Functional Regulatory T Cells Accumulate in Aged Hosts and Promote Chronic Infectious Disease Reactivation

Abstract

Declines in immune function are well described in the elderly, and are considered to contribute significantly to disease burden in this population. Regulatory T cells (T_regs), 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 T_regs, especially in aged hosts, we characterized T_regs by expression of FOXP3, a transcription factor crucial for T_reg differentiation and function. The proportion of FOXP3⁺CD4⁺ T_regs 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⁺ T_regs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral T_regs from elderly humans improves effector T cell responses in most subjects. Importantly, T_regs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young T_regs. The increased proportion of T_regs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old T_regs efficiently suppressed the production of IFN-gamma by effector T cells. Finally, in vivo depletion of T_regs in old mice attenuated disease severity. Accumulation of functional T_regs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of T_reg numbers and/or activity may be envisioned to enhance control of infectious diseases in this fragile population.

Introduction

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 (1-4)). 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 (7-10), but the most consistent and significant alterations are seen in the T lymphocyte compartment (11, 12), particularly within CD4⁺ T cells (8, 13-15).

CD4⁺ regulatory T cells (T_regs) maintain self-tolerance in the periphery (16-18) and play a role in the control of autoimmunity and tumor immunity (18-20). They have been shown to decrease the level of activation, proliferation and cytokine production of effector T cells (T_effs) in mice and humans (21-24), as well as control the immune function of dendritic cells (DCs) (25, 26). T_regs were first characterized by their expression of the IL-2Rα chain (CD25) (16). Additional molecules have been associated to T_reg function, such as cytotoxic T lymphocyte associated antigen (CTLA)-4 (27) and the glucocorticoid-induced tumor necrosis factor receptor (GITR) (28). More recently, the transcriptional factor FoxP3 (Forkhead box P3) has been shown to play a crucial role in many aspects of murine T_reg biology, namely their differentiation, function and maintenance (29-32). In humans, FOXP3 is also crucial for T_reg function, as evidenced by the acquisition of T_reg activity following de novo FOXP3 expression in non-T_regs (33).

Previous studies have shown increased numbers of CD25⁺CD4⁺ T_regs 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 (37-40). Although, FOXP3 expression has recently been used to assess the proportion of T_regs in aged humans (41), it remains unclear whether T_regs maintain their suppressive activity in aged hosts. Indeed, some studies show maintenance of suppressive activity of T_regs in aged mice (34, 36) and elderly people (37, 39), whereas some studies reported decreased T_reg-mediated suppression in aged mice (35) and humans (42).

During the acute phase of the infection by L. major, activation of T_effs 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 T_effs (CD4⁺CD25⁻ T cells, producing IFN-γ) and T_regs 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 T_reg:T_eff balance at the local site induces parasite multiplication and subsequently reappearance of the lesion (45)

In this report, we show in that: (i) FoxP3⁺ T_regs accumulate in aged mice and elderly humans; (ii) T_regs from aged mice and elderly humans are functional; and (iii) depletion of T_regs in vitro and/or in vivo increases Teff responses. Together, these data suggest that T_reg accumulation in aged hosts contributes to the immune suppression associated with aging.

Materials and Methods

Human subjects

Healthy elderly individuals (≥ 70-year old) were recruited in a retirement community in the Cincinnati 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-year old) were recruited at Cincinnati Children's Hospital Medical Center 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.

Mice

6-8-week old C57BL/6 mice were purchased from Charles River (Wilmington, MA) or Taconic. 20-month old C57BL/6 mice were purchased from Harlan (Chicago, IL) 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 Children's Hospital Research Foundation Animal Facility or NIH 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).

Cell preparation

For humans, blood samples (40 ml) were collected on sodium heparin. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation on Ficoll Paque™ Plus (GE Healthcare, Piscataway, NJ) within 4 hours of sample collection and frozen in fetal calf serum (FCS) with 10% dimethyl sulphoxide (DMSO; SIGMA, St. Louis, MO).

For mice, single cell suspensions were prepared from spleen, peripheral (retromaxillar and popliteal) lymph nodes (pLNs) and mesenteric LNs (mLNs). Blood was collected on heparin. Erythrocytes were lyzed by incubating the spleen or blood cells with 1 ml ACK (Cambrex, Walkersville, MD) for 2 min, on ice.

