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The Journal of Immunology, 2003, 170: 1675-1682.
Copyright © 2003 by The American Association of Immunologists

CD4+CD25- T Cells in Aged Mice Are Hyporesponsive and Exhibit Suppressive Activity 1

Jun Shimizu2,* and Eiko Moriizumi{dagger}

* Immunological Aging Research Group, Division of Physiology and Aging, and {dagger} Molecular Gerontology Research Group, Division of Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies on humans and rodents have established that functional deterioration of CD4 T cells occurs with aging. We report in this study that ~70% of CD4+CD25- T cell preparations from individual 24-mo-old mice are hyporesponsive to in vitro stimulation with anti-CD3 Ab. The remaining 30% of CD4+CD25- T cell preparations showing the intermediate or normal responsiveness in the primary stimulation also exhibit the hyporesponsive properties after primary stimulation. Both of these hyporesponsive aged CD4+CD25- T cells could inhibit the proliferation of cocultured CD4+CD25- T cells from young mice, like CD4+CD25+ T cells, which have recently been demonstrated as an immune regulator in young mice. Another experiment revealed that hyporesponsive aged CD4+CD25- T cells arrest the cell division of cocultured young CD4+CD25- T cells. The suppressive activity observed in aged CD4+CD25- T cells is aging-dependent, not mediated by humoral factors, cell-contact dependent, and broken by the addition of IL-2 or anti-GITR Ab, but not by anti-CTLA-4 Ab. These studies show that aging not only leads to a decline in the ability to mount CD4+CD25- T cell responses, but at the same time, also renders these aged CD4+CD25- T cells suppressive.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is widely accepted that peripheral tolerance is maintained by a population of regulatory T cells, which prevent the activation of pathogenic self-reactive T cells (1, 2, 3, 4). Recently, it was demonstrated that one group of those regulatory T cells, CD4+CD25+ T cells, plays an important role in the maintenance of self tolerance in in vivo experiments (5). One of the most interesting properties of these CD4+CD25+ T cells revealed from in vitro analyses is that they are naturally anergic and suppressive for the activation of other CD4+CD25- T cells containing self-reactive T cells (6, 7). It has also been previously reported that anergized T cells can mediate regulatory function on other T cells in vitro and in vivo (8, 9, 10). These data prompted us to investigate the function of CD4 T cells in aged mice, because it is well known that aging leads to a decline in the ability to mount normal CD4 T cell responses (11, 12, 13). That is, we may be able to regard these hyporesponsive CD4 T cells in aged mice as other naturally anergized CD4 T cells. These hyporesponsive CD4 T cells in aged mice may only be functionally deteriorated (14) or may have other functional aspects. In this study, we examined the latter possibility using an in vitro culture system.

In accordance with previous reports, our experiments confirmed that CD4 T cells in aged mice are hyporesponsive against stimulation by anti-CD3 Ab. Interestingly, these hyporesponsive CD4 T cells from aged mice also suppressed the activation of young normal CD4 T cells. One important finding is that these hyporesponsive/suppressive properties of aged CD4 T cells were found on CD4+CD25- T cell fractions. These data show that aging leads not only to hyporesponsiveness of CD4+CD25- T cells but also a gain in the suppressive activity of CD4+CD25- T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male C57BL/6 (B6) mice were purchased from Japan SLC (Shizuoka, Japan). All young and aged mice used in our experiments were maintained in a specific pathogen-free animal facility and treated in accord with the institutional guidelines for animal care. Aged mice with evidence of gross pathology were excluded from the study.

Cell preparation

Whole splenic CD4 T cells were purified using magnetic beads conjugated with anti-CD4 Ab and a magnetic column (Miltenyi Biotec, Bergisch Gladbach, Germany) (>98% purity). In some experiments, splenic cells were incubated at 5 x 107/ml for 45 min at 37°C with the culture supernatant (SN)3 of the hybridoma cells secreting anti-CD25 Ab (7D4), and rabbit complement diluted to a final concentration of 1/10 (Cedarlane Laboratories, Hornby, Ontario, Canada) (15). The treatment was repeated twice, and the resulting cells were then used for the preparation of CD4+CD25- T cells with anti-CD4 magnetic beads as mentioned above. The purity of the final CD4+CD25- T cell preparation from 2- or 24-mo-old mice was typically >98% CD4+ and <0.2% CD25+, or >96% CD4+ and <0.1% CD25+, respectively.

