Originally published online as doi:10.1189/jlb.0907627 on September 9, 2008

Published online before print September 9, 2008

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(Journal of Leukocyte Biology. 2008;84:1454-1461.)
© 2008 by Society for Leukocyte Biology

The frequency of regulatory CD3⁺CD8⁺CD28^–CD25⁺ T lymphocytes in human peripheral blood increases with age

Rita Simone^*,1, Anna Zicca^,2 and Daniele Saverino^*,3

^* Department of Experimental Medicine, Section of Human Anatomy, University of Genova, Genova, Italy; and
Department of Biomorphology and Biotechnologies, University of Messina, Messina, Italy

³ Correspondence: University of Genova, Department of Experimental Medicine, Section of Human Anatomy, Via De Toni, 14, 16132 Genova, Italy. E-mail: daniele.saverino{at}unige.it

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Aging is commonly associated with immune deficiency and dysregulation. The aging of the immune system involves a progressive reduction in naïve T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. We have investigated frequency, phenotype, and function of CD3⁺CD8⁺CD28^–CD25⁺ T cells in healthy volunteers over a wide age range. We demonstrate that the frequency of CD3⁺CD8⁺CD28^–CD25⁺ T cells in healthy volunteers increases with age. Peripheral CD3⁺CD8⁺CD28^–CD25⁺ T cells share phenotypic and functional features with CD3⁺CD4⁺CD25⁺ regulatory T cells (Tregs): In particular, they strongly express CTLA-4 and forkhead box P3. We observed that in vitro, functional titration assays of CD3⁺CD8⁺CD28^–CD25⁺ T cells show equivalent regulatory function in young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. Finally, CD3⁺CD8⁺CD28^–CD25⁺ T cells seem to specifically express the CD122 receptor. Altogether, these observations demonstrate an increase in peripheral blood CD8⁺ Tregs associated with aging.

Key Words: aging • Treg • CD4⁺CD25⁺

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Normal aging is associated with the development of a series of events, collectively called immune senescence, and that leads to a reduced efficiency of the immune system responses. In fact, a reduced response to recall antigens, as well as an increased sensibility to infections and malignancy can be observed [¹ , ² ]. In addition, an impaired self-tolerance can be evident, as can be deduced by the increased incidence of autoimmune diseases observed with aging. However, the mechanisms underlying these clinical effects remain poorly understood. Thymic involution in adults is associated with a marked reduction in thymic T cell output, as measured by decreased TCR excision circle⁺ T cells in subjects aged over 65 years and a reduction in the number of naïve CD45RA⁺ T cells [³ , ⁴ ]. It is likely, therefore, that maintenance of the peripheral T cell pool in the elderly will depend on an increase in proliferation of existing T cells through peripheral expansion of T cells with affinity for foreign and self-antigen [⁵ ]. This phenomenon may at least partly explain the contraction of the TCR repertoire and the emergence of clonal T cell populations as seen in the memory compartment in elderly subjects [⁶ , ⁷ ]. Impaired central tolerance and a reduction in the diversity of the T cell repertoire may be key factors in immune dysregulation in the elderly, and peripheral mechanisms for immune regulation may become increasingly important.

In the early 1970s, R. K. Gershon [⁸ ] suggested that T lymphocytes could act as regulatory cells, suppressing immune responses. However, these cells and the molecular mechanisms responsible for suppression were difficult to be characterized. Recently, advances in the identification of CD4⁺ T cell subpopulations, together with the use of genetically modified mice, have led to a renaissance of this field, and regulatory T cells (Tregs) are now thought to be an additional mechanism by which peripheral self-tolerance is maintained, alongside T cell deletion and T cell anergy [^{9 10 11} ]. The concept of Tregs is attractive, as it could explain how tolerance can be adoptively transferred by T cells, how pathological responses to self and harmless foreign antigens are prevented, and how bystander tissue insult is avoided during normal immune responses.

The existence of thymus-derived Tregs was suggested initially by the onset of autoimmune diseases in mice after thymectomy on Day 3 of life [¹² , ¹³ ]. These disorders were found to be a result of a loss of peripheral CD4⁺ T cells that constitutively express IL-2R⁺ (CD25), which appears late in the periphery after birth [¹⁴ ]. Physiologically generated CD4⁺CD25⁺ T cells inhibit a wide range of autoimmune and inflammatory disorders such as gastritis, oophoritis, orchitis, thyroiditis, colitis, and spontaneous autoimmune diabetes [^{15 16 17 18} ].

