Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0506364 on March 16, 2007

Published online before print March 16, 2007
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(Journal of Leukocyte Biology. 2007;81:1386-1394.)
© 2007 by Society for Leukocyte Biology

Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice

Liang Zhao, Liguang Sun, Hongjun Wang, Haixia Ma, Guangwei Liu and Yong Zhao1

Transplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

1 Correspondence: Transplantation Biology Research Division, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beisihuan Xi Road 25, Beijing, China 100080. E-mail: zhaoy{at}ioz.ac.cn

ABSTRACT

A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4+CD25+ Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4+CD25+ Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vß family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4+CD25+ Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-{gamma}) but not cell proliferation of effector T cells, as compared with CD4+CD25+ Treg cells of young mice. In addition, the percentages of CD4+CD8–CD25+ Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4+CD25+ Treg cells were altered significantly with aging in mice. The functional defects of CD4+CD25+ Treg cells may shed light on the role of CD4+CD25+ Treg cells in the increased sensitivity to autoimmune diseases of aged populations.

Key Words: aging • immune tolerance

INTRODUCTION

A progressive decline in the integrity of the immune system is one of the physiologic changes during mammalian aging. The aging-associated immunity alterations occur in every component of the immune system, including T, B, and NK cells, monocytes, dendritic cells, macrophages, granulocytes, and erythrocytes [1 2 3 4 5 ]. In aged individuals, T cells shift from naïve to memory phenotypes (decreased numbers of naive T cells) and from Th1 to Th2 cytokine productions, increase the proportion of T cells expressing NK markers or receptors, and produce more proinflammatory cytokines [6 7 8 ]. Numerous studies have demonstrated a deficiency in the ability of splenocytes from aged mice to respond to antigens in respect to antibody products, cell proliferation, cell death, and the generation of cytotoxic cells [9 10 11 12 ].

A panel of immune disorders including autoimmune diseases, chronic infections, and cancer has been linked closely with quantitative and/or qualitative defects of regulatory T cells (Treg cells) [13 14 15 16 17 ]. Today, there is increasing evidence for an active and "dominant" tolerance mediated by Treg cells [18 ]. So far, several subtypes of Treg cells have been described, including CD4+CD25+ Treg cells, IL-10-producing CD4+ Treg cells-1, TGF-ß-secreting Th3 cells, CD4+CD45RBlow T cells, CD4+CD62Lhigh T cells, CD8+CD25+ Treg cells, CD8+CD28– Treg cells, {gamma}{delta} T cells, and NKT cells [19 20 21 22 ]. As autoimmune diseases occur more often in the aging population [23 , 24 ], the question arises as to whether aging alters the occurrence and/or function of the Treg cells in aged individuals. It is surprising that limited studies about Treg cells or suppressor cells have been performed in the aging population so far [25 , 26 ].

Mice, which are older than 18–24 months, are generally recognized as aged mice [1 , 12 ]. Thus, the present studies about aging-related CD4+CD25+ Treg cell changes were performed on more than 20-month-old Balb/c mice. Our studies have shown that the percentages of CD4+CD25+ Treg cells in the periphery and thymi of aged mice changed significantly. The phenotypes and Vß family expression of the CD4+CD25+ Treg cells in aged mice were modified too. In addition, a significantly decreased, immunosuppressive function of CD4+CD25+ Treg cells was detected in aged mice. The changes of CD4+CD25+ forkhead box p3 (Foxp3)+ Treg cells in aged mice may shed new light on the causes of age-dependent, immune disorders and partially explain the increased susceptibility for autoimmune responses and chronic inflammatory diseases in the elderly.

