Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype

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1 International Immunology, Vol. 20, No. 3, pp doi: /intimm/dxm151 ª The Japanese Society for Immunology All rights reserved. For permissions, please Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype Brigitte Santner-Nanan 1, Nabila Seddiki 2, Erhua Zhu 3, Verena Quent 1, Anthony Kelleher 2, Barbara Fazekas de St Groth 3, * and Ralph Nanan 1, * 1 Discipline of Paediatrics, Nepean Clinical School, University of Sydney, Penrith 2751, New South Wales, Australia 2 Centre for Immunology, St Vincents Hospital, Darlinghurst and National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, Kensington 2052, New South Wales, Australia 3 Centenary Institute of Cancer Medicine and Cell Biology, Faculty of Medicine, University of Sydney, Locked Bag 6, Newtown 2042, New South Wales, Australia Keywords: CD4+ T cell differentiation, immune regulation Abstract We and others recently described a method for isolating viable forkhead boxp3 (FoxP3 1 ) T regulatory cells (Tregs) by means of the surface phenotype CD4 1 CD127 lo CD25 1. In this study, we used the new strategy to measure Treg numbers, phenotype and function at different ages. Mean percentages of CD4 1 CD127 lo CD25 1 Tregs increased only slightly throughout life, from 6.10% in cord blood to 7.22% in PBMC from adults between 20 and 25 years and 7.50% in PBMC from adults over the age of 60. In all age groups, a higher proportion of Tregs had acquired a CD45RA 2 memory phenotype compared with CD4 1 Foxp3 2 conventional T cells. This increase was entirely attributable to increased Tregs with an effector memory phenotype, whereas central memory phenotype cells were comparably represented within the Treg and conventional CD4 1 T-cell populations. Expression of CD95 also differed between Tregs and conventional CD4 1 T cells at all ages. However there was no difference in the suppressive capacity of the different naive and memory Treg subsets. These results suggest that, compared with their conventional CD4 1 T-cell counterparts, Tregs undergo preferential differentiation from a naive to an effector memory phenotype, driven by their specificity for self- rather than foreign antigen. However, number and function are remarkably stable throughout life. Introduction T regulatory cells (Tregs) are a distinct population of CD4 + T cells that protect against autoimmunity by maintaining peripheral self-tolerance (1 4). In contrast to conventional (non-regulatory) T cells that require a period of differentiation in order to become functional, Tregs are believed to exit the thymus in a fully functional state (5 7). However, several recent studies have defined further developmental stages in human Treg populations, with predominantly naive phenotype (CD45RA + ) Tregs in cord blood (CB) and a mixture of naive and memory phenotype (CD45RO + ) Treg sub-populations in adult blood (8, 9). In contrast to conventional memory T cells, which have been categorized into effector and central memory sub-populations on the basis of chemokine receptor 7 (CCR7) expression and the speed at which they develop effector function in vitro (10), distinct subsets of memory phenotype Tregs have not yet been identified. In addition, while the cytokine IL-7 is essential for the transition of conventional CD4 + T cells from effector to memory compartments (11 13), we and others have recently shown that Tregs express only low levels of the IL-7 receptor (CD127) (14, 15). Thus, Tregs with a memory phenotype may also differ from conventional memory T cells in regards to their cytokine requirements. In this study, we utilized intracellular forkhead boxp3 (FoxP3) staining and the newly described CD4/CD25/CD127 staining method to assess number, function and phenotype of the Treg subset from early infancy to late adulthood. Our data indicate that the pool of Tregs is maintained within a narrow range throughout adult life. Furthermore, comparison of the expression of memory/activation markers by Tregs and their non-regulatory counterparts showed that effector but not central memory phenotype cells constitute a larger proportion of the Treg pool in all age groups. Further distinctions in regards to expression of CD95 by Tregs and conventional T cells suggest that number and phenotype within *These authors contributed equally to this study. Correspondence to: R. Nanan; ralphn@med.usyd.edu.au Received 19 August 2007, accepted 14 December 2007 Transmitting editor: T. Huenig Advance Access publication 14 January 2008