Despite numerous studies, the mechanisms by which CD4⁺CD25⁺ T cells exert their regulatory function are unclear. Some studies have shown that regulation in vivo depends on the production of suppressive cytokines such as IL-10 and TGF-β and cell-surface molecules such as CD152 [^{19 20 21} ]. In vitro experiments, aimed at further dissecting the mechanisms by which T cells exert their regulatory function, have given controversial results. Indeed, in contrast to findings in vivo, neither soluble cytokines nor CD152 seem to be required for the suppressive effects of CD4⁺CD25⁺ cells in vitro [^{22 23 24} ]. Taken together, in vitro studies of CD4⁺CD25⁺ T cells support a cell contact-dependent, cytokine-independent mechanism of suppression [²⁴ ]. In addition, there is evidence of an increasing of CD4⁺CD25⁺ T cell subset in the peripheral blood with aging [^{25 26 27 28} ].

CD8⁺ T cells have been reported to be essential in vivo to prevent experimental autoimmune encephalomyelitis and to participate in oral tolerance [^{29 30 31} ]. Nevertheless, the existence of a CD8⁺ Treg subset has received less attention, despite earlier studies that identified CD8 populations with a suppressive effect. Otherwise, it has been shown that regulatory/suppressor CD8⁺CD28^– T cells can be generated/expanded in vitro by multiple rounds of stimulation with allogenic, xenogenic, or antigen-pulsed, syngenic APCs [³² , ³³ ]. In addition, it has been suggested that CD8⁺ Tregs can act by regulating acute or established inflammation [³⁴ ] and that they are present in the tumor-infiltrating lymphocyte population of cancer patients [³⁵ ].

Although several aspects of the biology, mechanism of action, and role of these cells remain to be elucidated, there is evidence that a Treg subset can be involved in cancer patients [^{34 35 36 37 38 39 40} ].

In this paper, we have analyzed CD8⁺CD28^–CD25⁺ T cell (indicated from here as CD8⁺CD28^– Treg) frequency, phenotype, and function in healthy volunteer donors over a wide age range. We found marked increases in CD8⁺CD28^– T cell frequencies with increasing age. Direct, functional assays demonstrated suppressive effects in vitro. In fact, CD4⁺CD25⁺ and CD8⁺CD28^– Tregs from young and elderly subjects suppress proliferation and cytokine production in response to polyclonal T cell stimulation.

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Subjects
Forty healthy volunteers between the ages of 20 and 81 years were recruited into the study after informed consent. The donors were defined as "healthy" after answering a questionnaire excluding evident pathologies (i.e., autoimmune diseases, infectious diseases, acute inflammation, and cancer). The research was approved by the Ethical Committee of the University of Genova and San Martino Hospital (Italy) and was in accordance with the principles of the Helsinki II Declaration.

Flow cytometry
PBMC were isolated from peripheral blood samples by density centrifugation over Ficoll-Hypaque gradient. Antibodies used for flow cytometry were PE-, PE-CY5.5-, allophycocyanin-CY5-, or FITC-conjugated anti-CD3 (IgG1, clone UCHT1), anti-CD4 (IgG1, RPA-T4), anti-CD8 (IgG1 RPA.Tg), anti-CD25 (IgG1, BC96), anti-CD28 (IgG1, CD28.2), and anti-CD127 (IgG1, eBioRDR5; eBiosciences, San Diego, CA, USA); anti-CD62 ligand (CD62L; IgG1, LT-TD180), anti-CD45RO (IgG1, UCHL1), anti-CD38 (IgG1, HIT1), and anti-HLA-DR (IgG1, 1E5; ImmunoTools, Friesoythe, Germany); and anti-CD122 (IgG1, MIK-β3; BD Biosciences, Hamburg, Germany). Appropriate fluorochrome-labeled, isotype-matched mAb were used as controls. For intracellular staining, immunomagnetic-sorted cells were fixed in 2% paraformaldehyde and permeabilized with 0.1% saponin before staining with anti-CD152 (IgG2a, 14D3), anti-forkhead box P3 (FoxP3; IgG2a, PCH101; eBiosciences), or isotype-matched control antibody. Flow cytometry was performed on a FACSCalibur (BD Biosciences). Unconjugated anti-CD28 mAb was a gift by Daniel Olive (Université de la Méditerranée, Cancer and Immunology Institute of Marseille, Marseille, France).

