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* Section of Immunology, Department of Mechanism of Aging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Aichi, Japan;
Division of Gene Function in Animals, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan;
Department of Experimental Pathology, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan; and
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The results mentioned above were obtained through studies using young mice. In contrast, studies with humans and rodents have established that the functional deterioration of immune responses with aging is largely due to age-associated changes in the T cell compartment (20, 21, 22). It is unclear, however, whether functional changes (deterioration or augmentation) in CD4+CD25+ T cells occur with aging. In this report, we examined the functional potential of CD4+CD25+ T cells and CD4+CD25– T cells using various aged C57BL/6 (B6) mice. In vitro analyses revealed that, even in aged mice, CD4+CD25+ T cells maintained their suppressive properties without any functional deterioration or augmentation. In contrast with CD4+CD25+ T cells, whole CD4+CD25– T cells from aged mice showed diminished proliferative responses. Furthermore, our in vitro analyses showed that aged CD4+CD25– T cells included a Foxp3+ suppressive CD4 T cell population.
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Materials and Methods |
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Male C57BL/6 mice (2 mo old) were purchased from Japan SLC. All mice were maintained in a specific pathogen-free animal facility and treated in accordance with institutional guidelines for animal care. Aged mice with evidence of gross pathology were excluded from the study.
Preparation of lymphocytes
To purify CD4+CD25+ or CD4+CD25– T cells, B6 spleen cells stained with PE-anti-CD4 (H129.19) and FITC-anti-CD25 (7D4) Ab (all purchased from BD Pharmingen) were sorted with a cell sorter, JSAN (Bay Bioscience). In some experiments, cells were stained with FITC-anti-CD4 Ab, biotinylated anti-CD25 Ab, and PE-conjugated streptavidin (eBioscience). The purity of sorted CD4+CD25+ and CD4+CD25– T cells was >94 and >98%, respectively.
Spleen cells were depleted of CD25+ cells as described previously (8). Briefly, spleen cells were incubated at 5 x 107/ml for 45 min at 37°C with the culture supernatant (SN)4 of the hybridoma cells secreting anti-CD25 (7D4) Ab and rabbit complement 1/10-diluted at the final concentration (Cedarlane Laboratories). The treatment was repeated twice, and the resulting CD25+ cell-depleted cell fraction was then used for the preparation of CD4+CD25– T cells using magnetic beads conjugated with anti-CD4 (GK1.5) Ab and a magnetic column (Miltenyi Biotec). In all experiments, the efficiency of cell depletion and the purity of cells were analyzed with FACSCalibur (BD Biosciences) or JSAN after incubation with anti-CD25 (PC61) Ab plus FITC-anti-rat IgG (Caltag Laboratories) or PE-anti-CD4 (H129.19) Ab. Abs were all purchased from BD Pharmingen. The purity of the final CD4+CD25– T cell preparation was typically >98% CD4+ and <0.2% CD25+.
In vitro proliferation assay
The sorted T cells (1 x 104/well) were stimulated for 3 days with anti-CD3 (145–2C11) Ab SN, along with irradiated (20 Gy) young B6 spleen cells as APC (5 x 104/well), in 96-well round-bottom plates in DMEM supplemented with 10% FCS. The incorporation of [3H]thymidine (37 kBq/well) (DuPont/NEN) by proliferating T cells (triplicate cultures) during the last 6 h of the culture was measured. Data are the mean ± SD for triplicate wells.
Cell staining
The intracellular staining of mouse splenocytes with anti-Foxp3 Ab was done with the use of an anti-mouse Foxp3 staining set (eBioscience) according to the manufacturer’s directions. For rhodamine-123 (R123; Molecular Probes) staining, cells were incubated with 12.5 mM R123 for 10 min at 37°C, washed three times with cold PBS, incubated for an additional 30 min at 37°C, and washed. In some experiments, the stained cells were further stained with PE-anti-CD103 Ab (BD Pharmingen).
RNA analysis
For the quantitative analysis of mRNA expression, RNA was extracted from the samples using Isogen reagent (Nippon Gene), and cDNA was synthesized using standard procedures. The expression of Foxp-3 and hypoxanthine phosphoribosyltransferase (HPRT) was determined by quantitative real-time PCR using the primers below. Expression was normalized to the level of HPRT in each sample: Foxp-3, 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCACTTG-3'; and HPRT, 5'-TGAAGAGCTACTGTAATGATCAGTCAAC-3' and 5'-AGCAAGCTTGCAACCTTAACCA-3'.
