of July 16, 2018.This information is current as

CD45RA or CD45RORegulatory T Cells That Express either

+Migratory Characteristics of Human CD4Different Proliferative Potential and

N. Akbar and Milica Vukmanovic-StejicFrancesco Falciani, Kwee Yong, Malcolm H. Rustin, ArneKissane, Elaine Agius, Sarah E. Jackson, Mike Salmon, Nicola J. Booth, Arthur J. McQuaid, Toni Sobande, Steve

http://www.jimmunol.org/content/184/8/4317doi: 10.4049/jimmunol.0903781March 2010;

2010; 184:4317-4326; Prepublished online 15J Immunol 

MaterialSupplementary

1.DC1http://www.jimmunol.org/content/suppl/2010/03/15/jimmunol.090378

Referenceshttp://www.jimmunol.org/content/184/8/4317.full#ref-list-1

, 29 of which you can access for free at: cites 50 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on July 16, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Different Proliferative Potential and MigratoryCharacteristics of Human CD4+ Regulatory T Cells ThatExpress either CD45RA or CD45RO

Nicola J. Booth,* Arthur J. McQuaid,* Toni Sobande,* Steve Kissane,†

Elaine Agius,*,‡ Sarah E. Jackson,* Mike Salmon,† Francesco Falciani,†

Kwee Yong,x Malcolm H. Rustin,‡ Arne N. Akbar,* and Milica Vukmanovic-Stejic*

Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with

previous reports that the (∼80%) majority of natural Tregs in adults are CD45RO+. The proportion of CD45RA+ Tregs decreases,

whereas CD45RO+ Tregs increase significantly with age. Nevertheless, a small proportion of CD45RA+ Tregs are found even in old

(>80 y) adults and a proportion of these express CD31, a marker for recent thymic emigrants. We found that CD45RO+ Tregs were

highly proliferative compared with their CD45RA+ counterparts. This was due in part to the conversion of CD45RA Tregs to

CD45RO expression after activation. Another difference between these two Treg populations was their preferential migration to

different tissues in vivo. Whereas CD45RA+ Tregs were preferentially located in the bone marrow, associated with increased

CXCR4 expression, CD45RO+ Tregs were preferentially located in the skin, and this was associated with their increased expres-

sion of CLA and CCR4. Our studies therefore show that proliferation features strongly in maintenance of the adult Treg pool in

humans and that the thymus may make a minor contribution to the maintenance of the peripheral pool of these cells, even in older

adults. Furthermore, the different tissue compartmentalization of these cells suggests that different Treg niches exist in vivo, which

may have important roles for their maturation and function. The Journal of Immunology, 2010, 184: 4317–4326.

Natural regulatory T cells (nTregs), defined as CD4+

CD25hiFoxp3+, play an essential role in the control ofimmune responses (1). They are crucial for maintaining

peripheral tolerance and protection against autoimmunity, andthey also modulate immunity to infections and tumors (1).Therefore, to maintain controlled immunity, it is paramount thatthis regulatory population is maintained throughout life. CD4+

Foxp3+ Tregs are often considered to be a distinct lineage ofthymic origin (reviewed in Refs. 2 and 3). However, the thymusinvolutes with age (4), which suggests that the number of nTregmight be reduced during aging. However, we and others haveshown that Treg numbers are increased in older animals and hu-mans (5–8). Hence, the number of nTregs throughout life must

either be sustained through extensive proliferation or by genera-tion from another, extrathymic, source, such as the CD4+ T cellresponder pool (2, 3, 7, 9).Most Foxp3+ Tregs (90–95%) in the peripheral blood of adult