In other experiments, to purify CD4+CD25+ or CD4+CD25- T cells, B6 spleen cells were stained with PE-labeled anti-CD4 (H129.19) and FITC-labeled anti-CD25 (7D4) Ab (all purchased from BD PharMingen, San Diego, CA), and sorted by cell sorter (EPICS ELITE; Beckman Coulter, Fullerton, CA). The purity of sorted CD4+CD25+ and CD4+CD25- T cells was >94% and >98%, respectively.

Flow cytometric analysis

The following Abs and fluorescent reagents were used: biotin-anti-CD44 (IM7), PE-anti-CD45RB (16A), FITC-anti-CD4 (RM4-5) (all purchased from BD PharMingen), and PE-streptavidin (BioMeda, Foster City, CA). For the analysis of CD44 and CD45RB expression, the purified CD4+CD25- T cells were stained with the appropriate Abs and analyzed with an EPICS ELITE.

CFSE labeling

For the labeling of CD4 T cells with CFSE (Molecular Probes, Eugene, OR), the cells were washed with ice-cold PBS. CFSE was then added to a final concentration of 0.6 µM and incubated for 10 min at room temperature, and washed three times. On days 0 and 2 of culture, the cells were harvested and analyzed with an EPICS ELITE.

Proliferation assays

CD4 T cells (1 x 104/well) purified from spleen cells were stimulated with 5% of SN of anti-CD3 Ab (145-2C11), which can induce a maximum proliferation, in the presence of mitomycin C (MMC)-treated spleen cells as APC in 96-well round-bottom plates in DMEM containing 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 µM 2-ME. In some experiments, spleen cells were depleted of CD4+ or CD8+ cells using anti-CD4 (RL172.4), anti-CD8 (3.155) Abs, and complement, treated with MMC, then the resulting cells were used as APC. The proliferation of T cells (triplicate cultures) was determined by measuring the incorporation of [3H]TdR (37 kBq/well) for the final 6 h of the 2- or 3-day culture. In other experiments, anti-IL-4 Ab (11B11), anti-IL-10 Ab (JES5-2A5) (both purchased from BD PharMingen), anti-TGF-{beta} Ab (polyclonal Ab and 1D11; both purchased from R&D Systems, Minneapolis, MN), or murine rIL-2 (donated by Shionogi, Osaka, Japan) were added to the cell culture. Fab of anti-CTLA-4 Ab (UC10-4F10-11) were prepared with the use of immobilized papain (Pierce, Rockford, IL) according to the manufacturer’s instructions. Anti-glucocorticoid-induced TNFR family-related gene (GITR) Ab (DTA-1) (16) was purified from ascites in SCID mice by 50% ammonium sulfate precipitation and protein G columns.

Transwell experiments

Transwell experiments were performed in 24-well plates (Corning Glass, Corning, NY). In the outer well, 10 x 104 young or aged CD4+CD25- T cells with 15 x 104 APC, and in the inner well, 1 x 104 young CD4+CD25- T cells and 4 x 104 aged CD4+CD25- T cells with 2.5 x 104 young APC were cultured along with anti-CD3 Ab. On day 3, the cells in the inner wells were transferred to 96-well plates and pulsed with [3H]TdR for 6 h.

Cytokine assays

Cytokines in culture SNs were measured with commercially available ELISA kits for mouse IL-2, IL-4, IL-10, IFN-{gamma} (BD PharMingen), and TGF-{beta}1 (Promega, Madison, WI) according to the manufacturer’s protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aged CD4 T cells are hyporesponsive and suppress the activation of young CD4 T cells

First, we compared the ability of CD4 T cells from young (2-mo-old) or aged (24-mo-old) B6 mice to proliferate in response to TCR stimulation by anti-CD3 Ab. As shown in Fig. 1A, ~70% of the CD4 T cell preparations from aged mice mounted <30% response when compared with the responses by young CD4 T cells. The remaining 30% of CD4 T cell preparations from aged mice still exhibited a normal or comparable proliferative response with the young ones. In the case of hyporesponsive aged CD4 T cells, by increasing the number of APC in the stimulation culture, the responses were slightly recovered, but still with a significant difference in response-magnitude compared with young CD4 T cells (Fig. 1B). These data demonstrate that CD4 T cell proliferation tends to decline with donor age. This result provided a hint that these hyporesponsive aged CD4 T cells might suppress the activation of other T cells. This is because it has been reported that CD4+CD25+ T cells in young mice are immunoregulatory T cells having hyporesponsive (anergic) and suppressive properties (6, 7). These data led us to next test the possibility that hyporesponsive aged CD4 T cells have other suppressive functions.



