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MUCOSAL BIOLOGY

Naturally arising CD4⁺CD25⁺ regulatory T cells suppress the expansion of colitogenic CD4⁺CD44^highCD62L^– effector memory T cells

Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Submitted 12 September 2005 ; accepted in final form 15 December 2005

	ABSTRACT

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Naturally arising CD4⁺CD25⁺ regulatory T (T_R) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4⁺ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (T_EM) CD4⁺ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4⁺CD44^highCD62L^– lamina propria (LP) T cells obtained from colitic CD4⁺CD45RB^high T cell-transferred mice, we have shown in the present study that CD4⁺CD25⁺ T_R cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4⁺ T_EM cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4⁺CD25⁺ T_R cells proliferated in the presence of IL-2-producing colitogenic LP CD4⁺ T_EM cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4⁺CD25⁺ T_R cells and the their suppressive activity on colitogenic LP CD4⁺ T_EM cells.

murine colitis model; interleukin-2

CD4⁺CD25⁺ T cells have been shown to be potent T_R cells in a number of murine models as well as in rats and humans (12). Functional analysis of murine CD4⁺CD25⁺ T_R cells has shown that those cells, which constitutively express inhibitory cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), glucocorticoid-induced TNF receptor (GITR), and Foxp3 transcription factor (Forkhead box Foxp3 transcriptional isoform) (10, 12, 17), failed to proliferate or secret cytokines in response to polyclonal or antigen-specific stimulation in the in vitro system (13, 18). CD4⁺CD25⁺ T_R cells suppress the proliferation of responder CD4⁺CD25^– T cells in a cell contact-dependent manner (13, 18). However, their effect in vivo appears to depend in some but not all systems on IL-10 and/or TGF- expression (1, 14). An advantage of these T_R cells is their ability to act as bystander suppressors and dampen the inflammation (19), which was recently followed by the demonstration that naturally arising CD4⁺CD25⁺ T_R not only prevented the development of colitis induced by the adoptive transfer of CD4⁺CD45RB^high naive T cells into severe combined immunodeficient (SCID) mice (10) but also cured the established colitis (6, 8). Although these studies suggest that manipulation of CD4⁺CD25⁺ T_R cells may be beneficial in the treatment of patients with IBD, it is still unclear whether the late administration of CD4⁺CD25⁺ T_R cells (10 days–4.5 wk after transfer of CD4⁺CD45RB^high cells) suppresses the pathogenic effector memory T (T_EM) cells or the residual CD4⁺CD45RB^high naive T cells in the gastrointestinal system.

In this study, we have attempted to clarify the exact nature of the suppressive activity of CD4⁺CD25⁺ T_R cells against pathogenic/colitogenic memory CD4⁺ T cells using the colitogenic lamina propria (LP) CD4⁺ effector T_EM-mediated colitis model induced by adoptive transfer of the colitogenic CD4⁺CD62L^–CD44^high T cells obtained from established colitic CD4⁺CD45RB^high T-cell-transferred mice.

	MATERIALS AND METHODS

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Mice. Female BALB/c, C.B-17 scid/scid (SCID), C57BL/6-Ly5.2, and C57/BL6 recombinase-activating gene (RAG)-2-knockout mice (RAG-2-KO; Ly5.2) were purchased from Japan Clear (Tokyo, Japan). C57BL/6-Ly5.1 mice were obtained from The Jackson laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Tokyo Medical and Dental University. Donors and recipients were used at 6–12 wk of age according to the guidelines of the Institutional Committee on Animal Research in Tokyo Medical and Dental University and were approved by the committee.

Antibodies. The following MAbs were obtained from BD Pharmingen (San Diego, CA) and used for purification of cell populations and flow cytometric analysis: Fc- (CD16/CD32)-blocking MAb (2.4G2); FITC-, phenylephrine (PE)-, and CyChrome-conjugated anti-mouse CD4 (RM4-5); FITC- and PE-conjugated anti-mouse CD25 (7D4); FITC-conjugated anti-mouse CD45RB (16A); FITC-conjugated anti-mouse CD44 (IM7); FITC-conjugated anti-mouse CD62L (MEL-14); purified anti-murine CD3e (145-2C11); purified anti-murine CD28 (37.51); purified anti-murine IL-2 (JES6-5H4); PE-conjugated streptavidin; biotin-conjugated rat IgG2; PE-conjugated mouse IgG; and PE-conjugated rat IgG.