humans express CD45RO (10), suggesting these cells have beenpreviously activated and induced to proliferate (11). In youngeradults, ∼10% of Foxp3+ Tregs express CD45RA and are consideredan unprimed or naive Treg population (12, 13). We have previouslyshown that CD45RO+ Tregs turnover very rapidly in both young andold donors and are susceptible to apoptosis and replicative senes-cence, indicating that they have a limited capacity for self-renewal(7, 10). Therefore, the inherent thymic involution suggests thatnTregs may also be derived from other sources in adulthood.However, the source of nTregs in older subjects has yet to be fullydelineated. Because CD45RO+ Tregs are the largest component ofthe adult Treg population (10), most work on Tregs to date has beencentered on this subset. However, CD45RA+ Tregs are more prev-alent in early life, with a high proportion of cord blood Tregs (∼75%in full-term cord blood) expressing this phenotype (14). It is of in-terest that human CD45RA+ Tregs expand readily in vitro, givingrise to a homogeneous Treg population, whereas some CD45RO+

Tregs lose their Foxp3 expression and suppressive capacity (15),suggesting that naive Tregs might play an important role in themaintenance of the Treg pool in adults. Recent work has suggestedthat CD45RA-expressing Tregs may be subdivided further into re-cent thymic emigrants (RTEs), which express the adhesionmoleculeCD31, and those that do not: these may have experienced someactivation and/or proliferation in the periphery (16).In this study, we set out to extend our previous work on the

turnover of human Tregs in vivo (7, 9, 17) and to further explorethe mechanisms of Treg maintenance in old humans. We madea number of novel observations: first, although CD45RO+ Tregsare extremely proliferative, with ∼20% in cycle at any given time,

*Division of Infection and Immunity, Department of Immunology, and xDepartmentof Hematology, University College London; ‡Department of Dermatology, Royal FreeHospital, London; and †Medical Research Council Center for Immune Regulation,University of Birmingham, Birmingham, United Kingdom

Received for publication November 25, 2009. Accepted for publication February 16,2010.

This work was supported by grants from the Biotechnological and Biological Scien-ces Research Council (to A.N.A. and M.V.-S.), the British Skin Foundation (to A.N.A.and M.H.R.), and Dermatrust (to A.N.A. and M.H.R.).

The microarray data presented in this article have been submitted to the EuropeanBioinformatics Institute (www.ebi.ac.uk/arrayexpress) under accession numberE-MEXP-2372.

Address correspondence and reprint requests to Dr. Milica Vukmanovic-Stejic, De-partment of Immunology, University College London, 46 Cleveland Street, LondonW1T 4JF, United Kingdom. E-mail address: m.vukmanovic-stejic@ucl.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this paper: DAVID, Database for Annotation, Visualization,and Integrated Discovery; HDMEC, human dermal microvascular endothelial cell;nTreg, natural regulatory T cell; RTE, recent thymic emigrant; Treg, regulatoryT cell; Tresp, responder T cell.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903781

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

a far smaller proportion of CD45RA+ Tregs (∼2%) are in cycle. Thisis likely to be because the majority of proliferating CD45RA+ Tregsconvert to CD45RO+ phenotype after activation. Second, we foundthat CD45RA+ and CD45RO+ Tregs have very distinct gene ex-pression patterns, suggesting they may not be simply the same cellsat different stages of differentiation. Finally, we show that CD45RA+

and CD45RO+ Tregs preferentially localize in different tissues, andthis is related to their expression of different tissue-specific che-mokine receptors. Taken together, these data suggest that humanCD4+Foxp3+ Tregs in adults represent a heterogeneous populationcontaining a mixture of thymic and peripherally induced regulatorycells. Furthermore, the composition of this pool shifts toward pe-ripherally induced nTregs during aging. Finally, the different lo-calization of these subsets in vivo indicates that they may exert theirfunction at distinct sites in the body and that agingmay be associatedwith preferential localization of nTregs in certain organs, such as theskin, that has been reported previously (18).

Materials and MethodsSubjects

Human peripheral blood was obtained directly from healthy donors withinthe age range 20–91 y or in the form of buffy coats (National BloodService, Colindale, U.K.). Blood and bone marrow samples were obtainedfrom healthy bone marrow donors by the Department of Hematology,University College London. All donors gave written informed consent andthe work was approved by the Ethics Committee of the Royal Free Hos-pital (London, U.K.).