Isolation of Tregs
For functional assays, CD8⁺CD28^– and CD8⁺CD28⁺ cells as well as CD4⁺CD25⁺ and CD4⁺CD25^– were isolated by immunomagnetic cell sorting with specific mAb-coated microbeads (Miltenyi Biotec, Milan, Italy). CD8⁺ T cells were separated by negative selection using the CD8 T cell isolation kit (Miltenyi Biotec) and then incubated with the FITC-conjugated anti-CD28 mAb and with anti-FITC MicroBeads (Miltenyi Biotec). CD8⁺CD28^– and CD8⁺CD28⁺ T cell subsets were obtained by negative (flow-through) and positive selection (eluted, column-retained cells), respectively. The purity of each subset, as assessed by flow cytometry, was consistently >95%, and viability was greater than 99% by the trypan blue exclusion test (not shown). CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25^– were isolated using the CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotec). Briefly, CD4⁺ cells were negatively purified, CD4⁺CD25^– cells were obtained by negative selection, and CD4⁺CD25⁺ cells were positively selected. Using this procedure, we selected a population containing the CD4⁺CD25^high fraction accompanied by varying numbers of the CD25^low subset. The artificial activation of cells was excluded by cytofluorimetric analysis of surface expression of CD69, which was always <0.5% (not shown). The purity of CD4⁺CD25⁺ and CD4⁺CD25^– subsets was greater than 90–95%, and viability was greater than 99% by the trypan blue exclusion test (not shown).

Proliferation assays
RPMI-1640 medium supplemented with L-glutamine (2 mM), penicillin (100 IU/ml)/streptomycin (100 µg/ml), and 10% human AB serum (all from Invitrogen, Milan, Italy) was used in all assays. Direct suppression "add-back" experiments were performed as described previously [⁴¹ ]. Briefly, 1 x 10⁴ CD8⁺CD28^–, CD4⁺CD25⁺, or CD4⁺CD25^– cells were incubated in the presence of PHA (Sigma Chemical Co., St. Louis, MO, USA) at 2 µg/ml and 1 x 10⁴-irradiated (30 Gy), autologous PBMC that had been depleted of T cells (CD3 microbeads, Miltenyi Biotec). Incubations were performed with CD8⁺CD28^– or CD4⁺CD25^– fractions, alone or in coculture (CD8⁺CD28^–/CD4⁺CD25^–, CD4⁺CD25^–/CD4⁺CD25^–), at a ratio of 1:1. Similarly, incubations were performed with CD4⁺CD25⁺ fractions, alone or in coculture (CD4⁺CD25⁺/CD4⁺CD25^–, CD4⁺CD25^–/CD4⁺CD25^–), at a ratio of 1:1. In some experiments, titrations of CD8⁺CD28^– or CD4⁺CD25⁺ were added to CD4⁺CD25^– cells (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:512). All incubations were run in triplicate in 96-well plates with a final volume of 200 µl. At 72 h, 1 µCi [3H]-thymidine was added to each well, and proliferation by [3H]-thymidine incorporation was assessed after a further 16 h.

Cytokine assays
CD8⁺CD28^–, CD4⁺CD25⁺, or CD4⁺CD25^– T cells were cocultured as above, and supernatants were evaluated after 72 h for IFN-, IL-4, IL-10, and TGF-β (Bender, Milan, Italy) production by ELISA.

Statistics
Statistical analysis was performed using GraphPad Prism software 4.0 (GraphPad Software Inc., San Diego, CA, USA). The nonparametric Spearman’s rank correlation coefficient with two-tailed P value was used to assess the significance of correlation between frequency of CD8⁺CD28^– T cells or CD4⁺CD25⁺ T cells and subject age. ANOVA analyses were performed to evaluate differences among the Treg frequency and the different age groups. Mann-Whitney analyses were performed to assess differences in young and elderly donor cell marker expression.