Induction of colitis induced by the adoptive transfer of CD4⁺CD45RB^high T cells. Colitis was induced in SCID mice by adoptive transfer of CD4⁺CD45RB^high T cells as described previously (9). Briefly, CD4⁺ T cells were isolated from splenocytes from BALB/c mice using the anti-CD4 (L3T4) MACS magnetic separation system (Miltenyi Biotec, Auburn, CA). Enriched CD4⁺ T cells were labeled with PE-conjugated anti-mouse CD4 MAb and FITC-conjugated anti-CD45RB MAb and sorted into CD45RB^high (highest staining 30%) and CD45RB^low (lowest staining 30%) fractions on a FACSVantage (Becton Dickinson, Sunnyvale, CA). Each SCID mouse underwent intraperitoneal injection with 3 x 10⁵ CD4⁺CD45RB^high T cells. The colitic CD4⁺CD45RB^high T-cell-transferred SCID mice were killed 6–8 wk after transfer to isolate the colitogenic LP CD4⁺ T_EM cells (20). The entire length of colon was opened longitudinally, washed with PBS, and cut into small pieces. The dissected mucosa was incubated with Ca²⁺- and Mg²⁺-free HBSS containing 1 mM DTT (Sigma-Aldrich, St. Louis, MO) for 45 min to remove mucus and then treated with 2.0 mg/ml collagenase (Worthington Biomedical, Freehold, NJ) and 0.01% DNase (Worthington Biochemical) for 2 h. The cells were pelleted twice through a 40% isotonic Percoll gradient solution and then subjected to Ficoll-Hypaque density gradient centrifugation (40%-75%). Enriched CD4⁺ LP T cells were obtained by positive selection using anti-CD4 (L3T4) MACS magnetic beads. The resultant cells analyzed using FACSCalibur contained >96% CD4⁺ cells.

Flow cytometry. Flow cytometric two-color analysis was performed as described previously (4). Isolated LP mononuclear cells (LPMCs) from colitic CD4⁺CD45RB^high cell-transferred SCID mice or age-matched normal BALB/c mice were preincubated with Fc- receptor-blocking MAb for 20 min, followed by incubation with CyChrome-conjugated anti-mouse CD4 MAb and PE-conjugated anti-CD45RB, anti-CD44, anti-CD62L, or anti-CD69 MAb for 30 min on ice. After cells were stained, flow cytometry and data analysis were performed using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA).

In vitro proliferation assay. As antigen-presenting cells (APCs), CD4^– cells were prepared from BALB/c splenocytes by depleting CD4⁺ cells with anti-CD4 MACS beads and treated with 50 µg/ml mitomycin C (MMC) for 45 min at 37°C. To obtain splenic CD4⁺CD25⁺ T cells, enriched CD4⁺ splenocytes were stained with PE-conjugated anti-mouse CD25 MAb and FITC-conjugated anti-CD4 MAb and sorted into CD4⁺CD25⁺ and CD4⁺CD25^– subpopulations on a FACSVantage. In coculture experiments, splenic CD4⁺CD25⁺ or CD4⁺CD25^– T cells (0, 1.0, 2.0, or 4.0 x 10⁴ as T_R or control non-T_R cells) were cultured with splenic CD4⁺CD25^– T cells (1 x 10⁴ as control responders) or colitogenic memory CD4⁺ T cells (1 x 10⁴ as responders) and MMC-treated CD4^– cells (1 x 10⁵ as APCs) in round-bottomed 96-well plates in RPMI 1640 medium containing 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol (Complete medium) supplemented with soluble anti-CD3 MAb (145-2C11, 50 ng/ml colitogenic memory LP CD4⁺ cells, 5 µg/ml CD4⁺CD25^– cells; BD Pharmingen). In some experiments, blocking MAb against IL-2 was added at 0, 2, or 10 µg/ml from the beginning of culture. To determine proliferation, each well was pulsed with 1.0 µCi of [³H]thymidine (NEN, Boston, MA) for the last 9 h of 48- or 96-h culture. For cytokine assay, the supernatants, which were removed before addition of [³H]thymidine at proliferation assays, were collected and analyzed by a specific ELISA (R&D Systems, Minneapolis, MN). In some experiments, cells were labeled with 1.0 ml of PBS-1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) for 10 min at 37°C, followed by addition of 1.0 ml of FCS for 2 min, and then were washed three times in PBS. CFSE-labeled Ly5.1⁺CD4⁺ responder cells (1 x 10⁵) were cocultured with CFSE-labeled Ly5.2⁺CD4⁺CD25⁺ T_R cells (1 x 10⁵) with APC (5 x 10⁵) and anti-CD3 (50 ng/ml for LP CD4⁺ responders) in 96-well round-bottomed plates for 120 h in triplicate experiments. After incubation, cells were collected, stained for Ly5.1 or Ly5.2, and analyzed using FACS. Propidium iodide was added to exclude dead cells. Proliferation analysis was based on the division times of CFSE⁺CD4⁺ T cells.