Isolation of cells

BloodwasdilutedwithHBSS (Sigma-Aldrich,St.Louis,MO)and layeredontoFicoll-Paque (GE Healthcare, Buckinghamshire, U.K.) before centrifuging toisolate PBMCs. For sorting, CD4+ T cells were isolated from PBMCs usinganti–CD4-conjugated magnetic beads and a MACS column (Miltenyi Biotec,Auburn, CA); the cells were then stained for CD25 and CD45RO and sortedusing a FACSAria (BD Biosciences, San Jose, CA). Alternatively, CD4+

CD25hi Tregs were isolated from PBMCs using a Treg isolation kit (MiltenyiBiotec), which was adjusted by inserting an extra step to remove memory(CD45RO+; directly conjugated microbeads), naive (CD45RA+; directly con-jugated microbeads), or CD39+ (anti-CD39 FITC Ab [Ancell, Bayport, MN],5ml/100ml, followed by anti-FITCmicrobeads) cells prior toCD25hi isolation.

Flow cytometry

Anti-human fluorescently conjugated mAbs were purchased from Abcam(Cambridge, U.K.) or Ancell (CD39 FITC), BD Biosciences (CD4 PerCP,CD25 PE-Cy7, CD45RA FITC, Ki67 FITC, CD31 PE, CCR4 PE, CLAFITC, CXCR4 PE, CTLA-4 PE, and IFN-g allophycocyanin), CaltagLaboratories (Burlingame, CA) (CD45RA allophycocyanin), DakoCyto-mation (Carpinteria, CA) (CD45RO PE, CD45RO FITC), eBioscience(San Diego, CA), or Miltenyi Biotec (FOXP3 allophycocyanin) and R&DSystems (Minneapolis, MN) (IL-2 PE). Whole PBMCs or bone marrowwas stained on ice with surface Abs; intracellular staining for Ki67,CTLA-4, and FOXP3 was performed using the eBioscience FOXP3staining buffer kit to permeabilize the cells. Cells were then fixed in 2%paraformaldehyde and acquired on a FACSCalibur or BD LSR I (BDBiosciences). For Treg analysis, samples were first gated on CD4 andFoxp3 to identify Tregs and then subdivided into naive and memory basedon the staining with either CD45RA or CD45RO. Analysis was performedusing CellQuest (BD Biosciences) or FlowJo software.

Suppression assays

A total of 50,000 CD4+CD252 T cells were plated per well, in triplicate,alone or with an equal number of CD25hi Tregs added, in a 96-well round-bottomed plate. For suppression assays comparing naive andmemory Tregs,CD45RA+CD25hi cells and CD45RO+CD25hi cells were isolated and addedto CD4+CD252 at various ratios. Triplicates of 50,000 CD4+ cells aloneand 50,000 CD4+CD25hi Tregs alone were also cultured for comparison.Cells were stimulated with either anti–CD3-/anti–CD28-coated beads(T cell expander beads; Dynal, Invitrogen Life Technologies, Carlsbad, CA)at 50,000 beads per well, or 1 mg/ml plate-bound anti-CD3 (OKT3) withirradiated PBMCs to provide costimulation. To investigate suppressionof proliferation, after 3 d of culture at 37˚C and 5% CO2 in complete me-dium (RPMI 1640 with 10% human serum), the plates were pulsed with

[methyl-3H]thymidine (MPBiomedicals, Illkirch, France) and incubated for16 h before being harvested onto filter mats and counted using a betascintillation counter. To investigate suppression of cytokine production incocultures, CD4+CD252 were first labeled with CFSE (1 mM) to allow thedistinction between responder T cell (Tresp) and Treg populations andstimulated for 2 d as outlined above. A total of 50 ng/ml ionomycin and 250ng/ml PMA were then added, and the cells were incubated for 3 h beforebrefeldin A (5 mg/ml) was added, and the cells incubated for an additional 2h. The cells were then stained for IFN-g and IL-2, before being acquired ona flow cytometer (FACSCalibur; BD Biosciences), using the gating onCFSE+ cells to examine cytokines produced by the responder population.