2 376 Phenotype and function of Tregs throughout life Treg subsets are controlled independently of the pool of conventional T cells. Despite these age-dependent phenotypic changes, Treg functional capacity as assessed by in vitro suppression assays remains stable throughout life. Methods Human subjects Peripheral blood from healthy adult volunteers (ages and >60 years) was obtained by venous puncture and collected into lithium heparin tubes. Volunteers did not suffer from any obvious intercurrent illnesses, e.g. common colds. Individuals on immunosuppressive drugs, e.g. steroids or chemotherapy, were excluded. CB samples from healthy full-term neonates born at the Nepean Hospital were acquired immediately after delivery from the clamped umbilical cord. Mononuclear cells (MNCs) were isolated by Ficoll-Hypaque (Amersham Pharmacia, Piscataway, NJ, USA) gradient centrifugation. All samples were obtained after the donors or their guardians gave informed consent. The Ethics Committee of the Sydney West Area Health Service has approved this project according to the Declaration of Helsinki. Cell staining and flow cytometry MNCs were suspended in PBS containing 0.1% FCS and 0.02% sodium azide. Surface antibody staining was performed as described previously (9). The following antibodies were used: FITC-, PE- or biotin-conjugated anti-cd4 (BD Biosciences, Mountain View, CA, USA and ebioscience, San Diego, CA, USA), anti-cd25-fitc, -PE or -allophycocyanin (APC) (BD PharMingen and Miltenyi Biotec, Gladbach, Germany), anti-cd27-fitc or APC (BD PharMingen and ebioscience), anti-cd38-fitc (BD PharMingen), anti- CD127 unconjugated or -PE (BD PharMingen) and -FITC (ebioscience), anti-cd45ra-biotin, anti-cd45ro-pe (BD Phar- Mingen), anti-cd95-pe-cy5 or -APC (BD PharMingen) and anti-ccr7-alexafluor647 (BD PharMingen). Biotin-conjugates were developed with streptavidin conjugated with peridinin chlorophyll protein (PerCP) (BD PharMingen). FITC-conjugated anti-mouse mab (BD PharMingen) was used to detect unconjugated anti-cd127. Intracellular staining for Foxp3 was performed using the anti-human staining set and protocol from ebioscience. Briefly, after cell-surface staining, the cell pellet was re-suspended by pulse vortexing and adding cold Fix/ Perm buffer. The cell suspension was incubated at 4 C for 30 min in the dark and washed twice with permeabilization buffer. To block non-specific binding to permeabilized cells, 2% normal rat serum was added at room temperature for 15 min, followed by anti-human Foxp3 antibody incubated at 4 C for 30 min in the dark. Cells were then washed twice with permeabilization buffer and finally re-suspended in Flow Cytometry Staining Buffer. Data collection was performed on a FACSCalibur (Becton Dickinson) and data files analyzed using CellQuest and FlowJo software (Treestar, San Carlos, CA, USA). Cell enrichment and sorting CD4 + T cells were first enriched from adult or cord PBMC by negative selection using the T cell Isolation Kit II from Miltenyi Biotech. For flow cytometric sorting, bead-selected CD4 cells were stained using a combination of anti-cd4-fitc, anti-cd127-pe and anti-cd25-apc or anti-cd25-fitc, anti- CD127-PE, anti-cd45ra-percp and anti-ccr7-alexafluor647. Cells were sorted on a FACSAria TM cell sorter using the indicated gates. Purity of the sorted populations was >95%. In vitro suppression assay All suppression assays were performed in 96-well roundbottom plates (Becton Dickinson) in a final volume of 200 ll per well. The culture medium used in all experiments was RPMI 1640 medium containing 2 mm L-glutamine supplemented with 10% heat inactivated fetal bovine serum, 100 U ml 1 penicillin and 100 lg ml 1 streptomycin. Responder (sorted CD4 + CD127 hi CD25 ) and suppressor (sorted CD4 + CD127 lo CD25 + ) cells were cultured in the presence of irradiated allogeneic T-cell-depleted PBMCs as antigen-presenting cells and anti-cd3 (Hit3a, BD PharMingen, at 0.25 lg ml 1 ). The responder cells were plated at per well and co-cultured with suppressor cells at various ratios in three to five replicate wells per ratio. In initial experiments, titrations of suppressor cells from 1:1 to 0.1:1 were performed. Comparison of the resulting dose response curves indicated that the higher dilutions gave results proportional to the 1:1 cultures, so later experiments used only the 1:1 ratio. In control cultures, responders were added instead of suppressor cells at a 1:1 ratio. [ 3 H]thymidine ([ 3 H]TdR) was added at 72 h for the final 16 h of culture. The cells were then harvested onto glass fiber filters and assessed for uptake of the labeled thymidine by liquid scintillation counting. [ 3 H]TdR incorporation in the suppressor:- responder cultures was expressed as a percentage of the responder:responder controls. Statistical analysis Statistical analysis was performed using Prism 4.0 software (GraphPad, San Diego, CA, USA). A one-way analysis of variance (ANOVA) followed by a Newman Keuls multiple comparison test was used to estimate the significance of differences between