Naturally anergic and suppressive CD25+CD4+ T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation
Yuhshi Kuniyasu1,2,
Takeshi Takahashi1,3,
Misako Itoh1,
Jun Shimizu1,
Gotaroh Toda2 and
Shimon Sakaguchi1,3
1 Department of Immunopathology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
2 Department of Medicine, Jikei Medical University, Tokyo 305-0006, Japan
3 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
Correspondence to:
S Sakaguchi
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Abstract
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A CD4+ T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4+ T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25+CD4+ T cells are functionally distinct from activated or anergy-induced T cells derived from CD25CD4+ T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25 spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25+CD4+ T cells, do not. Third, upon polyclonal T cell stimulation, CD25+CD4+ T cells express CD25 at higher levels and more persistently than CD25CD4+ T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25+CD4+ T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.
Keywords: anergy, autoimmune disease, immunoregulation, self-tolerance
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Introduction
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There is accumulating evidence that, besides clonal deletion or anergy, T cell-mediated dominant control of self-reactive lymphocytes also contributes to the maintenance of immunologic self-tolerance (13). For example, development of T cell-mediated autoimmune diseases [such as thyroiditis, gastritis and insulin-dependent diabetes mellitus (IDDM)] can be prevented in various animal models by inoculating CD4+ T cells or a subpopulation of CD4+ T cells (such as CD5high, CD45RBlow, CD62Lhigh or CD25+CD4+ T cells) prepared from histocompatible normal animals (417). Furthermore, various autoimmune diseases including thyroiditis, gastritis and IDDM can be de novo produced in normal rodents by simply removing a CD5high, CD45RBlow or CD25+ subpopulation of CD4+ T cells without exogenous immunization with self-antigens, and reconstitution of the removed cell population prevented the development of these autoimmune diseases (411). It remains obscure, however, whether such an immunoregulatory CD4+ T cell population is functionally and phenotypically a single entity of regulatory T cells irrespective of various cell surface phenotypes reported to date for the population(s); whether they are functionally distinct from other immunoregulatory T cells, such as anergy-induced suppressive T cells or regulatory T cells secreting a particular immunoregulatory cytokine (1824); and whether they are functionally and phenotypically stable in the normal immune system if they are engaged in the maintenance of immunologic self-tolerance at all.
We have previously shown that CD25+ T cells, which constitute 510% of CD4+ T cells and <1% of CD8+ T cells in the periphery of normal naive mice, are able to prevent autoimmune disease in vivo and suppress immune responses to non-self antigens in general (911). Interestingly, the CD25+CD4+ T cells are naturally unresponsive (anergic) to TCR stimulation in vitro (2527), if one defines anergy as a reversible anti-proliferative state (25). Furthermore, upon TCR stimulation, they potently suppress the activation/proliferation of other CD4+ T cells and CD8+ T cells presumably through inhibiting IL-2 formation (2527). This in vitro CD25+CD4+ T cell-mediated suppression depends on cellcell interactions on antigen-presenting cells (APC) and does not appear to be mediated by far-reaching or long-lasting humoral factors or apoptosis-inducing signals (25, 27). Similar findings were also made with CD38+CD45RBlowCD4+ T cells (28). Furthermore, the normal thymus is continuously producing CD25+CD4+ T cells as a functionally mature immunoregulatory T cell subpopulation (26).
In this report, we have attempted to further characterize the immunoregulatory CD4+ T cells concerned with the maintenance of immunologic self-tolerance. We show that, compared with other markers (such as CD45RB, CD62L or CD38), the expression of CD25 is highly specific for this immunoregulatory T cell population and that other T cells can hardly acquire the in vivo autoimmune-preventive activity or the in vitro anergic/suppressive property when activated to a CD25+ state or experimentally rendered anergic in vitro. Moreover, the expression of CD25 is highly stable on these CD4+ regulatory T cells, in contrast to transient CD25 expression on activated T cells in general. Our results indicate that the CD25+CD4+ T cell population present in normal naive mice is a functionally and phenotypically unique immunoregulatory population, which plays a key role in maintaining immunologic self-tolerance and controlling immune responses in general.
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Methods
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Mice
Both 8-week-old BALB/c or and 6-week-old BALB/c athymic nude (nu/nu) mice were purchased from SLC (Shizuoka, Japan). To obtain BALB/c nu/+ or +/+ mice, BALB/c nu/+ mice were mated in our animal facility. BALB/c-Thy-1a congenic mice were established in our laboratory as previously described (10). DO11.10 transgenic mice expressing ovalbumin (OVA)-specific transgenic TCR were the kind gift of Dr D. Y. Loh (Hoffman-La Roche (29). All these mice were maintained in our animal facility and cared for in accordance with the institutional guidelines for animal welfare.