MATERIALS AND METHODS

Animals
C57BL/6 (B6, H-2b) and Balb/c (H-2d) mice were purchased from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Beijing, China). All mice were maintained in a specific pathogen-free facility and were housed in microisolator cages containing sterilized feed and autoclaved bedding and were given water until their use at the age of ~3 months (young) and 20 months (aged). All experimental manipulations were undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals.

mAb and chemical reagents
The following mAb were purchased from BD Biosciences PharMingen (San Diego, CA, USA): FITC-conjugated rat antimouse CD4 mAb (RM4-5; IgG2a), FITC-labeled rat antimouse CD8 mAb (53-6.7; IgG2a), FITC-labeled rat antimouse CD25 mAb (7D4; IgM), FITC-labeled rat antimouse glucocorticoid-induced TNF receptor (GITR) mAb (DTA-1; IgG2b), FITC-labeled hamster antimouse TCR-ß chain mAb (H57-597; IgG2a), FITC-labeled antimouse Vß5.1/5.2 mAb (MR9-4; mIgG1), FITC-labeled antimouse Vß6 mAb (RR4-7; rat IgG2b), FITC-labeled antimouse Vß7 mAb (TR310; rat IgG2b), FITC-labeled antimouse Vß8.1/8.2 mAb (MR5-2; mIgG2a), FITC-labeled antimouse Vß9 mAb (MR10-2; mIgG1), FITC-labeled antimouse Vß11 mAb (RR3-15; rat IgG2b), PE-labeled rat antimouse CD4 mAb, PE-labeled rat antimouse CD8{alpha} mAb (53-6.7; IgG2a), and Cy5-labeled antimouse CD25 or anti-CD4 mAb.

In addition, FITC-labeled antimouse Foxp3 mAb (FJK-16 s) and its staining kit were obtained from eBioscience (San Diego, CA, USA). Rat antimouse FcR mAb (2.4G2; IgG2b) was produced by 2.4G2 hybridoma (American Type Culture Collection, Manassas, VA, USA) in our laboratory. OVA was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mitomycin C (C15H18N4O5) was obtained from Kyowa Hakko Co., Ltd. (Tokyo, Japan).

Immunofluorescence staining and flow cytometry (FCM)
Splenocytes (5x105) were prepared and washed once with FACS buffer (PBS, pH 7.2, containing 0.1% NaN3 and 0.5% BSA) as described before [27 ]. For two-color staining, cells were stained with PE-labeled antimouse CD4 mAb versus FITC-labeled antimouse TCR, CD25 mAb, or the nonspecific staining control mAb, respectively. For three-color staining, cells were stained with PE-labeled antimouse CD4 mAb and Cy5-labeled antimouse CD25 mAb versus FITC-labeled antimouse CTLA-4, GITR, TCR Vß3, Vß5.1/5.2, Vß6, Vß7, Vß8.1/8.2, Vß9, and Vß11 mAb, or the nonspecific staining control mAb. Nonspecific FcR binding was blocked by antimouse FcR mAb 2.4G2. At least 10,000 cells for two-color FCM and 30,000 cells for three-color were assayed using a FASCalibur FCM (Becton Dickinson, Mountain View, CA, USA), and data were analyzed with CellQuest software (Becton Dickinson). Nonviable cells were excluded using the vital nucleic acid stain propidium iodide. The percentage of cells stained with a particular reagent or reagents was determined by subtracting the percentage of cells stained nonspecifically with the negative control mAb from staining in the same dot-plot region with the antimouse mAb. Certain molecule expression levels were determined as the median fluorescence intensity (MFI) of the cells positively stained with the specific mAb.

To determine the intracellular expression of Treg cell-specific transcription factor Foxp3 in CD4+CD25+ cells, mouse splenocytes were first surface-stained with PE-labeled antimouse CD4 and Cy5-labeled antimouse CD25 mAb per standard practice. These cells were subsequently stained with FITC-labeled antimouse Foxp3 mAb (FJK-16 s, Cat. No. 11-5773, eBioscience) or FITC-labeled rat IgG2a nonspecific isotype control mAb, according to the manufacturer’s recommendations with a FITC-conjugated mouse Foxp3 staining set (eBioscience).