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  		FIGURE 1. Whole CD4 T cells prepared from aged mice are hyporesponsive and suppressive. Whole CD4 T cells (1 x 104/well) purified from aged (24-mo-old, {triangleup}) or young (2-mo-old, {circ}) B6 mice, or a mixture of both at a ratio of 1:1 ({blacktriangleup}) were stimulated with anti-CD3 Ab in the presence of MMC-treated young B6 spleen cells (2.5 x 104/well) as APC (A) or the titrated number of APC (B and C). Three days later, cell proliferation was measured. A, Each symbol represents the individual cell preparation. The vertical axis: percentage of response of aged CD4 T cells = 100 x ((cpm by aged CD4 T cells)/(cpm by young CD4 T cells)). B and C, Data points represent the mean of triplicate cultures ± SD. Each experiment is representative of four (B) or five (C) independent assays.

 
Aging renders CD4+CD25- T cells suppressive

Surprisingly, hyporesponsive aged CD4 T cells suppressed [3H]thymidine uptake by young CD4 T cells when cocultured in vitro with young CD4 T cells at a 1:1 ratio (Fig. 1C). These CD4 T cell preparations contained the suppressive CD4+CD25+ T cells. We next investigated whether or not the suppressive activity observed on whole CD4 T cells from aged mice is ascribed to the contaminating CD4+CD25+ T cells. To preclude the contribution of CD4+CD25+ T cells in our experimental system, CD25+ T cells were depleted from the spleen cells of young or aged mice, and then CD4 T cells were purified from CD25+ cell-depleted spleen cells. Interestingly, even after the depletion of CD25+ T cells, aged CD4+CD25- T cells were hyporesponsive and inhibited the proliferation of young CD4+CD25- T cells, irrespective of the use of young or aged APC in the stimulation cultures (Fig. 2), in a dose-dependent manner (Fig. 3A). In other coculture experiments, we used CD4 T cell- and CD8 T cell-depleted, MMC-treated spleen cells as APC. Even on these APC, the proliferation of young CD4+CD25- T cells was inhibited by coexisting aged CD4+CD25- T cells (young CD4+CD25- T cells, 32,609 cpm; young and aged CD4+CD25- T cells at 1:1 ratio, 2,759 cpm), demonstrating that the possible effects of CD4+CD25+ T cells and IL-2 release by T cells, both contained in the whole spleen cells used as APC, are negligible.



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  		FIGURE 2. Hyporesponsive and suppressive properties of CD4+CD25- T cells prepared from aged mice. The CD4 T cells were purified from CD25-positive cell-depleted young (2-mo-old) or aged (24-mo-old) B6 mice. The resulting cells are designated as CD4+CD25- T cells. CD4+CD25- T cells (1 x 104/well) prepared from young ({circ}) or aged ({triangleup}), or a mixture of both at a ratio of 1:1 ({blacktriangleup}) were stimulated with anti-CD3 Ab in the presence of the titrated number of MMC-treated young (A) or aged (B) B6 spleen cells as APC. The proliferation on day 3 of culture was measured. Experiments were done in triplicate cultures. Each experiment is representative of five (A) or three (B) independent assays.

 


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  		FIGURE 3. Suppressive activity of aged CD4+CD25- T cells is a dose- and aging-dependent. CD4+CD25- T cells prepared from young (2-mo-old, {circ}) or aged (24-mo-old, {triangleup}), or a mixture of both at a ratio of 1:1 ({blacktriangleup}) were stimulated with anti-CD3 Ab in the presence of MMC-treated young B6 spleen cells (2.5 x 104/well) as APC. A, Young CD4+CD25- T cells (1 x 104/well) were cultured with the titrated number of aged CD4+CD25- T cells as indicated. Data are representative of three experiments. B, Aged CD4+CD25- T cells were prepared from 18- or 24-mo-old B6 mice, then cultured with young (2-mo-old) CD4+CD25- T cells at a ratio of 1:1 for 3 days. The vertical axis: percentage of response of a mixture of young and aged CD4+CD25- T cells = 100 x ((cpm by a mixture of young and aged CD4+CD25- T cells)/(cpm by young CD4+CD25- T cells)). Each {blacktriangleup} represents a different experiment using the individual cell preparations. Mean and SDs are shown.