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CD8⁺ Tregs have a typical cell-surface phenotype
PBMC from healthy donors were separated by immuno-beads cell sorting in two different populations expressing CD3⁺CD8⁺CD28⁺ (defined as cytotoxic) and CD3⁺CD8⁺CD28^– (defined as regulatory). In an attempt to identify markers characteristic for CD8⁺ cytotoxic and CD8⁺ Treg lymphocytes, we first compared their immunophenotype using a panel of mAb as shown in Figure 1 . This figure represents a staining of CD8⁺ Tregs from a single donor. These patterns of expression were characteristic of this subpopulation of T cells, independently of the age donor.

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Figure 1. Analysis CD8+ Treg surface phenotype. To better identify markers characteristic for CD3+CD8+ cytotoxic (empty histograms) and CD3+CD8+ Treg lymphocytes (shaded histograms), we compared their immunophenotypes using a panel of mAb. The graphs represent a staining on T lymphocytes isolated from a single donor representative of the cohort of donors enrolled in this study. To this purpose, two healthy donors were chosen from each aging group. Numbers indicate mean fluorescence intensity (shaded histograms) with an identical setting of the instrument for all cells analyzed ± SD. Differences among these patterns of expression were characteristic of this subpopulation of T cells and independent to age donor (P>0.05). An irrelevant mAb (isotype-matched IgG1) was used as a negative control. Shaded and dashed profiles indicate, respectively, CD3+CD8+ cytotoxic and CD3+CD8+ Treg lymphocytes staining. For intracellular staining with anti-CD152 and anti-FoxP3, the irrelevant, isotype-matched mAb was IgG2a.

Cytotoxic and Tregs express comparable levels of HLA-DR and CD8. On the contrary, CD28 expression was restricted to the cytotoxic T cell population. The lymphonode homing receptor CD62L was expressed slightly more in the cytotoxic T cell population, whereas Treg expressed a higher level of CD45RO and a lower level of CD44 than cytotoxic T cells (expressing an effector/memory immunophenotype). In addition, CD8⁺ Tregs showed a higher expression of CD38. As expected, membrane expression of CD25 and cytoplasm expression of CD152 and FoxP3 molecules associated with CD4⁺CD25⁺ Tregs were slightly higher in CD8⁺ Tregs than in cytotoxic cells. Of interest, all cells expressing CD3⁺CD8⁺CD28^–CD152⁺ were positive for FoxP3 and negative or roughly negative for CD127 (data not shown). In fact, it has been shown that down-regulation of CD127 (the IL-7R) distinguishes Tregs from activated T cells, facilitating Treg purification and their functional characterization in human diseases [^{42 43 44} ].

The final part of our phenotypic analysis focused on the expression of CD122 (the receptor for IL-2/IL-15Rβ). CD122 binds two of the major maintenance and/or survival cytokines. We found that CD122 (IL-2/IL-15Rβ) was preferentially expressed on the CD8⁺CD28^– Treg population (Fig. 1) .

Tregs increase in peripheral blood with increasing age
Forty healthy volunteer donors were recruited into the study with an age range of 20–81 years: 20–30 years (n=7), 31–40 years (n=7), 41–50 years (n=7), 51–60 years (n=7), 61–70 years (n=6), and over 70 years (n=6). The frequency of CD8⁺CD28^– T cells and of CD4⁺CD25⁺ (combined CD25^int- and CD25^high-expressing cells) within PBMC was performed by flow cytometry. Appropriate isotype controls were performed with each sample (Fig. 1) , and samples were run repeatedly with each batch to ensure consistency in the protocol. The percentage of CD8⁺CD28^– expressing T cells showed a steady rise in association with increasing subject age (r=0.9244, P<0.0001), and this effect was observed without a plateau (Fig. 2A ). Similarly, the percentage of lymphocytes expressing CD4⁺CD25⁺ increased significantly with age (r=0.9591; P<0.0001; Fig. 2B ). No correlation was observed between subject age and the percentage of CD8⁺ and CD4⁺ T cells in peripheral blood or the absolute peripheral blood lymphocyte count (data not shown). As can be depicted from Figures 2 C and D , the comparison of single age classes shows an increment of the percentage of CD8⁺ and CD4⁺ Tregs, proportional to the age of the donor, although some of these differences result in not significant when analyzed by the ANOVA method (compared 20–30 with 31–40 and 41–50 groups and 31–40 with 41–50 in Fig. 2C for CD8⁺ Treg and 20–30 with 31–40 and 41–50 groups and 51–60 with 61–70 in Fig. 2D for CD4⁺ Tregs). Otherwise, the proportion of CD8⁺CD28^– was higher in older donors than younger: 9.91% (±1.72 SD) and 1.03% (±0.77), respectively, for donors aged >70 and for donors aged 20–30 (P=0.006; Fig. 1E ). Consistently, the proportion of CD4⁺CD25⁺ was higher in older donors with the median percentage of CD4⁺CD25⁺ cells in gated lymphocytes of 25.07% (±4.73) for donors aged >70 and 1.77% (±0.93) for donors aged 20–30 (P=0.006; Fig. 2F ).