Cell purification
Mouse CD4+CD25+ Treg cell populations were isolated from mouse splenocyte suspension using a CD4+CD25+ Treg isolation kit with the MidiMACSTM separator, according to the manufacturer’s protocols (Miltenyi, Bergisch Gladbach, Germany). Briefly, erythrocyte-depleted splenocytes were suspended in PBS containing 0.5% BSA and 2 mM EDTA (pH=7.2) and then incubated with a biotin-antibody cocktail against CD8{alpha} (Ly2), CD11b (membrane-activated complex 1), CD45R (B220), CD49B (DX5), and Ter-119 for 20 min at 4°C, and then microbead-conjugated, antibiotin mAb (Clone Bio318E7.2) was added to deplete non-CD4+ T cells. In parallel, the cells were stained with PE-labeled anti-CD25 mAb. The cell suspension was loaded on a LD column, which is placed in the magnetic field of a MACS separator, and then the unlabeled splenocytes were run through. The remaining fraction in the column is the enriched CD4+ T cells. For the isolation of CD4+CD25+ T cells, the PE-labeled CD25+ T cells in the enriched CD4+ T cell fraction were labeled magnetically with anti-PE MicroBeads and separated by MACS sorting. Positively sorted CD4+CD25+ T populations were always >95%, as confirmed by FCM each time.

MLR assay
Murine splenic CD4+CD25+ Treg cells were isolated from young Balb/c mice or aged Balb/c mice using a MACS sorting isolation kit as described above. CD4+CD25– T cells, which were used as responder T cells, were purified from young and aged Balb/c mice separately. C57BL/6 splenocytes were treated with mitomycin C at the concentration of 30 µg/ml, at 37°C for 30 min, and then washed three times. These cells were suspended in complete RPMI-1640 medium and were to be used as allogeneic stimulator cells [28 ]. In general, 8 x 104 responder cells (Balb/c CD4+CD25– T cells) and 8 x 104 stimulator cells (allogeneic C57BL/6 splenocytes) per well in RPMI-1640 medium supplemented with 10% FCS were added in 96-well, round-bottomed plates. CD4+CD25+ Treg cells were added subsequently to each well according to the ratio responder:Treg cells. Cells were cocultured in complete medium at 37°C and 5% CO2 for 96 h, and 0.5 µCi 3H-thymidine (radioactivity, 185GBq/mmol, Atomic Energy Research Establishment, China) was added during the last 18 h. Cells were harvested onto glass fiber filters with an automatic cell harvester (Tomtec, Toku, Finland). The radioactivity of each sample was assayed in a liquid scintillation analyzer (Beckman Instruments, Fullerton, CA, USA). Values are expressed as cpm from triplicate wells.

Detection of IL-2 and IFN-{gamma} production by ELISA
CD4+CD25– T cells (8x104) were cultured with allogeneic stimulators in the presence of 8 x 104 CD4+CD25+ Treg cells separated from young or aged mice for 3 days as mentioned above. The IL-2 and IFN-{gamma} levels in the culture medium were determined by IL-2 and IFN-{gamma} ELISA kits as per the manufacturer’s instruction.

Delayed-type hypersensitivity (DTH)
Sensitized effector T cells were generated by immunizing Balb/c mice with allogeneic C57BL/6 splenocytes. Ten days after immunization, Balb/c CD4+ T cells were enriched using the negative-selecting MACS kit for CD4+ T lymphocytes (BD Biosciences PharMingen). Allogeneic C57BL/6 splenic macrophages (SPMs) were used as stimulator cells. Sensitized Balb/c effector CD4+ T cells and allogeneic (C57BL/6) macrophage stimulators (5x105 cells/each) were injected intradermally into the pinnate of naïve Balb/c mice. Mice, which received sensitized Balb/c effector CD4+ T cells or allogeneic stimulator cells alone, were used as the negative control. In some recipient Balb/c mice, the CD4+CD25+ Treg cells from young or aged Balb/c mice were coinjected with effector T cells and stimulator cells intradermally at the same time. The changes in ear thickness were measured using an engineer’s micrometer at 24 or 48 h after the challenge, as reported previously [29 ]. The ear-thickness change was calculated by subtracting the thickness of the ear before injection from the thickness of the same ear after injection.

Statistical analysis
All data are presented as the mean ± SD. Student’s unpaired t-test for comparison of means was used to compare groups. A P value less than 0.05 was considered statistically significant.

