (CD16/32; 2.4G2; BD PharMingen) for 20 min, followed by incubation with specific FITC-, PE-, or biotin-labeled Abs for 30 min on ice. The mAbs used were anti-CD4 mAb, anti-CD25 mAb, anti-CD45RB, anti-GITR, anti-TCR, anti-TCR, anti-NK1.1, and anti-pan NK cell (DX5) mAbs. Biotinylated Abs were detected with PE or CyChrome streptavidin. Standard two- or three-color flow cytometric analyses were obtained using the FACSCalibur utilizing CellQuest software. Background fluorescence was assessed by staining with control isotype-matched mAbs.
Cytokine ELISA
To measure cytokine production, 1 x 105 LP CD4+ T cells were cultured in 200 µl culture medium at 37°C in a humidified atmosphere containing 5% CO2 in 96-well plates (Costar, Cambridge, MA) precoated with 5 µg/ml hamster anti-mouse CD3
mAb (145-2C11; BD PharMingen) and hamster 2 µg/ml anti-mouse CD28 mAb (37.51; BD PharMingen) in PBS overnight at 4°C. Culture supernatants were removed after 48 h and assayed for cytokine production. Cytokine concentrations were determined by specific ELISA per manufacturer’s recommendation (R&D Systems, Minneapolis, MN).
In vitro regulatory T cell function in CD4+CD25-GITR+ T cells
Spleen cells from BALB/c mice were separated into unfractioned whole CD4+ T cells, CD4+CD25+GITR+ T cells, and CD4+CD25-GITR+ T cells using the anti-CD4 (L3T4) MACS magnetic separation system and FACSVantage, as described above. Cells (5 x 104) and x-irradiated (20 Gy) BALB/c CD4- cells (2 x 105), as APCs, were cultured for 72 h in round-bottom 96-well plates in RPMI supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME. Cells were stimulated with Con A (5 µg/ml) in the presence or absence of human rIL-2 (100 U/ml). In coculture experiments, 2-fold numbers of CD4+CD25+GITR+ cells, CD4+CD25-GITR+ cells, or whole CD4+ cells (as a control) (1 x 105) were added into wells with the fixed dose of whole CD4+ cells (5 x 104) and x-irradiated (20 Gy) CD4- cells (2 x 105), as APCs. Incorporation of [3H]thymidine (1 µCi/well) by proliferating cells was measured during the last 9 h of culture. For cytokine assays, purified CD4+CD25+GITR+, CD4+CD25-GITR+ T cells, or whole CD4+ cells were cultured in complete medium consisting of RPMI 1640 (or serum-free medium (Nutridoma SP; Roche Molecular, Mannheim, Germany) in the case of TGF-) in flat-bottom 96-well plates (200 µl) at 2 x 105 cells/well and stimulated with 10 µg/ml plate-bound anti-CD3 mAb plus soluble anti-CD28 mAb (5 µg/ml). Supernatants were collected after 24 h for IL-2; 48 h for IL-4, IL-10, and IFN-; and 72 h for TGF-. Cytokines secreted into culture fluid were assayed by ELISA kits. Levels of TGF- in acidified supernatants were determined by TGF-1 Emax Immunoassay Kit (Promega, Madison, WI), according to the manufacturer’s instructions. Other cytokines were measured by specific ELISA per manufacturer’s recommendation (R&D Systems). For the analysis of CTLA-4 expression, CD4+ cells were sorted by FITC-conjugated anti-CD25 and biotinylated anti-GITR, followed by CyChrome-conjugated streptavidin. After sorting cells using a FACSVantage, cells were stained with PE-conjugated anti-CTLA-4 mAb. Before staining with anti-CTLA-4 mAb, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen) at 4°C for 30 min. Staining and washing were performed in Perm/Wash Buffer (BD PharMingen), and cells were washed once in PBS before analysis.
Statistical analysis
The results were expressed as the mean ± SD. Groups of data were compared by Mann-Whitney U test. Differences were considered to be statistically significant when p < 0.05.