Preparation of lymphocytes
To eliminate CD25+ cells, spleen and lymph node cell suspensions (5x107) were incubated in 12x75 mm glass tubes (Corning, Corning, NY) with 100 µl of 1:10-diluted ascites of anti-CD25 antibody (7D4, rat IgM) (30) or anti-CD8 antibody (3.155) (rat IgM) for 45 min on ice, washed once with HBSS (Gibco/BRL, Gaithersburg, MD), incubated with 1.0 ml of non-toxic rabbit serum [as complement (C) source] (Cederlane, Hornby, Ontario, Canada) 1:5-diluted with Medium 199 (Gibco) for 30 min in a 37°C water bath with occasional vigorous shakings, with 100 µg of DNase I (Sigma, St. Louis, MO) added for the last 5 min of the incubation, washed twice with HBSS, and then i.v. injected into 6- to 8-week-old female nude mice (9). To eliminate CD8+ cells or CD4+ cells, cell suspensions (5x107) were treated with anti-CD4 antibody (RL172.4) or anti-CD8 antibody (3.155) as previously described (9).
Serological analysis
For flow cytometric analysis, 1x106 cells were incubated with FITC-labeled or biotinylated mAb, with phycoerythrin (PE)streptavidin (BioMeda, Foster City, CA) as the secondary reagent for biotinylated antibody and analyzed by a flow cytometer (Epics-XL; Coulter, Miami, FL) with exclusion of dead cells by propidium iodide staining. FITC-labeled or biotinylated anti-CD25 antibody (7D4), and biotinylated antibody for CD4 (H129.19), CD8 (53-6.7), CD45RB (16A) (31), CD62L (L-selectin) (Mel-14) (32), CD90.2 (Thy-1.2) (30-H12) and CD90.1 (Thy-1.1) were purchased from PharMingen (San Diego, CA). Biotinylated anti-CD38 antibody was a gift from Dr K. Miyake (Saga Medical School, Saga, Japan) (33).
Cell sorting
Spleen and lymph node cell suspensions or thymocyte suspensions prepared from 8-week-old BALB/c mice were stained with FITC-conjugated anti-CD25 antibody (7D4) and PE-conjugated anti-CD4 antibody (H129.19), and sorted by a FACS (Epics Elite; Coulter), as previously described (25). Purity of the CD25+ and CD25CD4+ populations was >90 and ~99% respectively. In some experiments, CD4+ T cells were first enriched from spleen and lymph node cells by removing B cells, CD8+ T cells and adherent cells by panning on antibody-coated plastic dishes, as previously described (9), and then stained with FITCanti-CD25 antibody along with biotinylated anti-CD62L, anti-CD45RB or anti-CD38 antibody with PEstreptavidin as the secondary reagent. CD25+CD4+ T cells thus enriched were sorted into a CD62Lhigh or CD62L low, CD45RBhigh or CD45RB low, or CD38high or CD45RBlow population (25).
Cell culture
Lymph node and spleen cells (2.02.5x104), sorted as described above, and red blood cell-lysed, X-irradiated (20 Gy)-treated BALB/c spleen cells (5x104) as APC were cultured for 3 days in 96-well round-bottom plates (Costar) in RPMI 1640 medium supplemented with 10% FCS (Gibco/BRL), penicillin (100 U/ml) (Gibco/BRL), streptomycin (100 µg/ml) (Gibco/BRL) and 50 µM 2-mercaptoethanol (Sigma) (25,26). Anti-CD3 antibody (145-2C11) (34) (Cederlane) at a final concentration of 10 µg/ml, Con A at 1.0 µg/ml or OVA peptides (residue 323339) (29) at 0.3 µM, were added to the culture for stimulation (25). Incorporation of [3H]thymidine (1 µCi/well) by proliferating lymphocytes during the last 6 hours of the culture was measured.
Recombinant murine IL-2 (rIL-2) (3.89x106 U/mg) was a gift of Shionogi (Osaka, Japan) (25). To prepare Con A blasts for in vivo transfer, spleen and lymph node cells (5x106/ml) were cultured with 5 µg/ml of Con A for 3 days and washed twice with HBSS.
To render normal spleen cells anergic in vitro, BALB/c spleen cells were cultured with 1 µM ionomycin (IM; Sigma) overnight, as described by Jenkins et al. (35).
Histology and serology
Stomachs and other organs were fixed with 10% formalin and processed for hematoxylin & eosin staining. Serum titers of autoantibodies specific for the gastric parietal cells were assayed by ELISA (36). Gastritis was graded 0 to 2+ depending on macroscopic and histological severity: 0, the gastric mucosa was histologically intact; 1+, gastritis with histologically evident destruction of parietal cells and chief cells with cellular infiltration of the gastric mucosa; 2+, severe destruction of the gastric mucosa accompanying the formation of giant rugae due to compensatory hyperplasia of mucous-secreting cells (4,9).