Immunohistochemistry

Punch biopsies (5 mm) of normal skin were obtained from the volar flexoralaspect of the forearm, cut, fixed in acetone, and frozen. Frozen sections wereallowed to come to room temperature, washed in PBS, and then blockedusing a protein block from DakoCytomation. Primary Abs (mouse IgG1anti-CD4 and anti-Foxp3 biotin) were added in PBS and incubated over-night at 4˚C in the dark. After additional washes, secondary fluorochrome-conjugated Abs (anti-mouse IgG-Alexa488 and strep-Cy3) were added inPBS and incubated for 45 min at room temperature in the dark. Slides werewashed and mounted with Vectashield containing DAPI (Vector Labora-tories, Burlingame, CA). Images were acquired using the appropriate filterson a Leica DMLB microscope with a 340 objective, in conjunction witha Cool SNAP-Pro cf Monochrome Media Cybernetics camera and Im-agePro PLUS 6.2 software.

T lymphocyte transendothelial migration

Normal nontransformed human dermal microvascular endothelial cells(HDMECs) (PromoCell, Heidelberg, Germany) derived from young humandermis were cultured at 37˚C in a humidified 5% CO2, in endothelial cellbasal medium supplemented with 5% FCS (supplement pack C39220;PromoCell, Heidelberg, Germany). Migration assays were performed asdescribed previously (18). HDMEC monolayers were grown to confluencyin 96-well plates and stimulated with TNF-a (110,000 U/ml) and IFN-g(100 U/ml) for 24 h prior to migration assay. The T cell subsets to beinvestigated were isolated the day before the migration assay using MACStechnology and incubated overnight, without stimulation, at 37˚C and 5%CO2. They were then resuspended in serum-free medium (AIM-V; In-vitrogen Life Technologies) at a concentration of 200,000 cells/ml. Toperform the migration assays, endothelial monolayers were first washedwith HBSS, then 100 ml cell suspension was added and incubated at 37˚Cfor 2–4 h with a minimum of 6 wells per condition per experiment. Afterthe requisite time had passed, the plate was placed in a culture chamber(37˚C, 5% CO2) attached to a Zeiss Axiovert 200M light microscope.Time-lapse videos were obtained using a Hamamatsu Orca-ER digitalcamera and Simple PCI software, with images being taken every 10 s for 5min. Recordings were replayed at 160 times normal speed, and lympho-cytes that had migrated through the monolayer were identified and coun-ted. Lymphocytes on the surface of the monolayer were identified by theirhighly refractive morphology (phase-bright) and rounded or partiallyspread appearance. In contrast, cells that had migrated through themonolayer were phase-dark and highly attenuated. Data were expressed asthe percentage of total lymphocytes within a field that had migratedthrough the monolayer and a mean of six replicates was calculated.

Microarray analysis

RNA was extracted from six donor populations using the Ambion RNA-queous kit according to manufacturer’s instructions. The RNAwas reversetranscribed and amplified by the SMART amplification method (BDClontech, Palo Alto, CA). A microarray consisting of the Human GenomeArray Ready Oligo Set version 4.0 (Operon catalog number 810532) wasconstructed using UltraGAPS slides (Corning Glass, Corning, NY). Datawere acquired using a ScanArray Gx Plus (PerkinElmer, Wellesley, MA).All samples were run in parallel to a Universal Human Reference RNAsample (Stratagene, La Jolla, CA), which had been previously reversetranscribed. This facilitated comparison between samples. Donor samplesand reference were labeled with Cy3 and Cy5, respectively (BioprimeLabeling Kit; Invitrogen Life Technologies). Hybridizations were carriedout within Corning Hybridization Chambers overnight, using Lifter Slips(VWR International, West Chester, PA). Data normalization was per-formed using the Gene Expression Pattern Analysis Suite (www.gepas.org)(19), and the data were filtered by removing both the 20% lowest expressedgenes and those showing an unsatisfactory coefficient of variation (.0.4).