groups. Bartlett s test indicated that variances did not differ significantly between groups in any of the ANOVAs. The significance of the differences in phenotype between Foxp3 + Tregs and Foxp3 conventional T cells within each blood sample in Figs 3 and 4(B and D) was calculated using a paired two-tailed t-test. For all tests, P values <0.05 were considered significant. Results Percentages of FoxP3 + and CD25 + CD127 lo Tregs in CB and adult blood To investigate changes in the number of circulating Tregs throughout life, we used gating for CD4 + Foxp3 + versus CD4 + CD127 lo CD25 + to measure the percentage of Tregs within the CD4 + T cell subset. Gating strategies are shown in Fig. 1(A). As previously reported for CB and PBMCs of adult blood (9, 14), both flow cytometry gating strategies gave very similar Treg frequencies in the different age groups (Fig. 1B). Mean percentages of CD4 + FoxP3 + and

3 Phenotype and function of Tregs throughout life 377 Fig. 1. Treg percentages at different ages, comparing gating for Foxp3 + versus CD127 lo CD25 +. (A) CB and adult PBMCs from young and older donors were stained with CD4 and Foxp3 or CD4, CD127 and CD25 and gated as illustrated. (B) Left panel: CD127 lo Foxp3 + cells as a percentage of CD4 + cells; right panel: CD127 lo CD25 + cells as a percentage of CD4 + cells. Gray bars indicate CB, hatched bars adults between the age of years and white bars adults >60 years, and the number of samples in each group is indicated on the graph. The data are represented as mean 6 SEM. P values were calculated using one-way ANOVA followed by a Newman Keuls test. CD4 + CD127 lo CD25 + were 5.54% (61.71) versus 6.10% (61.99), respectively, in CB, 6.89% (61.75) versus 7.22% (61.76) in PBMC from young adults and 6.95% (61.71) versus 7.50% (61.88) in PBMC from older adults. While the percentage of Tregs increased slightly with age, only the difference between newborns and adults reached statistical significance. In vitro functional capacity of CD127 lo CD25 + cells at different ages Treg function was examined using Tregs sorted after staining with mab to CD4, CD25 and CD127. PBMCs from CB or adult blood were sorted into two subsets according to the gating strategy illustrated in Fig. 2(A). Sorted cell subpopulations from each individual were then co-cultured at a 1:1 ratio as described in Methods and the suppressive capacity of the Treg population was assessed using [ 3 H]TdR uptake. The data from five to six individuals in each age group are shown in Fig. 2(B). In every case, the sorted CD4 + CD127 lo CD25 + cells potently suppressed T-cell proliferation and the degree of suppression per cell did not change significantly with age. Differential expression of activation and memory markers by Treg and conventional CD4 + T cells as a function of age We recently showed that Tregs can be subdivided into at least two sub-populations based on surface expression of activation and memory markers such as CD45RA, CCR7, CD27, CD38 and CD95 (9). In the present study, we looked for differential expression of these markers by CD4 + Tregs versus non-tregs at different ages, using intracellular Foxp3 to identify Tregs (Fig. 3). As expected, most CB Tregs and

4 378 Phenotype and function of Tregs throughout life Fig. 2. Suppressive capacity of sorted CD127 lo CD25 + Tregs from donors of different ages. Negatively selected CD4 + T cells from CB or PBMC from donors of the indicated ages were stained for CD4, CD127 and CD25 and sorted on a FACSAria TM. (A) Gating strategy for sorting the CD4 + CD127 lo CD25 + Treg population (labeled 1). Responder cells were sorted autologous CD4 + CD127 hi CD25 cells (labeled 2). (B) Suppression assays were set up at multiple suppressor:responder ratios as described in Methods. The percentage of control proliferation from the cultures at a 1:1 ratio is shown. The bars represent mean 6 SEM of five to six independent experiments for each age group, with two to five replicate cultures per experiment. The difference between the groups did not reach statistical significance using one-way ANOVA. non-tregs expressed a naive CD45RA + phenotype. A progressive loss of CD45RA expression was seen with age in both Tregs and non-tregs. However, at all ages, a higher proportion of Tregs had switched to a CD45RA phenotype compared with conventional CD4 + T cells (Fig. 3). For both cell sub-populations, expression of CCR7 was significantly higher in CB than in adult blood, but there was no significant difference between younger and older adults. However, a higher proportion of Tregs than conventional CD4 + T cells had lost CCR7 expression in all age groups. The most striking difference between Tregs and their conventional counterparts was in the expression of CD95. In CB 21.19% (63.01) of Tregs were CD95 + compared with