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Results
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Cell surface phenotype of naturally anergic and suppressive CD25+CD4+ T cells in normal naive mice
We have previously shown that CD25+CD4+ T cells present in normal naive mice contained cells expressing various levels of CD45RB or CD62L, although the expression levels of these molecules might change depending on the environment under which the mice were maintained (26). Others have also shown that autoimmune-preventive CD4+ T cells can be CD38+CD45RBlow (28). To determine then whether the anergic property or the suppressive activity, or both, of the CD25+CD4+ population in normal naive mice can be attributed to a subpopulation included in it, the CD25+CD4+ population was further dissected into smaller populations by expression levels of CD45RB, CD62L or CD38 and each population was assessed in vitro for proliferative response to Con A stimulation or for suppressive activity on the activation/proliferation of co-cultured CD25CD4+ T cells. Figure 1
shows that, when the CD25+CD4+ T cell population was dissected to a CD45RBhigh or CD45RBlow (Fig. 1B
), CD62Lhigh or CD62Llow (Fig. 1C
), or CD38high or CD38low subpopulation (Fig. 1D
) by cell sorting, every subpopulation was virtually non-proliferative to the stimulation and potently suppressed the proliferation of co-cultured CD25CD4+ T cells. The result indicates that the anergic and suppressive property of the CD25+CD4+ T cell population cannot be reduced to a smaller subpopulation defined by an expression level of CD45RB, CD62L or CD38. Furthermore, high proliferative responses of CD25CD4+ T cells (Fig. 1A
) imply that CD38high or CD38low, CD62Lhigh or CD62Llow, or CD45RBhigh or CD45RBlow cells included in the CD25CD4+ T cell population (Fig. 1BD
) may not be sufficiently suppressive, if at all, even if such a subpopulation should contain suppressive T cells. Thus, expression of CD25 is highly specific for naturally anergic and suppressive CD4+ T cells present in normal naive mice.
Intensity and the pattern of CD25 expression are different between CD25+CD4+ T cells in normal naive mice and those activated from CD25CD4+ T cells
Since CD25 is generally expressed on activated T cells (30,37), we examined the intensity and the pattern of CD25 expression on CD25+ cell-depleted or non-depleted splenic CD4+ T cells prepared from normal naive BALB/c mice when the cells were stimulated with Con A (Fig. 2A
). Staining of stimulated (day 3 or 7) or pre-stimulated (day 0) spleen cells with FITCanti-CD4 antibody and PEanti-CD25 antibody showed that the CD25+CD4+ T cells present before stimulation increased their CD25 expression levels by day 3 and retained higher CD25 expression levels than the CD25CD4+ T cell-derived Con A blasts (or CD8+ Con A blasts) even on day 7 when the CD25 expression levels on the CD25CD4+ T cell-derived cells had already declined from the maximum levels (as judged from their mean fluorescence intensity). Furthermore, with a decline of the stimulation, the CD25 expression levels on the CD25+CD4+ T cell-derived blasts reverted to the pre-stimulation levels, whereas the CD25CD4+ T cell-derived blasts virtually lost CD25 expression. This difference in the time course and the intensity of CD25 expression was confirmed by preparing CD25+CD4+ T cells or CD25CD4+ T cells from Thy-1-congenic strains of mice (Fig. 2B
), i.e. CD25+CD4+ T cells prepared from BALB/c-Thy-1a congenic mice (which express CD90.1) and CD25CD4+ T cells from BALB/c mice (which express CD90.2) were mixed at 1:9 ratio as in normal mice [in which ~10% of CD4+ T cells are CD25+ (Fig. 1A
)] and stimulated with Con A (Fig. 2B
). Indeed, the stimulated CD25+CD4+ (CD90.1+) T cells showed higher levels of CD25 expression during the stimulation than the CD25CD4+ (CD90.2+) T cell-derived blasts. Thus, CD25+CD4+ T cells present in normal naive mice are distinct from CD25 T cell-derived activated T cells in intensity, stability and pattern of CD25 expression when stimulated.

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Fig. 2. CD25 expression on CD25 or uneliminated CD4+ T cells upon Con A stimulation. (A) CD25 or uneliminated BALB/c spleen cells were stimulated with Con A and stained with FITCanti-CD4 (ordinate) and PEanti-CD25 (abscissa) on day 3 or 7 of culture. Percentage of cells in each quadrant is also shown. (B) CD25+CD4+ T cells were purified by FACS from BALB-Thy-1a mice as shown in Fig. 1 , mixed with CD25CD4+ T cells similarly prepared from BALB/c mice (which are Thy-1b) at 1:9 ratio and stimulated with Con A. The cells were stained with FITCanti-Thy-1.1 or anti-Thy-1.2 and PEanti-CD25 on day 3 or 7 of culture. A representative result of three independent experiments is shown.
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Con A-activated T cells from normal CD4+ T cells can prevent autoimmune disease but those from TCR transgenic mice or activated CD8+ T cells cannot
CD4+ T cells, especially CD25+CD4+ T cells, in normal naive mice bear the autoimmune-preventive activity (911). To determine whether Con A-activated CD4+ T cells, the majority of which express CD25 as shown in Fig. 2
, can prevent autoimmune disease as well, we transferred to BALB/c athymic nude mice the cell mixtures containing a fixed number of CD25 cells and graded numbers of CD4+ T cell blasts prepared by Con A treatment of normal BALB/c spleen cells. The mice were examined 3 months later for the incidence of histologically evident autoimmune disease in various organs (Table 1
), for the histological severity of gastritis and for the titers of anti-parietal cell autoantibody (Fig. 3
). Con A blasts from CD4+ T cells were able to prevent the development of autoimmune diseases in a dose-dependent fashion, but an equivalent dose of Con A blasts from CD8+ T cells did not, although the preventive activity of the CD4+ blasts was slightly lower when compared with the same number of unstimulated CD4+ splenic T cells. By contrast, Con A blasts from CD25CD4+ T cells showed no autoimmune-preventive activity at all.

