Declines in immune function are well described in the elderly and are considered to contribute significantly to the disease burden in this population. Regulatory T cells (Tregs), a CD4+ T cell subset usually characterized by high CD25 expression, control the intensity of immune responses both in rodents and humans. However, because CD25 expression does not define all Tregs, especially in aged hosts, we characterized Tregs by the expression of FOXP3, a transcription factor crucial for Treg differentiation and function. The proportion of FOXP3+CD4+ Tregs increased in the blood of the elderly and the lymphoid tissues of aged mice. The expression of functional markers, such as CTLA-4 and GITR, was either preserved or increased on FOXP3+ Tregs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral Tregs from elderly humans improves effector T cell responses in most subjects. Importantly, Tregs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young Tregs. The increased proportion of Tregs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old Tregs efficiently suppressed the production of IFN-γ by effector T cells. Finally, in vivo depletion of Tregs in old mice attenuated disease severity. Accumulation of functional Tregs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of Treg numbers and/or activity may be envisioned to enhance the control of infectious diseases in this fragile population.

During aging, the integrity of the immune system progressively declines. In particular, the ability to fight off infections is decreased as evidenced by increased numbers of infections, more severe symptoms, prolonged duration, and poorer diagnosis (reviewed in Refs. 1, 2, 3, 4). Furthermore, reactivation of chronic infections occurs at a higher frequency in aged humans and mice (5, 6). These dysfunctions arise from alterations in every component of the immune system (7, 8, 9, 10), but the most consistent and significant alterations are seen in the T lymphocyte compartment (11, 12), particularly within CD4+ T cells (8, 13, 14, 15).

CD4+ regulatory T cells (Tregs)6 maintain self-tolerance in the periphery (16, 17, 18) and play a role in the control of autoimmunity and tumor immunity (18, 19, 20). They have been shown to decrease the levels of activation, proliferation, and cytokine production of effector T cells (Teffs) in mice and humans (21, 22, 23, 24), as well as control the immune function of dendritic cells (DCs) (25, 26). Tregs were first characterized by their expression of the IL-2R α-chain (CD25) (16). Additional molecules have been associated with Treg function, such as CTLA-4 (27) and the glucocorticoid-induced TNFR family-related protein (GITR) (28). More recently, the transcriptional factor FoxP3 (Forkhead box P3) has been shown to play a crucial role in many aspects of murine Treg biology, namely differentiation, function, and maintenance (29, 30, 31, 32). In humans, FOXP3 is also crucial for Treg function as evidenced by the acquisition of Treg activity following de novo FOXP3 expression in non-Tregs (33).

Previous studies have shown increased numbers of CD25+CD4+ Tregs in the periphery of aged BALB/c (34, 35) or C57BL/6 mice (36). Similar increases were also reported in the peripheral blood of elderly people (37, 38, 39, 40). Although, FOXP3 expression has recently been used to assess the proportion of Tregs in aged humans (41), it remains unclear whether Tregs maintain their suppressive activity in aged hosts. Indeed, some studies show maintenance of the suppressive activity of Tregs in aged mice (34, 36) and elderly people (37, 39), whereas other studies report decreased Treg-mediated suppression in aged mice (35) and humans (42).

During the acute phase of the infection by Leishmania major, activation of Teffs leads to the development of a small cutaneous lesion that heals spontaneously after few weeks (43). We have previously shown that during the chronic phase of the infection a high number of both Teffs (CD4+CD25 T cells, producing IFN-γ) and Tregs accumulate at sites of infection (44). A tight equilibrium between the two populations is responsible for the parasite persistence at the site of inoculation (44). Importantly, changes in the Treg:Teff balance at the local site induces parasite multiplication and subsequently reappearance of the lesion (45).

In this report, we show the following: 1) FoxP3+ Tregs accumulate in aged mice and elderly humans; 2) Tregs from aged mice and elderly humans are functional; and 3) depletion of Tregs in vitro and/or in vivo increases Teff responses. Together, these data suggest that Treg accumulation in aged hosts contributes to the immune suppression associated with aging.

Healthy elderly individuals (≥70 years old) were recruited in a retirement community in the Cincinnati, OH area. People in the upper third of functional status and with two or less comorbidities were eligible for enrollment. Enrolled individuals were not receiving immunosuppressive medication and had no chronic infection, known malignancy, or cognitive impairment. Volunteers with mild chronic conditions not thought to affect immune function were not excluded. Young healthy donors (≤30 years old) were recruited at Cincinnati Children’s Hospital Medical Center (Cincinnati, OH) with the same exclusion criteria as those used in the recruitment of elderly subjects. All subjects provided written informed consent to protocols approved by the corresponding Institutional Review Boards.

Six- to 8-wk-old C57BL/6 mice were purchased from Charles River Laboratories or Taconic Farms. Twenty-month-old C57BL/6 mice were purchased from Harlan through the National Institute on Aging contract. FoxP3-GFP knock-in C57BL/6 reporter mice were obtained from Dr. M. Oukka, Harvard Medical School, Cambridge, MA (46). Mice were maintained at the Children’s Hospital Research Foundation’s animal facility or the National Institutes of Health animal house facility under pathogen-free conditions. All experiments on mice were performed in accordance with institutional guidelines (Cincinnati Children’s Hospital Medical Center and National Institute of Allergy and Infectious Diseases).

For humans, blood samples (40 ml) were collected on sodium heparin. PBMCs were isolated by density centrifugation on Ficoll Paque Plus (GE Healthcare) within 4 h of sample collection and frozen in FCS with 10% DMSO (Sigma-Aldrich).

For mice, single cell suspensions were prepared from spleen, peripheral (retromaxillary and popliteal) lymph nodes (pLNs) and mesenteric lymph nodes (mLNs). Blood was collected on heparin. Erythrocytes were lysed by incubating the spleen or blood cells with 1 ml of ACK buffer (ammonium chloride-potassium; Cambrex) for 2 min on ice.

Thawed human PBMCs were incubated on ice for 5 min with human IgG (Sigma-Aldrich) to block Fc receptors and stained for 30 min for cell surface markers with a combination of the following Abs: anti-CD3-PerCP-Cy5.5 (clone SK7), anti-CD4-Pacific Blue or PE-Cy7 (clone RPA-T4), anti-CD25-allophycocyanin (clone M-A251), anti-CD69-allophycocyanin-Cy7 (clone FN50), anti-CCR5-PE-Cy7 (clone 2D7/CCR5), anti-CCR7-PE-Cy7 (clone 3D12), anti-integrin α4-allophycocyanin (clone 9F10), and anti-integrin β7-PE (clone FIB504) from BD Pharmingen; anti-CD27-allophycocyanin-Cy7 (clone O323) and anti-integrin β1-PE (clone MEM-101A) from eBioscience; anti-GITR-PE (clone 110416) and anti-TGFβRII-PE (clone 25508) from R&D Systems; anti-CD127-PE (clone IM1980) from Immunotech; and anti-CD45RA-Pacific Blue (clone MEM-56) and anti-CXCR4-allophycocyanin (clone 44717) from Caltag Laboratories. For intracellular staining, the eBioscience protocol for FOXP3 staining and the following Abs were used: anti-FOXP3-FITC (clone PCH101) and anti-CTLA-4-PE (clone 14D3) from eBioscience; anti-programmed death-1 (PD-1; clone MIH4) and anti-granzyme A-PE (clone CB9) from BD; anti-granzyme B-allophycocyanin (clone GB12) from Caltag Laboratories. Two hundred thousand events/sample were collected on a LSR II cytometer using the FACSDiva software (BD Biosciences). The CD25high, FOXP3+, GITR+, and CTLA-4+ gates were determined in relation to the staining on non-CD4+ T cells. The gate for granzyme B, CD127, CD45RA, or CD27 expression was set up according to the biphasic distribution of the particular marker. For all other markers the appropriate isotype-matched control Abs were used to define the positive gates within the CD4+CD3+ T cells.

Freshly isolated or cultured murine cells were incubated with the 24G2 cell line culture supernatant to block Fc receptors. Cells were stained for 20 min on ice with the following fluorochrome-conjugated Abs: anti-CD4-PE-Cy5 (clone RM4-5), anti-TCR-β-chain-allophycocyanin-Alexa Fluor 750 (clone H57-597), anti-CD25-allophycocyanin (clone PC61.5), anti-CD69-PE (clone H1.2F3), anti-CD103-FITC (clone 2E7), anti-CD27-allophycocyanin (clone LG.7F9), anti-CCR7-PE (clone 4B12), anti-GITR-allophycocyanin (clone DTA-1), and anti-PD-1-FITC (clone J43). The isotype controls used were rat IgG1 (clone R3-34), rat IgG2a (clone eBR2a), rat IgG2b (clone KLH/G2b-1-2), and hamster IgG (clone Ha4/8). Cells were washed with PBS and 1% FCS and then stained with anti-FoxP3-Pacific Blue (clone FJK-16s) and anti-CTLA-4-PE (clone UC10-4F10-11) using the FoxP3 staining set reagents and protocol from eBioscience. All Abs and controls were purchased from BD Pharmingen, eBioscience, or BioLegend. Cell acquisition was performed on a FACSCalibur cytometer using CellQuest Pro software or a LSRII cytometer using the FACSDiva software (BD Biosciences). Analysis was performed after gating on CD4+TCR-β+ cells, using CellQuest Pro software or FlowJo software (Tree Star).