 
To examine when this suppressive activity is conferred on CD4+CD25- T cells, we prepared CD4+CD25- T cells from 2-, 18-, and 24-mo-old B6 mice, and then stimulated these cells with anti-CD3 Ab in the presence of young APC. When stimulated separately, 73% of CD4+CD25- T cell preparations from 24-mo-old mice (11 of 15 mice) and 52% of those from 18-mo-old mice (9 of 17 mice) exhibited <30% response of CD4+CD25- T cells from 2-mo-old mice. When these hyporesponsive CD4+CD25- T cells were mixed at a 1:1 ratio with young (2-mo-old) CD4+CD25- T cells (total 2 x 104 T cells/well) and then stimulated, CD4+CD25- T cells from 24-mo-old mice showed more significant suppressive activity than those from 18-mo-old mice (Fig. 3B). As a control, a 2-fold cell number (2 x 104/well) of CD4+CD25- T cells from 2-mo-old mice exhibited 1.3- to 2.4-fold responses compared with the response by 1 x 104 CD4+CD25- T cells from 2-mo-old mice. CD4+CD25- T cells alone from 18- or 24-mo-old mice exhibiting the intermediate or normal responses had no suppressive activity in coculture experiments (data not shown). No significant differences were observed in the proportion of CD4+CD25- T cells from the spleens of 2-mo-old (22.0 ± 4.5%, n = 7), 12-mo-old (17.0 ± 3.4%, n = 10), or 24-mo-old (19.4 ± 5.3%, n = 10) B6 mice. Taken together, these results demonstrate that aging leads to a proliferative defect on CD4+CD25- T cells, and induces a suppressive function on CD4+CD25- T cells.

In contrast to CD4+CD25+ T cells showing unresponsiveness in vitro to TCR stimulation, CD4+CD25- T cells from aged mice showed, upon stimulation, hyporesponsiveness. This might be a confusing factor in interpreting data from proliferation assays. To rule out the involvement of proliferation by hyporesponsive aged CD4+CD25- T cells in proliferation assays, isolated CD4+CD25- T cells from young or aged mice were divided into two groups. One was treated with MMC, and the other was left untreated. After this treatment, these CD4+CD25- T cells were used in coculture experiments with young CD4+CD25- T cells as responder cells. As shown in Fig. 4A, MMC-treated aged CD4+CD25- T cells, which maintained the ability to produce cytokines with some decrement, had a similar suppressive capacity to their viable counterpart. In contrast, MMC-treated young CD4+CD25- T cells showed no suppressive function at all.



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  		FIGURE 4. Aged CD4+CD25- T cells arrest the proliferation of young CD4+CD25- T cells. A, CD4+CD25- T cells were prepared from young (2-mo-old) or aged (24-mo-old) B6 mice, aliquots were MMC-treated, then stimulated with anti-CD3 Ab and APC at the indicated combinations for 3 days. B, Young CD4+CD25- T cells were labeled with the dye CFSE and then stimulated alone or along with MMC-treated young or aged CD4+CD25- T cells. At the indicated days, cells were collected and assayed for CFSE expression on a flow cytometer. On day 2, cell proliferation was measured. Each experiment is representative of three independent assays.

 
To further analyze the suppressive activity of hyporesponsive aged CD4+CD25- T cells, young CD4+CD25- T cells as responder cells were labeled with CFSE, young or aged CD4+CD25- T cells as suppressor cells were treated with MMC, and then these cells were cocultured. Two days later, the cells were measured for proliferation and, at the same time, analyzed by flow cytometry (Fig. 4B). Young CD4+CD25- T cells labeled with CFSE entered cell division irrespective of the absence or presence of young CD4+CD25- T cells treated with MMC (32,250 or 27,487 cpm, respectively, in the proliferation assay), whereas in the presence of aged CD4+CD25- T cells, the same CD4+CD25- T cells as responder were arrested in the middle of cell division (405 cpm in the proliferation assay), which resulted in a slightly shifted single peak compared with the histogram on day 0. These results show that CD4+CD25- T cells from aged mice exhibiting hyporesponsiveness in TCR-mediated stimulation suppress the activation of normal young CD4 T cells in vitro.

The phenotype of CD4+CD25- T cells from young and aged mice

One of the effects of aging is the accumulation of memory T cells (11, 17). To evaluate the phenotype of young or aged CD4+CD25- T cells, we examined the levels of expression of CD44 and CD45RB. Results of a typical experiment are shown in Fig. 5A. Young CD4+CD25- T cells expressed a relatively naive phenotype, i.e., CD44lowCD45RBhigh. In contrast, in aged CD4+CD25- T cells, the shift to a memory phenotype, i.e., CD44highCD45RBlow, was observed. Next, to further evaluate whether the suppressive activity of aged CD4+CD25- T cells is due to an accumulation of T cells having memory phenotype, we separated aged CD4+CD25- T cells into CD44low and CD44high, or CD45RBlow and CD45RBhigh populations. These separated T cell populations were cocultured with young CD4+CD25- T cells, and evaluated their suppressive activity. As shown in Fig. 5B, all populations exerted the suppressive activity. Therefore, we found no evidence that the suppressive activity by aged CD4+CD25- T cells is responsible for the particular populations. Obviously, these experiments cannot preclude the possibility that these CD4+CD25- T cells are still heterogeneous.