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Results |
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Efforts to delineate regulatory T cell population have revealed that CD4+CD25+ T cell population in mice and humans retains regulatory T cell function (12, 30, 31, 32, 33). However, accumulating evidence has shown that CD4+CD25- T cell population also possesses regulatory activity (19, 20, 21, 22, 23, 24), although specific markers for CD4+CD25- regulatory T cells remain unclear. Recently, Shimizu et al. (25) have reported that GITR, a member of TNF/TNFR family, is a functional specific marker as regulatory T cells. In this study, we first postulated that both CD4+CD25+ and CD4+CD25- regulatory T cells express GITR. Thus, we assessed the correlation among the expression of GITR, CD25, and CD45RB on freshly isolated splenocytes. First, GITR was mainly expressed on CD4+ T cells (Fig. 1A, lower left panel). As expected, CD25+ cells expressed GITR (Fig. 1A, upper right panel). Interestingly, GITR-expressing cells also existed to some degree in CD25- subpopulation (Fig. 1A, upper right panel). Importantly, GITR was exclusively expressed on CD45RBlow cells (Fig. 1A, lower right panel). Three-color flow cytometric analysis revealed that GITR+ cells in MACS-sorted CD4+ T cells contain both CD25+ (60.3 ± 6.4%) and CD25- (39.3 ± 4.9%) fractions, although CD4+GITR- T cells were mostly CD25- (Fig. 1B). In addition, this analysis confirmed that CD4+GITR+ T cells are mostly CD45RBlow (Fig. 1B). These data indicate that GITR expression is a possible clue of the correlation between CD4+CD25+ (12) and CD4+CD45RBlow (8) regulatory T cells. Finally, we confirmed that approximately one-half of CD4+CD45RBlow T cell fraction was GITR+ T cells, although CD4+CD45RBhigh T cells were mostly GITR- (Fig. 1C).
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To analyze the functional role of CD4+GITR+ or CD4+GITR- subset in vivo, we first tested the regulatory activity of CD4+GITR+ T cells using the well-described CD4+CD45RBhigh T cell SCID-transferred colitis model (26). C.B-17 SCID mice were injected i.p. with one or two subpopulations of sorted CD4+ T cell in PBS: 1) CD4+CD45RBhigh alone (3 x 105 per body, as a positive control); 2) CD4+CD45RBhigh + CD4+CD45RBlow (each 3 x 105 per body, as a negative control); 3) CD4+CD45RBhigh + CD4+GITRhigh (each 3 x 105 per body); or 4) CD4+GITRlow (3 x 105 per body) (Fig. 2A). The results clearly demonstrated that control of intestinal inflammation resided predominantly within CD4+GITR+ fraction, as these cells significantly inhibited the development of wasting disease and colitis (Fig. 2, B–E). Colons from mice reconstituted with a mixture of CD4+CD45RBhigh and CD4+GITR+ cells exhibited no detectable pathological changes, and were indistinguishable from colons of mice reconstituted with a mixture of CD4+CD45RBhigh + CD4+CD45RBlow cells (Fig. 2D). In contrast, mice reconstituted with CD4+CD45RBhigh or CD4+GITR- cells alone did develop wasting disease and severe colitis (Fig. 2, B and D). The clinical and histological scorings also confirmed these results (Fig. 2, C and E). A further quantitative evaluation of CD4+ T cell infiltration was made by isolating LPMCs from the resected bowels. Only a few CD4+ T cells were recovered from the colonic tissue of mice reconstituted with CD4+CD45RBhigh and CD4+GITR+ cells as compared with mice reconstituted with CD4+CD45RBhigh or CD4+GITR- cells alone (Fig. 2F). Furthermore, the number of CD4+ splenocytes from mice reconstituted with CD4+CD45RBhigh and CD4+GITR+ cells was significantly less than that from mice reconstituted with CD4+CD45RBhigh alone (Fig. 2F). Interestingly, CD4+GITR--transferred mice developed more severe wasting disease as compared with mice transferred with CD4+CD45RBhigh T cells (Fig. 2B), albeit histological scores of these mice were similar (Fig. 2E). Consistent with this, CD4+ T cell recovery from spleen of CD4+GITR--transferred mice was significantly higher than that of CD4+CD45RBhigh-transferred mice (Fig. 2F), indicating peripheral expansion of CD4+ T cells of CD4+GITR--transferred mice was more severe as compared with that of CD4+CD45RBhigh-transferred mice.