Phenotypic analysis of cells

Thawed human PBMCs were incubated on ice for 5 min with human IgG (SIGMA) to block Fc receptors and stained for 30 min for cell surface markers with a combination of the following antibodies: anti-CD3-PerCP-Cy5.5 (clone SK7), anti-CD4-Pacific Blue or PE-Cy7 (clone RPA-T4), anti-CD25-APC (clone M-A251), anti-CD69-APC-Cy7 (clone FN50), anti-CCR5-PE-Cy7 (clone 2D7/CCR5), anti-CCR7-PE-Cy7 (clone 3D12), anti-integrin α4-APC (clone 9F10), anti-integrin β7-PE (clone FIB504) from BD Pharmingen (San Diego, CA); anti-CD27-APC-Cy7 (clone O323) and anti-integrin β1-PE (clone MEM-101A) from eBioscience (San Diego, CA); anti-GITR-PE (clone 110416) and anti-TGFβRII-PE (clone 25508) from R&D System (Minneapolis, MN); anti-CD127-PE (clone IM1980) from Immunotech (Marseille, France); anti-CD45RA-Pacific Blue (clone MEM-56) and anti-CXCR4-APC (clone 44717) from Caltag (Burlingame, CA). For intracellular stainings, the eBioscience's protocol for FOXP3 staining and the following antibodies were used: anti-FOXP3-FITC (clone PCH101) and anti-CTLA-4-PE (clone 14D3) from eBioscience; anti-PD-1 (clone MIH4) and anti-Granzyme A-PE (clone CB9) from BD; anti-Granzyme B-APC (clone GB12) from Caltag. 200,000 events/sample were collected on a LSRII™ cytometer using the FACSDiva™ software (BD Biosciences). The CD25^hi, 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 and CD27 expression was set up according to the biphasic distribution of this marker. For all the other markers, the appropriate isotype-matched control antibodies were used to define the positive gates within the CD4⁺CD3⁺ T cells.

Freshly isolated or cultured murine cells were incubated with 24G2 cell line culture supernatant to block Fc receptors. Cells were stained for 20 min on ice with the following fluorochrome-conjugated antibodies: anti-CD4-PE-Cy5 (clone RM4-5), anti-TCR-βchain-APC-Alexa Fluor 750 (clone H57-597), anti-CD25-APC (clone PC61.5), anti- CD69-PE (clone H1.2F3), anti-CD103-FITC (clone 2E7), anti-CD27-APC (clone LG.7F9), anti-CCR7-PE (clone 4B12), anti-GITR-APC (clone DTA-1) and anti-PD-1- FITC (clone J43). The isotype controls used were rat IgG₁ (clone R3-34), rat IgG_2a (clone eBR2a), rat IgG_2b (clone KLH/G2b-1-2), and hamster IgG (clone Ha4/8). Cells were washed with PBS, 1% FCS, 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 antibodies and controls were purchased from BD Pharmingen, eBioscience or BioLegend (San Diego, CA). Cell acquisition was performed on a FACSCalibur™ cytometer using the 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, Inc., Ashland, OR).

Human CD4⁺ T cell stimulation

Monocytes were isolated from PBMCs by positive selection using CD14 MicroBeads according to the manufacturer's instruction (Miltenyi Biotec, Auburn, CA), 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 manufacturer's instruction (Miltenyi Biotec). CD4⁺ T cells were depleted of CD25⁺ cells using CD25 MicroBeads (Miltenyi Biotec). As depletion of CD25⁺ cells by MicroBeads induces non-specific 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).

T_reg-depleted CD4⁺ and total CD4⁺ fractions were labeled for 5 min at room temperature with 0.625 μM of carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, OR) in PBS. 5 × 10⁵ CFSE-labeled T cells were cultured with 2 × 10⁵ CD14⁺ monocytes in RPMI supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml L-glutamine (Invitrogen, Gibco^®, Carlsbad, CA) and 10% FCS, and stimulated or not with phytohemagglutinin (PHA; 2 μg/ml; SIGMA) 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 were stained 30 min with anti-CD4-PE (clone RPAT4; BD), anti-CD3-PerCP-Cy5.5, anti-CD69-APC (clone FN50; BD) and anti-CD95- Pacific Blue (clone DX2; Caltag). After washes and formaldehyde fixation, up to 50,000 events/sample were collected on a LSR-II™ cytometer with the FACSDiva™ software. Data were analyzed by using FlowJo™ software.

In vitro T cell suppression assay

GFP⁻CD4⁺ (T_effs) and GFP⁺CD4⁺ (T_regs) cells were sorted from spleens or pLNs of FoxP3-GFP knock-in C57BL/6 mice as described below for CD25^hiCD4⁺ and CD25⁻CD4⁺ T cells. 5 × 10⁴ T_effs 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 ratio of T_regs to T_effs. 48 hours later, 1 μCi [³H]-thymidine was added into each well for 22 hours. Plates were harvested and radioactivity measured on a Wallac Trilux MicroBeta scintillation counter.

L. major infection

Promastigotes (metacyclics) of L. major clone V1 (MHOM/IL/80/Friedlin) were isolated as previously described (47). 8-10-week old mice were infected in the ear dermis with 10³ L. major metacyclic promastigotes using a 27½ G needle in a volume of 10 μl.

In vitro restimulation assay of lymphocytes from L. major-infected mice

A single-cell suspension was prepared from the retromaxillar 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). CD25^hiCD4⁺ and CD25⁻CD4⁺ 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. 5 × 10⁴ T cells were incubated with 1.4 × 10⁵ bone marrow-derived dendritic cells (BMDCs) in 200 μl RPMI containing 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, 55 μM 2-mercaptoethanol, 10% FCS, as previously described (48). BMDCs were previously incubated overnight with or without L. major metacyclics (parasites:BMDCs 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).