Using log-normalized ratios, the TIGR Multiexperiment Viewer ver-sion 4.0 (2) was used to perform significance analysis of microarrays.

4318 CHARACTERISTICS OF HUMAN CD45RO+ and CD45RA+ Tregs

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Hierarchical clustering and principal component analysis were performedon the resultant gene list. Gene clusters were annotated by use of Databasefor Annotation, Visualization, and Integrated Discovery (DAVID) (20).Microarray data are available at www.ebi.ac.uk/arrayexpress; accessionnumber E-MEXP-2372.

Statistics

The Student t test was used to analyze the differences in the means of datasets, paired where cells from the same donor were being compared orunpaired to compare between donors. Linear regression analysis wasperformed on age correlations.

ResultsCD45RA+ (naive) Tregs decrease with age

To investigate the effects of aging on the Treg pool, we harvestedPBMCs from healthy adult volunteers and investigated the pro-portion of Tregs expressing CD45RA and CD45RO receptors(henceforth, these will be referred to as naive and memory Tregs,respectively, for simplicity). In agreement with other studies, wefind that in adults a low proportion of Tregs express CD45RA (Fig.1A, 1B). Identifying Tregs by CD25hi (Fig. 1A) and Foxp3+ cells(Fig. 1B) expression gave similar results. It is of note that theintensity of Foxp3 expression is slightly lower on the CD45RA+

population (Supplemental Fig. 1) (21, 22). Both naive and mem-ory Tregs express low levels of CD127 (Fig. 1C), a phenotypiccharacteristic of Tregs (13, 23). To further investigate the com-position of the Treg pool in adult humans, we examined the ex-

pression of CD31, an adhesion molecule that has been proposed asa marker for RTEs (24). Although the naive nTreg pool is verysmall in adults, the majority (mean 70.3 6 3.3%, n = 31) of theseexpress CD31 (Fig. 1C), suggesting that a considerable proportionof these cells have been generated in the thymus.The total proportion of the nTregs identified as CD4+CD25hi or as

CD4+Foxp3+ increased significantly with age (Supplemental Fig. 2),which is in agreement with previous studies by ourselves and others(7, 8). However, the proportion of naive nTregs decreased duringaging (age range 20–91 y, n = 53; p = 0.0018) (Fig. 2A). This mirrorsthe changes observed in the non-Treg pool (data not shown). Therewas a concomitant increase in the proportion of memory Tregsduring aging (Fig. 2B). Although mean percentage of CD31-expressing cells within the naive Treg subset was 63% and did notsignificantly change with age (data not shown), the overall numberof CD45RA+CD31+ Tregs decreased sharply, virtually disappearingby the age of 80 y (Fig. 2C). These data suggest that a proportion ofcirculating Tregs, even in older adults, are generated directly fromthe thymus; however, this population decreases significantly withage, presumably as a consequence of thymic involution.

Naive and memory Tregs are highly proliferative

The significant decrease in the numbers of naive Tregs with in-creasing age raises the issue of the contribution of these cells to themaintenance of the Treg pool in older individuals. It is clear that newnaive Tregs cannot be produced at the same levels throughout life;therefore, the maintenance of the total Treg pool can be achieved byextensive turnover of the existing Tregs or by peripheral conversionof non-Tregs into Tregs (or the combination of these two mecha-nisms).We have previously shown that thememory Treg population

FIGURE 1. The CD4+CD25hi Treg population contains both naive

(CD45RA+) and memory (CD45RO+) cells. A, Dot plots showing repre-

sentative staining of CD45RA+ and CD45RO+ cells within CD4+CD252

and CD4+CD25hi subsets. CD4+CD25hi cells are gated by selecting CD4+

cells with brighter CD25 expression than the brightest staining seen on

non-CD4s (data not shown). B, Representative dot plot showing CD45RO

and Foxp3 expression among CD4+ T cells. C, left panel, Representative

histograms showing expression of the IL-7R (CD127) on CD45RA+ and

CD45RO+ subsets of CD4+ Tregs (filled histograms) and responder CD4

T cells (unfilled histograms). Right panel, Representative histograms in-

dicating expression of CD31 in CD45RA+ and CD45RO+ subsets of Tregs

and responder CD4 T cells.