only 1.08% (60.84) of conventional CD4 + T cells. Expression of CD95 by Tregs and conventional CD4 + T cells increased progressively with age, but reached only 50% within the conventional population, compared with 90% in the Treg compartment. CB Tregs expressed significantly less CD38 than conventional CD4 + T cells. Marked loss of CD38 expression was seen in both Treg and conventional populations in adult donors with Tregs from donors over the age of 60 years showing slightly higher expression of CD38 compared with conventional CD4 + T cells. CB Tregs expressed significantly less CD27 than conventional CD4 + T cells but the level of expression remained constant throughout life, whereas it declined slightly between birth and adulthood in the conventional CD4 + T-cell population. Age-dependent comparison of a naive, central and effector memory phenotype within Tregs and conventional CD4 + T cells By combining the staining for CD4, FoxP3, CD45RA and CCR7, we defined changes in naive (CD45RA + CCR7 + ), central memory (CD45RA CCR7 + ) and effector memory (CCR7 CD45RA ) subsets (10) within CD4 + FoxP3 + Tregs and FoxP3 conventional CD4 + T cells (Fig. 4A and B). The proportion of cells with a naive CD45RA + CCR7 + phenotype was significantly lower in the Treg subset compared with the conventional T-cell subset in all age groups with a reciprocal increase of Tregs with an effector memory phenotype. In CB as well as in young adult blood, the proportion of Tregs with a central memory phenotype was slightly increased compared with conventional CD4 + T cells, whereas the proportion in older adults was the same. Moreover, this proportion remained at a similar level throughout adult life, while cells of naive phenotype decreased and effector memory phenotype cells increased. These data indicate that acquisition of an effector memory phenotype is differentially controlled in the Tregs and conventional CD4 + T-cell pools, whereas the central memory phenotype is expressed comparably by adult Tregs and non-treg CD4 + T cells and is remarkably stable throughout adult life. To further characterize memory Tregs, we stained with a combination of antibodies to CD4, Foxp3, CD45RA and CD27, which divides cells with a memory phenotype into CD27 + and CD27 subsets (16). As expected from the data in Figs 3 and 4(A and B), the shift from naive to conventional

5 Phenotype and function of Tregs throughout life 379 Fig. 3. Expression of activation and differentiation markers by human Foxp3 + CD4 + T cells from CB and adult donors of different ages. MNCs derived from CB or adult peripheral blood were surface stained for expression of CD4, CD25 and CD45RA, CCR7, CD95, CD38 or CD27. Cells were then fixed, permeabilized and stained for expression of Foxp3. Mean percentages (6SEM) of Foxp3 + (open bars) versus Foxp3 (filled bars) cells expressing each of the surface markers (data from 10 to 14 independent experiments, each with a single donor). For each age group, the significance of differences in expression of each of the markers within Foxp3 + Tregs versus Foxp3 conventional T cells was evaluated using a paired two-tailed t-test. P values are indicated for each age group. The significance of age-related changes in expression of each marker within either Foxp3 + or Foxp3 cells was estimated using a one-way ANOVA followed by a Newman Keuls test. The calculated P values were significant in every case apart from CD27 expression by Foxp3 + cells. Pairwise comparisons were also significant in every case except for CCR7 expression by Foxp3 + cells for versus >60 years of age, CCR7 expression by Foxp3 cells for versus >60 years of age and CD38 expression by Foxp3 + cells for versus >60 years of age. CD27 + memory cells was more prominent in the Treg compartment than the conventional CD4 + T-cell compartment at all ages (Fig. 4C and D). The small subset of cells with a CD27 CD45RA phenotype generally increased with age but was not significantly different between Tregs and conventional CD4 + T cells within each age group. In vitro suppressive capacity of naive, central and effector memory Treg subsets To compare the suppressive capacity of Tregs with naive, central or effector memory phenotype, adult CD4 + T cells were stained for expression of CD25, CD127, CD45RA and CCR7 and sorted according to the scheme illustrated in Fig. 5(A). Responder cells were mixed with suppressor cells at a 1:1 ratio and suppressive capacity was measured using thymidine incorporation. Suppression did not differ significantly between the three populations (Fig. 5B), indicating that changes in Treg surface phenotype do not correlate with suppressive activity in this assay. Discussion The percentage of circulating CD4 + CD25 hi T cells has been reported to increase significantly with age (17), while their suppressive