CD25⁺ T cell depletion

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

Cytokine assays

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

Statistical analysis

Statistical analysis was performed using Prism (GraphPad Software Inc., San Diego, CA). For mice, group comparisons were made using the two-group Student's t-test. For human, the distribution of the values in the different groups was tested by 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 test. A p value < 0.05 was considered statistically significant. The Box-Cox transformation and the subsequent statistical analyses were conducted using SAS v9.

Results

The proportion of circulating T_regs is increased in healthy elderly subjects

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

The proportion of FOXP3⁺ cells was significantly increased within CD4⁺ T cells from elderly compared to young subjects (5.8 ± 0.4% versus 4.4 ± 0.4%; p = 0.03, unpaired t-test; N = 16/group; Fig. 1A and B). Because the level of FOXP3 per cell is an important factor determining T_reg activity (49), we evaluated the mean fluorescence intensities (MFI) of FOXP3 staining, which were similar in elderly and young donors (Fig. 1C). High level of CD25 expression is also described as a characteristic feature of T_regs. The number of CD25^hiCD4⁺ T cells was determined using a stringent gate to define CD25^hi expression (Fig. 1A). The proportion of CD25^hi cells in the CD4⁺ T cell population was not significantly different in elderly and young subjects, although there was a trend towards increased proportion in the elderly (Fig. 1A and B; p = 0.1, Mann-Whitney test). FOXP3⁺CD4⁺ T cells were more frequent than CD25^hiCD4⁺ T cells, in both young and elderly subjects (Fig. 1A and B; both p < 0.001). It has been recently described that T_regs expressed low levels of the IL-7 receptor, CD127, and that this expression pattern better defines T_regs in humans (50, 51). We therefore analyzed the percentage of CD25⁺CD127^loCD4⁺ T cells and found an increased percentage of those cells in the elderly (6.5 ± 0.8% versus 3.9 ± 0.4% in elderly and young subjects, respectively; p = 0.01, t-test). Thus, whether assessed by FOXP3 or CD25, CD127 expression, the frequency of T_regs is significantly increased in elderly humans.

FIGURE 1. T_reg frequency is increased in the blood of elderly individuals.

T_reg frequency was analyzed in PBMCs from 16 young (≤ 30-year old) and 16 elderly (≥ 70- year old) subjects. For FOXP3 staining, CD4⁻CD3⁺ 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 CD25^hi cells within the CD3- cells. (A) Representative expression of FOXP3 and CD25 in young and elderly subjects. The percentages of FOXP3⁺CD4⁺ and CD25^hiCD4⁺ cells in gated CD3⁺ T cells in a representative young and an elderly subject are shown in the upper and middle plot 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 CD25^hiCD4⁺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 line) and a 72-year old (unfilled line) donor.

T_regs from elderly and young subjects express the same functional markers

We then assessed the expression of markers on FOXP3⁺CD4⁺ T cells that have been previously associated with T_reg phenotype and function (52), such as CD25, CTLA- 4 and GITR. Expression of CD25, CTLA-4 and GITR by FOXP3⁺CD4⁺ T_regs was similar in young and elderly subjects (Table I; all p > 0.05). In both groups of subjects, FOXP3⁺CD4⁺ T_regs expressed also higher levels of CTLA-4 than FOXP3-CD4⁺ T cells (~ 40% versus 4%; p < 0.0001) (Table I). Similar data were obtained when CD25^hi expression was analyzed (Table I). GITR expression was very low in all groups (< 7%), but FOXP3⁺CD4⁺ T_regs expressed significantly higher levels than FOXP3⁻CD4⁺ T cells, in both groups of subjects (Table I). We also analyzed the proportion of naïve T_regs based on their expression of CD45RA. The proportion of naïve T_regs was lower in young individuals than the proportion of naïve T_effs, 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 which have been reported to correlate with regulatory activity in human or mice T_regs, such as CD27 (54-56), PD-1 (57, 58), TGFβRII (59), Granzymes A and B (60-62). CD27 expression by FOXP3⁺CD4⁺ T_regs 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 T_regs was also similar between young and old T_regs (Table I).

Table 1.