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Because of the involvement of GITR in regulatory T cell-mediated suppression (25), we next attempted to assess whether anti-GITR mAb (DTA-1) could abrogate the regulatory activity in intestinal inflammation, and induce a wasting disease in animals restored with a mixture of CD4+CD45RBhigh and CD4+CD45RBlow T cell fractions (Fig. 3A). As a positive control, mice restored with CD4+CD45RBhigh T cells alone developed a wasting disease that became evident 3–5 wk postreconstitution (Fig. 3B) and showed clinical and histological evidence of severe chronic colitis (Fig. 3, C–E). As a negative control, mice restored with both CD4+CD45RBhigh and CD4+CD45RBlow T cell populations and administrated with control IgG by i.p. injection (1 mg weekly up to 6 wk) did not develop wasting disease and colitis at all (Fig. 3, B–E). In contrast, administration of anti-GITR mAb (DTA-1) to mice restored with both CD4+CD45RBhigh and CD4+CD45RBlow T cell populations by i.p. injection (1 mg weekly up to 6 wk) induced, by 4 wk after T cell transfer, wasting disease (Fig. 3, B and C) and showed histological evidence of chronic intestinal inflammation (Fig. 3, D and E). In addition, LP CD4+ T cells from mice transferred with both CD4+CD45RBhigh and CD4+CD45RBlow T cell populations with DTA-1 administration produced significantly more IFN- as compared with those from mice transferred with both CD4+CD45RBhigh and CD4+CD45RBlow T cell populations with control IgG administration (data not shown). In contrast, production of IL-4 or IL-10 was not significantly affected (data not shown).
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Having evidence that the CD4+GITR+ T cell population has regulatory activity in vivo, and CD4+GITR+ T cells also exist in CD4+CD25- subpopulation, we sorted strictly CD4+GITR+ T cells into CD4+CD25+GITR+ and CD4+CD25-GITR+ T cell fractions, and tested for their ability to inhibit the colitis induced by transfer of CD4+CD45RBhigh cells (Fig. 4A). Mice restored with the CD4+CD45RBhigh subset alone developed a wasting disease and severe chronic colitis (Fig. 4, B and C). The results clearly demonstrated that regulation of intestinal inflammation resided predominantly within both the CD4+CD25+GITR+ and CD4+CD25-GITR+ fractions, as these cells significantly inhibited the development of wasting disease and colitis (Fig. 4, B–E), although the wasting disease by the cotransfer of CD4+CD25+GITR+ T cells was significantly milder than that by the cotransfer of CD4+CD25-GITR+ T cells, as indexed by the percentage of weight changes (Fig. 4B). However, the clinical and histological scores, the suppression of LP CD4+ T cell infiltration, and IFN- production by LP CD4+ T cells from mice cotransferred with CD4+CD25-GITR+ were apt to be milder, but not significant when compared with mice cotransferred with CD4+CD25+GITR+ T cells (Fig. 4, C–G). This indicates that regulatory T cells are not only present in CD4+CD25-GITR+ T cell population as well as CD4+CD25+GITR+ T cell population in vivo, but also dependent on the expression of GITR rather than that of CD25.