function in vitro decreases (18). However, recent advances in the identification of Tregs have suggested that these conclusions may not apply to the entire Treg population, which includes naive Tregs whose numbers decline markedly with age (8, 9). In addition, the age-dependent increase in CD25 hi non-treg effector/memory cells, which often contaminate the conventional CD25 hi Treg gate, may explain the age-dependent decrease in suppressive activity per cell within purified CD4 + CD25 hi T-cell populations. In this study, we used expression of FoxP3 as the definitive Treg marker to measure the frequency of Tregs in different age groups. We showed that FoxP3 + Tregs in human peripheral blood lie within a range of % of CD4 + T cells, with a mean value of 5.54% (61.71) in CB, 6.89% (61.75) in young adult blood (20 25 years of age) and 6.95% (61.71) in blood from adults over the age of 60 years (Fig. 1). Thus, the total representation of Tregs within circulating CD4 + T cells is remarkably stable throughout life. These data indicate that the pool size of human Tregs appears to be strictly regulated. Tregs are known to exist in resting and activated forms (8, 9), but only limited information is currently available regarding phenotypic distinctions within Tregs expressing the human effector/memory marker CD45RO. For conventional T cells, the changes in expression of CCR7, CD62L, CD27 and CD45RA/RO observed in vitro and in vivo are believed to correspond to different stages of T-cell differentiation (10, 19). In particular, the CD45RA CCR7 + central memory and CD45RA CCR7 effector memory subsets differ from each other and from CD45RA + CCR7 + naive cells in the speed and pattern of cytokines secreted after TCR ligation (10) and in their proliferative and differentiative responses to

6 380 Phenotype and function of Tregs throughout life Fig. 4. Age-dependent changes in the distribution of naive and memory subsets in CD4 + Foxp3 + versus Foxp3 cells. (A and B) CB and peripheral MNCs from adult blood were stained for the expression of CD4, CCR7 and CD45RA. After fixation, they were stained intracellularly for Foxp3. (A) Representative profiles of CD45RA versus CCR7, gated on CD4 + cells (upper panels), CD4 + Foxp3 cells (middle panels) and CD4 + Foxp3 + cells (lower panels). Numbers indicate the relative percentages in each quadrant. (B) Percentages of cells with a naive (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA ) or effector memory (CCR7 CD45RA ) phenotype within Foxp3 + cells (open bars) versus Foxp3 cell (filled bars). Each group consists of independent donors. (C and D) MNCs from subjects in each age group were stained for

7 Phenotype and function of Tregs throughout life 381 common gamma chain cytokines in the absence of TCR ligation (20). Our results show that Tregs undergo a comparable age-related switch to central and effector memory phenotypes (Fig. 4B). However, their acquisition of effector but not central memory markers is generally accelerated when compared with their non-treg counterparts. Thus, ;10% of CB Tregs have an effector memory (CD4 + CD45RA CCR7 ) phenotype compared with only 2.3% of conventional T cells. Throughout life, the proportion of effector memory phenotype cells within the Treg compartment increases by 5-fold, and at all ages is significantly higher than that in the conventional CD4 + T-cell compartment. In contrast, the proportion of cells with a central memory phenotype (CD4 + CD45RA CCR7 + ) increases only 2-fold between birth and 60 years of age, and this phenotype is not only comparably represented within Treg and conventional CD4 + cells in adult life but also remain at a similar level between 20 and 60 years of age. In the conventional memory T-cell pool, the strict homeostatic control of T-cell numbers is believed to be due, at least in part, to competition for limited resources such as the survival cytokines IL-7 and IL-15. For memory CD4 + T cells, IL-7 is a major homeostatic factor required for maintenance. Thus, treatment with mab specific for IL-7 or its receptor CD127 leads to progressive loss of memory CD4 + T cells (12, 13). Interestingly, we have previously shown that expression of CD127 allows an unambiguous distinction between CD127 lo Tregs and CD127 hi conventional T cells in peripheral blood (14, 15). We have now shown that Tregs with a central and effector memory phenotype are both contained within the CD127 lo population, indicating that their cytokine requirements are different from those of conventional CD4 + T cells. Our findings suggest that the Treg and conventional CD4 + T cell naive, central and effector memory pool sizes are controlled independently, as they show different patterns of change with age (Fig. 4A and B). However, why this process should be biased toward the