Phenotypic characterization of T_regs and Teffs in young and elderly subjects^a

	Young					Elderly
	Nb	Treg	Teff	Pc	ratiod	Nb	Treg	Teff	Pc	ratiod	pTrege	pTefff
CD25hi	16	42.5 ± 2.4	0.6 ± 0.1	**		16	49.1 ± 2.9	1.0 ± 0.3	**		0.10	0.32
CD25+	16	75.4 ± 1.8	8.5 ± 0.8	**		16	78.8 ± 1.9	11.8 ± 1.8	**		0.20	0.45
CD25+ CD127lo	9	58.8 ± 4.3	2.2 ± 0.2	**		11	69.0 ± 3.1	3.4 ± 0.6	**		0.07	0.21
CTLA-4+	16	44.6 ± 3.9	3.9 ± 0.6	**		13	39.8 ± 3.9	3.7 ± 0.4	**		0.41	0.96
GITR+	16	5.2 ± 0.4	1.3 ± 0.1	**		16	6.4 ± 0.7	1.7 ± 0.3	**		0.16	0.24
CD45RA+	10	14.7 ± 2.5	39.0 ± 4.3	**	2.65	10	9.5 ± 1.4	21.5 ± 3.0	*	2.26	0.09	< 0.01
CD27+	16	92.1 ± 0.9	88.5 ± 1.5	*		13	93.3 ± 0.9	81.2 ± 4.6	*		0.38	0.17
PD-1+	9	22.9 ± 7.9	21.9 ± 9.2	ns		11	22.8 ± 6.8	25.1 ± 7.6	ns		0.82	0.59
TGFβRII+	8	6.8 ± 2.6	2.2 ± 0.6	ns		10	4.9 ± 1.1	2.0 ± 0.4	*		1.00	0.82
GranzA+	9	3.7 ± 1.1	4.1 ± 1.0	ns		9	4.6 ± 2.0	7.2 ± 3.0	ns		1.00	1.00
GranzB+	9	4.0 ± 1.5	4.1 ± 1.3	ns		9	4.2 ± 1.6	12.6 ± 4.5	*		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+	9	27.5 ± 4.3	9.4 ± 1.2	**	0.3	11	28.6 ± 3.2	15.1 ± 1.9	*	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+	9	28.2 ± 3.7	37.4 ± 3.6	ns	1.3	11	15.8 ± 2.0	33.2 ± 2.1	**	2.1	< 0.01	0.31
α4+β1+	18	44.2 ± 2.0	65.2 ± 2.6	**	1.4	19	38.5 ± 2.1	63.1 ± 2.2	**	1.6	0.06	0.54
α4+β7+	18	12.0 ± 1.2	41.5 ± 2.4	**	3.5	19	7.6 ± 0.6	29.0 ± 2.5	**	3.8	0.02	0.001

^aPBMCs were obtained from healthy elderly individuals (≥ 70-year old) and young donors (≤ 30-year old). T_regs were defined as FOXP3+CD4+, Teffs were defined as FOXP3- CD4+ T cells. Values represent the mean (± SEM) percentage of positive cells for each marker.

^bN is the number of tested samples.

^cp values compare the percentage of T_reg and Teff expressing each marker. ** p<0.0001; * p<0.05; ns: not significant (p>0.05).

^dratio was calculated as the proportion of T_effs expressing each marker divided by the proportion of T_regs expressing it.

^ep_Treg values compare the percentage of T_regs expressing each marker in young versus elderly subjects.

^fp_Teff values compare the percentage of T_effs expressing each marker in young versus elderly subjects.

FOXP3, CD25, GITR and CTLA-4 are not only markers for T_regs, but also are transiently upregulated on human non-T_regs 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⁺ T_regs from both young and elderly, and was similar between the 2 groups (Table I). Together, these data indicate that the phenotype of circulating FOXP3⁺CD4⁺ T_regs 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 ratio of expression (T_effs versus T_regs) of homing markers is similar between old and young subjects

The increased proportion of T_regs 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 T_regs and T_effs of several T cell homing markers, namely CCR7, which mediates T cell entry into secondary lymphoid organs (66), α4β1 integrin, CXCR4 and CCR5, which allows T cell migration to inflamed tissues, and the gut-associated α4β7 integrin (67-72).

FOXP3⁺CD4⁺ T_regs 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⁺ T_regs expressed lower levels of α4β7 in aged subjects compared to those in young subjects (Table I). However, the same change in expression pattern was observed in T_effs from old subjects compared with young T_effs (Table I). Indeed, when the ratio of expression (T_effs versus T_regs) 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 T_regs. Interestingly, CXCR4 has been associated with T_reg migration and maintenance in the bone marrow (71). All together, these data suggest that the increased proportion of circulating T_regs in the elderly is not likely due to a selective dysregulation of T_reg homing to tissues, although we cannot rule out a role for decreased CXCR4 in the retention of T_regs in the blood.

The proportion of T_regs is increased in aged mice

Further determination of T_reg dynamics in humans being difficult due to the obvious restricted access to tissues, we pursued our analysis through the characterization of T_reg markers in multiple lymphoid organs from aged (≥ 20-month old) and young adult C57BL/6 mice (≤ 3-month old). In all tissues, the proportion of FoxP3⁺CD4⁺TCR⁺ cells was significantly higher in aged mice compared to young mice (Fig. 2A). FoxP3 level per cell was identical in cells from aged and young mice (Fig. 2B). In contrast, the proportion of circulating FoxP3⁺CD4⁺TCR⁺ cell was the same in the blood of aged and young mice (Fig. 2A). When the proportion of CD25^hiCD4⁺TCR⁺ cells was analyzed, we found a significant accumulation of these cells in peripheral (pLNs) and mesenteric lymph nodes (mLNs), but not in the spleen or the blood, of aged mice compared to young mice (Fig. 2C). The proportion of CD25^hiCD4⁺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 T_regs expressing CD103, a specific marker for natural T_regs (73, 74), was increased in aged mice (Fig. 2D).

FIGURE 2. Increased proportion of T_regs in aged mice.