Monocytes were isolated from PBMCs by positive selection using CD14 microbeads according to the manufacturer’s instructions (Miltenyi Biotec), achieving a purity of >87% as determined by flow cytometry. CD4+ T cells (>85% pure) were obtained by negative selection of CD14 cells using CD4+ T cell isolation kit II according to the manufacturer’s instructions (Miltenyi Biotec). CD4+ T cells were depleted of CD25+ cells using CD25 microbeads (Miltenyi Biotec). As depletion of CD25+ cells by microbeads induces nonspecific cell loss in the magnetic column, we controlled for that loss in the total CD4+ T cell fraction by using irrelevant microbeads (CD14 microbeads) according to manufacturer’s instructions. Passage of the CD4+ fraction through those CD14 microbeads did not change the percentage of CD25+ or FOXP3+CD4+ T cells (results not shown).

Treg-depleted CD4+ and total CD4+ fractions were labeled for 5 min at room temperature with 0.625 μM CFSE (Molecular Probes) in PBS. CFSE-labeled T cells (5 × 105) were cultured with 2 × 105 CD14+ monocytes in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml l-glutamine (Invitrogen), and 10% FCS and stimulated or not stimulated with PHA (2 μg/ml; Sigma-Aldrich) at 37°C in 5% CO2. After 3 days of stimulation, the recovered cells were incubated on ice for 5 min with human IgG to block Fc receptors and stained for 30 min with anti-CD4-PE (clone RPA-T4; BD Biosciences), anti-CD3-PerCP-Cy5.5, anti-CD69-allophycocyanin (clone FN50; BD Biosciences), and anti-CD95-Pacific Blue (clone DX2; Caltag Laboratories). After washes and formaldehyde fixation, up to 50,000 events per sample were collected on a LSR II cytometer with the FACSDiva software. Data were analyzed by using FlowJo software.

GFPCD4+ (Teffs) and GFP+CD4+ (Tregs) cells were sorted from spleens or pLNs of FoxP3-GFP knock-in C57BL/6 mice as described below for CD25highCD4+ and CD25CD4+ T cells. Teffs (5 × 104) were stimulated with 0.5 μg/ml anti-CD3 (clone 145-2C11; BD Biosciences) in the presence of 1 × 105 CD90+ cell-depleted and 3,000-rad γ-irradiated spleen cells. Cultures were set up in triplicate in 96-well U-bottom plates with different ratios of Tregs to Teffs. Forty-eight hours later, 1 μCi of [3H]thymidine was added into each well for 22 h. Plates were harvested and radioactivity measured on a Wallac TriLux MicroBeta scintillation counter.

Promastigotes (metacyclics) of L. major clone V1 (MHOM/IL/80/Friedlin) were isolated as previously described (47). Eight- to 10-wk old mice were infected in the ear dermis with 103L. major metacyclic promastigotes using a 27.5-gauge needle in a volume of 10 μl.

A single-cell suspension was prepared from the retromaxillary lymph nodes (LNs) of L. major-infected mice as described (43). CD4+ T cells were pre-enriched by negative selection using magnetic beads (CD4+ T cell isolation kit; Miltenyi Biotec). CD25highCD4+ and CD25CD4+ T cells were then purified using a FACSVantage cell sorter as previously described (21). The T cell subsets were >98% pure as analyzed by flow cytometry. Isolated cells were labeled for 5 min at room temperature with 1.25 μM CFSE in PBS. T cells (5 × 104) were incubated with 1.4 × 105 bone marrow-derived dendritic cells (BMDCs) in 200 μl of RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, 55 μM 2-ME, and 10% FCS as previously described (48). BMDCs were previously incubated overnight with or without L. major metacyclics (parasite:BMDC ratio = 5:1) and washed before culture with T cells. After 4 days of stimulation at 37°C in 5% CO2, cells were analyzed by flow cytometry (see above) and culture supernatants were collected for cytokine assays (see below).

Mice were injected with 1 mg of anti-CD25 (clone PC6C1; American Type Culture Collection) or isotype control (clone A1101-1) Abs as described previously (45). Abs were produced using serum-free medium (BD Biosciences) and a CELLine device (BD Biosciences) according to the manufacturer’s instructions. Abs were purified by protein G affinity chromatography (Pierce Chemical). The efficiency of depletion was shown to be >80%.

Mouse IFN-γ, IL-2, IL-10, and GM-CSF were quantified in culture supernatants using the DuoSet ELISA system (R&D Systems). Alternatively, a multiplex assay (Linco Research) was used following the manufacturer’s protocol.

Statistical analysis was performed using Prism (GraphPad Software). For mice, group comparisons were made using the two-group Student’s t test. For humans, the distribution of the values in the different groups was tested by a Shapiro-Wilk normality test. If the distribution was normal, group comparisons were made using the two-group Student’s t test. If the distribution was not normal, a Box-Cox transformation was made and transformed values were tested again for their normality. If the distribution became normal after transformation, a two-group Student’s t test was made; otherwise, group comparisons were made using a Mann-Whitney U test. A value of p < 0.05 was considered statistically significant. The Box-Cox transformation and the subsequent statistical analyses were conducted using SAS version 9.

FOXP3 has been identified as the most specific marker of Tregs in humans (32). We therefore investigated whether the number of FOXP3+CD4+ T cells was altered in elderly individuals (≥70 years old) compared with young adults (≤30 years old). Healthy donors were enrolled, applying criteria detailed in Materials and Methods. Mean ages of the elderly and young subjects were 82.8 ± 7.2 and 25.8 ± 2.7 years old, respectively.

The proportion of FOXP3+ cells was significantly increased within CD4+ T cells from elderly subjects compared with those from young subjects (5.8 ± 0.4 vs 4.4 ± 0.4%; p = 0.03, unpaired t test; n = 16/group; Fig. 1, A and B). Because the level of FOXP3 per cell is an important factor in determining Treg activity (49), we evaluated the mean fluorescence intensities (MFI) of FOXP3 staining, which were similar in elderly and young donors (Fig. 1,C). A high level of CD25 expression is also described as a characteristic feature of Tregs. The number of CD25highCD4+ T cells was determined using a stringent gate to define CD25high expression (Fig. 1,A). The proportion of CD25high cells in the CD4+ T cell population was not significantly different in elderly and young subjects, although there was a trend toward increased proportion in the elderly (Fig. 1, A and B; p = 0.1, Mann-Whitney U test). FOXP3+CD4+ T cells were more frequent than CD25highCD4+ T cells in both young and elderly subjects (Fig. 1, A and B; both p < 0.001). It has been recently described that Tregs expressed low levels of the IL-7 receptor CD127 and that this expression pattern better defines Tregs in humans (50, 51). We therefore analyzed the percentage of CD25+CD127lowCD4+ T cells and found an increased percentage of those cells in the elderly (6.5 ± 0.8 vs 3.9 ± 0.4% in elderly and young subjects, respectively; p = 0.01, t test). Thus, whether assessed by FOXP3, CD25, or CD127 expression, the frequency of Tregs is significantly increased in elderly humans.

FIGURE 1.

Treg frequency is increased in the blood of elderly individuals. Treg frequency was analyzed in PBMCs from 16 young (≤30-year old) and 16 elderly (≥70-year old) subjects. For FOXP3 staining, CD4CD3+ cells were used as negative control to determine the positivity threshold in CD4+CD3+ T cells. High expression of CD25 in CD4+ T cells was determined based on the absence of CD25high cells within the CD3 cells. A, Representative expression of FOXP3 and CD25 in young and elderly subjects. The percentages of FOXP3+CD4+ and CD25highCD4+ cells in gated CD3+ T cells in a representative young and an elderly subject are shown in the upper and middle plots, respectively. Expression of FOXP3 and CD25 in gated CD4+CD3+ T cells in a representative young and an elderly subject is shown in the lower plot. B, Percentages of FOXP3+ and CD25highCD4+CD3+ T cells in young and elderly subjects. Horizontal lines represent the mean values for each group. C, Representative FOXP3 expression in gated CD4+CD3+ T cells from a 23-year-old (filled histogram) and a 72-year-old (open histogram) donor.

FIGURE 1.