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Fig. 3. Development of autoimmune gastritis in nude mice transferred with CD25 spleen T cells and its prevention by co-transfer of normal T cells or Con A blasts. As shown in Table 1 , BALB/c nude mice were transferred with indicated cell suspensions, and histologically and serologically examined 3 months later. *CD4+ Con A blasts prepared from CD25 spleen cells. Solid circles, grade 2 gastritis; shaded circles, grade 1 gastritis; open circles, intact gastric mucosa. See Methods for histological grading of gastritis.
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DO11.10 TCR transgenic mice [in which the majority of CD4+ T cells express TCR specific for an OVA peptide (29)] harbor CD25+CD4+ T cells in the thymus and periphery, although their proportion is slightly smaller (~5% of the CD4+ population) than non-transgenic littermates (~10%) (26). To determine then whether CD4+ T cells activated by a specific antigen can prevent autoimmune disease, we transferred to BALB/c nude mice normal CD25 splenic cells mixed with splenic T cells from DO11.10 transgenic mice and subsequently immunized the nude mice with OVA (Table 2
and Fig. 4
). A group of nude mice received the mixture of CD25 cells and the transgenic T cells that had been activated in vitro with OVA peptides. The inoculated transgenic T cells showed no significant autoimmune-preventive activity in terms of the incidence of various autoimmune diseases (Table 2
), the severity of gastritis and the titers of anti-parietal cell autoantibodies (Fig. 4
), whether the co-transferred transgenic CD4+ T cells were activated in vivo or in vitro. By contrast, CD4+ T cells from non-transgenic littermates efficiently prevented autoimmune disease.

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Fig. 4. Failure of CD4+ T cells from TCR transgenic mice to prevent autoimmune gastritis in co-transfer with CD25 spleen cells. As shown in Table 2 , BALB/c nude mice were transferred with indicated cell suspensions and then immunized with OVA once a week 4 times or non-immunized. A group of nude mice was transferred with CD25 spleen cells mixed with transgenic CD4+ T cells that had been stimulated with OVA in vitro, which were then immunized with OVA as other groups of mice. These nude mice were histologically and serologically examined 3 months later. Solid circles, grade 2 gastritis; shaded circles, grade 1 gastritis; open circles, intact gastric mucosa.
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Thus, the autoimmune-preventive activity is in the CD25+CD4+ population present in normal naive mice and polyclonally activated CD25CD4+ T cells or homogenous antigen-specific CD4+ transgenic T cells activated by a specific antigen bear no significant autoimmune-preventive activity. The results indicate that mere activation of CD4+ or CD8+ T cells to CD25+ states is insufficient for conferring on T cells an autoimmune-preventive activity and that the autoimmune-preventive activity of CD25+CD4+ T cells in normal naive mice is not significantly affected by T cell activation.
IM-induced anergic T cells are unable to exert suppressive activity
CD25+CD4+ T cells present in normal naive mice are anergic to TCR stimulation (Fig. 1
). To determine then whether any anergic T cells are suppressive to other T cells, we rendered CD25CD4+ T cells anergic by treating them in vitro with IM overnight and examined their in vitro suppressive activity on anti-CD3 antibody-stimulated proliferation of freshly prepared CD25CD4+ T cells (Fig. 5A
) and their in vivo autoimmune-suppressive activity (Fig. 6
and Table 3
, experiment G). Such anergy-induced T cells derived from CD25CD4+ T cells failed to suppress the responses of CD25CD4+ T cells in vitro or to prevent the development of autoimmune disease in vivo. Exogenously added IL-2 partially restored the response of IM-treated CD25CD4+ T cells, exerting no significant effects on their suppressive activity (Fig. 5B
). Furthermore, transfer of IM-treated CD25 spleen cells to nude mice produced autoimmune diseases at similar incidences and with similar severities as the transfer of non-treated CD25 spleen cells (Table 3
, experiment D). The results indicate that the anergic state of IM-treated cells may be limited in duration and hardly alter the autoimmune-inducing activity of the CD25CD4+ population.

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Fig. 5. Failure of anergy-broken CD25+CD4+ T cells or anergized CD25CD4+ T cells to suppress the activation/proliferation of other T cells in vitro. (A) CD25 spleen cells prepared as described in the legend to Table 1 were treated with IM for 3 days in vitro and then stimulated with Con A along with fresh APC. Such IM-treated cells or non-treated cells were also mixed with freshly prepared CD25CD4+ T cells at an equal ratio and similarly stimulated. (B) A high dose of rIL-2 (100 U/ml) was also added to the Con A-stimulated culture of freshly prepared CD25+CD4+ T cells, IM-treated or non-treated CD25 cells, or the mixture of these cells and freshly prepared CD25CD4+ T cells. The means of duplicate cultures are shown and the SEMs were all within 10% of the mean. A representative result of two independent experiments is shown.