Induction of colitis induced by adoptive transfer of colitogenic LP CD4⁺CD44^highCD62L^– T_EM cells. SCID mice underwent intraperitoneal injection with sorted CD4⁺ T cell subpopulations in PBS. These mice were administered 4 x 10⁵ colitogenic LP CD4⁺CD44^highCD62L^– T_EM cells obtained from inflamed mucosa of CD4⁺CD45RB^high cell-transferred SCID mice alone or in combination with splenic 4 x 10⁵ CD4⁺CD25⁺ T_R cells. As controls, mice were administered 4 x 10⁵ splenic or mesenteric lymph node CD4⁺CD44^highCD62L^– T cells obtained from CD4⁺CD45RB^high cell-transferred SCID mice. After this cell transfer was performed, the recipient SCID mice were weighed initially and then three times per week afterward. They also were observed for clinical signs such as hunched posture, piloerection, diarrhea, and blood in the stool. Mice were killed 4 wk after T cell transfer and assessed for clinical scores (20) representing 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; and 3, extensive thickening); and stool consistency, 0–3 (0, normal beaded stool; 1, soft stool; 2, diarrhea; and 3, bloody stool).

Tissue samples were fixed in PBS containing 6% neutral buffered formalin. Paraffin-embedded sections (5 µm thick) were stained with hematoxylin and eosin. 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 most affected area was graded according to 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 (4). After lipoprotein lipases (LPLs) and splenocytes were isolated using the above-mentioned method, these cells were stained with FITC-conjugated anti-CD3 and PE-conjugated anti-CD4⁺ MAbs and the percentage of CD3⁺CD4⁺ T cells were analyzed using FACSCalibur, followed by calculation of the total number of CD4⁺ T-cells.

To measure cytokine production, 1 x 10⁵ freshly isolated LP CD4⁺ T cells were cultured in 200 µl of culture medium at 37°C in a humidified atmosphere containing 5% CO₂ in 96-well plates precoated with 5 µg/ml anti-mouse CD3 MAb (145-2C11) and 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 performing specific ELISA (IL-10) (R&D Systems, Minneapolis, MN) or mouse a Th1/Th2 cytokine CBA kit (IL-2, IL-4, IL-5, TNF-, and IFN-; BD Biosciences, San Jose, CA) according to the manufacturer's recommendations.

Statistical analysis. The results are expressed as means ± SD. Groups of data were compared using the Mann-Whitney U-test. Differences were considered statistically significant at P < 0.05.