Treg frequency is increased in the blood of elderly individuals. Treg frequency was analyzed in PBMCs from 16 young (≤30-year old) and 16 elderly (≥70-year old) subjects. For FOXP3 staining, CD4CD3+ cells were used as negative control to determine the positivity threshold in CD4+CD3+ T cells. High expression of CD25 in CD4+ T cells was determined based on the absence of CD25high cells within the CD3 cells. A, Representative expression of FOXP3 and CD25 in young and elderly subjects. The percentages of FOXP3+CD4+ and CD25highCD4+ cells in gated CD3+ T cells in a representative young and an elderly subject are shown in the upper and middle plots, respectively. Expression of FOXP3 and CD25 in gated CD4+CD3+ T cells in a representative young and an elderly subject is shown in the lower plot. B, Percentages of FOXP3+ and CD25highCD4+CD3+ T cells in young and elderly subjects. Horizontal lines represent the mean values for each group. C, Representative FOXP3 expression in gated CD4+CD3+ T cells from a 23-year-old (filled histogram) and a 72-year-old (open histogram) donor.

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We then assessed the expression of markers on FOXP3+CD4+ T cells that have been previously associated with Treg phenotype and function (52), such as CD25, CTLA-4, and GITR. Expression of CD25, CTLA-4, and GITR by FOXP3+CD4+ Tregs was similar in young and elderly subjects (Table I; all p > 0.05). In both groups of subjects, FOXP3+CD4+ Tregs also expressed higher levels of CTLA-4 than FOXP3CD4+ T cells (∼40 vs 4%; p < 0.0001) (Table I). Similar data were obtained when CD25high expression was analyzed (Table I). GITR expression was very low in all groups (<7%), but FOXP3+CD4+ Tregs expressed significantly higher levels than FOXP3CD4+ T cells in both groups of subjects (Table I). We also analyzed the proportion of naive Tregs based on their expression of CD45RA. The proportion of naive Tregs was lower in young individuals than the proportion of naive Teffs, as previously reported (53). This proportion was further decreased in elderly individuals, although the difference did not reach statistical significance (p = 0.09; Table I). We also analyzed the expression of several molecules that have been reported to correlate with regulatory activity in human or mice Tregs, such as CD27 (54, 55, 56), PD-1 (57, 58), TGFβRII (59), and granzymes A and B (60, 61, 62). CD27 expression by FOXP3+CD4+ Tregs was similar in young and elderly subjects (Table I), in agreement with a recent publication (41). PD-1, TGFβRII, and granzyme A/B expression by Tregs was also similar between young and old Tregs (Table I).

Table I.

Phenotypic characterization of Tregs and Teffs in young and elderly subjectsa

YoungElderlypTreggpTeffh
NbTregTeffpcRatiofNbTregTeffpcRatiof
CD25high 16 42.5 ± 2.4 0.6 ± 0.1 e  16 49.1 ± 2.9 1.0 ± 0.3 e  0.10 0.32 
CD25+ 16 75.4 ± 1.8 8.5 ± 0.8 e  16 78.8 ± 1.9 11.8 ± 1.8 e  0.20 0.45 
CD25+ 58.8 ± 4.3 2.2 ± 0.2 e  11 69.0 ± 3.1 3.4 ± 0.6 e  0.07 0.21 
CD127low             
CTLA-4+ 16 44.6 ± 3.9 3.9 ± 0.6 e  13 39.8 ± 3.9 3.7 ± 0.4 e  0.41 0.96 
GITR+ 16 5.2 ± 0.4 1.3 ± 0.1 e  16 6.4 ± 0.7 1.7 ± 0.3 e  0.16 0.24 
CD45RA+ 10 14.7 ± 2.5 39.0 ± 4.3 e 2.6 10 9.5 ± 1.4 21.5 ± 3.0 d 2.3 0.09 <0.01 
CD27+ 16 92.1 ± 0.9 88.5 ± 1.5 d  13 93.3 ± 0.9 81.2 ± 4.6 d  0.38 0.17 
PD-1+ 22.9 ± 7.9 21.9 ± 9.2 NS  11 22.8 ± 6.8 25.1 ± 7.6 NS  0.82 0.59 
TGFβRII+ 6.8 ± 2.6 2.2 ± 0.6 NS  10 4.9 ± 1.1 2.0 ± 0.4 d  1.00 0.82 
Granzyme A+ 3.7 ± 1.1 4.1 ± 1.0 NS  4.6 ± 2.0 7.2 ± 3.0 NS  1.00 1.00 
Granzyme B+ 4.0 ± 1.5 4.1 ± 1.3 NS  4.2 ± 1.6 12.6 ± 4.5 d  0.55 0.05 
CD69+ 15 4.3 ± 0.9 2.7 ± 0.4 NS  15 4.4 ± 0.9 2.4 ± 0.3 NS  0.98 0.80 
CCR5+ 27.5 ± 4.3 9.4 ± 1.2 e 0.3 11 28.6 ± 3.2 15.1 ± 1.9 d 0.5 0.85 0.02 
CCR7+ 18 29.4 ± 3.6 41.0 ± 5.2 NS 1.3 19 38.4 ± 3.9 49.8 ± 5.2 NS 1.3 0.10 0.23 
CXCR4+ 28.2 ± 3.7 37.4 ± 3.6 NS 1.3 11 15.8 ± 2.0 33.2 ± 2.1 e 2.1 <0.01 0.31 
α41+ 18 44.2 ± 2.0 65.2 ± 2.6 e 1.4 19 38.5 ± 2.1 63.1 ± 2.2 e 1.6 0.06 0.54 
α47+ 18 12.0 ± 1.2 41.5 ± 2.4 e 3.5 19 7.6 ± 0.6 29.0 ± 2.5 e 3.8 0.02 0.01 
YoungElderlypTreggpTeffh
NbTregTeffpcRatiofNbTregTeffpcRatiof
CD25high 16 42.5 ± 2.4 0.6 ± 0.1 e  16 49.1 ± 2.9 1.0 ± 0.3 e  0.10 0.32 
CD25+ 16 75.4 ± 1.8 8.5 ± 0.8 e  16 78.8 ± 1.9 11.8 ± 1.8 e  0.20 0.45 
CD25+ 58.8 ± 4.3 2.2 ± 0.2 e  11 69.0 ± 3.1 3.4 ± 0.6 e  0.07 0.21 
CD127low             
CTLA-4+ 16 44.6 ± 3.9 3.9 ± 0.6 e  13 39.8 ± 3.9 3.7 ± 0.4 e  0.41 0.96 
GITR+ 16 5.2 ± 0.4 1.3 ± 0.1 e  16 6.4 ± 0.7 1.7 ± 0.3 e  0.16 0.24 
CD45RA+ 10 14.7 ± 2.5 39.0 ± 4.3 e 2.6 10 9.5 ± 1.4 21.5 ± 3.0 d 2.3 0.09 <0.01 
CD27+ 16 92.1 ± 0.9 88.5 ± 1.5 d  13 93.3 ± 0.9 81.2 ± 4.6 d  0.38 0.17 
PD-1+ 22.9 ± 7.9 21.9 ± 9.2 NS  11 22.8 ± 6.8 25.1 ± 7.6 NS  0.82 0.59 
TGFβRII+ 6.8 ± 2.6 2.2 ± 0.6 NS  10 4.9 ± 1.1 2.0 ± 0.4 d  1.00 0.82 
Granzyme A+ 3.7 ± 1.1 4.1 ± 1.0 NS  4.6 ± 2.0 7.2 ± 3.0 NS  1.00 1.00 
Granzyme B+ 4.0 ± 1.5 4.1 ± 1.3 NS  4.2 ± 1.6 12.6 ± 4.5 d  0.55 0.05 
CD69+ 15 4.3 ± 0.9 2.7 ± 0.4 NS  15 4.4 ± 0.9 2.4 ± 0.3 NS  0.98 0.80 
CCR5+ 27.5 ± 4.3 9.4 ± 1.2 e 0.3 11 28.6 ± 3.2 15.1 ± 1.9 d 0.5 0.85 0.02 
CCR7+ 18 29.4 ± 3.6 41.0 ± 5.2 NS 1.3 19 38.4 ± 3.9 49.8 ± 5.2 NS 1.3 0.10 0.23 
CXCR4+ 28.2 ± 3.7 37.4 ± 3.6 NS 1.3 11 15.8 ± 2.0 33.2 ± 2.1 e 2.1 <0.01 0.31 
α41+ 18 44.2 ± 2.0 65.2 ± 2.6 e 1.4 19 38.5 ± 2.1 63.1 ± 2.2 e 1.6 0.06 0.54 
α47+ 18 12.0 ± 1.2 41.5 ± 2.4 e 3.5 19 7.6 ± 0.6 29.0 ± 2.5 e 3.8 0.02 0.01 
a

PBMCs were obtained from healthy elderly individuals (≥70 years old) and young donors (≤30 years old). Tregs were defined as FOXP3+CD4+and Teffs as FOXP3CD4+T cells. Values represent the mean (±SEM) percentage of positive cells for each marker.

b

N is the number of tested samples.

c

The p values compare the percentage of Tref and Teff expressing each marker.

d

p < 0.0001;

e

p < 0.05; NS, not significant (p > 0.05).

f

Ratio was calculated as the proportion of Teffs expressing each marker divided by the proportion of Tregs expressing it.

g

The pTreg values compare the percentage of Tregs expressing each marker in young versus elderly subjects.

h

The pTeff values compare the percentage of Teffs expressing each marker in young versus elderly subjects.