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Figure 2. Analysis of peripheral blood frequency of CD3+CD8+CD28–CD25+ and CD3+CD4+CD25+ Tregs with age. Analysis of Treg populations was performed on peripheral blood obtained by several healthy donors. The percentages of CD3+CD8+CD28– T lymphocytes, which are CD25+ (A), and that of CD3+CD4+ T lymphocytes, which are CD25+ (B), were plotted against subject age. Linear regression analysis is shown (solid line) with 95% confidence intervals (dotted lines). Scatter-plots of peripheral blood percentage of CD3+CD8+CD28–CD25+ (C) and CD3+CD4+CD25+ (D) Treg lymphocytes among different groups of subjects are represented. The one-way ANOVA nonparametric test (Kruskal-Wallis test) was used to compare the differences, and significances are shown. Scatter-plots of plasma percentage of CD3+CD8+CD28–CD25+ (E) and CD3+CD4+CD25+ (F) Treg lymphocytes between different groups of subjects (young vs. elderly) are represented. The Mann-Whitney rank sum test was used to compare the differences, and its significance is shown. N.S., Not significant.

Tregs in young and elderly subjects suppress immune responses in vitro
CD8⁺CD28^–/CD4⁺CD25⁺ and CD4⁺CD25^– T cell fractions were isolated at high purity (>97% for each fraction; not shown) from young (n=3; aged 20 and 31) and elderly (n=3; aged 71 and 80) donors. The ability of CD8⁺ and CD4⁺ Tregs to suppress proliferation of CD4⁺CD25^– T cells was determined by adding CD8⁺CD28^– and CD4⁺CD25⁺ T cells at a variable ratio to CD4⁺CD25^– T cells in the presence of polyclonal stimulation with PHA and autologous-irradiated, CD3-depleted APCs for 72 h. As shown in Figure 3A , equivalent suppression of CD4⁺CD25^– T cell proliferation in response to PHA stimulation was observed in young and elderly subjects when CD8⁺CD28^– Tregs were added. Furthermore, there was no significant difference in the degree of suppression mediated for each reducing titration of CD8⁺CD28^– T cells on CD4⁺CD25^– T cells, with an equivalent, dose-dependent effect demonstrated for CD8⁺CD28^– T cell-mediated suppression in young and elderly individuals (Fig. 3A) . Similarly, there were no significant differences in the degree of CD4⁺CD25^– T cell suppression mediated for each reducing titration of CD4⁺CD25⁺ Tregs (Fig. 3B) .

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Figure 3. Results of add-back suppression experiments in young and elderly subjects. Magnetic-sorted CD3+CD8+CD28– and CD3+CD4+CD25+ Tregs were isolated from young (n=3; age 20, 25, and 31) and elderly (n=3; age 71, 75, and 80), healthy donors. CD3+CD8+ Treg-mediated inhibition of proliferation in young () and elderly () donors is shown (A); results are mean percentage for three separate donors in each age group (mean with error bars are shown). Similar results in inhibiting T cell proliferation mediated by the adding of CD4+ Tregs are represented (B). Results are mean percentage for three separate donors in each age group (mean with error bars are shown). Representative plots of production of IFN- are shown in young (C, left) and elderly (C, right) donors. Irradiated PBMCs (irr. PBMC) were used as internal control for stimulator cells–IFN- production. Results are mean percentage for three separate donors in each age group (mean with error bars are shown).

Finally, production of IFN- by CD4⁺CD25^– T cells in the above PHA stimulation was also inhibited by the addition in coculture of CD8⁺CD28^– Tregs, and this effect was observed in young and elderly donors, respectively (Fig. 3C) .