	RESULTS

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Interaction between CD4⁺CD25⁺ T_R cells and colitogenic T_EM cells. Efforts to delineate T_R cell population have revealed that CD4⁺CD25⁺ T cell populations in mice and humans retain T_R function (12). However, the mechanism by which naturally arising CD4⁺CD25⁺ T_R cells control pathogenic CD4⁺ T_EM cells in autoimmune diseases is not fully understood. To clarify this mechanism, we assessed the in vivo T_R activity of CD4⁺CD25⁺ T_R cells from normal BALB/c spleen against the isolated colitogenic memory CD4⁺ T_EM cells. To study CD4⁺ T_EM cells, we first isolated LP CD4⁺ T cells of Th1-mediated colitic C.B-17 SCID mice by inducing the adoptive transfer of BALB/c splenic CD4⁺CD45RB^high T cells (9). As shown in Fig. 1, flow cytometric analysis revealed that the colitic LP CD4⁺ T_EM cells were CD44^highCD69⁺CD62L^–, indicating that they were activated T_EM cells. In contrast, normal splenic CD4⁺ T cells express CD44^high–lowCD69^–CD62L^+/–.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Scatterplots showing the phenotypic characterization of normal splenic CD4+ T cells and colitic lamina propria (LP) CD4+ T cells induced by adoptive transfer of normal splenic CD4+CD45RBhigh T cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Different timing of the suppressive activity of CD4+CD25+ regulatory T (TR) cells plotted against colitogenic memory LP CD4+ colitogenic effector memory (TEM) cells. CD4+CD25+ cells suppressed the proliferation of LP CD4+ TEM cells in 96-h coculture assay (B) but not in 48-h coculture assay (A), whereas CD4+CD25+ TR cells suppressed the proliferation of normal splenic CD4+CD25– responders in both 48-h (A) and 96-h (B) coculture assays. Colitogenic LP CD4+ TEM cells or CD4+CD25– responders were incubated with soluble anti-CD3 MAb (50 ng/ml concentration for colitogenic LP CD4+ TEM cells and 5 µg/ml concentration for normal splenic CD4+CD25– T cells) in the presence of antigen-presenting cells (APCs) and the indicated ratio of CD4+CD25+ TR cells. Results are expressed as means ± SD of triplicate cultures. *P < 0.05.

View larger version (13K): [in this window] [in a new window]   Fig. 3. CD4+CD25+ TR cells extensively proliferated in coculture with colitogenic LP CD4+ TEM cells. A: 1 x 105 total normal splenic CD4+CD25– or colitogenic LP CD4+ TEM cells (Ly5.2) and 1 x 105 CD4+CD25+ TR cells (Ly5.1) were cultured alone or were cocultured with 5 x 105 APCs in presence of anti-CD3 MAb. Proliferation of the CD4+ cells was measured by dilution with carboxyfluorescein diacetate succinimidyl ester (CFSE) 96 h after culture. B: colitogenic LP CD4+ TEM cells produced a large amount of IL-2, whereas the counterpart CD4+CD25+ TR cells did not. SP, spleen. C: effects of blocking MAb against IL-2 on colitogenic LP CD4+ TEM cell-mediated proliferation of CD4+CD25+ TR cells. An equal number (1 x 104) of colitogenic LP CD4+ TEM cells and normal CD4+CD25+ TR cells were cocultured and stimulated with anti-CD3 (50 ng/ml) and 5 x 104 APCs in the presence or absence of indicated MAbs. *P < 0.05.

CD4⁺CD25⁺ T cells inhibit development of T_EM cell-mediated colitis. To investigate the suppressor activity of splenic CD4⁺CD25⁺ T_R cells in a T_EM-cell-mediated chronic colitis model, we first transferred CD4⁺ T_EM cells obtained from various tissues of colitic CD4⁺CD45RB^high T cell-transferred SCID mice into new SCID mice (Fig. 4A). SCID mice that underwent transfer of colitic spleen (SP), mesenteric lymph node (MLN), or LP CD4⁺ T cells developed severe wasting diseases (Fig. 4, B and C) 5 wk after transfer, in contrast to normal SCID mice that were administered PBS. Histological examination showed prominent epithelial hyperplasia with glandular elongation and a massive infiltration of mononuclear cells in the LP of the colon in SCID mice that had undergone transfer with colitic SP, MLN, or LP CD4⁺ T cells, but not in SCID mice that did not undergo cell transfer (Fig. 4D). This difference was confirmed by histological scoring of multiple colon sections (Fig. 4E). A further quantitative evaluation of CD4⁺ T cell infiltration was performed by isolating LPL from resected bowels. Only a few CD4⁺ T cells were recovered from colonic tissue of SCID mice that had not undergone transfer compared with SCID mice that had undergone transfer with colitic SP, MLN, or LP CD4⁺ T cells (Fig. 4F).