FOXP3, CD25, GITR, and CTLA-4 are not only markers for Tregs but are also transiently up-regulated on human non-Tregs upon activation (22, 63, 64). Therefore, we investigated whether the increased percentage of FOXP3+CD4+ T cells in elderly subjects reflects increased numbers of activated T cells. To do that, we looked at the expression of CD69 by FOXP3+ cells (65). Low CD69 expression (<5%) was observed in FOXP3+CD4+ Tregs from both young and elderly and was similar between the two groups (Table I). Together, these data indicate that the phenotype of circulating FOXP3+CD4+ Tregs is similar between young and elderly subjects, as assessed by multiple markers. Moreover, it appears unlikely that the increased proportion of FOXP3+CD4+ T cells in elderly individuals reflect recent activation, because it was not associated with the expression of a classical activation marker.

The increased proportion of Tregs in the blood from elderly subjects could be the consequence of their altered homing to tissues. To address this issue, we analyzed the expression by Tregs and Teffs of several T cell homing markers, namely CCR7, which mediates T cell entry into secondary lymphoid organs (66), α4β1 integrin, CXCR4, and CCR5, which allow T cell migration to inflamed tissues, and the gut-associated α4β7 integrin (67, 68, 69, 70, 71, 72).

FOXP3+CD4+ Tregs expressed similar levels of CCR5, CCR7, and α4β1 in young and old subjects (Table I), as recently reported for CCR7 (41). In contrast, FOXP3+CD4+ Tregs expressed lower levels of α4β7 in aged subjects compared with those in young subjects (Table I). However, the same change in expression pattern was observed in Teffs from old subjects compared with young Teffs (Table I). Indeed, when the ratio of expression (Teffs vs Tregs) was calculated for homing markers, no difference was found between old and young subjects (Table I). Only CXCR4 expression was different, with a specific decrease on old Tregs. Interestingly, CXCR4 has been associated with Treg migration and maintenance in the bone marrow (71). Altogether, these data suggest that the increased proportion of circulating Tregs in the elderly is not likely due to a selective dysregulation of Treg homing to tissues, although we cannot rule out a role for decreased CXCR4 in the retention of Tregs in the blood.

With further determination of Treg dynamics in humans being difficult due to the obviously restricted access to tissues, we pursued our analysis through the characterization of Treg markers in multiple lymphoid organs from aged (≥20 mo old) and young adult C57BL/6 mice (≤3 mo old). In all tissues, the proportion of FoxP3+CD4+TCR+ cells was significantly higher in aged mice compared with young mice (Fig. 2,A). FoxP3 levels per cell were identical in cells from aged and young mice (Fig. 2,B). In contrast, the proportion of circulating FoxP3+CD4+TCR+ cells was the same in the blood of aged and young mice (Fig. 2,A). When the proportion of CD25highCD4+TCR+ cells was analyzed, we found a significant accumulation of these cells in pLNs and mLNs, but not in the spleen or the blood, of aged mice compared with young mice (Fig. 2,C). The proportion of CD25highCD4+TCR+ cells was always lower than the proportion of FoxP3+CD4+TCR+ cells in all tissues in young and in aged mice (all p < 0.05). Furthermore, the proportion of Tregs expressing CD103, a specific marker for natural Tregs (73, 74), was increased in aged mice (Fig. 2 D).

FIGURE 2.

Increased proportion of Tregs in aged mice. Single cell suspensions from spleens, pLNs, mLNs, LNs, and blood were first stained for the surface markers CD4, TCR, CD25, CD69, CD103, and PD-1, followed by staining for the intracellular marker FoxP3. Flow cytometry analysis on gated CD4+TCR+ cells is shown. A, Percentages of FoxP3+ cells in the CD4+TCR+ cell populations from 2- to 3-mo-old (closed circles) or 20- to 28-mo-old (open circles) mice. Horizontal lines represent the mean values for each group. B, Representative overlay of FoxP3 expression in spleen, pLN, and mLN cells from a 3-mo-old (filled histogram) and a 28-mo-old (open histogram) old mouse. C, Percentages of CD25high cells in the CD4+TCR+ cell populations from 2- to 3-mo-old (closed circles) or 20- to 28-mo-old (open circles) mice. Horizontal lines represent the mean values for each group. D, Expression of FoxP3 with CD69, CD103, and PD-1 is shown for splenic cells of a 3-mo-old (left panel) or 28-mo-old (right panel) mouse, representative of six mice each. Values represent the percentages of each population in the indicated quadrant. Values in parenthesis are the percentages of gated FoxP3 (left) or FoxP3+ (right) cells that are positive for the y-axis marker.

FIGURE 2.

Increased proportion of Tregs in aged mice. Single cell suspensions from spleens, pLNs, mLNs, LNs, and blood were first stained for the surface markers CD4, TCR, CD25, CD69, CD103, and PD-1, followed by staining for the intracellular marker FoxP3. Flow cytometry analysis on gated CD4+TCR+ cells is shown. A, Percentages of FoxP3+ cells in the CD4+TCR+ cell populations from 2- to 3-mo-old (closed circles) or 20- to 28-mo-old (open circles) mice. Horizontal lines represent the mean values for each group. B, Representative overlay of FoxP3 expression in spleen, pLN, and mLN cells from a 3-mo-old (filled histogram) and a 28-mo-old (open histogram) old mouse. C, Percentages of CD25high cells in the CD4+TCR+ cell populations from 2- to 3-mo-old (closed circles) or 20- to 28-mo-old (open circles) mice. Horizontal lines represent the mean values for each group. D, Expression of FoxP3 with CD69, CD103, and PD-1 is shown for splenic cells of a 3-mo-old (left panel) or 28-mo-old (right panel) mouse, representative of six mice each. Values represent the percentages of each population in the indicated quadrant. Values in parenthesis are the percentages of gated FoxP3 (left) or FoxP3+ (right) cells that are positive for the y-axis marker.

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We also analyzed the expression by FoxP3+CD4+ T cells of markers associated with Treg function. FoxP3+CD4+TCR+ Tregs express high levels of GITR, CTLA-4, and PD-1 in both groups of animals, with a trend toward higher expression in aged mice depending on the tissue analyzed (Table II and Fig. 2,D). The activation marker CD69 was more expressed by old FoxP3+ cells than young FoxP3+CD4+TCR+ cells in all analyzed tissues (Table II and Fig. 2,D). However, there was a trend toward decreased CD69 expression on circulating Tregs from aged mice (p = 0.07, Table II). The trend toward decreased proportion of CD27 and CCR7 expression in old Tregs (Table II) also suggests increased Treg differentiation in aged mice.

Table II.

Expression of markers in FoxP3+CD4+TCR+ cells from young and aged micea

TissueCD25+GITR+CTLA-4+
YoungAgedpbYoungAgedpbYoungAgedpb
Spleenc 62.6 ± 1.8 35.6 ± 4.4 <0.01 93.1 ± 1.0 97.5 ± 0.7 <0.01 51.5 ± 5.1 65.6 ± 6.3 0.11 
PLNc 72.6 ± 1.1 65.3 ± 5.2 0.19 ND ND ND 55.0 ± 5.2 71.8 ± 5.7 0.05 
MLNc 72.0 ± 1.1 58.6 ± 4.5 0.02 93.1 ± 1.5 95.5 ± 1.3 0.24 55.6 ± 5.7 76.6 ± 6.2 0.03 
Bloodd 37.2 ± 2.7 33.2 ± 2.3 0.33 83.9 ± 5.2 94.0 ± 1.2 0.13 26.7 ± 5.0 26.1 ± 1.2 0.92 
TissueCD25+GITR+CTLA-4+
YoungAgedpbYoungAgedpbYoungAgedpb
Spleenc 62.6 ± 1.8 35.6 ± 4.4 <0.01 93.1 ± 1.0 97.5 ± 0.7 <0.01 51.5 ± 5.1 65.6 ± 6.3 0.11 
PLNc 72.6 ± 1.1 65.3 ± 5.2 0.19 ND ND ND 55.0 ± 5.2 71.8 ± 5.7 0.05 
MLNc 72.0 ± 1.1 58.6 ± 4.5 0.02 93.1 ± 1.5 95.5 ± 1.3 0.24 55.6 ± 5.7 76.6 ± 6.2 0.03 
Bloodd 37.2 ± 2.7 33.2 ± 2.3 0.33 83.9 ± 5.2 94.0 ± 1.2 0.13 26.7 ± 5.0 26.1 ± 1.2 0.92 
CD27+CCR7+CD69+
TissueYoungAgedpbYoungAgedpbYoungAgedpb
Spleend 87.0 ± 1.9 70.1 ± 3.7 0.02 5.2 ± 0.6 1.5 ± 0.2 0.02 14.8 ± 0.5 50.2 ± 1.6 <0.01 
pLNd 85.4 ± 1.6 75.5 ± 3.5 0.06 3.7 ± 0.8 1.5 ± 0.5 0.08 20.1 ± 0.9 38.4 ± 1.7 <0.01 
mLNd 86.3 ± 0.8 73.0 ± 1.3 <0.01 4.4 ± 0.6 2.0 ± 0.5 0.03 25.8 ± 0.0 59.2 ± 5.4 <0.01 
Bloodd 27.0 ± 6.6 11.2 ± 1.8 0.08 15.4 ± 7.1 3.5 ± 1.2 0.18 19.2 ± 5.7 5.2 ± 1.0 0.07 
CD27+CCR7+CD69+
TissueYoungAgedpbYoungAgedpbYoungAgedpb
Spleend 87.0 ± 1.9 70.1 ± 3.7 0.02 5.2 ± 0.6 1.5 ± 0.2 0.02 14.8 ± 0.5 50.2 ± 1.6 <0.01 
pLNd 85.4 ± 1.6 75.5 ± 3.5 0.06 3.7 ± 0.8 1.5 ± 0.5 0.08 20.1 ± 0.9 38.4 ± 1.7 <0.01 
mLNd 86.3 ± 0.8 73.0 ± 1.3 <0.01 4.4 ± 0.6 2.0 ± 0.5 0.03 25.8 ± 0.0 59.2 ± 5.4 <0.01 
Bloodd 27.0 ± 6.6 11.2 ± 1.8 0.08 15.4 ± 7.1 3.5 ± 1.2 0.18 19.2 ± 5.7 5.2 ± 1.0 0.07 
a