FIGURE 2. Alterations in the composition of the Treg pool with age.

PBMCs from healthy volunteers (n = 59, age range 21–91 y) were stained

for surface marker expression and analyzed using flow cytometry. A, Graph

of age-related change in the proportion of CD4+ Tregs expressing

CD45RA. B, Graph showing change with age in the proportion of CD4+

Tregs expressing CD45RO. C, Graph showing change with age in the

proportion of Tregs with RTE phenotype (CD45RA+CD31+).

The Journal of Immunology 4319

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

is highly proliferative in vivo using deuterium labeling (7). How-ever, small numbers of naive Tregs precluded the study of thissubset using this method. In the current study, we extended ourprevious observations by measuring the proliferative activity ofindividual Treg subsets by their expression of Ki67, a protein ex-pressed in the nucleus of cells during all stages of the cell cycle(17). PBMCs were stained ex vivo with CD4, Foxp3, CD45RO,and Ki67, gated on CD4+Foxp3+, and further subdivided intomemory and naive subsets based on CD45RO staining (CD45RO2

cells were assumed to be naive). In some experiments, CD25staining was also included. We found that a very high percentage(mean 16.6 6 1.3%, n = 18) of memory Tregs expressed Ki67,confirming that a large proportion of these cells were dividing atany given time (Fig. 3A, 3B). A smaller percentage of naive Tregswere also in cycle (mean 2.2 6 0.4%, n = 18), whichwas significantly lower than the memory Treg pool. These resultsextend our previous observations on the proliferative rate of human

Tregs considerably (7). A significantly lower proportion of CD4+

CD45RO+ responder cells expressed Ki67 (mean 2.0 6 0.2%,n=18) comparedwithCD45RO+Tregs,whereasKi67 expression innaive CD4+ responder cells was virtually absent (mean 0.46 0.1%,n = 18). Next, we investigated the proliferation of CD31+ andCD312 populations within the CD45RA+ Tregs; the CD45RA+

CD31+ subset proliferated significantly more than the CD45RA+

CD312 population (Fig. 3A, 3B) (means 2.87 and 1.54%, re-spectively, n = 18; p = 0.0427). One of the possibilities may be thatthe higher proliferation in the RTEs may be residual Ki67 ex-pression resulting from turnover in the thymus.When we examined the proliferation of Treg subsets in different

age groups, we found a decreased proportion of memory Tregsexpressing Ki67 in the old (age range 71–91 y) compared with theyoung (age range 22–35 y) subjects (old, mean 13.66 1.3%, n = 10;young, mean 19.7 6 1.6%, n = 9) (Fig. 3B, right panel). In-terestingly, there was significantly increased proliferation in the

FIGURE 3. Both memory and naive Tregs are highly proliferative. PBMCs from healthy donors were stained for expression of the nuclear proliferation

marker Ki67. Tregs were defined by gating on CD4+FOXP3+ cells. CD45RO+ cells were designated as memory and CD45RO- cells were assumed to be

CD45RA+ and designated as naive. A, Representative dot plots showing Ki67 expression by CD45RO+, CD45RA+, CD45RA+CD31+, and CD45RA+CD312

Tregs and responders. B, left panel, Cumulative data showing the proportion of CD45RO+ and naive regulatory and responder cells in all age groups that

stained positive for Ki67 (n = 37). Right panel, Ki67 expression by Treg subsets (CD45RO+, CD45RA+CD31+, and CD45RA+CD312) in young (ages 22–35 y)

and old (ages 71–91 y) (n = 9). C, Ki67 expression in Treg and responder subsets (CD45RO+ and CD45RA+) in three donors (ages 27, 31, and 38 y) over time.