Single cell suspensions from spleens, peripheral (pLNs), mesenteric (mLNs) lymph nodes and blood were first stained for the surface markers CD4, TCR, CD25, CD69, CD103 and PD-1, followed by staining for the intracellular markers FoxP3. Flow cytometry analysis on gated CD4⁺TCR⁺ cells is shown. (A) Percentages of FoxP3⁺ cells in the CD4⁺TCR⁺ cell populations from 2-3- month old (closed circles) or 20-28-month old (open circles) mice. Horizontal lines represent the mean values for each group. (B) Representative overlay of Foxp3 expression in spleen, pLNs and mLNs cells from a 3-month (filled line) and a 28-month (unfilled line) old mouse. (C) Percentages of CD25^hi cells in the CD4⁺TCR⁺ cell populations from 2-3-month old (closed circles) or 20-28-month 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-month (left panel) or 28-month (right panel) old mouse, representative of 6 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.

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

Table 2.

Expression of markers in FoxP3⁺CD4⁺TCR⁺ cells from young and aged mice^a

	CD25+			GITR+			CTLA-4+
Tissue	Young	Aged	pd	Young	Aged	pd	Young	Aged	pd
Spleenb	62.6 ± 1.8	35.6 ± 4.4	< 0.01	93 ± 1	97.5 ± 0.7	< 0.01	51.5 ± 5.1	65.6 ± 6.3	0.11
pLNb	72.6 ± 1.1	65.3 ± 5.2	0.19	ND	ND	ND	55.0 ± 5.2	71.8 ± 5.7	0.05
mLNb	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
Bloodc	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+
Tissue	Young	Aged	pd	Young	Aged	pd	Young	Aged	pd
Spleenc	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
pLNc	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
mLNc	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
Bloodc	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

^aSingle 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 month old and aged mice were 20-28 month old.

^b6 mice/group were analyzed. ND: not determined.

^c3 mice/group were analyzed.

^dp values (t-test) compare proportions in young and old mice.

T_reg depletion increases in vitro T_eff function in the elderly

Our phenotypic data show increased proportion of cells with T_reg characteristics in aged humans and mice. However, because FOXP3⁺ or CD25^hi cells are not always functionally suppressive, we further characterized T_reg function in elderly humans by analyzing the effect of T_reg 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. 3A). In absence of stimulation, CD4⁺ T cells, with or without T_regs, did not proliferate or express activation markers (data not shown). After 3 days of PHA stimulation, T_reg- depleted cells proliferated better than total CD4⁺ T cells (not depleted of T_regs) in 4 of the 7 tested individuals (group A: subjects $\square ,\Delta \text{,\hspace{0.25em}}\nabla ,\text{\hspace{0.25em}+}$; Fig. 3B). In contrast, in 3 individuals, T_reg depletion did not result in increased proliferation after PHA stimulation (group B: subjects ◊, ○, ×; Fig. 3B).

FIGURE 3. Depletion of CD25^hiCD4⁺ T cells from the blood of elderly individuals increased CD4 T cell function.

5 × 105 CFSE-labeled total CD4⁺ or T_reg-depleted CD4⁺ (CD25-CD4⁺) T cells from 7 elderly individuals (≥ 70-year old) were cultured with 2 × 10⁵ autologous CD14⁺ monocytes and 2 μg/ml PHA for 3 days. (A) Percentage of FOXP3⁺ cells before CD25^hi depletion (Total CD4⁺, left) and after CD25^hi depletion (T_reg-depleted CD4⁺, right). The values from the same subject are linked by a line. Group A is composed of the 4 individuals in whom T_reg depletion led to increased proliferation, whereas group B comprised the 3 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 X. (B) CFSE dilution and (C) the expression of CD69 and CD95 markers 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 T_reg-depleted CD4⁺ T cells, are shown in (B). Percentages of CD69⁺ and CD95⁺ cells, in total or T_reg-depleted CD4⁺ T cells, are shown in (C). Each individual is represented by the same symbol in panels (A), (B) and (C).

Because proliferation does not recapitulate overall CD4 T cell function, particularly for memory cells, we also analyzed other read-outs of T cell activation, such as expression of the activation markers CD69 and CD95 (Fig. 3C). PHA stimulation induced CD69 and CD95 up-regulation and cytokine production in all PHA-stimulated cultures, compared with unstimulated cultures (data not shown). In 3/4 samples of the group A, CD69 and CD95 expression both increased following T_reg depletion (Fig. 3C). In the 4^th subject from that group (subject +), T_reg depletion led to decreased CD69 expression, but stable and high CD95 expression (Fig. 3C). Of note, this individual exhibited the highest level of proliferation, before and after T_reg depletion. In group B individuals, depletion did not change CD95 expression and increased CD69 expression in only one individual (subject ○) (Fig. 3C). Depletion of T_regs therefore increased activation and/or proliferation of CD4⁺ T_effs in 5/7 elderly after 3 days of culture with PHA. Of note, this percentage of responders is similar to that reported in young individuals, in whom T_reg depletion led to increased proliferation in response to PHA in 4/6 individuals (75). These data suggest that T_regs in elderly humans are functional in most individuals and may be able to inhibit T_eff responses in vitro.