Single-cell suspensions from spleens, pLNs, mLNs, and blood were stained for surface expression of CD4, TCR, CD25, CD27, CD69, CCR7, and GITR, followed by intracellular staining for FoxP3 and CTLA-4. Results are expressed as mean (±SEM) percentages of FoxP3+CD4+TCR+ cells expressing each marker. Young mice were 2–3 mo old and aged mice were 20–28 mo old.

b

The p values (t test) compare proportions in young and old mice.

c

Six mice/group were analyzed; ND, Not determined.

d

Three mice per group were analyzed.

Our phenotypic data show an increased proportion of cells with Treg characteristics in aged humans and mice. However, because FOXP3+ or CD25high cells are not always functionally suppressive, we further characterized Treg function in elderly humans by analyzing the effect of Treg depletion on CD4 function. Because of its intracellular localization, FOXP3 expression cannot be used to deplete cells. However, the number of FOXP3+ cells was significantly reduced following the depletion of CD25+ cells from total CD4+ T cells and no significant difference was observed between individuals (Fig. 3,A). In the absence of stimulation, CD4+ T cells, with or without Tregs, did not proliferate or express activation markers (data not shown). After 3 days of PHA stimulation, Treg-depleted cells proliferated better than total CD4+ T cells (not depleted of Tregs) in four of the seven tested individuals (Fig. 3,B, group A, subjects □, ▵, ▿, and +). In contrast, in three individuals Treg depletion did not result in increased proliferation after PHA stimulation (Fig. 3 B, group B, subjects ⋄, ○, and ×).

FIGURE 3.

Depletion of CD25highCD4+ T cells from the blood of elderly individuals increased CD4 T cell function. CFSE-labeled total CD4+ or Treg-depleted CD4+ (CD25CD4+) T cells (5 × 105) from seven elderly individuals (≥70 years old) were cultured with 2 × 105 autologous CD14+ monocytes and 2 μg/ml PHA for 3 days. A, Percentage of FOXP3+ cells before CD25high depletion (total CD4+; left) and after CD25high depletion (Treg-depleted CD4+; right). The values from the same subject are linked by a line. Group A is composed of the four individuals in whom Treg depletion led to increased proliferation, whereas group B comprised the three individuals who exhibited no increase in proliferation after PHA stimulation. The percentage of FOXP3+ cells following CD25 depletion was determined in all subjects, except subject ×. B and C, CFSE dilution (B) and the expression of CD69 and CD95 markers (C) were analyzed by flow cytometry on gated CD4+CD3+ T cells. Percentages of dividing cells (CFSElow, corresponding to cells that have divided at least once) in total or Treg-depleted CD4+ T cells are shown in B. Percentages of CD69+ and CD95+ cells in total or Treg-depleted CD4+ T cells are shown in C. Each individual is represented by the same symbol in all three panels.

FIGURE 3.

Depletion of CD25highCD4+ T cells from the blood of elderly individuals increased CD4 T cell function. CFSE-labeled total CD4+ or Treg-depleted CD4+ (CD25CD4+) T cells (5 × 105) from seven elderly individuals (≥70 years old) were cultured with 2 × 105 autologous CD14+ monocytes and 2 μg/ml PHA for 3 days. A, Percentage of FOXP3+ cells before CD25high depletion (total CD4+; left) and after CD25high depletion (Treg-depleted CD4+; right). The values from the same subject are linked by a line. Group A is composed of the four individuals in whom Treg depletion led to increased proliferation, whereas group B comprised the three individuals who exhibited no increase in proliferation after PHA stimulation. The percentage of FOXP3+ cells following CD25 depletion was determined in all subjects, except subject ×. B and C, CFSE dilution (B) and the expression of CD69 and CD95 markers (C) were analyzed by flow cytometry on gated CD4+CD3+ T cells. Percentages of dividing cells (CFSElow, corresponding to cells that have divided at least once) in total or Treg-depleted CD4+ T cells are shown in B. Percentages of CD69+ and CD95+ cells in total or Treg-depleted CD4+ T cells are shown in C. Each individual is represented by the same symbol in all three panels.

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Because proliferation does not recapitulate overall CD4 T cell function, particularly for memory cells, we also analyzed other readouts of T cell activation such as expression of the activation markers CD69 and CD95 (Fig. 3,C). PHA stimulation induced CD69 and CD95 up-regulation and cytokine production in all PHA-stimulated cultures compared with unstimulated cultures (data not shown). In three of four samples from group A, CD69 and CD95 expression both increased following Treg depletion (Fig. 3,C). In the fourth subject from that group (subject +), Treg depletion led to decreased CD69 expression but stable and high CD95 expression (Fig. 3,C). Of note, this individual exhibited the highest level of proliferation before and after Treg depletion. In group B individuals, depletion did not change CD95 expression and increased CD69 expression in only one individual (Fig. 3 C, group B, subject ○). Depletion of Tregs therefore increased activation and/or proliferation of CD4+ Teffs in five of seven elderly subjects after 3 days of culture with PHA. Of note, this percentage of responders is similar to that reported in young individuals in whom Treg depletion led to increased proliferation in response to PHA in four of six individuals (75). These data suggest that Tregs in elderly humans are functional in most individuals and may be able to inhibit Teff responses in vitro.

To more clearly delineate the functional activity of FoxP3+ cells during aging, we sorted FoxP3+ T cells from pLNs of aged (15–18 mo old) or young (2–4-mo old) FoxP3-GFP knock-in C57BL/6 mice. Teffs (CD4+GFP T cells) were sorted from pLNs of 2-mo-old mice, to eliminate the confounding effect of decreased responsiveness of aged Teffs (reviewed by Refs. 76 and 77). Tregs from pLNs of aged mice were more suppressive on a per cell basis than those from young mice, suppressing 80% of anti-CD3-stimulated Teff proliferation at a Teff:Treg ratio of 1:1 in comparison to the 54% suppression achieved by young Tregs (Fig. 4). At a Teff:Treg ratio of 10:1, 20% suppression was observed with old Tregs compared with the 3% induced by young Tregs (Fig. 4). Interestingly, Teffs from aged mice could be inhibited equally by Tregs from old and young mice (data not shown).

FIGURE 4.

FoxP3+CD4+ T cell suppressive function is intact in aged mice. GFPCD4+ T cells (Teffs) (5 × 104) were sorted from LNs of 2- to 4-mo-old FoxP3-GFP knock-in C57BL/6 mice and stimulated in triplicate with 0.5 μg/ml anti-CD3 and 1 × 105 irradiated T cell-depleted spleen cells from the same mice. GFP+CD4+ T cells (Tregs) were sorted from LNs of 2- to 4-mo-old (▪) or 15- to 18-mo-old (□) mice and cocultured with Teffs at different Teff:Treg ratios ranging from 1.3:1 to 101:1. Proliferation was measured by thymidine incorporation in the last 22 h of a 3-day culture. In the absence of Tregs, 21,800 ± 2,075 cpm were counted.

FIGURE 4.