4320 CHARACTERISTICS OF HUMAN CD45RO+ and CD45RA+ Tregs

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

CD45RA+CD31+ Treg subset in the old (old, mean 4.1 6 0.9%,n = 9; young, mean 1.7 6 0.3%, n = 9) (Fig. 3B, right panel).In addition, we followed three healthy donors over a period of3–4 mo and found a consistent level of Ki67 expression in allTreg and Tresp subsets over time (Fig. 3C).This suggests thata very high proportion of CD45RO+ Tregs are always in cellcycle, whereas CD45RA+ Tregs divide at a slower rate. Ourdata cannot exclude the possibility that although a high pro-portion of memory Tregs proliferate, this pool may also containsome cells that divide very slowly (25, 26).We investigated whether CD45RA-expressing Tregs convert to

CD45RO expression when they proliferate, as has been shown forresponder cells (11). After several days of stimulation, naive Tregsbegin to express CD45RO and concomitantly lose expression ofCD45RA (Fig. 4A, 4B). By day 4 of stimulation,.70%of both Tregsand Tresps change their phenotype from exclusively CD45RA+ toCD45RO+ phenotype. This shows for the first time that upon entryinto cell cycle after activation, naive Tregs lose their expression ofCD45RA and become CD45RO+ and part of the memory Treg pool.Therefore, although present in small numbers in adults, naive Tregscan proliferate and contribute toward themaintenance of thememoryTreg pool. However, the extent towhich this occurs in old individualsis not clear at present.

Naive and memory Tregs show distinct gene expressionpatterns

Thepresentdata andpreviousobservations indicate that both thymus-derived naive Tregs and conventional CD4 T cells can contribute tothe repopulation of thememory Treg pool in adult humans (2, 3, 9). Itis currently not possible to distinguish between nTregs from pe-ripherally induced Treg populations, and the relative contribution ofcells from either of these sources to the total Treg pool is unclear (2,3, 9). To further characterize the relationship between naive andmemory Tregs, as well as Tregs and Tresps, we undertook globalgene expression profiling of the four different subsets.

CD4 T cells were sorted into CD25hiCD45RA+ (naive Treg),CD25hiCD45RO+ (memory Treg), CD252CD45RA+ (naive re-sponder), and CD252CD45RO+ (memory responder) populations.In these studies, naive Tregs were not further fractionated intoCD31+ and CD312 populations because of the low cell numbers.RNA was extracted and amplified, and microarray analysis wasperformed. A significance analysis for microarrays was performed(using TIGR Multiexperiment Viewer, version 4.0) between allfour populations eliciting a 121-gene list, with a false-discoveryrate of 10%. Hierarchical clustering of the gene list provided fivedistinct gene clusters. The functions of the genes within theseclusters were probed by applying the cluster lists to DAVID (20)annotating each cluster with details of Gene Ontology pathwayassociations. The CD45RA+ Treg population was clearly distinctfrom all of the other tested populations. Memory Tregs, memoryresponders, and naive responders all showed similar gene ex-pression profiles to each other. Naive Tregs showed activation ofgenes in cluster A and inactivation of genes in clusters B and D(Fig. 5). All three other subsets showed approximately the reverseof this pattern, with naive responders showing the highest acti-vation of clusters B and D. This suggests that naive Tregs may bea distinct population, whereas the other three subsets are related toeach other. This is consistent with our previous hypothesis thata large proportion of memory Tregs in adults may be derived fromTresps (9), whereas CD45RA+ Tregs represent a distinct thymi-cally derived population. We are currently investigating thefunctional implications of changes in expression of particulargenes between the different Treg subsets.