Tregs in young and elderly subjects modulate cytokine production in vitro
The amount of IL-4, IL-10, and TGF-β released in the culture supernatant was tested in the same subjects investigated in proliferation experiments. The levels of cytokines released in the culture supernatant are shown in Table 1 .

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Table 1. Cytokine Levels in Culture Supernatant before and after CD8+ Treg Addition

Before Treg addition in elderly donors, the level of IL-4, IL-10, and TGF-β secreted by CD4⁺CD25^– was slightly higher than in young donors.

In young and elderly donors, the direct addition of CD8⁺CD28⁺ Tregs to CD4⁺CD25^– T cells resulted in an increase of IL-4, IL-10, and TGF-β in the supernatant. Although the changes in IL-4 were similar in young and elderly, the increase in IL-10 and TGF-β levels was significantly higher in the elderly (15.6- and 12.4-fold, respectively) than in the young (10.1- and 7.8-fold, respectively).

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The interest in regulation of antigen-specific immune responses has led to the identification of T cell subsets that have suppressive properties. Many experimental models have been used to define Treg/suppressor T cells, which have been identified as CD4⁺, CD8⁺, or CD4/CD8 double-negative and shown to use myriad mechanisms from anti-idiotypic recognition and cytotoxicity to immune deviation, cytokine secretion, and "prohibition" of APCs. The best-characterized populations of Tregs are naturally occurring CD4⁺CD25⁺ Tregs from which a growing list of phenotypic markers, presumed to allow the unequivocal identification of Tregs, has been derived [⁴⁰ ]. Moreover, evidence indicates that CD8⁺ cells can also be divided into effector and regulatory subsets. CD8⁺ effector cells have cytotoxic activity and can produce IFN- or IL-4. CD8⁺ Tregs, by contrast, lack cytotoxic activity and produce IL-10 or TGF-β. In addition, they are hyporesponsive to secondary stimulation [⁴⁵ ].

The mechanism of action of CD8⁺ Tregs bears many similarities to that of CD4⁺CD25⁺ Tregs, including inhibition of IL-2 production in target cells, dependence on cell contact, and abrogation of suppression with PMA/ionomycin. Differently from CD4⁺CD25⁺-mediated suppression, reversed by exogenous IL-2, CD8⁺ Treg-mediated inhibition is reversed by ligation of CD28 or TCR-mediated stimuli, but not IL-2. The lack of response to IL-2 is presumably a result of the observed blockade of CD25 up-regulation, which provides a high-affinity IL-2R. Studies of CD4⁺CD25⁺ cells and human CD8⁺ Tregs suggest that suppression is mediated through APCs via down-regulation of costimulatory molecules or up-regulation of inhibitory receptors. CD4⁺CD25⁺ Tregs also suppress via direct T–T contact [⁴⁶ ].

Human CD4⁺CD25⁺ T cells are known to be functionally heterogeneous and include cells with suppressive function and recently activated effector CD4⁺ T cells [¹⁶ ]. However, characterization of CD4⁺CD25⁺ T cells in elderly and young donors demonstrated no differences in terms of membrane activation markers or expression of CD152 [²⁵ ]. The only phenotypic difference was the increased expression of CD45RO in CD4⁺CD25^high T cells in the elderly cohort, a phenomenon well described previously for CD4⁺ T cells [¹⁷ ]. These observations suggest that the increase in CD4⁺CD25⁺ T cells in the elderly is not a result of expansion of activated effector T cells and furthermore, that CD4⁺CD25⁺ T cells in this age group have a phenotype consistent with that reported previously for Tregs [²⁵ ]. Together, these observations indicate that there is an increase in CD4⁺CD25⁺ Tregs in peripheral blood with progressive aging.

Similarly, experiments conducted on animals indicated that higher pools of CD4⁺CD25⁺FoxP3⁺ and CD8⁺CD25⁺FoxP3⁺ cells were accumulated in spleens and lymph nodes from old mice when compared with young animals [²⁷ ]. In addition, it has been shown that the levels, phenotypes, and function of CD4⁺CD25⁺ Tregs were altered significantly with aging in mice [²⁸ ].