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Fig. 6. Effects of breaking the anergic and suppressive state of regulatory T cells, or inducing anergy in other T cells, on induction of autoimmune disease. BALB/c spleen cells treated as in Table 3 were transferred to BALB/c nude mice, which were histologically and serologically examined 3 months later. Solid circles, grade 2 gastritis; shaded circles, grade 1 gastritis; open circles, intact gastric mucosa.
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Table 3. Failure of anergy-broken CD25+CD4+ T cells or anergy-induced CD25CD4+ T cells to prevent autoimmune diseasea
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Besides IM treatment, we attempted to induce anergy in purified CD25CD4+ T cells (or T cell lines derived from CD25CD4+ T cells) by culturing them on anti-CD3 antibody-bound plates (38). The anergy thus induced was, however, transient and incomplete when compared with IM treatment, and not significantly suppressive on the activation/proliferation of CD25CD4+ T cells (data not shown).
CD25+ cells activated with Con A alone are suppressive in vivo and in vitro but those activated with Con A and IL-2 are not
In contrast with the treatment of CD25+CD4+ T cells with Con A alone (Fig. 1A
), treatment of CD25+CD4+ T cells with Con A and a high dose of IL-2 broke their anergic state and simultaneously abrogated their suppressive activity in vitro (Fig. 5B
) (25). Furthermore, BALB/c splenic cells similarly treated with Con A and IL-2 not only failed to prevent autoimmune disease in nude mice when co-transferred with CD25 spleen cells (Table 3
, experiment F); they themselves also produced autoimmune disease in nude mice (Table 3
, experiment C, and Fig. 6
) (25). As the controls of these experiments, spleen cells treated with Con A alone elicited few autoimmune diseases (Table 3
, experiment B), whereas Con A-treated CD25 spleen cells efficiently produced various autoimmune diseases at high incidences (data not shown). Thus, treatment of normal spleen cells, especially CD4+ T cells, with a high dose of IL-2 along with TCR stimulation can abrogate not only the in vitro suppressive activity but also the in vivo autoimmune-preventive activity of CD25+CD4+ T cells.
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Discussion
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The immunoregulatory CD4+ T cells concerned with the maintenance of natural immunologic self-tolerance appear to be, at least in part, CD5high, RT6.1+, CD45RBlow, CD38+, CD62L+ or CD25+ (417,2528). Our previous report showed that the immunoregulatory activity of the CD5high or CD45RBlow CD4+ T cell population could be attributed to CD25+ T cells included in it (9,25,26). In addition to this finding, the following results in this report indicate that the expression of CD25 is highly specific, when compared with other molecules, for the immunoregulatory CD4+ T cells present in the periphery of normal naive mice. First, the anergic and suppressive property of the CD25+CD4+ T cell population could not be further reduced to a smaller subpopulation expressing CD45RB, CD62L or CD38 at a high or low level. Second, a high proliferative response of the CD25CD4+ T cell population, which contains T cells expressing CD38, CD62L or CD45RB at various levels (Fig. 1
), indicates that, even if the population should contain any suppressive T cells defined by the expression levels of these cell surface molecules, they may not be sufficiently suppressive. Third, as discussed below, CD25+CD4+ T cells in normal naive mice are continuously and stably expressing CD25 even in the absence of exogenous T cell stimulation, whereas other T cells lose CD25 when the stimulation is abrogated (Fig. 2
).
High expression of CD25 and intermediate to low expression of CD45RB on the immunoregulatory CD25+CD4+ T cells (as shown in Fig. 1
) suggest that they might be in an activated or primed state (3941). The CD25+CD4+ T cells present in normal naive mice are indeed CD5high, CD44high, CD11a/CD18high and CD54high, as observed with activated, primed or memory T cells in general (9,25,26,3941). However, they appear to be distinct from the usual activated T cells for the following reasons. First, the development of autoimmune disease was prevented by Con A blasts from uneliminated CD4+ T cells, but not by Con A-activated CD4 T cell blasts prepared from CD25CD4+ T cells. The latter rather produced severe autoimmune diseases when transferred to athymic nude mice (data not shown). Second, CD25+CD4+ T cells that had differentiated in a few months from the CD25CD4+ T cells inoculated into nude mice hardly suppressed the in vitro activation/proliferation of CD25CD4+ T cells prepared from the same nude mice, as previously shown (26). Third, T cell blasts prepared from TCR-transgenic mice, which are homogeneous in terms of TCR specificity, failed to prevent autoimmune disease even when stimulated in vivo or pre-stimulated in vitro with a specific antigen. The result apparently contrasts with our former finding that DO11.10 transgenic mice harbor CD25+CD4+ T cells, which are anergic and exert potent suppression in vitro upon stimulation with an OVA peptide (25,26). This discrepancy could be attributed in part to a smaller number of OVA-activated CD25+CD4+ regulatory T cells contained in the CD4+ T cell inocula for autoimmune prevention, i.e. the size of the CD25+CD4+ T cell population in DO11.10 was about half that of normal mice. In addition, a sizable number of the transgenic CD25+CD4+ T cells expressed endogenous TCR
chains in association with transgenic TCR ß chains, resulting in altered antigen specificities, hence inefficient activation of CD25+CD4+ regulatory T cells with OVA (26). Alternatively, but not exclusively, the discrepancy could be due to a possible failure of OVA-specific CD25+CD4+ regulatory T cells to be efficiently guided to the APC expressing specific self-antigen peptides, hence failure of controlling self-reactive T cells there (25).