Cytotoxicity assay
Spleen cells (5 x 106 cells in 2 ml) were cultured for given periods in 24-well plates (Costar). Viable cells were harvested and used as effectors in the 51Cr release cytotoxicity assay, in which target cells (1 x 106) were labeled with 3.7 MBq of sodium chromate (DuPont/NEN) for 60 min at 37°C, washed three times, and incubated at 1 x 104 cells/well with indicated numbers of effector cells in 96-well round-bottom plates (Costar) for 6 h at 37°C. The mean percentage specific lysis of duplicate cultures was calculated from the radioactivity of the SN: % specific lysis = 100 x (cpm experimental release – cpm spontaneous release)/(cpm maximal release – cpm spontaneous release). Spontaneous release from the target cells incubated in medium alone was always <15% of the maximal release obtained by adding 1 N HCl to the labeled target cells.
Tumor cells
B16 (B6-derived melanoma) (23) and RLmale1 (BALB/c-derived radiation leukemia) (24) used as target cells in cytotoxicity assays were gifts from Drs. H. Fujiwara (Osaka University, Osaka, Japan) and E. Nakayama (Okayama University, Okayama, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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It is well recognized that aging leads to a decline in the ability to mount normal CD4 T cell responses (20, 21, 22). This observation raises the possibility that the functional deterioration of whole CD4 T cells may result from a functional imbalance (qualitative or quantitative) of CD4+CD25+ T cells in aged hosts, which were recently reported as a T cell-subpopulation functioning to down-modulate immune responses (1, 2, 3). To investigate this possibility, we first compared the number of CD4+CD25+ T cells from B6 mice of different ages. The staining of B6 (2 mo old) spleen cells with anti-CD4 and anti-CD25 Ab is shown in Fig. 1A. In aged mice, we observed slightly increased total numbers of spleen cells (2 mo old: 901 x 105 ± 371 x 105, n = 5; 12 mo old: 982 x 105 ± 194 x 105, n = 7; 24 mo old: 1083 x 105 ± 287 x 105, n = 6). No significant differences were observed in the proportion of total CD4+ T cells from the spleens of 2-mo-old (24.2 ± 4.8%, n = 7), 12-mo-old (20.5 ± 5.0%, n = 10), or 24-mo-old (22.3 ± 3.8%, n = 9) B6 mice. In contrast, as shown in Fig. 1B, the proportion of CD25-expressing cells (R2 in Fig. 1A) among CD4+ T cells from 24-mo-old mice was slightly larger (
1.7-fold), as compared with that among CD4+ T cells from 2-mo-old mice (2 mo old: 9.1 ± 1.0%, n = 7; 12 mo old: 11.7 ± 1.9%, n = 10; 24 mo old: 15.6 ± 2.5%, n = 9).
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Next, we examined whether the CD4+CD25highFoxp3+ T cells from aged mice were functionally suppressive or not. The sorted CD4+CD25high T cells from young or aged mice were mixed with young CD4+CD25– T cells as responder cells at various ratios and cultured along with APC and anti-CD3 Ab. As shown in Fig. 1D, we observed the same level of suppression by CD4+CD25high T cells from young or aged mice. Both CD4+CD25high T cells exhibited an anergic state following polyclonal stimulation. Taken together, these results demonstrate that CD4+CD25highFoxp3+ T cells retain their suppressive function even in aged hosts.
Functional comparison of CD4+CD25– T cells prepared from young or aged B6 mice
In the absence of suppressive CD4+CD25+ T cells, the remaining CD4+CD25– T cells exhibit a more efficient activation in response to polyclonal stimulation (4, 6). However, we found that this was not the case for aged CD4+CD25– T cells. CD4+CD25– T cells from young and aged mice were stimulated in various culture conditions (Fig. 2A). Young CD4+CD25– T cells were stimulated more efficiently under better culture conditions (higher concentration of anti-CD3 Ab SN and/or optimal number of APC per well). However, aged CD4+CD25– T cells exhibited poor responsiveness even in the best culture conditions for young CD4+CD25– T cells, contrasting to the maintenance of function in aged CD4+CD25high T cells (Fig. 1D).