T_regs from old mice suppress anti-CD3-induced proliferation of T_effs

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

FIGURE 4. FoxP3⁺CD4⁺ T cell suppressive function is intact in aged mice.

5 × 10⁴ GFP⁻CD4⁺ T cells (T_effs) were sorted from LNs of 2-4-month 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 (T_regs) were sorted from LNs of 2-4-month (closed squares) or 15-18-month (open squares) old mice and co-cultured with T_effs at different T_eff:T_reg ratios, ranging from 1.3:1 to 101:1. Proliferation was measured by thymidine incorporation in the last 22 hours of a 3-day culture. In the absence of T_regs, 21,800 ± 2,075 cpm were counted.

L. major infection spontaneously reactivates in aged mice

We next reasoned that, if T_reg 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 T_regs 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 weeks post-inoculation, although a few viable parasites persist in the site of the former lesion and in the draining LNs (44). 8 weeks post-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 months post-healing (Fig. 5), suggesting increased T_reg activity in aged L. major-infected mice.

FIGURE 5. L. major spontaneously reactivates in aged mice.

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

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

FIGURE 6. T_regs from aged mice respond to L. major.

8- to 10-week old C57BL/6 mice were inoculated in the ear dermis with L. major. 5 or 21 months later, CD25⁺CD4⁺ T_regs were purified by FACS from the draining LNs. 5 × 10⁴ CFSE-labeled T cells 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 CFSE^low cells. Values in parenthesis are the CFSE mean fluorescence intensities (MFI) within the CFSE^low cells. (B) Cytokines were quantified in T_reg cultures with uninfected BMDCs (open bars) or L. major-infected BMDCs (black bars). Results are representative of 6 independent experiments. nd, not detected.

CD25⁻CD4⁺ T_effs 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. 7A). Importantly, the co-culture of T_effs and T_regs clearly decreased the production of both IL-2 and GM-CSF in aged mice, as well as in young mice (Fig. 7A), confirming the maintained suppressive capacity of T_regs 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 T_reg:T_eff co-cultures, likely due to the IL- 10 production by T_regs, but levels were similar between aged and young T cells (Fig. 7A). Because T_reg purification on the basis of CD25 expression may lead to their contamination by T_effs, we confirmed those data by purifying T_regs from old FoxP3-GFP knock-in mice that had been infected by L. major when they were young. As shown in Fig. 7B, significant suppression of IFN-γ production by T_effs was achieved when increasing numbers of T_regs were added to T_eff culture stimulated with L. major-infected BMDCs. No major effect was observed for IL-10 production (data not shown).

FIGURE 7. Cytokine production by T_effs from L. major-infected mice in response to L. major antigens is blocked by T_regs.

(A) 8- to 10-week old C57BL/6 mice were inoculated in the ear dermis with L. major. 5 or 21 months later, CD25⁺CD4⁺ (T_regs) and CD25-CD4⁺ (T_effs) were purified from the draining LNs (see Fig. 6). T_effs were cultured with uninfected BMDCs (open bars), L. major-infected BMDCs (black bars), or cocultured with T_regs (T_eff:T_reg ratio of 5:4) and L. major-infected BMDCs (grey bars). Cytokines were measured by ELISA. Results are representative of 6 independent experiments. nd, not detected. (B) FoxP3⁺ CD4⁺ T cells were sorted from FoxP3-GFP knock-in animals of 57-week old L. major-infected mice that were infected when young (9-10-week old). FoxP3⁺ cells were then mixed at different T_eff:T_reg ratios with T_effs (CD4⁺CD25-CD62L-) purified from a 22-week old infected mice in presence of L. major-infected BMDCs. IFN-_ was measured by ELISA in 4 day-supernatant.

T_regs play a critical role in L. major reactivation in aged mice

To directly address the role played by T_regs in L. major reactivation in old mice, we treated old L. major-infected mice (> 48-week old, which 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 T_regs 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. 8B). However, IL-10 production did not change.

FIGURE 8. In vivo depletion of T_regs in old L. major-infected mice increases the production of IFN-γ by T_effs at the infection site.

(A) Old L. major-infected mice (> 48 week old, which had been infected when they were 8-week old) or young L. major-infected mice (16-week old, which had been infected when they were 8-week old) were treated with anti-CD25 Ab or isotype control (N = 4/group) (1 mg for 3 weeks, 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.

T_regs play a critical role in the exacerbation of L. major infection in aged mice

Another important and related question is whether the increased proportion of functionally suppressive T_regs 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. 9A, primary infection with L. major induces a more severe course of disease in old mice, compared with young mice. Indeed, lesions appeared 3 weeks post-infection in both groups, but tended to be larger in old animals already at 4 weeks post-infection. Disease severity was clearly apparent at all the following time points. Moreover, 1 out of the 3 old infected animals lost the infected ear at 7 weeks post-infection, 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 T_regs had played a major role in such an exacerbated pathological process, we treated old mice (> 43-week old) with anti-CD25 Ab or isotypematched 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. 9B, treatment with anti-CD25 Ab significantly reduced the lesion size at 4 and 6 weeks, suggesting the importance of T_regs in the increased severity of L. major infection in aged animals.