Another important characteristic of the immunoregulatory CD25+CD4+ population is that it is naturally anergic to TCR stimulation (Figs 1 and 5
) (2527). This raises the question whether any T cells rendered anergic can also be suppressive on the activation/proliferation of other T cells (1820). Indeed, recent studies by others have shown that anergic T cells exert suppression not via humoral factors including immunoregulatory cytokines, but through cellcell interactions on APC apparently in a manner similar to naturally anergic and suppressive CD25+CD4+ T cells (19,20). These experimentally induced anergic T cells are also CD25+ in general (4244). Our present experiments, however, showed that when CD25CD4+ T cells were experimentally rendered anergic in vitro by IM treatment, they were ineffective in inhibiting the development of autoimmune disease in vivo and in suppressing the activation/proliferation of other T cells in vitro (Table 3
and Fig. 5
). This difference between our results and those of others (1820) could be attributed, at least in part, to their use of T cell clones, which may be homogenous not only in antigen specificity but also in the state of activation and the pattern of cytokine formation. Alternatively, the difference could be due to different ways of inducing anergy, e.g. our use of IM and their use of specific peptides or plate-bound anti-CD3 antibody (see Results). Furthermore, the following characteristics of CD25+CD4+ naturally anergic/suppressive T cells also make them distinct from experimentally induced anergic and suppressive T cells (1820). First and foremost, the anergic and suppressive state of the former is the basal and default condition for them, e.g. the CD25+CD4+ T cells whose anergic state had been abrogated by TCR stimulation along with a high dose of IL-2 or anti-CD28 antibody ligating the CD28 molecules spontaneously revert to the original anergic/suppressive state upon removal of IL-2 or anti-CD28 antibody (25,26). Indeed, T cell clones and lines that can be prepared from the CD25+CD4+ T cell population in normal naive mice and maintained by intermittent stimulations with anti-CD3 antibody and IL-2 show the anergic/suppressive property when exogenous IL-2 is withdrawn (25,26) (J. Shimizu and S. Sakaguchi, manuscript in preparation). This markedly contrasts with usual anergic T cells, which will never revert to an anergic state spontaneously once their anergic state is abrogated (42). Second, the suppression by the CD25+CD4+ T cells is antigen-nonspecific in the effector phase (i.e. they suppress not only T cells with the same antigen specificity but also those with other specificities as well, as shown in 25), which is similar to linked suppression (45). In addition, they suppress the activation/proliferation of CD4+ T cells as well as CD8+ T cells (25,26). This contrasts with antigen-specific or -restricted suppression by experimentally induced anergy (1820). Third, the normal thymus appears to be continuously producing the immunoregulatory CD25+CD4+ T cells which are already anergic and suppressive in the normal thymus (26). Although T cell clones or lines can be prepared which are anergic and suppressive in vitro, it remains to be determined whether normal T cells can be somehow converted to anergic and suppressive T cells in the periphery when exposed to self-antigens without co-stimulation in vivo (1,1820).
In conclusion, the present results indicate that naturally anergic and suppressive CD25+CD4+ T cells present in normal naive mice are distinct from other activated, anergic or regulatory T cells in phenotypic and functional characteristics. The phenotype of CD25+CD4+ immunoregulatory T cells, on the other hand, suggests that they themselves may be continuously stimulated with self-antigens, hence in a CD25+ activated state, in the normal internal environment and continuously exerting suppressive control of other T cells (including self-reactive T cells) by raising their activation thresholds (25,26). Further characterization of this unique regulatory T cell population is required to elucidate the mechanism of T cell-mediated dominant immunoregulation and thereby to develop ways for effectively treating or preventing autoimmune disease (9), eliciting tumor immunity (46) or inducing tolerance to allo-transplants (9), by manipulating the mechanism.
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Acknowledgments
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We thank Dr D. Y. Loh for the TCR transgenic mice, Dr K. Miyake for anti-CD38 mAb and Ms E. Moriizumi for preparing the histology. This work was supported by grants-in-aid from the Ministry of Human Welfare and the Organization for Pharmaceutical Safety and Research of Japan.
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Abbreviations
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APC antigen-presenting cell |
Con A concanavalin A |
IDDM insulin-dependent diabetes mellitus |
IM ionomycin |
OVA ovalbumin |
PE phycoerythrin |
 |
Notes
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Transmitting editor: K. Okumura
Received 6 March 2000,
accepted 17 April 2000.