RESULTS

Significantly enhanced percentages of peripheral CD4+CD25+ Treg cells in aged mice
It has been reported that there are significant changes in the levels and phenotypes of different immune cells in mice, which are more than 18 months old [30 ]. To determine whether the alteration of CD4+CD25+ Treg cells occurs in aged mice, we compared the levels of CD4+CD25+ T cells in the periphery of young and aged Balb/c mice using FCM. Significantly lower percentages of CD4+ T cells were observed in aged mice rather than in young mice (data not shown), as reported earlier [11 ]. It is surprising that compared with young mice, a significantly enhanced CD4+CD25+ T cell subpopulation in PBLs was observed in mice, which were 20 months old (P<0.05, Fig. 1A and 1B ). The percentages of CD4+CD25+ T cells in CD4+ PBLs of aged Balb/c mice were markedly higher than those of young Balb/c mice (Fig. 1B) .


Figure 1
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  		Figure 1. Significantly enhanced levels of CD4+CD25+ Treg cells in the periphery of aged Balb/c mice. (A) One representative of CD4+CD25+ T cells in young (a) and aged (b) mice assessed by FCM is shown. SSC, Side-scatter; FSC, forward-scatter. (B) The enhanced percentages of CD4+CD25+ T cells gated in PBLs (a) as well as in CD4+ PBLs (b) of aged mice. (C) Significantly increased percentages of CD4+CD25+ T cells in the spleens (SPL) and lymph nodes (LN) of aged mice. (a) The enhanced percentages of CD4+CD25+ T cells in spleens and lymph nodes of aged mice; (b) the increased percentages of CD4+CD25+ T cells gated in CD4+ cells of spleens and lymph nodes in aged mice; (c) the percentages of CD4+ T cells in spleens and lymph nodes of aged mice; (d) the total cell numbers in spleens and lymph nodes of aged mice. Data are a summary of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the corresponding groups. More than eight mice in each group were examined.

 
Consistently with the peripheral blood, significantly higher percentages of CD4+CD25+ T cells in spleens and lymph nodes of aged mice were observed when compared with the young Balb/c mice (P<0.05 or P<0.01, respectively, Fig. 1C ). The percentages of CD4+CD25+ T cells among CD4+ T cells in spleens and lymph nodes of aged, Balb/c mice were significantly higher than those of young Balb/c mice, whereas the percentages of CD4+ T cells in spleens or lymph nodes of aged mice were the same as or a little lower than those of young Balb/c mice (Fig. 1C) . The total cell numbers of CD4+ T cells in spleens or lymph nodes did not reveal statistically significant differences between young and aged Balb/c mice (Fig. 1C) . These data indicate that the percentage of CD4+CD25+ T cells in the peripheral immune tissue was enhanced markedly in aging mice.

To confirm whether the CD4+CD25+ T cells in aged mice are Treg cells, the expression of Foxp3, a transcript factor specifically expressed in Treg cells [31 32 33 ], was detected by intracellular staining FCM. The majority of mouse CD4+CD25+ T cells expressed high levels of Foxp3 (Fig. 2A and 2B ). No significant difference for Foxp3 expression in CD4+CD25+ T cells in spleens or lymph nodes was observed in aged and young mice (Fig. 2B and 2C) . In addition, few of CD4+CD25– T cells expressed Foxp3 in young and aged Balb/c mice (Fig. 2D) . Although the percentage of Foxp3+cells in CD4+CD25– T cells in aged Balb/c (4.02+2.18%) was higher than those of young mice (2.62+1.71%), no statistical significance was achieved (P>0.05).


Figure 2
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  		Figure 2. The expression of Foxp3 in CD4+CD25+ T cells of aged Balb/c mice. Three-color, intracellular staining FCM was used to detect the expression of Foxp3 in different subsets of splenic T cells. (A) One representative of FCM profiles was shown for the Foxp3 staining gated in CD4+CD25+ cells in young and aged mice. (B) The mean percentages of Foxp3+ cells gated in CD4+CD25+ cells. No statistical difference was achieved among identical groups. (C) The MFI of Foxp3 staining gated in CD4+CD25+ T cells in aged and young mice was summarized. Data are shown as mean ± SD. More than eight mice in each group were examined. (D) One representative of FCM profiles was shown for the Foxp3 staining gated in CD4+CD25– cells in young and aged Balb/c mice. Few CD4+CD25– cells express Foxp3. Eight mice in each group were examined.