FoxP3+CD4+ T cell suppressive function is intact in aged mice. GFPCD4+ T cells (Teffs) (5 × 104) were sorted from LNs of 2- to 4-mo-old FoxP3-GFP knock-in C57BL/6 mice and stimulated in triplicate with 0.5 μg/ml anti-CD3 and 1 × 105 irradiated T cell-depleted spleen cells from the same mice. GFP+CD4+ T cells (Tregs) were sorted from LNs of 2- to 4-mo-old (▪) or 15- to 18-mo-old (□) mice and cocultured with Teffs at different Teff:Treg ratios ranging from 1.3:1 to 101:1. Proliferation was measured by thymidine incorporation in the last 22 h of a 3-day culture. In the absence of Tregs, 21,800 ± 2,075 cpm were counted.

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We next reasoned that, if Treg function and proportion were increased with age, it would interfere with the ability of the aged hosts to control chronic infections. To test this hypothesis, we used the L. major model in which Tregs have been shown to play a major role in lesion reactivation (44, 45). After inoculation of 103 metacyclic promastigotes of L. major into the ear dermis, young C57BL/6 mice develop a small lesion that resolves spontaneously within 12 wk postinoculation, although a few viable parasites persist in the site of the former lesion and in the draining LNs (44). Eight weeks after healing, 5% of the infected mice exhibited clinical signs of lesion reactivation (Fig. 5). Importantly, spontaneous reactivation increased with aging until 75% of the mice had reactivated at 24 mo after healing (Fig. 5), suggesting increased Treg activity in aged L. major-infected mice.

FIGURE 5.

L. major spontaneously reactivates in aged mice. Eight-week-old C57BL/6 mice (n = 20) were inoculated in the ear dermis with 103L. major metacyclic promastigotes. After the lesions were resolved 12 wk later, mice were monitored for clinical signs of ear swelling and inflammation indicating a reactivation of the lesions.

FIGURE 5.

L. major spontaneously reactivates in aged mice. Eight-week-old C57BL/6 mice (n = 20) were inoculated in the ear dermis with 103L. major metacyclic promastigotes. After the lesions were resolved 12 wk later, mice were monitored for clinical signs of ear swelling and inflammation indicating a reactivation of the lesions.

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We previously reported that Tregs from L. major-infected young mice are able to respond specifically to L. major (78). Therefore, we analyzed whether L. major-specific Treg activity was detectable at the time when spontaneous disease reactivation occurs. CD25+CD4+ Tregs were purified from draining LNs of aged mice or young mice that had all been infected when they were 2-mo old and were restimulated in vitro with BMDCs infected or not infected with L. major metacyclic promastigotes. In aged mice, extensive proliferation of Tregs was detected in response to both uninfected and infected BMDCs (Fig. 6,A). The fact that old Tregs from infected mice proliferate in the presence of uninfected BMDCs may reflect their higher state of in vivo activation, a finding in agreement with their higher expression of several activation markers on Tregs from noninfected mice (Table II and Fig. 2). However, the lower MFI observed in the culture with infected BMDCs suggests a more sustained proliferation of Tregs in this condition. Tregs from aged mice also produced cytokines in response to infected BMDCs, although at a lower level than young Tregs (Fig. 6 B). Taken together, these results suggest that the Treg activation level is higher in aged mice, potentially through Ag-independent pathways, but the proportion of L. major-specific Tregs and/or their per cell basis capacity to respond is lower. There are several potential explanations for these results. First, as Ag load increases, more Tregs are stimulated by L. major Ag in vivo and continue to proliferate when they are removed from the animal, seemingly nonspecifically. Second, Tregs that are not specific for L. major are expanded in vivo and recognize and respond to endogenous Ags displayed on BMDCs. Third, the proliferation of aged Tregs in response to uninfected BMDCs has nothing to do with Ag recognition by Tregs but could be due to soluble factors released from BMDCs that cause Tregs to proliferate. Without a L. major-specific MHC class II tetramer, it is difficult to address the frequency of L. major-specific Tregs or how this frequency changes with age and whether exogenous/endogenous Ags vs soluble factors drive Treg proliferation.

FIGURE 6.

Tregs from aged mice respond to L. major. Eight- to 10-wk old C57BL/6 mice were inoculated in the ear dermis with L. major. Five or 21 mo later CD25+CD4+ Tregs were purified by FACS from the draining LNs. CFSE-labeled T cells (5 × 104) were restimulated with 1.4 × 105 uninfected or L. major-infected BMDCs for 4 days. A, CFSE dilution was analyzed by flow cytometry on gated CD4+TCR+ cells. Values in top left quadrants are the percentages of CFSElow cells. Values in parenthesis are the CFSE MFIs within the CFSElow cells. B, Cytokines were quantified in Treg cultures with uninfected BMDCs (open bars) or L. major-infected BMDCs (black bars). Results are representative of six independent experiments; nd, Not detected.

FIGURE 6.

Tregs from aged mice respond to L. major. Eight- to 10-wk old C57BL/6 mice were inoculated in the ear dermis with L. major. Five or 21 mo later CD25+CD4+ Tregs were purified by FACS from the draining LNs. CFSE-labeled T cells (5 × 104) were restimulated with 1.4 × 105 uninfected or L. major-infected BMDCs for 4 days. A, CFSE dilution was analyzed by flow cytometry on gated CD4+TCR+ cells. Values in top left quadrants are the percentages of CFSElow cells. Values in parenthesis are the CFSE MFIs within the CFSElow cells. B, Cytokines were quantified in Treg cultures with uninfected BMDCs (open bars) or L. major-infected BMDCs (black bars). Results are representative of six independent experiments; nd, Not detected.

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CD25CD4+ Teffs from aged mice produced IL-2 and GM-CSF after stimulation with infected BMDCs, albeit at a lower level than those from young mice, and a similar amount of IL-10 (Fig. 7,A). Importantly, the coculture of Teffs and Tregs clearly decreased the production of both IL-2 and GM-CSF in aged mice as well as in young mice (Fig. 7,A), confirming the maintained suppressive capacity of Tregs in aged mice and their potential to suppress protective immune responses in aged L. major-infected mice. Increased IL-10 production was observed in the Treg:Teff cocultures, likely due to the IL-10 production by Tregs, but levels were similar between aged and young T cells (Fig. 7,A). Because purification of Tregs on the basis of CD25 expression may lead to their contamination by Teffs, we confirmed those data by purifying Tregs from old FoxP3-GFP knock-in mice that had been infected by L. major when they were young. As shown in Fig. 7 B, significant suppression of IFN-γ production by Teffs was achieved when increasing numbers of Tregs were added to a Teff culture stimulated with L. major-infected BMDCs. No major effect was observed for IL-10 production (data not shown).

FIGURE 7.

Cytokine production by Teffs from L. major-infected mice in response to L. major Ags is blocked by Tregs. A, Eight- to 10-wk-old C57BL/6 mice were inoculated in the ear dermis with L. major. Five or 21 mo later, CD25+CD4+ (Tregs) and CD25CD4+ (Teffs) were purified from the draining LNs (see Fig. 6). Teffs were cultured with uninfected BMDCs (open bars) or L. major-infected BMDCs (filled bars) or cocultured with Tregs (Teff:Treg ratio of 5:4) and L. major-infected BMDCs (gray bars). Cytokines were measured by ELISA. Results are representative of six independent experiments; nd, Not detected. B, FoxP3+CD4+ T cells were sorted from FoxP3-GFP knock-in animals of 57-wk-old L. major-infected mice that were infected when young (9–10 wk old). FoxP3+ cells were then mixed at different Teff:Treg ratios with Teffs (CD4+CD25CD62L) purified from 22-wk-old infected mice in presence of L. major-infected BMDCs. IFN-γ was measured by ELISA in a 4-day supernatant.

FIGURE 7.

Cytokine production by Teffs from L. major-infected mice in response to L. major Ags is blocked by Tregs. A, Eight- to 10-wk-old C57BL/6 mice were inoculated in the ear dermis with L. major. Five or 21 mo later, CD25+CD4+ (Tregs) and CD25CD4+ (Teffs) were purified from the draining LNs (see Fig. 6). Teffs were cultured with uninfected BMDCs (open bars) or L. major-infected BMDCs (filled bars) or cocultured with Tregs (Teff:Treg ratio of 5:4) and L. major-infected BMDCs (gray bars). Cytokines were measured by ELISA. Results are representative of six independent experiments; nd, Not detected. B, FoxP3+CD4+ T cells were sorted from FoxP3-GFP knock-in animals of 57-wk-old L. major-infected mice that were infected when young (9–10 wk old). FoxP3+ cells were then mixed at different Teff:Treg ratios with Teffs (CD4+CD25CD62L) purified from 22-wk-old infected mice in presence of L. major-infected BMDCs. IFN-γ was measured by ELISA in a 4-day supernatant.

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To directly address the role played by Tregs in L. major reactivation in old mice, we treated old L. major-infected mice (>48 wk old, who had been infected when they were young) with anti-CD25 Ab or isotype control following the experimental protocol that we had previously used (45). As shown in Fig. 8, in vivo depletion of Tregs in the anti-CD25-treated group significantly increased IFN-γ production by the T cells purified from the infection site and draining LNs. As expected, anti-CD25 treatment significantly increased IFN-γ production in young infected mice (Fig. 8 B). However, IL-10 production did not change.