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References
|
---|
-
Modigliani, Y., Bandeira, A. and Coutinho, A. 1996. A model of developmentally acquired thymus-dependent tolerance to central and peripheral antigens. Immunol. Rev. 149:155.[ISI][Medline]
-
Fowell, D., McKnight, A. J., Powrie, F., Dyke, R. and Mason, D. 1991. Subsets of CD4+ T cells and their roles in the induction and prevention of autoimmunity. Immunol. Rev. 123:37.[ISI][Medline]
-
Sakaguchi, S. and Sakaguchi, N. 1994. Thymus, T cells and autoimmunity: various causes but a common mechanism of autoimmune disease. In Coutinho, A. and Kazatchkine, M., eds, Autoimmunity: Physiology and Disease, p. 203. Wiley-Liss, New York.
-
Sakaguchi, S., Fukuma, K., Kuribayashi, K. and Masuda, T. 1985. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance: deficit of a T cell subset as a possible cause of autoimmune disease. J. Exp. Med. 161:72.[Abstract]
-
Sugihara, S., Izumi, Y., Yoshioka, T., Yagi, H., Tsujimura, T., Tarutani, O., Kohno, Y., Murakami, S., Hamaoka, T. and Fujiwara, H. 1988. Autoimmune thyroiditis induced in mice depleted of particular T cell subsets. I. Requirement of Lyt-1dull L3T4bright normal T cells for the induction of thyroiditis. J. Immunol. 141:105.[Abstract/Free Full Text]
-
Smith, H., Lou, Y.-H., Lacy, P. and Tung, K. S. K. 1992. Tolerance mechanism in experimental ovarian and gastric autoimmune disease. J. Immunol. 149:2212.[Abstract/Free Full Text]
-
McKeever, U., Mordes, J. P., Greiner, D. L., Appel, M. C., Rozing, J., Handler, E. S. and Rossini, A. A. 1990. Adoptive transfer of autoimmune diabetes and thyroiditis to athymic rats. Proc. Natl Acad. Sci. USA 87:7618.[Abstract]
-
Powrie, F. and Mason, D. 1990. OX-22high CD4+ T cells induce wasting disease with multiple organ pathology: prevention by OX-22low subset. J. Exp. Med. 172:1701.[Abstract]
-
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. Immunologic tolerance maintained by activated T cells expressing IL-2 receptor
-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
-
Asano, M., Toda, M., Sakaguchi, N. and Sakaguchi, S. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184:387.[Abstract]
-
Suri-Payer, E., Amar, A. Z., Thornton, A. M. and Shevach, E. M. 1998. CD4+ CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J. Immunol. 160:1212.[Abstract/Free Full Text]
-
Mordes, J. P., Gallina, D. L., Handler, E. S., Gleiner, D. L., Nakamura, N., Pelletier, A. and Rossini, A. A. 1987. Transfusions enriched for W3/25+ helper/inducer T lymphocytes prevent spontaneous diabetes in the BB/W rats. Diabetologia 30:22.[ISI][Medline]
-
Boitard, C., Yasunami, R., Dardenne, M. and Bach, J. F. 1989. T cell-mediated inhibition of the transfer of autoimmune diabetes in NOD mice. J. Exp. Med. 169:1669.[Abstract]
-
Sakaguchi, N., Miyai, K. and Sakaguchi, S. 1994. Ionizing radiation and autoimmunity: induction of autoimmune disease in mice by high dose fractionated total lymphoid irradiation and its prevention by inoculating normal T cells. J. Immunol. 152:2586.[Abstract/Free Full Text]
-
Fowell, D. and Mason, D. 1993. Evidence that the T cell repertoire of normal rats contains cell with the potential to cause diabetes. Characterization of the CD4+ T cell subset that inhibits this autoimmune potential. J. Exp. Med. 177:627.[Abstract]
-
Seddon, B., Saoudi, A., Nicholson, M. and Mason, D. 1996. CD4+ CD8 thymocytes that express L-selectin protect rats from diabetes upon adoptive transfer. Eur. J. Immunol. 26:2702.[ISI][Medline]
-
Herbelin, A., Gombert, J.-M., Lepault, F., Bach, J. F. and Chatenoud, L. 1998. Mature mainstream TCR
ß+ CD4+ thymocytes expressing L-selectin mediate `active tolerance' in the nonobese diabetic mice. J. Immunol. 161:2620.[Abstract/Free Full Text]
-
Lombardi, G., Sidhu, S., Batchelor, R. and Lechler, R. 1994. Anergic T cells as suppressor cells in vitro. Science 264:1587.[ISI][Medline]
-
Taams, L. S., van Rensen, A. J. M. L., M. Poelen, M. C., van Els, C. A. C. M., Besseling, A. C., Wagenaar, J. P. A., van Eden, W. and Wauben, M. H. M. 1998. Anergic T cells actively suppress T cell responses via the antigen-presenting cell. Eur. J. Immunol. 28:2902.[ISI][Medline]
-
Chai, J.-G., Bartok, I., Chandler, P., Vendetti, S., Antoniou, A., Dyson, J. and Lechler, R. 1999. Anergic T cells act as suppressor cells in vitro and in vivo. Eur. J. Immunol. 29:686.[ISI][Medline]
-
Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A. and Weiner, H. L. 1994. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalitis. Science 265:1237.[ISI][Medline]
-
Rapoport, M. J., Jaramillo, A., Zipris, D., Lazarus, A. H., Serreze, D. V., Leiter, E. H., Cyopick, P., Danska, J. S. and Delovitch, T. L. 1993. Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice. J. Exp. Med. 178:87.[Abstract]
-
Powrie, F., Carlino, J., Leach, M. W., Mauze, S. and Coffman, R. L. 1996. A critical role for transforming growth factor-b but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells. J. Exp. Med. 183:2669.[Abstract]
-
Groux, H., O'Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. and Roncarolo, M. G. 1997. A CD4+ T cell subset inhibits antigen-specific T cell responses and prevents colitis. Nature 389:737.[ISI][Medline]
-
Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. and Sakaguchi, S. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells. Int. Immunol. 10:1969.[Abstract]
-
Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S. 1999. Thymus and autoimmunity: Production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
-
Thornton, A. and Shevach, E. M. 1998. CD4+ CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
-
Read, S., Mauze, S., Asseman, C., Bean, A., Coffman, R. and Powrie, F. 1998. CD38+ CD45RBlow CD4+ T cells: a population of T cells with immune regulatory activities in vitro. Eur. J. Immunol. 28:3435.