 
The phenotype changes of peripheral CD4+CD25+ Treg cells in aged mice
It has been reported that the phenotype of T cells shifted from naïve to memory T cells with aging [34 ]. Naïve mouse CD4+ T cells displayed as CD45RBhighCD62LhighCD44low cells, whereas memory CD4+ T cells showed as CD45RBlowCD62LlowCD44high cells. As expected, significantly decreased percentages of CD45RBhighCD62Lhigh cells and enhanced CD44high cells in gated CD4+CD25– splenocytes were detected in aged Balb/c mice compared with those in young Balb/c mice (P<0.01, Fig. 3A ), indicating that CD4+CD25– T cells shift from naïve to memory phenotype in aged Balb/c mice. In addition, increased percentages of CD4+CD25– T cells expressing GITR or CD69 molecules were detected in aged Balb/c mice (P<0.01, Fig. 3A ). It is unexpected that markedly lower percentages of CD4+CD25+ T cells expressing CD45RBhighCD62Lhigh were detected in aged Balb/c mice (Fig. 3B) , whereas the expression of CD44 molecules on these cells was unchanged.


Figure 3
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  		Figure 3. The phenotype of CD4+CD25+ Treg cells in aged Balb/c mice Three-color staining FCM was used to detect the expression of GITR, CD44, CD45 receptor B (CD45RB), CD62 ligand (CD62L), and CD69 on CD4+CD25+ splenocytes. (A) One representative of FCM profiles was shown for GITR, CD44, CD45RB, CD62L, and CD69 staining gated in CD4+CD25– or CD4+CD25+ cells in young and aged mice. The gray line represents young Balb/c mice, and the solid line is for aged Balb/c mice. (B) The mean percentages of GITR+, CD44high, CD45RBhigh, CD62Lhigh, and CD69+ cells gated in CD4+CD25– cells of young and aged mice. (C) The mean percentages of GITR+, CD44high, CD45RBhigh, CD62Lhigh, and CD69+ cells gated in CD4+CD25+ cells of young and aged mice. One representative of three independent experiments with identical results is shown. **, P < 0.01; ***, P < 0.001, compared with the corresponding groups. Eight mice in each group were examined.

 
The Vß family changes of CD4+CD25+ Treg cells in aged mice
TCR diversity in the peripheral immune system alters during aging [35 , 36 ]. The expression of Vß families on CD4+CD25+ T cells or CD4+CD25– T cells in aged mice was detected. Significantly lower percentages of Vß7+ cells and Vß11+ cells and higher percentages of Vß8.1/8.2+ cells on CD4+CD25– T cells were observed in spleens of aged Balb/c mice when compared with those in young Balb/c mice (Fig. 4A ). The percentages of other Vß families expressed on these T cells were unchanged during aging. The percentages of Vß5.1/5.2+, Vß7+, and Vß11+ cells in CD4+CD25+ cells were decreased significantly in aged Balb/c mice, and the percentages of Vß6+ and Vß8.1/8.2+ cells in CD4+CD25+ cells were enhanced significantly in these Balb/c mice compared with the young Balb/c mice (Fig. 4B) . Thus, the Vß distribution of TCR of Treg or non-Treg cells changed significantly with aging.


Figure 4
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  		Figure 4. The TCR Vß repertoire of CD4+CD25+ Treg cells in aged mice Three-color staining FCM was used to detect the Vß expression on CD4+CD25+ or CD4+CD25– cells in each mice. Thirty thousand cells were analyzed for each sample. (A) The Vß expression on mouse CD4+CD25– cells. (B) The Vß expression on CD4+CD25+ cells. Results are presented as mean ± SD. Six mice in each group were studied. *, P < 0.05; **, P < 0.01, compared with young Balb/c mice.