generation of effector rather than central memory phenotype Tregs remains unclear. The strikingly similar representation of the central memory phenotype within Tregs and conventional CD4 + T cells, and its stability throughout adult life (Fig. 4B), certainly suggests that central memory pool size is controlled independent of effector memory pool size for all CD4 + T cells. Our unpublished data describing acquisition of central memory phenotype during antigen-specific priming of CD4 + T cells in a murine model suggests that the central memory pool provides an early snapshot of the repertoire recruited into cell division, whereas cells with an effector memory phenotype are subject to continuous antigen-dependent selection, producing a narrower repertoire and a pool size that more closely reflects the size of the primary response (A. Spencer and B. Fazekas de St Groth, unpublished data). Alternatively, differences in the survival rate of the effector memory sub-populations between Tregs and conventional CD4 + T cells might account for the different pool sizes. Interestingly, the age-related increase in expression of the pro-apoptotic receptor CD95 (Fas) by FoxP3 + cells parallels the increase in FoxP3 + cells with an effector memory phenotype (Fig. 3), consistent with previous demonstrations of coordinated expression of CD45RO and CD95 in Tregs (9, 21, 22). High expression of CD95 by Tregs in fetal lymph nodes has been reported (23) and we have now demonstrated significantly higher expression in CB Tregs compared with their conventional counterparts (Fig. 3). Since the healthy fetus does not usually encounter foreign antigen in utero, activation and differentiation of Treg probably reflect the early interaction of circulating fetal Treg with self-antigens. During adult life, continuing self-antigen-specific activation of naive Treg emigrants from the thymus could not only trigger the immunosuppressive function of Treg but would drive differentiation, thereby constantly supplying the pool of effector memory phenotype Tregs. To date the function of human Tregs has been assessed by measuring their capacity to suppress responder cell proliferation in vitro. In this study, we have shown using purified CD127 lo CD25 + cells that Tregs maintain their in vitro suppressive activity throughout life. In particular, we have confirmed our previous data indicating that CB CD127 lo CD25 + cells exhibit comparable in vitro suppressive activity to their adult counterparts (9, 14) and have extended the analysis to show that the suppressive capacity of adult central and effector memory phenotype Tregs is also equivalent (Fig. 5B). These data contrast with the well-defined changes in function that accompany the acquisition of memory phenotype by conventional CD4 + T cells (24). Clearly, naive phenotype Tregs differ from their conventional counterparts in that they have undergone significant differentiation in the thymus and may therefore be pre-primed for function in the periphery. In addition, the conventional 3-day suppression assay may provide sufficient time for all FoxP3-expressing Tregs to achieve equivalent function. Thus, it is possible that subtle functional differences between naive, central and effector memory Tregs are obscured in the current assays but will be detected as more sophisticated assays are developed. For example, a recent publication has reported functional heterogeneity within Treg sub-populations defined by expression of the activation marker MHC class II (25). Alternatively, the phenotypic changes that correlate with changes in TCR-dependent functions in conventional T cells may occur independently of changes in suppressive surface expression of CD4, CD27 and CD45RA, and intracellular expression of Foxp3. (C) Representative profiles of CD45RA versus CD27 gated on CD4 + (upper panels), CD4 + Foxp3 (middle panels) and CD4 + Foxp3 + cells (lower panels). (D) Percentages of naive (CD27 + CD45RA + ), conventional memory (CD27 + CD45RA ) and CD27 memory (CD27 CD45RA ) cells within Foxp3 + (open bars) versus Foxp3 cells (filled bars). Each group consists of independent donors. For each age group, the significance of differences in expression of each of the markers within Foxp3 + Tregs versus Foxp3 conventional T cells was evaluated using a paired two-tailed t-test. P values are indicated for each age group. The significance of age-related changes in expression of each marker within either Foxp3 + or Foxp3 cells was estimated using a one-way ANOVA followed by a Newman Keuls test. The calculated P values were significant in every case. Pairwise comparisons were also significant except for CCR7 + CD45RA + Foxp3 cells for versus >60 years of age, CCR7 + CD45RA Foxp3 + cells for versus >60 years of age and CCR7 + CD45RA Foxp3 cells for versus >60 years of age.