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Appearance of CD4+CD25–Foxp3+ T cells in aged mice
We next examined whether all aged CD4+CD25– T cells suffer some loss of function. To this end, we first investigated the expression of Foxp3 in all spleen cells. Foxp3-expressing cells belonged to the CD4 T cell population in young and aged mice (Fig. 3A). The proportion of Foxp3+ cells in aged mice was increased significantly (Foxp3+ cells among young CD4 T cells: 14.1 ± 2.8%, n = 4; among aged CD4 T cells: 32.1 ± 5.1%, n = 4; p = 0.00079). Next, the expression of CD25 in Foxp3+ and Foxp3– CD4 T cells (R4 and R5 in Fig. 3A, respectively) was analyzed (Fig. 3B). Again, we confirmed that almost all young CD4+Foxp3+ T cells expressed CD25 (CD25+ cells: 87.3 ± 4.0%, n = 4) and that aged CD4+Foxp3– T cells include CD25+ and CD25– cells, suggesting that the aged CD4 T cell population contains temporarily activated CD4+CD25+Foxp3– T cells. In contrast with young CD4+Foxp3+ T cells, surprisingly, a significant proportion of CD4+Foxp3+ T cells from aged mice were CD25 negative (CD25-positive cells: 70.5 ± 6.1%, n = 4), suggesting the appearance and/or an increase in the number of CD4+CD25–Foxp3+ T cells with aging.
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We have previously shown that aged CD4+CD25– T cells could be divided into three populations based on staining with R123 and anti-CD103 Ab (25). Both CD4+CD25– T cells from young and aged mice can incorporate R123 during a short period of exposure. However, following incubation for 30 min at 37°C under R123-free conditions, an obvious difference was observed between young and aged CD4+CD25– T cells, i.e., the majority of the aged cells extruded R123 (referred to as R123low), whereas the majority of the young cells retained R123. In addition, we observed a significant number of CD103+ cells in aged CD4+CD25– T cells. These results are reconfirmed in Fig. 4A. Furthermore, we reported that aged CD4+CD25–R123lowCD103+ T cells (cells in R6 in Fig. 4A) were unresponsive (anergic) upon stimulation and exerted a suppressive effect in a proliferation assay in vitro (25). This is also confirmed in Fig. 4B, demonstrating the appearance of a suppressive and anergic cell population among CD4+CD25– T cells in aged mice. In contrast, CD4+CD25–R123lowCD103– T cells (cells in R7) were significant low responders, and CD4+CD25–R123high T cells (cells in R8) maintained a normal level of responsiveness with some variation among individual cell preparations from aged mice (Fig. 4C).
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10% of CD4+CD25–R123lowCD103– T cells are Foxp3+ (in R7: 13.8%), and that another population of cells (CD4+CD25–R123high T cells) are all negative (in R8: 1.8%). These results are consistent with our previous results and ones mentioned above: aged CD4+CD25–R123lowCD103+ T cells (R6) had a suppressive function (Fig. 4B); CD4+CD25–R123lowCD103– T cells (R7) exhibited hyporesponsiveness to polyclonal stimulation (Fig. 4C) and weak suppressive activity compared with CD4+CD25–R123lowCD103+ T cells (25); and CD4+CD25–R123high T cells (R8) maintained a normal level of responsiveness (Fig. 4C) and are not suppressive (25). We further examined the Foxp3 expression in young CD4+CD25– T cell subpopulations separated in terms of R123 and CD103 staining (Fig. 4E). In a representative experiment, young CD4+CD25–R123high T cells (R8) contained 0.2% Foxp3-positive cells. In contrast, young CD4+CD25–R123lowCD103+ T cells (R6) and CD4+CD25–R123lowCD103– T cells (R7) contained 16.1 and 5.2% Foxp3-positive cells, respectively. It is notable that young CD4+CD25–R123lowCD103+ T cells (R6) consist of mainly Foxp3– cells, contrasting to the Foxp3 expression in aged CD4+CD25–R123lowCD103+ T cells (R6 in Fig. 4D).
Expression of Foxp3 in thymocytes from aged mice
We further examined the expression of CD25 on thymocytes prepared from aged mice (Fig. 5A). Although the thymus was very small in aged mice compared with young mice, the profile of CD25 expression in aged mice was almost the same as that in young mice (the staining profile of young thymocytes is shown in Ref. 26), i.e., the CD4+CD8– and CD4–CD8– cell populations contained CD25+ cells; the intensity of CD25 expression was greater on cells in the CD4–CD8– fraction than the CD4 single-positive fraction.