FIGURE 8.

In vivo depletion of Tregs in old L. major-infected mice increases the production of IFN-γ by Teffs at the infection site. A, Old L. major-infected mice (>48 wk old, who had been infected when they were 8 wk old) or young L. major-infected mice (16 wk old, who had been infected when they were 8 wk old) were treated with anti-CD25 Ab or isotype control (n = 4/group) (1 mg for 3 wk, twice a week). After Ab treatment, mice were sacrificed. T cells were purified from the infection site and draining LNs and restimulated in vitro with L. major-infected BMDCs. B, IFN-γ and IL-10 were measured by ELISA in 4-day supernatants. p values correspond to the comparisons between mice treated with isotype control (open symbols) or anti-CD25 Ab (hatched symbols) using t tests.

FIGURE 8.

In vivo depletion of Tregs in old L. major-infected mice increases the production of IFN-γ by Teffs at the infection site. A, Old L. major-infected mice (>48 wk old, who had been infected when they were 8 wk old) or young L. major-infected mice (16 wk old, who had been infected when they were 8 wk old) were treated with anti-CD25 Ab or isotype control (n = 4/group) (1 mg for 3 wk, twice a week). After Ab treatment, mice were sacrificed. T cells were purified from the infection site and draining LNs and restimulated in vitro with L. major-infected BMDCs. B, IFN-γ and IL-10 were measured by ELISA in 4-day supernatants. p values correspond to the comparisons between mice treated with isotype control (open symbols) or anti-CD25 Ab (hatched symbols) using t tests.

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Another important and related question is whether the increased proportion of functionally suppressive Tregs in aged hosts plays a role in the increased disease severity observed for multiple infections in such aged hosts. We have previously shown that lesion size is a direct indication of parasite growth. As shown in Fig. 9,A, primary infection with L. major induces a more severe course of disease in old mice compared with young mice. Indeed, lesions appeared 3 wk postinfection in both groups but tended to be larger in old animals already at 4 wk postinfection. Disease severity was clearly apparent at all of the time points that followed. Moreover, one of the three old infected animals lost the infected ear at 7 wk postinfection through an acute necrotic process at a time when lesions were starting to heal in young animals. To determine whether the age-related increased proportion of Tregs had played a major role in such an exacerbated pathological process, we treated old mice (>43 wk old) with anti-CD25 Ab or isotype-matched Ab control at the time they were infected with L. major parasites and then twice a week. Infection outcome was then monitored by measuring the lesion size. As shown in Fig. 9 B, treatment with anti-CD25 Ab significantly reduced the lesion size at 4 and 6 wk, suggesting the importance of Tregs in the increased severity of L. major infection in aged animals.

FIGURE 9.

L. major causes exacerbated disease in old mice and Tregs contribute to such increased disease severity. A, Primary infection with L. major parasites. Four young mice (10 wk old) and three old C57BL/6 mice (57 wk old) were inoculated in the ear dermis with 103L. major metacyclic promastigotes. Lesion size was measured weekly in all animals. Mean (and SD) lesion sizes (in millimeters) are shown for young (filled symbols) and old (open symbols) mice. p values indicate differences at each time point (t test). At 7 wk postinfection one of the old mice lost an ear through an acute necrotic process. B, Effect of anti-CD25 Ab on primary L. major infection in old mice. Two 43-wk-old mice (filled circles) and two 55-wk old mice (filled squares) received anti-CD25 treatment (1 mg for 3 wk, twice a week) at the time of L. major infection. Three 43-wk-old mice (open circles) and three 55-wk-old mice (open squares) received the isotype control Ab following the same regimen. Lesion size (in millimeters) was measured weekly in all animals. p values correspond to the differences in lesion size between anti-CD25-treated and isotype-treated mice at the indicated time points.

FIGURE 9.

L. major causes exacerbated disease in old mice and Tregs contribute to such increased disease severity. A, Primary infection with L. major parasites. Four young mice (10 wk old) and three old C57BL/6 mice (57 wk old) were inoculated in the ear dermis with 103L. major metacyclic promastigotes. Lesion size was measured weekly in all animals. Mean (and SD) lesion sizes (in millimeters) are shown for young (filled symbols) and old (open symbols) mice. p values indicate differences at each time point (t test). At 7 wk postinfection one of the old mice lost an ear through an acute necrotic process. B, Effect of anti-CD25 Ab on primary L. major infection in old mice. Two 43-wk-old mice (filled circles) and two 55-wk old mice (filled squares) received anti-CD25 treatment (1 mg for 3 wk, twice a week) at the time of L. major infection. Three 43-wk-old mice (open circles) and three 55-wk-old mice (open squares) received the isotype control Ab following the same regimen. Lesion size (in millimeters) was measured weekly in all animals. p values correspond to the differences in lesion size between anti-CD25-treated and isotype-treated mice at the indicated time points.

Close modal

In this study we investigated the proportion, phenotype, and suppressive function of Tregs in aged mice and humans. Our study shows an increased proportion of FoxP3+CD4+ T cells in the tissues of aged C57BL/6 mice as well as in the blood of healthy elderly humans. Importantly, our in vitro and in vivo data strongly support our hypothesis that Tregs inhibit immune responses in aged hosts, contributing to reactivation of chronic infectious diseases.

Our data show a significantly increased proportion of FoxP3+CD4+ T cells in multiple lymphoid tissues from aged C57BL/6 mice compared with young mice as already reported in aged BALB/c mice (34). An increased proportion of CD25highCD4+ T cells in the lymphoid tissues of aged animals was also found in these animals in agreement with other studies (35, 36). Interestingly, the proportion of CD25highCD4+ T cells is always lower than that of FoxP3+CD4+ T cells in both aged and young hosts, supporting the notion that CD25 expression defines only a portion of Tregs (79). FoxP3 expression clearly plays a crucial role in the maintenance of Treg activity, with its ablation in adult mice leading to catastrophic autoimmune diseases (80). In contrast, CD25−/− mice exhibit reduced numbers of FoxP3+ cells, but those cells are fully able to suppress in vitro (81, 82).

An increased proportion of FOXP3+CD4+ T cells was also found in the blood of healthy elderly humans. There was a trend toward an increased proportion of CD25highCD4+ T cells in the blood of elderly donors, although the difference was not significant. It has recently been shown that a low level of the IL-7 receptor CD127 increases the specificity of Treg characterization for human cells (50, 51). A significantly increased proportion of CD25+CD127lowCD4+ T cells was also found in elderly humans. Previous studies using CD25high expression to characterize Tregs have reported similar (53, 83) or increased Treg proportions (37, 38, 39, 40) in elderly individuals. Discordances between studies may arise from differences in the phenotyping techniques and/or the characteristics of the studied populations (e.g., mean age, health status, or criteria of exclusion). In mice, the proportion of Tregs in the blood was not increased; in contrast, we observed an increased proportion of circulating FOXP3+CD4+ Tregs in elderly humans compared with young subjects. Differences between mice and humans could reflect the fact that FOXP3 is less specific of the Treg lineage in humans than in mice, because its expression is transitorily induced in Teffs following TCR activation (64). However, the low level of CD69 expression on FOXP3+ cells argues against that hypothesis. Alternatively, increased triggering of the Treg compartment in humans may come from the higher level of stimulation of the immune system exerted by constant exposure to pathogens. Of note, we have found that in young adults the proportion of FoxP3+CD4+ T cells was higher in tonsils than in blood (∼10 vs 4%; our unpublished data), similar to the murine data. Study of tissue Tregs in elderly humans has not yet been undertaken and will be essential to clarify this issue.

An important question raised by our data is how Tregs accumulate with age. Circulating Tregs in elderly humans do not express specifically altered patterns of homing receptors, suggesting that defective tissue homing is not likely to explain their increased proportion, which is confirmed by the fact that increased Treg proportion was found in all lymphoid tissues in aged mice. Peripheral Tregs could be derived either from Tregs that have developed in the thymus or from converted non-Tregs. In aged mice, the total number of CD25+CD4+ single-positive thymocytes decreased following the reduction in thymocyte numbers, although the percentage of CD25+ cells increased in CD4+ single-positive thymocytes (35), suggesting a reduced thymic Treg input in those mice. Tregs in aged hosts are mostly memory cells with a highly differentiated phenotype as shown in this study and by others (35, 41, 79, 84, 85). These data suggest that Tregs in elderly might come from thymic-derived Tregs that have proliferated in the periphery. Few data are available on Treg in vivo turnover, particularly in aging subjects. In young mice, the CD25+CD4+ Treg population is composed of two subsets with distinct homeostasis (86), one subset exhibiting a rapid proliferation rate whereas the other subset did not divide but was long-lived. These findings suggest that although Tregs can proliferate in vivo, resting thymic-derived Tregs may also persist for long time in vivo and this population may participate in the maintenance of peripheral Treg numbers. In healthy humans, young and elderly alike, CD45RO+ Tregs had a rapid doubling time compared with those of memory or naive CD4+ T cells (40). However, human CD45RO+ Tregs exhibited short telomere length in both young and elderly individuals and did not up-regulate telomerase after activation (40, 87), raising the question of whether peripheral proliferation of thymic-derived Tregs is sufficient to maintain the increased proportion of Tregs seen in aged hosts.