8 382 Phenotype and function of Tregs throughout life central memory phenotype is differentially regulated in both Treg and conventional CD4 + T-cell compartments is not yet understood. In this context, it would be interesting to test whether memory differentiation of Tregs manifests abnormalities in subjects with chronic immunopathologies, such as autoimmune disorders or allergies. Funding Australian Women and Children s Research Foundation and the National Health and Medical Research Council. Fig. 5. In vitro suppressive capacity of memory Treg subsets. Negatively selected CD4 + T cells were stained for CD45RA, CD127, CD25 and CCR7. (A) The sorting strategy is illustrated. Sorted subsets of cells (populations 2, 3 or 4) were co-cultured with CD127 + CD25 responder cells (population 1) in a 1:1 ratio as described in Methods. (B) The percentage of control proliferation is shown. The bars represent mean 6 SEM of three independent experiments with three to five replicate cultures per experiment. The difference between the groups did not reach statistical significance using one-way ANOVA. potency in Tregs. In this scenario, loss of CCR7 expression by a Treg would still indicate an inability to home to lymph nodes, but would not be accompanied by an increase in suppressive potency. The suppressive activity of CD4 + CD25 + population cells has previously been reported to be strongly age dependent (18). In that report, it was suggested that a shift of the suppressive T-cell population from an activated (CD25 + ) to a non-activated (CD25 ) state might account for the apparent absence of suppressive function in the CD4 + CD25 + population in older individuals. However, we could not detect suppressive function within the CD4 + CD25 population [data not shown and (14)]. In addition, we previously showed that the reported lack of suppressive activity within the CD4 + CD25 int population (26) was due to unrecognized contamination by non-suppressive CD127 hi CD25 + cells (14). Without the use of CD127 expression to exclude conventional CD4 + T-cell contamination of purified Treg populations, it has been difficult to test the function of all but the population expressing the highest levels of CD25 (26). It is therefore likely that the reported age-dependent loss of suppressive function by CD4 + CD25 + cells (18) can be attributed to the age-dependent increase in non-suppressive memory cells with a CD127 hi CD25 + phenotype. In summary, our data show that the age-related phenotypic shifts within the Treg population are distinct from those of their conventional CD4 + T-cell counterparts, with a strong bias toward acquisition of effector memory phenotype and expression of CD95. These changes are consistent with the availability of self-antigen to drive Treg differentiation, although why Acknowledgements The authors would like to thank the staff of the Centenary Institute Flow Cytometry Facility for their assistance with cell sorting. Author contributions: B.S.-N designed and performed experiments, assisted in writing the paper; N.S. assisted in designing experiments and writing paper; C.Z. performed experiments; V.Q. performed experiments; A.K. provided new reagents; B.F. designed experiments and wrote the paper and R.N. designed experiments, contributed clinical samples and wrote the paper. Abbreviations ANOVA analysis of variance APC allophycocyanin CB cord blood CCR7 chemokine receptor 7 Foxp3 forkhead boxp3 [ 3 H]TdR [ 3 H]thymidine MNC mononuclear cell PerCP peridinin chlorophyll protein Treg T regulatory cell References 1 Baecher-Allan, C., Viglietta, V. and Hafler, D. A Human CD4+CD25+ regulatory T cells. Semin. 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