-
Murphy, K. M., Heimberger, A. B. and Loh, D. Y. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlo thymocytes in vivo. Science 250:1720.[ISI][Medline]
-
Ortega-R, G., Robb, R. J., Shevach, E. M. and Malek, T. R. 1984. The murine IL-2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells. J. Immunol. 133:1970.[Abstract/Free Full Text]
-
Bottomly, K., Luqman, M., Greenbaum, L., Carding, S., West, J., Pasqualini, T. and Murphy, D. B. 1989. A monoclonal antibody to murine CD45R distinguished CD4 T cell populations that produce different cytokines. Eur. J. Immunol. 19:617.[ISI][Medline]
-
Reichert, R., Gallatin, M., Butcher, E. and Weissman, I. 1984. A homing receptor-bearing cortical thymocyte subset: implication for thymus cell migration and the nature of cortisone-resistant thymocytes. Cell 38:89.[ISI][Medline]
-
Kikuchi, Y., Yasue, T., Miyake, K., Kimoto, M. and Takatsu, K. 1995. CD38 ligation induces tyrosine phosphorylation of Bruton tyrosine kinase and enhanced expression of interleukin 5-receptor alpha chain: synergistic effects with interleukin-5. Proc. Natl Acad. Sci. USA 92:11814.[Abstract]
-
Leo, O., Foo, M., Sachs, D., Samelson, L.E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374.[Abstract]
-
Jenkins, M., Pardoll, D. W., Mizuguchi, J., Chused, T. M. and Schwartz, R. H. 1987. Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones. Proc. Natl Acad. Sci. USA 84:5409.[Abstract]
-
Sakaguchi, S. and Sakaguchi, N. 1989. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. V. Neonatal administration of cyclosporin A causes autoimmune disease. J. Immunol. 142:471.[Abstract/Free Full Text]
-
Uchiyama, T., Broder, S. and Waldmann, T. A. 1981. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac(+) cells. J. Immunol. 126:1393.[Free Full Text]
-
Quill, H. 1996. T cell anergy. In Herezenbeg, L. A., Weir, D. M., Herzenberg, L. A. and Blackwell, eds, Weir's Handbook of Experimental Immunology, p. 116.1, Blackwell Science, Oxford.
-
Vitetta, E. S., Berton, M. T., Burger, C., Kepron, M., Lee, W. T. and Yin, X-M. 1991. Memory B and T cells. Annu. Rev. Immunol. 9:193.[ISI][Medline]
-
Sprent, J. 1994. T and B memory cells. Cell 76:315.[ISI][Medline]
-
Dutton, R. W., Bradley, L. M. and Swain, S. L. 1998. T cell memory. Annu. Rev. Immunol. 16:201.[ISI][Medline]
-
Essery, G., Feldmann, M. and Lamb, J. R. 1988. Interleukin-2 can prevent and reverse antigen-induced unresponsiveness in cloned human T lymphocytes. Immunology 64:413.[ISI][Medline]
-
Jenkins, M. K. and Schwartz, R. H. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165:302.[Abstract]
-
Rammensee, H.-G., Kroschewski, R. and Frangoulis, B. 1989. Clonal anergy induced in mature Vß6+ T lymphocytes on immunizing Mls-1b mice with Mls-1a expressing cells. Nature 339:541.[ISI][Medline]
-
Davies, J. D., Leong, L. Y. W., Mellor, A., Cobbold, S. P. and Waldmann, H. 1996. T cell suppression in transplantation tolerance through linked recognition. J. Immunol. 156:3602.[Abstract]
-
Shimizu, J., Yamazaki, S. and Sakaguchi, S. 1999. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163:5211.[Abstract/Free Full Text]