 
The functional changes of CD4+CD25+ Treg cells in aged mice
After demonstrating the phenotype and percentage changes of CD4+CD25+ Treg cells in Balb/c mice, which were 20 months old, we studied their immunosuppressive functions in vitro and in vivo. As is shown in Figure 5 , CD4+CD25+ Treg cells of aged and young Balb/c mice showed identical, significant inhibition on the allogeneic, antigen-induced cell proliferation of syngeneic CD4+CD25– T cells. However, the inhibiting effect of CD4+CD25+ Treg cells from aged Balb/c mice on the IL-2 production of effector T cells was significantly lower than those from young Balb/c mice (P<0.01, Fig. 5B ). CD4+CD25+ Treg cells in aged Balb/c mice showed similar inhibiting ability on the IFN-{gamma} production of effector T cells induced by allogeneic antigens (Fig. 5C) . It is important that during the in vivo function study, CD4+CD25+ Treg cells of aged Balb/c mice showed significantly lower inhibiting ability on the syngeneic CD4+CD25– T cell-mediated DTH responses to allogeneic antigens compared with CD4+CD25+ Treg cells of young mice, and Treg cells of young and aged Balb/c mice had significant inhibition on the DTH reaction of syngeneic CD4+CD25– T cells (Fig. 5B) . Thus, the immunosuppressive function of CD4+CD25+ Treg cells was decreased markedly in aged Balb/c mice.


Figure 5
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  		Figure 5. The decreased, immunosuppressive effects of CD4+CD25+ Treg cells in aged Balb/c mice in vitro and in vivo. The cell proliferation was determined by 3H-TdR incorporation as described in Materials and Methods. (A) The inhibitory effects of CD4+CD25+ Treg cells from aged and young Balb/c mice on the responses of CD4+CD25– T cells to alloantigens. (B) The inhibitory effects of CD4+CD25+ Treg cells from aged and young mice on the IL-2 product of CD4+CD25–T cells stimulated with alloantigens. (C) The inhibitory effects of CD4+CD25+ Treg cells from aged and young mice on the IFN-{gamma} product of CD4+CD25– T cells stimulated with alloantigens. (D) The inhibiting effects of Treg cells were detected in an in vivo DTH assay as described in Materials and Methods. The ear-thickness changes at 24 h (a) or 48 h (b) after challenging with the sensitized T cells (Sens. Balb/c T cells). Results are expressed as the mean value of triplicates ± SD. One representative of two independent experiments with similar results is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the indicated group.

 
The changes of CD4+CD25+ Treg cells in the thymi of aged mice
It has been reported that the thymus serves as a central immune tissue and is critical for T cell development and declines with aging [37 ]. The process of thymic involution involves the progressive loss of normal organ architecture and cellular composition and a significant reduction in the output of mature T lymphocytes [23 , 37 ]. We found that total cell numbers of thymocytes and the subsets, including CD4/CD8 double-positive (DP), CD4/CD8 double-negative (DN), and CD4 or CD8 single-positive (SP) thymocytes, decreased significantly in Balb/c mice, which were 20 months old compared with young Balb/c mice (P<0.05, Fig. 6A 6B 6C ). Significantly higher percentages of CD4+CD8–CD25+ thymocytes among CD4SP thymocytes were observed in aged Balb/c mice rather than in those of young Balb/c mice (P<0.001, Fig. 6D ). However, the cell numbers of CD4+CD8–CD25+ thymocytes in aged Balb/c mice were remarkably lower than those in young Balb/c mice (Fig. 6E) . Consistently with the peripheral CD4+CD25+ Treg cells, thymic CD4+CD25+ T cells of aged Balb/c mice expressed similar levels of Foxp3 molecules, as determined by intracellular staining FCM (Fig. 6F and 6G) . These data indicate that CD4+CD8–CD25+ thymocytes decreased remarkably with aging and were more resistant to aging-induced changes compared with other subsets in the thymus.