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Discussion |
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10% Foxp3+ cells (Fig. 3A). However, the former exhibited a significant low responsiveness (Fig. 4C) compared with the normal responsiveness by the latter. Based on these results, it is likely that a remarkable low responsiveness of aged CD4+CD25–R123lowCD103– T cells results from hyporesponsiveness of Foxp3– cells in aged CD4+CD25–R123lowCD103– T cells and not from a suppressive function of Foxp3+ cells included in aged CD4+CD25–R123lowCD103– T cells. Regarding the relationship between the thymus and aging, dramatic changes in the thymic microenvironment with aging, referred to as thymic involution, have long been considered the most reasonable cause of the age-related decline in T cell function (20, 21, 22). However, of particular importance in this study is that, even in aged mice, CD4+CD25high T cells maintained the ability to suppress the activation of normal young CD4+CD25– T cells (Fig. 1D), demonstrating that the functional maintenance of CD4+CD25high T cells is independent of the age-related changes of the thymic microenvironment. It is presumable that the expression of Foxp3 in (Fig. 1C, right panel) and/or the self-reactivity of CD4+CD25high T cells (28) are important to maintain the suppressive function throughout aging.
In the young, most suppressive/regulatory CD4 T cells were characterized by the constitutive expression of CD25. Even upon polyclonal T cell stimulation, CD4+CD25+ T cells expressed CD25 at higher levels and more persistently than CD4+CD25– T cell-derived activated T cells (29). As mentioned, we observed that CD4+CD25high T cells from aged mice were Foxp3+ (Fig. 1C, right panel) and suppressive (Fig. 1D). CD4+CD25low T cells from aged mice were a mixture of Foxp3+ and Foxp3– cells (Fig. 1C, left panel), suggesting that these CD4+CD25low T cells contain activated/nonsuppressive T cells. Even in humans, the experiments using human peripheral blood lymphocytes revealed that CD4+CD25high T cells, but not CD4+CD25low T cells, exhibited the characteristics of regulatory cells such as hyporesponsiveness and suppression of proliferation (30). Therefore, a better marker of suppressive/regulatory CD4 T cells may be CD25high. In contrast, the CD4+CD25– T cells from aged mice included Foxp3+ suppressive cells (as CD4+CD25–R123lowCD103+ T cells) (Fig. 4, A, B, and D). Aged CD4+CD25–R123lowCD103– T cells also included some Foxp3+ cells (Fig. 4D). Taken together, these results demonstrate that Foxp3, but not CD25 nor CD103, is the best marker of suppressive T cells even in aged hosts and suggest the need for a specific cell surface marker of suppressive/regulatory CD4 T cells.
Regarding the relationship between the marker CD25 and suppressive function, it was demonstrated that CD4+CD25+ T cells transferred into syngeneic nude mice change the level of CD25 expression, i.e., some CD4+CD25+ T cells adopt the CD4+CD25– phenotype, although they were still Foxp3+ and suppressive (16). Furthermore, we have shown that the responsiveness of young CD4+CD25–R123low cells (a mixture of R6 and R7) was slightly lower than young CD4+CD25–R123high cells (25), although it was not a dramatic hyporesponsiveness like R6 cells and/or R7 cells in aged mice (Fig. 4, B and C), that both young and aged CD4+CD25–R123low T cells contained Foxp3+ cells (Fig. 4, D and E), and that young CD4+CD25+ T cells exhibited R123low phenotype (25). Therefore, it is possible that CD4+CD25–R123lowCD103+ (and/or CD103–) Foxp3+ T cells in aged mice are derived from CD4+CD25+ (and/or CD25–) Foxp3+ suppressive T cells in the young. The relation between young CD4+Foxp3+ T cells and aged CD4+CD25–R123low Foxp3+ T cells will need to be investigated further. In addition, although analyses in vitro revealed that aged CD4+CD25–R123lowCD103+Foxp3+ T cells were anergic and suppressive (Fig. 4B) similar to CD4+CD25+ T cells in the young, at present, it is not clear whether these aged CD4+CD25–R123low CD103+Foxp3+ T cells have a role as immunoregulatory cells to maintain self-tolerance. CD4+CD25high T cells maintained their suppressive function even in aged mice (Fig. 1D). Therefore, it is unlikely that, in addition to aged CD4+CD25high T cells, aged CD4+CD25–R123lowCD103+Foxp3+ T cells were actively required to maintain self-tolerance in aged hosts.