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  		FIGURE 5. Phenotype of young and aged CD4+CD25- T cells. A, The expression of CD44 and CD45RB on CD4+CD25- T cells from young (2-mo-old, dotted line) and aged (24-mo-old, bold line) mice are shown. Only control staining (sold line) is shown with cells from the young ones. The control staining with cells from the aged ones was similar. B, Aged CD4+CD25- T cells were sorted into two populations based on the levels of expression of CD44 or CD45RB. These aged T cells having the phenotype, as indicated in B, were cocultured with young CD4+CD25- T cells as responder cells. Two days later, cell proliferation was measured. Each experiment is representative of four (A) or three (B) independent assays.

 
The mechanisms of aged CD4+CD25- T cell-mediated suppression

It has been reported that IL-2 can abrogate the suppressive function of CD4+CD25+ T cells (6, 7). Therefore, we tested if this is the case for aged CD4+CD25- T cells. The addition of IL-2 augmented the response of young CD4+CD25- T cells themselves, and at the same time, the suppression by hyporesponsive aged CD4+CD25- T cells was abrogated, contrasting with the relatively low response of aged CD4+CD25- T cells even when a high amount of IL-2 was added (Fig. 6A). Interestingly, by washing IL-2 out from the primary stimulation culture of aged CD4+CD25- T cells along with IL-2 (50 U/ml) on days 7–14, the recovered CD4 T cells (designated as Aged (Primed) in Fig. 6B) were again hyporesponsive and suppressed the activation of the freshly prepared young CD4+CD25- T cells. In contrast, young CD4+CD25- T cells stimulated for 7–14 days with APC, anti-CD3 Ab, and IL-2 (50 U/ml), followed by washing out IL-2 and recovery (Young (Primed)), were neither hyporesponsive nor suppressive. Repetitive restimulation along with APC, anti-CD3 Ab, and IL-2 resulted in a stable expansion of young CD4+CD25- T cells. In contrast, even with the coexistence of a high amount of IL-2, aged CD4+CD25- T cells exhibited relatively weak proliferation in the primary culture (Fig. 6A), and weaker proliferation in the following restimulation culture. Therefore, it is not possible, so far, to maintain CD4 T cells derived from aged CD4+CD25- T cells for >1 mo.



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  		FIGURE 6. Ability of aged CD4+CD25- T cells after the primary stimulation. A, CD4+CD25- T cells prepared from young (2-mo-old, {circ}) or aged (24-mo-old, {triangleup}), or a mixture of both at a ratio of 1:1 ({blacktriangleup}) were stimulated with anti-CD3 Ab and APC in the presence or absence of the titrated amount of IL-2. B, Young or aged CD4+CD25- T cells prestimulated with anti-CD3 Ab, APC, and IL-2 (10 U/ml) for 10 days, as in A, were collected and washed. The recovered T cells are designated as Young (Primed) or Aged (Primed), respectively. These primed cells (1 x 104/well) were restimulated with anti-CD3 Ab and fresh APC in the presence or absence of freshly prepared young CD4+CD25- T cells (1 x 104/well), as indicated, for 3 days. A representative result of five independent experiments is shown in A, and of four in B. C, Freshly prepared young CD4+CD25- T cells were stimulated with anti-CD3 Ab in the presence of freshly prepared aged CD4+CD25- T cells (designated as Aged (Fresh)), Aged (Primed), or Young (Primed) at a ratio of 1:1. The vertical axis: percentage of response of a mixed culture = 100 x ((cpm by a mixture of freshly prepared young CD4+CD25- T cells and the indicated T cells)/(cpm by freshly prepared young CD4+CD25- T cells)). Each symbol represents the individual cell preparation. The same cell source is connected by a line.