View larger version (35K): [in this window] [in a new window]   Fig. 4. Severe combined immunodeficient (SCID) mice that underwent transfer of SP, mesenteric lymph node (MLN), and LP CD4+ T cells obtained from mice that had undergone classic CD4+CD45RBhigh colitic cell transfer developed colitis. A: C.B-17 SCID mice underwent intraperitoneal injection with normal splenic CD4+CD45RBhigh T cells. Six weeks after transfer, CD4+ T cells were isolated from SP, MLN, and LP and were injected into new SCID mice (1.5 x 105 cells/mouse). As a negative control, PBS without cells was injected into SCID mice. n = 5 mice in each group. All mice were killed 5 wk after undergoing transfer. B: mice that underwent transfer with the colitic SP, MLN, and LP CD4+ T cells did not gain weight. *P < 0.05. C: mice that underwent transfer with colitic SP, MLN, and LP CD4+ T cells showed signs of severe clinical colitis. *P < 0.05. Data are means ± SD of 5 mice in each group. *P < 0.05. D: histopathogical comparison of distal colon from the mice injected with the colitic SP, MLN, or LP CD4+ T cells, or PBS. Original magnification, x10. E: histograms were obtained to calculate histological scores determined 5 wk after mice underwent transfer as described in MATERIALS AND METHODS. Data are means ± SD from 4 mice in each group. *P < 0.05. F: LP CD4+ T cells were isolated from SCID mice that underwent injection with colitic SP, MLN, or LP CD4+ T cells or with PBS 5 wk after transfer, and the number of LP CD4+ cells was determined using flow cytometry. Data are means ± SD of 4 mice in each group. *P < 0.05.

View larger version (33K): [in this window] [in a new window]   Fig. 5. CD4+CD25+ TR cells inhibited development of TEM cell-mediated colitis induced by adoptive transfer of colitogenic memory LP CD4+ T cells into SCID mice as well as that induced by CD4+CD45RBhigh T cells. A: schematic of transfer protocols. Seven SCID mice in each group underwent intraperitoneal injection with the following T-cell subpopulations: 1) colitogenic LP TEM CD4+ cells alone (4 x 105 cells) or 2) colitogenic LP TEM CD4+ (4 x 105 cells) + CD4+CD25+ cells (4 x 105 cells). B: CD4+CD25+ TR cells inhibited a wasting disease. SCID mice administered CD4+CD25+ TR cells were weighed on the day of T-cell transfer and 3 times/wk thereafter. Data regarding colitogenic LP TEM CD4+ cells (–/–) and colitogenic LP TEM CD4+ + CD4+CD25+ TR cells () are shown. Statistical analysis was performed to compare slopes of weight changes between groups for colitogenic LP TEM CD4+ cells vs. colitogenic LP TEM CD4+ + CD4+CD25+ TR cells. *P < 0.05. C: gross appearance of colon from SCID mice transferred with colitogenic LP CD4+ TEM cells (top lane) and colitogenic LP CD4+ TEM + CD4+CD25+ TR cells (bottom lane). D: histopathological image showing distal colon 4 wk after transfer. Original magnification, x100. E: histological score. *P < 0.05. F: number of LP CD4+ T cells. Data are means ± SD of 7 mice in each group. *P < 0.05. G: cytokine production by LP CD4+ T cells. Isolated LP CD4+ T cells were stimulated with plate-coated anti-CD3 MAb and soluble anti-CD28 MAb for 72 h. Cytokines in supernatants were measured using ELISA. Data are means ± SD of 7 mice in each group. *P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In the present study, we have demonstrated that naturally arising CD4⁺CD25⁺ T_R cells suppressed the development of colitogenic CD4⁺ T_EM cell-mediated chronic colitis. Furthermore, in vitro suppression assay revealed that CD4⁺CD25⁺ T_R cells proliferated extensively in response to a high dose of IL-2 produced by colitogenic LP CD4⁺ counterpart T_EM cells at the early time point (48 h after coculture) but suppressed the proliferation of colitogenic memory CD4⁺ T_EM cells at the late time point (96 h) in accordance with the decreased production of IL-2. These results may indicate the exact strategy of CD4⁺CD25⁺ T_R cells against CD4⁺ T_EM cells in autoimmune disorders.