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We finally explored whether CD4+CD25-GITR+ subset retains regulatory activity in vitro. Unfractionated CD4+,CD4+CD25+GITR+, and CD4+CD25+GITR+ T cells were sorted (Fig. 5A). Recently, it has been shown that resting regulatory CD4+CD25+ T cells intracellularly express CTLA-4 (13, 14). Thus, we analyzed intracellular expression of this marker on freshly isolated CD4+CD25+GITR+ and CD4+CD25-GITR+ T cells. As shown in Fig. 5A, CD4+CD25-GITR+ T cells as well as CD4+CD25+GITR+ cells expressed elevated levels of CTLA-4 intracellularly, but this was expressed on only 0.5% of unfractionated CD4+ cells. CD4+CD25-GITR+ subsets were found to be hyporesponsive to stimulation with Con A (5 µg/ml) as compared with unfractionated CD4+ T cells (Fig. 5B), albeit more proliferative than CD4+CD25+GITR+ counterparts, indicating both CD4+CD25+GITR+ and CD4+CD25-GITR+ T cells were anergic. However, the addition of 100 U/ml exogenous IL-2 to the cultures with CD4+CD25+GITR+ T cells, but not CD4+CD25-GITR+ T cells, partially overcame the proliferative defect, suggesting that CD25 is a functional receptor for CD4+CD25+GITR+ regulatory T cells (Fig. 5B). Furthermore, CD4+CD25-GITR+ T cells were able to suppress the proliferation of CD4+ cells when cocultured in vitro, albeit less potent than that of their CD4+CD25+GITR+ counterparts (Fig. 5C), indicating that CD4+CD25-GITR+ T cells were also regulatory. Finally, supernatants after in vitro stimulation with anti-CD3/CD28 mAbs were analyzed by ELISA. As shown in Fig. 5D, unfractionated CD4+ cells produced IFN-, IL-4, IL-2, and IL-10, but not TGF-, whereas CD4+CD25+GITR+ cells and CD4+CD25-GITR+ cells produced less amounts of IFN- and IL-4, but significantly higher levels of IL-10 and TGF- as compared with whole CD4+ T cells. Interestingly, CD4+CD25-GITR+ T cells produced significantly higher level of IL-2 than CD4+CD25+GITR+ cells. These findings demonstrate CD4+CD25-GITR+ T cells as well as CD4+CD25+GITR+ cells are phenotypically and functionally identical with regulatory T cells in vitro, although the data of cytokine production are yet controversial.
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Because Lehmann et al. (22) have recently demonstrated that CD4+CD25- E+ (integrin E7) T cells in periphery display regulatory activity in vivo and in vitro, we then asked whether the expression of GITR has a correlation with that of E. As shown in Fig. 6, most of CD4+CD25-GITR+ T cells were not within E+ fraction, although very small part of these cells expressed E (4.3 ± 0.9% in whole CD4+CD25-GITR+ T cells), indicating the GITR marker for regulatory T cell does not correlate with expression of E. Consistent with the previous report (22), we also found that most of CD4+CD25+GITR+ T cells did not express E (20.9 ± 5.9% in whole CD4+CD25+GITR+ T cells) (Fig. 6). Based on another evidence that NKT cells are CD25- (34), and also possess regulatory function in some autoimmune disease models, such as diabetes model (35, 36), we next asked whether our CD4+CD25-GITR+ cells derived from C57BL/6 mice express NK 1.1, which is one of NKT markers. However, both CD4+CD25+GITR+ and CD4+CD25-GITR+ cells did not express NK 1.1 (data not shown), indicating our cells were not NKT cells. Additionally, we confirmed that CD4+CD25-GITR+ cells were also DX5- (data not shown), indicating our cells were neither NKT nor NK cells. Finally, we asked whether CD4+CD25-GITR+ T cells really express TCR, because it has become clear that TCR+ T cells play a key role in the regulation of mucosal immunity (37). However, all CD4+CD25-GITR+ cells as well as CD4+CD25+GITR+ cells were TCR+, but not TCR+. Taken together with these results, our CD4+CD25-GITR+ regulatory T cells should be a novel and unique subset, although CD4+CD25-E+ cells may overlap a small part of CD4+CD25-GITR+ regulatory T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At present, useful regulatory T cell surface markers for the discrimination between functional subsets of CD4+ cells are CD25 (12), CD45RB (26), E integrin (22), and GITR (25). The majority of the CD4+CD25+ cells were CD45RBlow and also GITR+ (Fig. 1). It is widely accepted that CD4+CD25+ T cells are the major population to contain regulatory T cells in several experimental systems. Hence, the relationship between CD4+CD25+ and CD4+CD45RBlow regulatory T cell subsets is not well established. In addition, there is increasing evidence that regulatory CD4+ T cells exist in the CD25- compartment as well (19, 22, 24). Although Shimizu et al. (25) have shown that GITR is indeed predominantly expressed on CD4+CD25+ spleen cells, as previously described, we found that a part of CD4+CD25- T cells also expressed GITR, whereas most of CD4+CD25- T cells did not (Fig. 1), raising a question of whether CD4+CD25-GITR+ T cells play any useful part in protective immune responses as regulatory T cells. In fact, we demonstrated that CD4+CD25-GITR+ T cells were anergic and regulatory, expressed CTLA-4 intracellularly, and produced both IL-10 and TGF- in vitro (Fig. 5), indicating these cells should be regulatory T cells. As expected, CD4+CD25-GITR+ T cells also inhibited the development of wasting disease and colitis in vivo (Fig. 4). Taken together with the fact that administration of anti-GITR mAb induced chronic colitis in mice restored with both CD4+CD45RBhigh and CD4+CD45RBlow T cells (Fig. 3), the present studies could demonstrate that CD4+CD45RBlow subset of regulatory T cells expresses GITR.