 
In the course of these experiments, we found that CD4+CD25- T cells from aged mice, which had no significant suppressive activity in the primary coculture with young CD4+CD25- T cells, gain suppressive properties after primary stimulation (Fig. 6C). As shown in Figs. 1A and 3B, some preparations of aged CD4+CD25- T cells maintained the intermediate or normal responsiveness when compared with those of young CD4+CD25- T cells. At the same time, these aged CD4+CD25- T cells have weak or no suppressive activity in coculture experiments (Fig. 6C, left panel). However, after primary stimulation of these aged CD4+CD25- T cells alone, the recovered CD4 T cells were almost hyporesponsive (data not shown) and suppressive (Fig. 6C, middle panel). In contrast, young CD4+CD25- T cells had no suppressive activity even after the same primary stimulation (Fig. 6C, right panel). These results suggest the possibility that aged CD4+CD25- T cells might be committed to exert the suppressive activity, and the stimulation of those cells might accelerate the appearance of hyporesponsive/suppressive properties.

In the next series of experiments, we investigated whether or not the suppressive activity by aged CD4+CD25- T cells is mediated by soluble factors or via cognate interaction with young CD4+CD25- T cells. To this end, first, the culture SNs from various combinations were examined to determine whether they represent an inhibitory effect by aged CD4+CD25- T cells. Aged CD4+CD25- T cells were confirmed to be suppressive in the primary culture (Fig. 7A). On day 2, the SNs from these cultures were harvested and added to another set of the stimulation cultures of the freshly prepared young CD4+CD25- T cells. As shown in Fig. 7B, none of the SNs had a suppressive effect. In another experiment, although we examined the capacity of the SNs prepared from a 5-fold higher cell number of aged CD4+CD25- T cells per well, no inhibitory effect was observed even with these SNs (data not shown).



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  		FIGURE 7. The suppressive activity of aged CD4+CD25- T cells is not mediated by soluble factors, but requires cell-cell contact. A, CD4+CD25- T cells prepared from young (2-mo-old) or aged (24-mo-old) B6 mice were cultured alone or cocultured at a 1:1 ratio in the presence of anti-CD3 Ab and APC. Three days later, cell proliferation was measured. From the same set of cultures, on day 2, culture SNs were harvested, then used for B and C. B, The freshly isolated young CD4+CD25- T cells were cultured with anti-CD3 Ab and APC in the presence or absence of SNs (final 50%) from A. Proliferation was measured on day 3. C, Concentrations of the indicated cytokines in the culture SNs from A were measured by ELISA. Results are mean from duplicate wells. D, CD4+CD25- T cells prepared from young (2-mo-old, {blacksquare}) or aged (24-mo-old, ), or a mixture of both at a ratio of 1:1 ({square}) were stimulated with anti-CD3 Ab and APC in the presence or absence of the indicated Ab (final 10 µg/ml each). Three days later, cell proliferation was measured. E, In Transwell culture system, young or aged CD4+CD25- T cells were cultured in the inner (In) or the outer (Out) wells, at the indicated combinations along with anti-CD3 Ab and APC. After 3 days of culture, cells in the inner well were harvested, transferred to 96-well plates, and pulsed with [3H]thymidine. Each experiment is representative of three (A–D) or four (E) independent assays.

 
Using the same SNs, we measured the concentrations of IL-2 and immunosuppressive cytokines such as IL-4, IL-10, TGF-{beta}, and IFN-{gamma} (Fig. 7C). In addition to the reports for CD4+CD25+ T cells (6, 7), suppressive aged CD4+CD25- T cells produced undetectable amounts of IL-2 (<3.0 pg/ml in ELISA), and reduced the IL-2 production in coculture with young CD4+CD25- T cells. Regarding IL-4 and IL-10, young CD4+CD25- T cell cytokine production was undetectable, whereas aged CD4+CD25- T cells produced significant amounts, and coexisting young CD4+CD25- T cells did not affect these productions. However, the addition of anti-IL-4 or anti-IL-10 Ab, even with their mixtures, at sufficient concentrations to neutralize the cytokines was not able to break the suppression (Fig. 7D). Productions of IFN-{gamma} in any of the culture combinations were under the detection level. Recently, it has been reported that CD4+CD25+ T cells, upon stimulation, express cell-bound TGF-{beta}, and that anti-TGF-{beta} Ab can abolish their suppressive activity (18). However, TGF-{beta} activity was not detected in any combinations of cultures in our experiments, even after acid treatment of SNs. In addition, the two kinds of anti-TGF-{beta} Ab used in our experiments were not able to abolish the suppression by aged CD4+CD25- T cells (Fig. 7D).

Next, we used Transwell chambers, which allow the movement of soluble factors but exclude direct cell contact between the inner and the outer well, to test the involvement of cell-cell contact. Young or aged CD4+CD25- T cells or both were stimulated in the inner wells along with APC. As shown in Fig. 7E, the proliferation of young CD4+CD25- T cells was inhibited by the coexistence of aged CD4+CD25- T cells in the same wells. However, the separation of aged CD4+CD25- T cells from young CD4+CD25- T cells, that is, the culturing of aged CD4+CD25- T cells in the outer wells along with APC and anti-CD3 Ab, could abrogate the suppressive effect by aged CD4+CD25- T cells. These results show that direct cell contact of young and aged CD4+CD25- T cells is critical for the exertion of suppression by aged CD4+CD25- T cells.