In humans, it is reported that, during the activation of CD4+CD25– T cells in an immune response, two populations of cells may arise, effector CD4+CD25+ cells and Foxp3-expressing regulatory CD4+CD25+ T cells (31). Therefore, we examined whether the activation of CD4+CD25– T cells results in the expression of Foxp3 in aged mice, accounting for the existence of CD4+CD25–Foxp3+ T cells in aged mice (Figs. 3B and 4D). We purified cells in R7 and R8 in Fig. 4A from aged mice, cultured each cells (1 x 104/well in 96-round-bottom plate) along with splenic APC (1 x 104/well), anti-CD3 Ab (5% SN), and IL-2 (10 U/ml). Seven days later, cells were harvested and used for a real-time PCR experiment to evaluate the amount of Foxp3 in aged CD4+CD25– T cells before and after an in vitro stimulation culture. However, the activation culture induced no changes in the amount of Foxp3. For example, the relative Foxp3 expression (Foxp3/HPRT) in aged CD4+CD25–R123lowCD103– T cells (cells in R7 in Fig. 4A) and CD4+CD25–R123high T cells (R8) was 0.15 and 0.11 before stimulation and 0.04 and 0.08 after stimulation, respectively (compared with 7.49 for CD4+CD25–R123lowCD103+ T cells). Therefore, it is likely that this population (CD4+CD25–Foxp3+ T cells in aged mice) accumulates over time and under specific conditions in vivo, independently of any stimulation.
Age-related defects in CD4 T cell function have been well documented (20, 22). The consequences of these defects are substantial and lead to an increased incidence of infectious diseases in the elderly. Therefore, it is very important to improve the function of CD4 T cells in the elderly. In that regard, the results in this report suggest the importance of studies focused on the function of each CD4 T cell subpopulation, not on the whole CD4 T cell population. Our results show that aged CD4 T cells can be divided into suppressive Foxp3+ cells (consisting of CD25+ and CD25– T cells), hyporesponsive Foxp3–R123low cells, and normal responsive Foxp3–R123high cells. By inhibiting the suppressive function of Foxp3+ cells, by augmenting the responsiveness of normal Foxp3–R123high cells included as a minor population among aged CD4 T cells, or by preventing the gain of low responsiveness in Foxp3–R123low cells as a major population, proper treatments for each CD4 T cell subpopulation, not for CD4 T cells overall, would be needed for improving the CD4 T cell function in aged hosts.
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Footnotes |
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1 This work was supported by a Grant-in-Aid for Scientific Research (B) from Japan Society for the Promotion of Science, the Mitsubishi Foundation, Mitsui Sumitomo Insurance Welfare Foundation, and a Research Grant for Longevity Sciences (16A-2) (to J.S.). 
2 T.N. and J.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jun Shimizu, Section of Immunology, Department of Mechanism of Aging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan. E-mail address: jun{at}nils.go.jp
4 Abbreviations used in this paper: SN, supernatant; R123, rhodamine-123; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication December 29, 2005. Accepted for publication March 7, 2006.
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-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]) monoclonal antibody. Cancer Res. 59: 3128-3133. This article has been cited by other articles:
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and 
, respectively) were cultured for 3 days with anti-CD3 Ab (final 5% of SN) in the presence of young B6 splenic APC (20 Gy irradiated). Cell proliferation was measured on culture day 3. Young CD4+CD25– T cells, young CD4+CD25high T cells, and aged CD4+CD25high T cells cultured as well gave 78,242, 126, and 633 cpm, respectively, in a representative experiment. A result representative of three independent experiments is shown.
) B6 mice were cultured along with anti-CD3 Ab SN and APC. The amount of anti-CD3 Ab or the number of APC was titrated. Cell proliferation was measured on culture day 3. B, CD25+ cell-depleted splenic cells (designated as CD25–, 5 x 106 cells in 2 ml) prepared from 2-, 12-, or 24-mo-old B6 mice (circle, square, or triangle, respectively) were cultured without tumor cells for various periods and assessed for activity to kill B16 target cells at an E:T ratio of 80. A result representative of four independent experiments is shown. C, CD25– or uneliminated (whole) B6 spleen cells were cultured for 9 days and assessed for activity to kill RLmale1 or B16 tumor cells (closed or open symbols, respectively) at an E:T ratio of 80. Each symbol represents the activity of a single culture prepared from an individual mouse.