IL-2 is required for in vivo and in vitro activation of CD4⁺CD25⁺ T_R cells and to sustain their CD25 expression (3). Because CD4⁺CD25⁺ T_R cells do not produce IL-2 (12, 18) (Fig. 3B), their proliferation seems to depend on IL-2 produced by their target T cells or activated dendritic cells. Until recently, CD4⁺CD25⁺ T_R cells were described as refractory to stimulation through the TCR and as nonproliferative in in vitro culture (13). In addition, under circumstances in which T_R cells were made to proliferate in vitro by the addition of exogenous IL-2, they lost their suppressive capacity (12). How is IL-2 involved in suppressing IL-2-producing responder T cells by CD4⁺CD25⁺ T_R cells that cannot produce IL-2 in the intestine under inflammatory conditions?

At the early time point (48 h after in vitro coculture), CD4⁺CD25⁺ T_R cells proliferated vigorously in response to a high amount of IL-2 produced by the colitogenic LP CD4⁺ T_EM cells. Indeed, anergy-breaking CD4⁺CD25⁺ T_R cells cannot suppress colitogenic LP CD4⁺ T_EM cells directly by intercellular interaction at this stage; however, CD4⁺CD25⁺ T_R cells may compete for IL-2 with LP CD4⁺ T_EM cells. The constitutive expression of all three chains of the high-affinity IL-2 receptor might be enable CD4⁺CD25⁺ T_R cells to take up IL-2 preferentially rather than LP CD4⁺ T_EM cells, which rarely can express high level of IL-2R- (CD25). Consistent with this hypothesis, it has been demonstrated in vitro and in vivo that competition for and/or consumption of IL-2 indeed occurs (5). During coculture of CD4⁺CD25⁺ T_R cells with responder cells, CD4⁺CD25⁺ T_R cells upregulate CD25, whereas induction of CD25 expression in responder T cells is conversely suppressed (5). Furthermore, upregulation of CD25 on CD4⁺CD25⁺ T_R cells is inhibited by the addition of anti-IL-2 antibody, whereas the addition of IL-2 conversely restores CD25 expression in responder cells, demonstrating that the differential expression of CD25 is regulated by IL-2 (2). Thus competition for and/or consumption of IL-2 by CD4⁺CD25⁺ T_R cells may be boosted by the positive feedback loop of IL-2 uptake and CD25 upregulation. The same inverse regulation of CD25 expression on CD4⁺CD25⁺ T_R cells vs. responder T cells has been observed in vivo upon adoptive cotransfer of T_R cells and responder cells (5). Although CD4⁺CD25⁺ T_R cells do not suppress the proliferation of the LP CD4⁺ T_EM cells directly, IL-2 consumption by CD4⁺CD25⁺ T_R cells might lead to an increased ratio of T_R to responder cells and inactivation of LP CD4⁺ T_EM cells because of the lack of IL-2 followed by decreased IL-2 production by LP CD4⁺ T_EM cells.

In contrast, CD4⁺CD25⁺ T_R cells might gain original suppressive activity to responder T cells in accordance with the decreased IL-2 concentrations at the late stage. In fact, [³H]thymidine uptake was significantly suppressed in a culture with both CD4⁺CD25⁺ T_R cells and colitogenic LP CD4⁺ T_EM cells at 96 h but not at 48 h after culture. This suppression might depend on intercellular contact but would also require IL-2 competition and/or consumption. Altogether, we hypothesize that the suppression of IL-2-producing LP CD4⁺ T_EM cells by CD4⁺CD25⁺ T_R cells occurs via the following two steps: 1) IL-2 consumption followed by the expansion of CD4⁺CD25⁺ T_R cells and the adjustment of the T_R-to-responder ratio and 2) regained suppressive activity via intercellular contact.