Figure 6
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  		Figure 6. The levels of CD4+CD25+ Treg cells and other subsets in the thymi of aged Balb/c mice. Thymocytes were stained with PE-Cy5-labeled anti-CD4 mAb, PE-labeled anti-CD25 mAb, and FITC-labeled anti-CD8 mAb and analyzed by FCM. (A) The total cell numbers of thymocytes in young and aged mice. (B) The mean percentages of DN, CD8SP, CD4SP, and DP thymocytes in the thymi of young and aged mice. (C) The cell numbers of DN, CD8SP, CD4SP, and DP thymocytes in the thymi of young and aged mice. (D) The mean percentages of CD4+CD25+ T cells gated in CD4SP cells in the thymi of young and aged mice. (E) The total cell numbers of CD4+CD25+ T cells in the thymi of young and aged mice. (F) One representative of FCM profiles was shown for the Foxp3 staining gated in CD4+CD25+ cells in young and aged mice. (G) The mean percentages of Foxp3+ cells gated in CD4+CD25+ thymocytes in aged and young mice. *, P < 0.05; ***, P < 0.001, compared with the identical groups. Results were shown as mean ± SD (n=6), which was one representative of three independent experiments.

 
DISCUSSION

It is well known that advancing age is associated with significant alternations in the function of human and mouse T cells [38 , 39 ]. Aging leads to a decrease in the ability to mount strong T responses to new antigens and to previously encountered recall antigens in mice and humans [4 ]. It has been proposed that the altered responses of T cells from aged animals result from the accumulation of memory T cells. The percentages of memory T cells increase with aging, which is supported by the markedly increased expression of CD44, as well as the decreased expression of CD45RB and CD62L on T cells in aged mice [40 , 41 ]. In accordance with previous reports, normal percentages and total cell numbers of CD4 or CD8 cells in spleens and lymph nodes and significantly lower percentages of CD4+ T cells in the peripheral blood were observed in 20-month-old mice. The majority of CD4+CD25– T cells displays a memory phenotype in the periphery of these mice. This observation was supported by a recent report that the major changes of peripheral blood leukocyte composition caused by aging are the reduced peripheral blood CD4+ lymphocyte population [42 ]. Therefore, a decrease in the proportion of peripheral blood CD4+ T cell subset is a sensitive parameter for the aging-related immune system alteration.

It is important that the percentages of CD4+CD25+ Treg cells expressing Treg-specific transcript factor Foxp3 in the periphery (blood, spleen, and lymph nodes) increased significantly in aged Balb/c mice. In addition, the size of the TCR repertoire and phenotypes of the peripheral CD4+CD25+ Treg cells seemly changed with aging, as determined by the Vß family and cell surface molecules. The modified Vß family distribution of the peripheral CD4+CD25+ Treg cells may be a result of the selectively clonal expansion during aging, which is supported by the decreased expression of CD45RB and CD62L on CD4+CD25+ Treg cells in aged mice.

Although CD4+CD25+ Treg cells in aged mice had normal suppression on the proliferation of effector T cells stimulated by alloantigens, CD4+CD25+ Treg cells in aged mice showed significantly decreased immunosuppressive function as determined by their in vitro inhibition on the cytokine products (IL-2 and IFN-{gamma}) of effector T cells and their inhibition on DTH response of sensitized effector T cells in vivo. Although the decreased IL-2 levels in the culture systems were likely a result of the inhibition of Treg cells on effector cells, the possibility that the IL-2 consumption of Treg cells could not be excluded with the present data. The reasons for the decreased function of CD4+CD25+ Treg cells of aged mice are unclear at this moment. The decreased immunosuppressive function of CD4+CD25+ Treg cells in aged mice is unlikely a result of the contamination of other memory/activated cells, as the cell purity of CD4+CD25+ T cells and the expression of Foxp3 in these cells were determined to be identical in young and aged mice. It has been reported that CD4+CD25+CD62Lhigh T cells have more potent, immunosuppressive ability than CD4+CD25+CD62L– or CD4+CD25+ T cells [43 ]. The changes of the phenotypes of these Treg cells in aged mice may be involved in their functional alteration. Our data showed that CD4+CD25+ T cells of aged mice expressed significantly lower levels of CD62L than those of young mice. These may partially explain the poor inhibiting ability of CD4+CD25+ Treg cells in aged mice, which needs to be addressed in the future.