FIGURE 9. L. major causes exacerbated disease in old mice, and T_regs contribute to such increased disease severity.

(A) Primary infection with L. major parasites: 4 young mice (10-week old) and 3 old C57BL/6 mice (57-week old) were inoculated in the ear dermis with 10³ L. major metacyclic promastigotes. Lesion size was measured weekly in all animals. Mean (and SD) lesion sizes (mm) are shown for young (black symbols) and old (white symbols) mice. P values indicate differences at each time point (t-test). At 7 weeks post-infection, 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-week old mice (black circles) and two 55-week old mice (black squares) received anti-CD25 treatment (1 mg for 3 weeks, twice a week) at the time of L. major infection. Three 43- week old mice (white circles) and three 55-week old mice (white squares) received the isotype control Ab following the same regimen. Lesion size (mm) 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.

Discussion

In this study, we investigated the proportion, phenotype and suppressive function of T_regs in aged mice and humans. Our study shows increased proportion of FoxP3⁺CD4⁺ T cells in 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 T_regs 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 to young mice, as already reported in aged Balb/c mice (34). Increased proportion of CD25^hiCD4⁺ 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 CD25^hiCD4⁺ 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 T_regs (79). FoxP3 expression clearly plays a crucial role in the maintenance of T_reg activity, 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).

Increased proportion of FOXP3⁺CD4⁺ T cells was also found in the blood of healthy elderly humans. There was a trend towards increased proportion of CD25^hiCD4⁺ T cells in the blood of elderly donors, although the difference was not significant. It has recently been shown that low level of the IL-7 receptor, CD127, increases the specificity of T_reg characterization for human cells (50, 51). Significantly increased proportion of CD25⁺CD127^loCD4⁺ T cells was also found in elderly humans. Previous studies using CD25^hi expression to characterize T_regs have reported similar (53, 83) or increased T_reg proportions (37-40) 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 T_regs in the blood was not increased; in contrast, we observed an increased proportion of circulating FOXP3⁺CD4⁺ T_regs in elderly humans compared to young subjects. Differences between mice and humans could reflect the fact that FoxP3 is less specific of the T_reg lineage in humans than in mice, because its expression is transitorily induced in T_effs following TCR activation (64). However, the low level of CD69 expression on FoxP3⁺ cells argues against that hypothesis. Alternatively, increased triggering of the T_reg 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 (approximately 10% versus 4%, unpublished data), similar to the murine data. Study of tissue T_regs 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 T_regs accumulate with age. Circulating T_regs 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 T_reg proportion was found in all lymphoid tissues in aged mice. Peripheral T_regs could derive either from T_regs that have developed in the thymus or converted non-T_regs. In aged mice, the total number of CD25⁺CD4⁺ single positive (SP) thymocytes decreased following the reduction in thymocyte numbers, although the percentage of CD25⁺ cells increased in CD4⁺ SP thymocytes (35), suggesting a reduced thymic T_reg input in those mice. T_regs 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 T_regs in elderly might come from thymic-derived T_regs that have proliferated in the periphery. Few data are available on T_reg in vivo turnover, particularly in aging. In young mice, the CD25⁺CD4⁺ T_reg 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 T_regs can proliferate in vivo, resting thymic-derived T_regs may also persist for long time in vivo and this population may participate in the maintenance of peripheral T_reg numbers. In healthy humans, young and elderly alike, CD45RO⁺ T_regs had a rapid doubling time compared with those of memory or naïve CD4⁺ T cells (40). However, human CD45RO⁺ T_regs exhibited short telomere length -in both young and elderly individuals-, and did not upregulate telomerase after activation (40, 87), raising the question of whether peripheral proliferation of thymic-derived T_regs is sufficient to maintain the increased proportion of T_regs seen in aged hosts.

T_regs might also be generated from CD25-CD4⁺ T cells in the periphery. In both mice and humans, transforming growth factor-β (TGF-β) induces CD25⁻FOXP3⁻CD4⁺ T cells to become FOXP3⁺ T_regs (reviewed by (88)), although the stability of such conversion, as well as the requirement for other molecules than TGF-β, is still debated (89-93). The state of activation of DCs, as well as the proportion of different DC subsets (mDC versus pDC), also plays a role in both T_reg 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 T_reg accumulation in elderly individuals. However, the contribution of such converted cells to the pool of circulating T_regs remains an open question, since no marker(s) have been found that distinguish between natural FoxP3⁺ T_regs and induced FoxP3⁺ T_regs. Of note, our published data support the idea that no neo-generation of T_regs occur during L. major infection (78).