Tregs might also be generated from CD25CD4+ T cells in the periphery. In both mice and humans, TGF-β induces CD25FOXP3CD4+ T cells to become FOXP3+ Tregs (reviewed in Ref. 88), although the stability of such conversion, as well as the requirement for other molecules than TGF-β, is still debated (89, 90, 91, 92, 93). The state of activation of DCs, as well as the proportion of different DC subsets (myeloid DCs vs plasmacytoid DCs), also plays a role in both Treg conversion and peripheral expansion (25, 26). Interestingly, DC subsets and maturation levels are changed during aging (94, 95), and this could play a role in Treg accumulation in elderly individuals. However, the contribution of such converted cells to the pool of circulating Tregs remains an open question, because no marker(s) have been found that distinguish between natural FoxP3+ Tregs and induced FoxP3+ Tregs. Of note, our published data support the idea that no neogeneration of Tregs occurs during L. major infection (78).

An extensive phenotypic characterization of Tregs (defined as FoxP3+CD4+ T cells) was performed in the old mice. Expression of CD25 by Tregs from aged animals was lower than that from their young counterparts, a result in agreement with previous studies (34, 36). The underlying mechanisms have not been completely elucidated and could include the loss of CD25 expression by FoxP3+CD4+ Tregs. In support of that argument, in vivo expanded Tregs become CD25low while maintaining similar levels of other Treg-associated molecules (96). Similarly, CD25 turnover may be increased in aging, as suggested by increased soluble CD25 levels in the serum of aged humans (97, 98). In contrast to decreased CD25 expression, expression of other markers associated with Treg function such as GITR, CTLA-4, or PD-1, was maintained or increased on FoxP3+CD4+ Tregs from aged mice, depending on the tissue analyzed. The activation marker CD69 was also expressed more by old FoxP3+CD4+ Tregs than by young Tregs in all analyzed tissues except in blood, where the frequency of CD69+ Tregs tended to decrease, suggesting an accumulation of activated Tregs in the tissues of aged mice. Similarly, we found a trend toward a decreased proportion of CD27 and CCR7 expression in old Tregs (Table II), suggesting increased Treg differentiation. Taken together, these data suggest that Tregs are more activated in tissues from aged mice than in young mice.

We also extensively characterized circulating Tregs in healthy elderly humans in comparison to young Tregs and did not find major age-related differences in the expression of molecules that have previously been associated with Treg function, such as CD27, PD-1, TGFβRII, or granzymes A and B. Similar to our data in old mice, there was a trend toward decreased expression of CD25 on FOXP3+CD4+ Tregs in elderly humans. Low and constant expression of CD69 was found in both young and old human blood Tregs in contrast to its decreased expression in circulating Tregs of aged mice, suggesting a difference in activation patterns between species.

Because phenotype does not recapitulate functional activity, we analyzed Treg-mediated suppression during aging. Interpretation of suppression assays in aging mice can be confounded by the fact that CD25 expression is not accurate in recapitulating FoxP3 expression (and Treg activity) in aged mice (35, 36, 79). Therefore, to circumvent this caveat, we used FoxP3-GFP knock-in mice and clearly show that FoxP3+ cells from aged mice have a greater in vitro suppressive activity on a per cell basis than their young counterparts. Old Tregs were also able to suppress Teffs from aged mice. The use of FoxP3-GFP cells in our study may explain the difference between our results and those in previous studies that showed similar or decreased suppressive activity of old CD25high Tregs compared with young Tregs. Because we show that many of the FoxP3+ cells in aged mice are CD25low, the previous studies sorting old Tregs based on CD25 expression may have been affected by contamination of the effector population with FoxP3+CD25 Tregs. In addition, CD25+ Tregs from aged, chronically L. major-infected mice also maintained their responsiveness to L. major Ags and their ability to suppress IFN-γ production by Teffs in response to L. major-infected DCs. In elderly humans Tregs appear functional, their in vitro depletion leading to increased CD4+ T cell function in most individuals, although the functional consequence of such depletion was modest. Several factors could have contributed to such a result, including the expected heterogeneity between human subjects, the use of old autologous Teffs, and the fact that human Tregs could not be depleted on the basis of their FOXP3 expression.

Increased proportion of functional Tregs in aging may translate into dampened immune responses in aged hosts. Strongly supporting this hypothesis, spontaneous reactivation of L. major lesions, which we have previously shown to result from Treg accumulation in the chronic infectious site (44, 45), occurred in the majority of aged infected mice. We also demonstrate a direct role of Tregs in such reactivation, because in vivo depletion of Tregs in old mice infected when young significantly increased the production of the effector cytokine IFN-γ by the Teffs purified from the infection site and draining LNs. Furthermore, our experiments show that Tregs play a critical role in the increased disease severity of L. major infection in old mice. Taken together, our data strongly suggest that age-associated Treg accumulation is likely to play a major role in the increased severity, as well as the reactivation, of chronic infections in aged mice and humans. Our data in an infectious model are thus in agreement with the recent study showing that Treg accumulation in aged mice plays a crucial role in inhibiting the activation of anti-tumor responses (34).

How Tregs act is still unclear and we have not formally ruled out the possibility that the mechanisms mediating such suppression may be different in old Tregs. Expression of activation markers by old tissue Tregs was increased whereas, on a per a cell basis, old Tregs produced less IL-10, which has been shown to play a role in Treg-mediated suppression in the L. major model (44). Tregs from old murine lymphoid tissues were clearly suppressive both in vivo and in vitro, although the mechanisms by which old Tregs suppress will need to be further investigated in the future.

An alternative explanation of increased severity and frequency of infectious diseases in aged hosts could be the decreased capacity of aged Teffs to proliferate and produce cytokines. Indeed, profound, diverse alterations in TCR-mediated activation have been described in T cells from aged mice and humans (99, 100). Accordingly, decreased cytokine production by old Teffs, including in response to L. major Ags, was observed in both our study (Fig. 6) and previous studies (36, 79). Similarly, in three of seven old individuals, depletion of CD25 resulted in partial or absent improvement of CD4 T cell function, suggesting intrinsic decreased responsiveness of the Teff subset in these individuals. However, when old memory TCR Tg CD4+ T cells were transferred into young animals, they function as well as their young counterparts (101), suggesting that antigenic-specific protective effector mechanisms are retained in old hosts. In support of that hypothesis, purified CD4+ Teffs from old animals produced large amounts of the effector cytokine IFN-γ after restimulation with L. major Ags, which was blocked by coculture with Tregs. Furthermore, Treg depletion in vivo allowed for better control of L. major, suggesting that aged Teffs had retained some function in such experimental conditions. Of note, in the model of infection with high doses of L. major, which is less sensitive to Treg regulation than our model, old mice controlled infection as well as young mice (102), further suggesting that Teff function is not completely abolished in old animals.

Taken together, our findings suggest that decreased T cell responsiveness in aged hosts results from both intrinsic defects and an altered balance between stimulatory/regulatory mechanisms, the exact contribution of each mechanism likely to be variable depending on the context (infectious diseases/autoimmunity/cancer). Manipulation of Treg numbers and/or activity may therefore be critical to enhance immune responses in the aged and may be envisioned to enhance the control of chronic infectious diseases, as well as vaccine efficiency, in this fragile population.

We thank all the study participants and Dr. M. Oukka, Harvard Medical School, Cambridge, MA for the kind gift of FoxP3-GFP knock-in C57BL/6 reporter mice. We also thank Dan Marmer (Cincinnati Children’s Hospital Research Foundation Sorting Core), Kevin Holmes and Carol Henry (National Institute of Allergy and Infectious Diseases Flow Cytometry Unit) for cell sorting, as well as Dr. Keller (Cincinnati Children’s Hospital Research Foundation) and Kim Beacht (National Institute of Allergy and Infectious Diseases) for help with mouse care, and Kris Orsborn (Cincinnati Children’s Hospital Research Foundation) for help with cell stainings.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by National Institutes of Health Grant AG025149 (to C.C.), the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (to Y.B.), and a Colciencias fellowship (to P.A.V.).

6

Abbreviations used in this paper: Treg, regulatory T cell; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; FoxP3, Forkhead box P3; GITR, glucocorticoid-induced TNFR-associated protein; LN, lymph node; mLN, mesenteric lymph node; MFI, mean fluorescence intensity; PD-1, programmed death-1; pLN, peripheral lymph node (retromaxillary and popliteal); Teff, effector T cell.

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