These observations raise another question of the relationship between CD25+ and CD25- regulatory CD4+ T cells and the origin of these cells. At this point, we cannot exclude that the two subsets differ in their development, function, and/or specificity. It is possible that regulatory CD25- T cells are descendants of thymic regulatory CD25+ T cells, and represent an alternative state of the same functional pool of peripheral regulatory T cells. Although further study is needed to address this, CD4+CD25+ T cells might be enriched for regulatory T cells simply because they are activated (effector state), and might become CD25- T cells in the absence of the appropriate stimuli (memory state). If so, GITR might strictly maintain to suppress the regulatory function in the resting conditions, because GITR signaling is shown to abolish regulatory function. Accordingly, GITR might be more reliable surface marker for regulatory T cells because both CD4+CD25- and CD4+CD25+ regulatory T cells retain GITR expression. Furthermore, this hypothesis seems to be favorable for the reason that CD4+CD25-GITR+ regulatory T cells retain less potent regulatory function as compared with CD4+CD25+GITR+ T cells (Fig. 5).
However, it is also well known that the transfer of CD4+CD45RBhigh T cells into syngeneic athymic nude mice does not induce the development of wasting disease and colitis (38), whereas the transfer into syngeneic SCID mice does (26). This contrasting evidence strongly suggests that there exists some regulatory T cell compartment in recipient athymic nude mice to protect the development of colitis. Indeed, several lines of evidence suggest that some extrathymic TCR T cells also exist in athymic nude mice (39, 40). These cells appear relatively late in ontogeny, randomly increase in numbers as a function of age, and encompass CD4-CD8-, CD4+CD8-, and CD4-CD8+ cells. In fact, one possible pathway of extrathymic selection of CD4+CD25- regulatory T cells has been demonstrated by elegant studies in which mice expressing a transgenic TCR that recognizes an influenza hemagglutinin (HA) peptide were crossed to two lineages expressing the HA peptide under the control of different promoters (21). In this study, expression of HA Ag by thymic epithelial cells produced mostly CD4+CD25+ regulatory T cells. In contrast, expression of HA Ag by nonactivated hemopoietic cells produced mostly CD4+CD25- regulatory T cells even in the absence of a functioning thymus. This indicates that distinct pathways can be exploited to interfere with unwanted immune responses. If so, some CD4+ T cells in athymic nude mice might retain regulatory function. Alternatively, it is also possible that SCID mice are poorly equipped to face DNA damage (DNA double-strand break repair system) induced by inflammation in the intestinal mucosa compared with nude mice (41, 42). Further study will be needed to determine whether the origin of CD4+CD25-GITR+ T cells is thymic or extrathymic.
Studies in various animal models provide strong evidence of a role for cytokines in the effector function of regulatory T cells in vivo; however, the cytokines involved vary depending on the model. In SCID-transferred IBD model, protection from colitis does not require IL-4, but is crucially dependent on TGF- and IL-10 production by T cells (17). Consistent with this, CD4+CD25-GITR+ T cells as well as CD4+CD25+GITR+ T cells produced significant levels of IL-10 and TGF-, but not IL-4. Further investigation will be needed to address whether neutralizing anti-IL-10R and anti-TGF- mAbs abrogate the protective effect of CD4+CD25-GITR+ T cells in vivo.