It has been shown that signaling through CTLA-4 (19, 20) and GITR molecules (16, 21), both expressed on CD4+CD25+ T cells, is able to influence the suppressive function by CD4+CD25+ T cells, i.e., active signals via CTLA-4 induce suppression, and active signaling through GITR is required for abrogating suppression. As shown in Fig. 8B, this was confirmed; blockade of CTLA-4-mediated signaling by the addition of anti-CTLA-4 Fab dampened suppression, whereas GITR stimulation by anti-GITR Ab whole molecule abrogated suppression. In contrast to CD4+CD25+ T cells, anti-CTLA-4 Fab had no or a negligible effect on aged CD4+CD25- T cell-mediated suppression, and the addition of anti-GITR Ab broke the hyporesponsiveness of aged CD4+CD25- T cells and elicited the proliferation of a mixture of aged and young CD4+CD25- T cells. This indicates that aged CD4+CD25- T cell-mediated suppression uses different mechanisms to those by CD4+CD25+ T cells.



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  		FIGURE 8. Suppression by aged CD4+CD25- T cells is abrogated by anti-GITR Ab, but not by anti-CTLA-4 Ab. A, CD4+CD25- T cells prepared from young (2-mo-old, {blacksquare}) or aged (24-mo-old, ), or a mixture of both at a ratio of 1:1 ({square}) were stimulated with anti-CD3 Ab and APC in the presence or absence of the indicated Ab (final 100 µg/ml). Three days later, cell proliferation was measured. B, CD4+CD25- T cells or CD4+CD25+ T cells were sorted from spleen cells of young B6 mice, and cultured as indicated in A. Each experiment is representative of four (A) or three (B) independent assays.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro analyses using CD4+CD25- T cells from aged mice revealed that freshly prepared aged CD4+CD25- T cells, or these cells after in vitro prestimulation, exhibit hyporesponsiveness to TCR stimulation and at the same time, suppress the activation of other normal CD4 T cells. Furthermore, the appearance of these hyporesponsive/suppressive CD4+CD25- T cells was aging-dependent. These results indicate that aging not only leads to a decline in T cell activation, but also confers a suppressive function on CD4+CD25- T cells.

Although it has been convincingly demonstrated that many of the T cells that are responsible for suppressing self-reactive T cells are naturally occurring CD4+CD25+ T cells (5, 6, 7, 22), several recent studies have provided evidence for the existence of suppressive CD4+CD25- T cells (23, 24, 25, 26). In the case of naturally unresponsive and suppressive CD4+CD25+ T cells, it has been shown that the specificity for self peptides with high affinity directs the selection of CD4+CD25+ T cells in the thymus (27). How are these suppressive CD4+CD25- T cells generated? The involvement of agonistic Ags may not be required for the generation of hyporesponsive CD4+CD25- T cells. Linton et al. (17) demonstrated that CD4 T cells that have most likely never encountered Ags undergo changes associated with aging, including a reduced proliferative capacity. This suggests that other additional intrinsic changes unrelated to Ag exposure occur with aging and are responsible for the hyporesponsiveness of aged CD4 T cells. However, there may be a different requirement for Ag stimulation. As shown in Fig. 6C, in vitro stimulation of aged CD4+CD25- T cells rendered nonhyporesponsive CD4+CD25- T cells hyporesponsive and suppressive, indicating that CD4+CD25- T cells in aged mice are already committed to be suppressive irrespective of their responsiveness. Therefore, as one possibility, Ag stimulation may facilitate the acquisition of the hyporesponsiveness-phenotype and the appearance of suppressive activity, whereas other intrinsic changes may be critical in committing CD4+CD25- T cells to be hyporesponsive/suppressive.