Although B and T lymphocytes have been shown to have a decline in response to antigenic stimulus with aging, paradoxically, there has been an increase in autoantibodies and the occurrence of autoimmunity [44 ]. Several types of diseases, including autoimmune disease, could be coupled with the general processes of aging [45 ]. The functional defect of CD4+CD25+ Treg cells in aged individuals may contribute partially to the higher frequencies of autoimmune diseases. It is interesting that the percentages of host superantigen-recognizing Vß5.1/5.2+ and Vß11+ T cells in CD4+CD25+ Treg cells were decreased markedly in aged mice. Thus, considering the antigen specificity for the immunosuppression of CD4+CD25+ Treg cells, the decreased CD4+CD25+ Treg cells expressing Vß5.1/5.2+ and Vß11+ TCRs might likely make host susceptible to autoimmunity.

It is well known that the thymus involutes progressively throughout life, beginning at approximately the sexual maturation in human beings [23 ]. In mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution [11 , 46 ]. The accumulation of the CD4/CD8 DN stage of thymocytes in 20-month-old mice was associated with a developmental block between the CD25–CD44+ and CD25+CD44+ stages [46 ]. In addition to supporting the previous reports, our present study showed that the percentages of CD4+CD8–CD25+ Treg or CD4+CD25+Foxp3+ Treg cells in the thymi of aged mice increased markedly, indicating that CD4+CD8–CD25+ Treg cells might be relatively resistant to aging compared with other subsets of thymocytes in mice. Conversely, the total cell numbers of CD4+CD8–CD25+ Treg cells in the thymi of aged mice were decreased markedly. It is known that CD4+CD25+ Treg cells could be produced in the thymus and in the periphery, although the mechanisms to keep the homeostatic state of peripheral CD4+CD25+ Treg cells have not been determined so far. Our present studies supported that the higher percentages of the peripheral CD4+CD25+ Treg cells might mainly be a result of the peripheral accumulation of CD4+CD25+ Treg cells.

Conversely, our studies showed that the majority of CD4+CD25+ T cells was Foxp3+, whereas few CD4+CD25– T cells express Foxp3 in young Balb/c mice. These data are consistent with the previous reports about the Foxp3 expression pattern in T cells in mice [32 , 47 ]. It seems that the Foxp3 expression pattern in T cells is different in mice and humans, as Foxp3 may express in human but not mouse CD4+ T cells during activation [48 ]. In addition, it was reported recently that aged C57BL/6 mice had a higher percentage of Foxp3+ cells in CD4+CD25– T cells [49 ]. However, our studies in aged Balb/c mice did not find the significant expression of Foxp3 in CD4+CD25– T cells in spleens and lymph nodes (<5%). The reasons for the somewhat inconsistent results are not clear at this moment. The possibility that different changes in Treg cells may occur in aged C57BL/6 and Balb/c mice cannot be excluded.

In summary, our present study indicated that the levels, phenotypes, Vß diversity, and function of CD4+CD25+ Treg cells were altered significantly with aging in mice. The declined ability of the thymus to produce Treg cells and the increased levels of the peripheral CD4+CD25+ Treg cells of aged mice suggest that the homeostasis of Treg cells in the periphery of aged mice was altered. The functional defects of CD4+CD25+ Treg cells in aged mice may shed light on the role of Treg cells in the increased sensitivity to autoimmune diseases of aged population.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Natural Science Foundation for Distinguished Young Scholars (C03020504 to Y. Z.), the National Basic Research Program (973 Program, 2003CB515501 to Y. Z.), 100 Quality Vocational Colleges of Chinese Academy of Sciences (2003-85 to Y. Z.), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars of State Education Ministry (2005-546 to Y. Z.). The authors thank Dr. David E. Corbin, Hong Shen, and Zeqing Niu for their kind review of the manuscript, Ms. Jing Wang, Mr. Yabing Liu, and Ms. Jianxia Peng for their expert technical assistance, Ms. Qinghuan Li for her excellent laboratory management, and Ms. Yuli Liu for her outstanding animal husbandry.

Received May 31, 2006; revised January 5, 2007; accepted January 18, 2007.

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