The reasons for expansion of CD8⁺CD25⁺ as well as CD4⁺CD25⁺ Tregs with age are unclear. Against a background of progressive thymic atrophy and reduced, naïve T cell output, it is unlikely that the expansion of CD8⁺CD25⁺ T cell numbers in the elderly is thymically derived, and therefore, the peripheral expansion of existing Tregs may be a predominant factor. In vitro, conventional CD4⁺CD25^– T cells may undergo induction to a Treg phenotype in the setting of impaired costimulation and under the influence of immunosuppressive cytokines such as IL-10 [¹⁸ , ¹⁹ ].

Recently, three different CD8⁺ T suppressor cell subpopulations have been identified and functionally characterized in humans. One of these subsets is represented by CD8⁺CD28^– T cells, which inhibit alloantigen, xeno-antigen, and nominal, antigen-specific CD4⁺ T cell responses. CD8⁺CD28^– T cells were generated in vitro by multiple rounds of stimulation of PBMC with allogeneic, xenogeneic, or antigen-pulsed, syngeneic APCs [³⁴ ]. Another type of CD8⁺ T cells was generated in vitro from purified, circulating CD8⁺ lymphocytes incubated for 1 week with monocytes, IL-2, and GM-CSF. These cells inhibited the proliferative response of T cells stimulated with specific antigens, anti-CD3 mAb, or mitogens. Furthermore, they also seem to suppress the lysis mediated by cytotoxic cells [³⁴ ]. A third subpopulation of CD8⁺ suppressor lymphocytes has been identified recently [⁴⁷ ]. These cells were generated by stimulating purified, naive CD8⁺ T lymphocytes with plasmacytoid dendritic cells (DC). Their generation depends on antigen presentation and secretion of IL-10 by DC. The mechanism of action of this subset of CD8⁺ suppressor cells is not yet understood completely, although it is clear that they exert their function through soluble factors without direct interaction with APCs. Each of these suppressor subsets acts in different ways to regulate antigen-specific T cell responses. Functional alterations were shown to be associated with the relapse of autoimmune diseases and onset of acute rejection episodes in transplanted recipients.

In the present study, we have demonstrated that the frequency of peripheral blood Tregs/suppressor T cells increases in association with aging. Our study included donors over 70 years of age, in whom particularly high numbers of CD8⁺CD28^– Tregs were observed with a ninefold increase compared with donors aged between 20 and 30 years.

It is possible that the increasing CD8⁺CD28^– together with CD4⁺CD25⁺ Treg frequencies observed in the elderly contribute to immune deficiency and plays a part in the broader decline in adaptive responses during immunosenescence.

In addition, it was observed that individuals with a "healthy immune system" might live longer than those whose immune systems produce autoantibodies [⁴⁸ ]. A possible explanation of these observations was that as healthy centenarians are a highly selected population (only 1 in 10,000 human beings reaches 100 years old), survival could be associated with an unusually efficient immune system, devoid of the age-related abnormalities often seen in the "younger" elderly. Studies in healthy centenarians support this idea by showing well-preserved immune functions such as NK cytotoxicity, proliferative responses to mitogenic stimuli, and ability to cope with oxidative stress [⁴⁸ ]. Thus, centenarians might also be characterized by a lower frequency of circulating Tregs than younger elderly. Obviously, such a hypothesis would require experimental support.

Finally, the high expression of CD122/IL-2/IL-15Rβ could suggest that high levels of this receptor may allow Tregs to act as a cytokine sink, taking important maintenance signals away from other cells and ensuring their own dominance in the aged T lymphocyte pool. If so, one would expect that their response to cytokines that induce proliferation may result in only low proliferation/survival signals. Testing this hypothesis should help to uncover the rules that govern population maintenance and the reasons for their deregulation in the aging immune system.

 
This work was supported by a grant from Fondazione CARIGE (Genova, Italy) and Drogetti di Ricerca di Ateneo, University of Genova to D. S. We are indebted to Prof. Marcello Bagnasco (Medical and Radiomethabolic Therapy Unit, Department of Internal Medicine, University of Genova, Italy) for helpful suggestion in writing the manuscript. We thank Rosa Piccardo for her secretarial assistance. All financial/commercial conflicts of interests are disclosed.

 
¹ Current address: Department of Clinical and Experimental Medicine, Section of Internal Medicine, University of Verona, Verona, Italy.

² Current address: Policlinico Universitario "Agostino Gemelli," Università Cattolica del Sacro Cuore, Rome, Italy

Received September 13, 2007; revised August 4, 2008; accepted August 14, 2008.

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