An extensive phenotypic characterization of T_regs (defined as FoxP3⁺CD4⁺ T cells) was performed in the old mice. Expression of CD25 by T_regs from aged animals was lower than in 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⁺ T_regs. In support of that argument, in vivo expanded T_regs become CD25^low while maintaining similar levels of other T_reg-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 T_reg function such as GITR, CTLA-4 or PD-1, was maintained or increased on FoxP3⁺CD4⁺ T_regs from aged mice, depending on the tissue analyzed. The activation marker CD69 was also more expressed by old FoxP3⁺CD4⁺ T_regs than young T_regs, and that in all analyzed tissues except in the blood in which the frequency of CD69⁺ T_regs tended to decrease, suggesting an accumulation of activated T_regs in tissues in aged mice. Similarly, we found a trend towards decreased proportion of CD27 and CCR7 expression in old T_regs (Table II), suggesting increased T_reg differentiation. Taken together, those data suggest that T_regs are more activated in tissues from aged mice than in young mice.

We also extensively characterized circulating T_regs in healthy elderly humans, in comparison to young T_regs, and did not find major age-related differences in the expression of molecules that have previously been associated with T_reg function, such as CD27, PD-1, TGFβRII or Granzymes (A and B). Similar to our data in old mice, there was a trend towards decreased expression of CD25 on FoxP3⁺CD4⁺ T_regs in elderly humans. Low and constant expression of CD69 was found on both young and old human blood T_regs, in contrast to its decreased expression in circulating T_regs of aged mice, suggesting a difference in activation patterns between species.

Because phenotype does not recapitulate functional activity, we analyzed T_reg-mediated suppression during aging. Interpretation of suppression assays in aging mice can be confounded by the fact that CD25 expression is not accurate to recapitulate FoxP3 expression (and T_reg 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 T_regs were also able to suppress T_effs from aged mice. The use of FoxP3-GFP cells in our study may explain the difference between our results and previous studies that showed similar or decreased suppressive activity of old CD25^hi T_regs compared to young T_regs. As we show that many of the FoxP3⁺ cells in aged mice are CD25^lo, the previous studies sorting old T_regs based on CD25 expression may have been by contamination of the effector population with FoxP3⁺CD25⁻ T_regs. In addition, CD25⁺ T_regs from aged chronically L. major-infected mice also maintained their responsiveness to L. major antigens and their ability to suppress IFN-γproduction by T_effs in response to L. major-infected DCs. In elderly humans, T_regs 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 T_effs and the fact that human T_regs could not be depleted on the basis of their FOXP3 expression.

Increased proportion of functional T_regs 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 T_reg accumulation in the chronic infectious site (44, 45), occurred in the majority of aged infected mice. We also demonstrate a direct role of T_regs in such reactivation, because in vivo depletion of T_regs in old mice, infected young, significantly increased the production of the effector cytokine IFN-γ by the T_effs purified from the infection site and draining LNs. Furthermore, our experiments show that T_regs play a critical role in the increased disease severity of L. major infection in old mice. Taken together, our data strongly suggest that the age-associated T_reg accumulation is likely to play a major role in the increased severity of chronic infections, 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 T_reg accumulation in aged mice plays a crucial role in inhibiting the activation of anti-tumor responses (34).

How T_regs act is still unclear and we have not formally ruled out that the mechanisms mediating such suppression may be different in old T_regs. Expression of activation markers by old tissue T_regs was increased, whereas, on a per a cell basis, old T_regs produced less IL-10, which has been shown to play a role in T_reg-mediated suppression in the L. major model (44). T_regs from old murine lymphoid tissues were clearly suppressive both in vivo and in vitro, although the mechanisms by which old T_regs 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 T_effs 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 T_effs, including in response to L. major antigens, was observed in both our study (Fig. 6) and previous studies (36, 79). Similarly, in 3/7 old individuals, depletion of CD25 resulted in partial or absent improvement of CD4 T cell function, suggesting intrinsic decreased responsiveness of the T_eff 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⁺ T_effs from old animals produced large amounts of the effector cytokine IFN-γ after restimulation with L. major-antigens, which was blocked by co-culture with T_regs. Furthermore, T_reg depletion in vivo allowed for better control of L. major, suggesting that aged T_effs 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 T_reg regulation than our model, old mice controlled infection as well as young mice (102), further suggesting that T_eff 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/auto-immunity/cancer). Manipulation of T_reg numbers and/or activity may therefore be critical to enhance immune responses in the aged, and may be envisioned to enhance control of chronic infectious diseases, as well as vaccine efficiency, in this fragile population.

Abbreviations

T_regs

regulatory T cells

T_effs

effector T cells

L. major

Leishmania major

FoxP3

Forkhead box P3

CTLA-4

Cytotoxic T Lymphocyte Associated antigen 4

GITR

Glucocorticoid-Induced Tumor necrosis factor Receptor

MFI

Mean Fluorescence Intensity

PHA

phytohemagglutinin

LNs

lymph nodes

pLNs

peripheral LNs (retromaxillar and popliteal)

mLNs

mesenteric LNs

PBMCs

Peripheral Blood Mononuclear Cells

FCS

Fetal Calf Serum

DMSO

Dimethyl sulphoxide

CFSE

Carboxyfluorescein diacetate, succinimidyl ester

ELISA

Enzyme Linked Immunosorbent Assay

DCs

Dendritic cells

BMDCs

Bone Marrowderived DCs

pDCs

plasmacytoid DCs

mDCs

myeloid DCs

TGF-β

Transforming Growth Factor-β