In a recent publication, Lehmann and colleagues (22) reported that the integrin E7, which recognizes epithelial cadherin, identified the most potent subpopulation of regulatory CD4+CD25+ T cells. In addition, they showed that CD4+CD25-E+ T cells displayed to protect the development of the SCID-transferred colitis, and also possessed regulatory function in vitro. However, it should be emphasized that these cells and our CD4+CD25-GITR+ T cells are distinct subpopulations of each other. Most importantly, CD4+CD25-GITR+ T cells produced fewer amounts of IFN- and IL-4, although CD4+CD25-E+ T cells did produce large amounts of these cytokines. Second, CD25 could be expressed on both E+ and E- T cells, while it was expressed on only GITR+ T cells. Third, the ratio of CD4+CD25-E+ T cells to total of CD4+CD25+E+, CD4+CD25+E-, and CD4+CD25-E+ was quite small, when compared with the ratio of CD4+CD25-GITR+ T cells to total of CD4+CD25+GITR+ and CD4+CD25-GITR+ T cells. Fourth, it is well known that one-third of lamina propria CD4+ T cells from normal mice express E7 integrin (44), while the population of GITR+LP CD4+ T cells is rather less (5–10% in CD4+ T cells; data not shown), similarly to that of splenic CD4+ T cells (Fig. 1). Finally, it has been reported that E-deficient mice developed autoimmune disease in skin (45); however, GITR-deficient mice did not (46), indicating E might be needed to maintain regulatory function, but conversely, GITR may be needed to suppress regulatory function. Taken together, these results support that GITR expresses on CD4+ T cells in independent manner with the expression of E7 integrin.
In conclusion, we showed that CD4+GITR+ T cell population, regardless of CD25 expression, should contain regulatory T cells. The evidence in this study may provide not only a clue of the exact correlation between CD4+CD45RBlow and CD4+CD25+ regulatory T cells, but also the tool to investigate the origin of CD4+CD25+ and CD4+CD25- regulatory T cells. Furthermore, the present study suggests that GITR could be beneficial as a specific marker for regulatory T cells controlling mucosal inflammation and as a target for treatment of IBD.
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Acknowledgments |
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Footnotes |
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2 K.U. and T.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Takanori Kanai, Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: taka.gast{at}tmd.ac.jp
4 Abbreviations used in this paper: IBD, inflammatory bowel disease; GITR, glucocorticoid-induced TNFR family-related gene; HA, hemagglutinin; HPF, high power field; IEL, intraepithelial cell; LP, lamina propria; LPMC, lamina propria mononuclear cell.
Received for publication February 19, 2003. Accepted for publication May 7, 2003.
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, CD4+CD45RBhigh + CD4+CD45RBlow; 
, CD4+CD45RBhigh + CD4+GITR+; and 
, CD4+GITR-. Statistical analysis was performed to compare the slopes of the weight change between the groups of mice that received CD4+CD45RBhigh + CD4+CD45RBlow or CD4+CD45RBhigh + CD4+GITR+ T cells with mice that received CD4+CD45RBhigh T cells. *, p < 0.05. C, CD4+GITR+ T cells inhibited a chronic colitis. Clinical scores were determined at 4 wk after transfer, as described in Materials and Methods. Data are indicated as the mean ± SEM of seven mice in each group. *, p < 0.05. D, Histopathogical comparison of distal colon from mice injected with CD4+CD45RBhigh alone, CD4+CD45RBhigh + CD4+CD45RBlow, CD4+CD45RBhigh + CD4+GITR+, or CD4+GITR-. Original magnification, x100. E, Histological scores were determined at 4 wk after transfer, as described in Materials and Methods. Data are indicated as the mean ± SEM of seven mice in each group. *, p < 0.05. F, LP cells and splenocytes were isolated at 4 wk after T cell transfer, and the number of CD4+ cells was determined by flow cytometry. Data are indicated as the mean ± SEM of seven mice in each group. *, p < 0.05.