Most recently, two groups of researchers have demonstrated that naturally arising CD4⁺CD25⁺ T_R cells cured the established colitis induced by the adoptive transfer of CD4⁺CD45RB^high T cells (6, 8). Although these reports suggest that manipulation of CD4⁺CD25⁺ T_R cells may be beneficial in view of clinical checkpoints for the treatment of IBDs, whether delayed administration of CD4⁺CD25⁺ T_R cells suppress pathogenic CD4⁺ T_EM cells or residual CD4⁺CD45RB^high naive T cells in their systems because of relatively early administration of CD4⁺CD25⁺ T cells (10 days–4.5 wk after transfer of CD4⁺CD45RB^high cells) is still unclear. Importantly, using T_EM-cell adoptive transfer experiments in the present study, we have demonstrated that CD4⁺CD25⁺ T_R cells suppress the expansion of pathogenic CD4⁺ T_EM cells as well as the development of chronic colitis without having any impact on naive T cells.

However, the question arises where CD4⁺CD25⁺ T_R cells suppress the pathogenic CD4⁺ T_EM cells in vivo. Because approximately two-thirds of CD4⁺CD25⁺ T_R cells express CD62L (12), which is essential for homing to lymph nodes, these CD4⁺CD25⁺ T_R cells appear to function in lymph nodes, mesenteric lymph nodes in this case, to suppress the activation and proliferation of naive T cells. However, it is also known not that one-third of CD4⁺CD25⁺ T_R cells obtained from normal spleen do not express CD62L (19) but also that CD4⁺CD25⁺ T_R cells can express integrin ₄₇ (16), which is a homing receptor for gut. Mottet et al. (8) recently demonstrated that CD4⁺CD25⁺ T_R cells acted in the intestine to regulate the effector function or the expansion of pathogenic T_EM cells as well as acting in the secondary lymph nodes in a murine colitis model induced by the adoptive transfer of normal CD4⁺CD45RB^high T cells into SCID mice. In fact, our group (7) has identified CD4⁺CD25^bright T cells with a regulatory phenotype in human colon obtained from normal individuals and from patients with IBD. Furthermore, LP CD4⁺ T_EM cells have a characteristic of the T_EM phenotype and tend to recirculate through nonlymphoid tissues (11). Collectively, it is possible that CD4⁺CD25⁺ T_R cells act in the gut in this setting. However, we have demonstrated that the transfer of colitic SP and MLN CD4⁺ T cells obtained from mice that underwent CD4⁺CD45RB^high T cell transfer into new SCID mice induced colitis as well as colitic LP CD4⁺ T cells. The evidence suggests that the SP and the MLN may play an important role as a reservoir for colitogenic CD4⁺ T_EM cells that can recirculate into the gut. Thus it might be possible that CD4⁺CD25⁺ T_R cells prevent the recruitment of recirculating colitogenic CD4⁺ T_EM cells in the SP and the MLN. Further studies are needed to assess this issue using splenectomized lymph node-null mice to exclude the role of lymph nodes.

In summary, we have demonstrated herein that CD4⁺CD25⁺ T_R cells suppress the expansion of pathogenic CD4⁺ T_EM cells as well as the development of chronic colitis without any impact on naive T cell activation on the basis of T_EM cell adoptive transfer experiments. Although many critical checkpoints remain to be passed, this study indicates that cell therapy using T_R cells as living immunosuppressants offers hope for the treatment of patients with autoimmune diseases such as IBDs.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported in part by grants-in-aid for Scientific Research, Scientific Research on Priority Areas, Exploratory Research and Creative Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor and Welfare; the Japan Medical Association; the Foundation for Advancement of International Science; the Terumo Life Science Foundation; the Ohyama Health Foundation; the Yakult Bio-Science Foundation; and the Research Fund of Mitsukoshi Health and Welfare Foundation.

	ACKNOWLEDGMENTS

 
We express special thanks to Profs. Hiroshi Kiyono and Hiromichi Ishikawa for helpful discussion, Drs. Tatsuji Nomura and Kenichi Tamaoki for providing RAG-2-KO mice, Dr. Masami Moriyama for providing rIL-7, Dr. Tetsuo Sudo for providing anti-IL-7R MAb, and Yuko Ito for manuscript preparation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Kanai, Dept. of Gastroenterology and Hepatology, Tokyo Medical and Dental Univ., 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (e-mail: taka.gast{at}tmd.ac.jp)

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	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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