There are many reports showing that, in addition to the naturally occurring CD4+CD25+ T cells (6, 7), artificially induced anergic or hyporesponsive T cells have suppressive properties (8, 9, 10, 28, 29, 30). Some of these T cells were induced by manipulating costimulatory molecules on APC, repetitive stimulation with immature dendritic cells, or expression of Ag by hemopoietic cells. The common point in these experimental systems resides in the interaction between T cells and APC, which leads to the acquisition of suppressive activity. Therefore, it is plausible that APC in aged mice have some implications in rendering aged CD4+CD25- T cells suppressive, although most studies have found little evidence of an effect of age on such cells’ ability to support T cell activation (11, 31). Recently, it has been reported that the expression of agonist Ag by nonactivated hemopoietic cells generates mostly CD4+CD25--suppressive T cells (30). These T cells were derived from mature T cells in the absence of a functioning thymus, and suppression was IL-10-independent and was overcome by IL-2. Some of these properties are also observed on aged CD4+CD25- T cells, suggesting a possibility that nonactivated hemopoietic cells as APC render aged CD4+CD25- T cells suppressive. Further studies will be needed to determine whether this is the case or not.

As shown in Fig. 7C, we observed that significant levels of IL-10 are produced by aged suppressive CD4+CD25- T cells without any influence by cocultured young CD4+CD25- T cells. This pattern of IL-10 production resembles that reported by Dieckmann et al. (32). They showed that CD4+CD25- T cells cocultured with CD4+CD25+ T cells are anergized (so-called infectious tolerance), and then become IL-10-producing and IL-10-dependent suppressor cells. Another group also reported the infectious tolerance by CD4+CD25+ T cells, although they showed that the infected CD4+CD25- T cells are TGF-{beta} producers (33). In our studies, the suppressive activity was mediated by neither IL-10 nor TGF-{beta}. The staining experiments of freshly prepared or primed aged CD4+CD25- T cells with anti-TGF-{beta} showed no or negligible levels of staining (data not shown). However, it is notable that the suppressive activity is transferable to other T cells (32, 33, 34). It is possible that aging may result in the accumulation of CD4+CD25- T cells anergized or infected by CD4+CD25+T cells during aging.

As well as CD4+CD25+ T cells, aged suppressive CD4+CD25- T cells inhibited the IL-2 production by cocultured young CD4+CD25- T cells (Fig. 7C). As one possibility, aged CD4+CD25- T cells exert the suppression by consuming the IL-2 produced by young CD4+CD25- T cells. However, this is unlikely because when young CD4+CD25- T cells were cocultured with young CD4+CD25- T cells as control, no suppression was observed (Fig. 4A). Moreover, even after the prestimulation, the primed T cells derived from young CD4+CD25- T cells had no suppressive effect on the freshly prepared young CD4+CD25- T cells as responder (Fig. 6, B and C). Taken together, these results indicate that the consumption of IL-2 is not the main cause of the suppression by aged CD4+CD25- T cells.

In contrast, as shown in Fig. 6A, by adding IL-2 to the coculture of young and aged CD4+CD25- T cells, the suppressive effect by aged CD4+CD25- T cells was not observed. Even with a high amount of IL-2, aged CD4+CD25- T cells kept a relative hyporesponsiveness when compared with young ones. Therefore, it still remains to be elucidated whether IL-2 is abrogating the suppressive activity of aged CD4+CD25- T cells or merely activating young CD4+CD25- T cells to overcome suppressive effects by aged CD4+CD25- T cells.

It remains to be determined whether suppressive CD4+CD25- T cells from aged mice are heterogeneous as subsets. These T cells may contain suppressive and suppressed (or infected) subpopulations. Furthermore, as shown previously, in the case of CD4+CD25+ T cells, it has been reported that CD4+CD25+ T cells before and after the stimulation exhibited a difference in sensitivity against fixation, i.e., activated-fixed CD4+CD25+ T cells had a suppressive activity, in contrast, resting-fixed ones had no suppressive activity at all (32, 33). Therefore, we need to consider the heterogeneity as a subpopulation, the stage of T cell activation, and whether aged CD4+CD25- T cells are infectious T cells or infected ones, in elucidating the molecular mechanisms of suppression by CD4+CD25- T cells from aged mice.


   Acknowledgments
 
We thank Dr. Shimon Sakaguchi (Kyoto University, Kyoto, Japan) for helpful discussion.


   Footnotes
 
1 This work was supported by a Research Grant for Longevity Sciences (12C-05) from the Ministry of Health and Welfare (to J.S.) and Japan Health Foundation for the prevention of chronic diseases and the improvement of quality of life of patients (to J.S.). Back

2 Address correspondence and reprint requests to Dr. Jun Shimizu, Immunological Aging Research Group, Division of Physiology and Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015, Japan. E-mail address: jun{at}tmig.or.jp Back

3 Abbreviations used in this paper: SN, supernatant; GITR, glucocorticoid-induced TNFR family-related gene; MMC, mitomycin C. Back

Received for publication September 26, 2002. Accepted for publication December 2, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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