Naive and memory Tregs have different phenotypes but haveequal suppressive capacities

Despite intensive research, the exact mechanism of suppressionused by Tregs has not been fully elucidated (1). Furthermore, it isnot clear whether different populations of Tregs have distinctfunctions. To assess the functional similarities between CD45RA+

FIGURE 4. CD45RA+ Tregs acquire expression of

CD45RO after stimulation in vitro. A, Dot plots

showing expression of CD45RA and CD45RO by

stimulated CD45RA+ Tregs and Tresps at days 0, 3,

and 4 of in vitro stimulation with anti–CD3- and anti–

CD28-coated beads. B, Graph showing CD45RO ex-

pression by CD45RA+ Tregs and responders over 7 d of

in vitro stimulation, as above.

The Journal of Immunology 4321

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

and CD45RO+ Tregs, we first compared their expression of sur-face molecules that have been previously shown to influencefunctional properties of Tregs. Numerous studies have implicatedCTLA-4 as a mediator of Treg function in both humans and mice(27, 28). We found that CTLA-4 was expressed preferentially byCD45RO+ Tregs compared with CD45RA+ Tregs (mean 12.1 61.22 and 4.2 6 1.57%, respectively, n = 7; p = 0.0009 pairedStudent t test) (Fig. 6A, Supplemental Fig. 3A).Recent studies suggest that CD39 and CD73 may play a role in

the mechanism of Treg suppression in both mice and humans (29,

30). We therefore investigated the expression of these moleculesby the different human Treg subsets. We found that a high pro-portion of CD4+Foxp3+ Tregs expressed CD39 (mean 60.6 64.0%, n = 17) (Fig. 6B). This was due to mainly to a large pro-portion (79.7 6 1.7%, n = 8) of CD45RO+ Tregs that expressCD39, whereas very few CD45RA+ human Tregs express thismolecule (mean of 12.8 6 2.8% [n = 8; p , 0.0001]) (Fig. 6B).Neither CD45RO+ nor CD45RA+ responder CD4 cells expressedsignificant levels of CD39 (CD45RO: mean 9.7 6 1.4%;CD45RA+: mean 1.5 6 0.6%; n = 8; data not shown). None of theCD4 T cell subsets expressed significant levels of CD73, and onlya very small proportion of Tregs coexpressed CD39 and CD73(Supplemental Fig. 3B). This is in contrast to previous studies inmice (30) and suggests that CD73 is unlikely to be involved inTreg function in humans.We hypothesized that differences in expression of CD39 and

CTLA-4 molecules in Treg function may result in differences in thesuppressive ability of the two subsets. To test this, Tresps (CD4+

CD252) were stimulated with beads coated with anti-CD3 andanti-CD28, alone or in the presence of CD45RA+ or CD45RO+

Tregs at different ratios, and proliferation was measured after 3 d.We found that despite the phenotypic differences, CD45RA+ andCD45RO+ Tregs are equally capable of suppressing the pro-liferation of Tresps (Fig. 6C), in agreement with previous studies(12, 13). We also investigated the capacity of CD45RA+ andCD45RO+ Tregs to suppress production of the cytokines IFN-gand IL-2 by Tresps (Fig. 6D). To do this, we cocultured the Tregsubsets with responders in the presence of beads coated in anti-CD3 and anti-CD28 for 2 d; we then restimulated the cells withPMA and ionomycin for 5 h on day 2, in the presence of brefeldinA. We then stained for the presence of intracellular cytokines andanalyzed the cells by FACS. No difference in suppression of eithercytokine was observed between CD45RO+ and CD45RA+ Tregs;hence, it appears that both subsets of Tregs can suppress responderCD4+ T cells equally. One possible explanation is that naive Tregsrapidly upregulate CTLA-4 and/or CD39 once stimulated (akin toeffector T cells, which upregulate these molecules following ac-tivation) and then exert suppressive activity. Alternatively, CD39and pericellular adenosine generated by CD39 and CD73 (29, 30)are not critical for suppressive activity in vitro; although, wecannot exclude that they may contribute to suppression in morephysiological conditions.

Naive and memory Tregs migrate to different tissues

The observation of such striking phenotypic differences, despitetheir apparent functional equivalence, raises the question to whatextent the two Treg subsets perform the same role but at differentsites in vivo. A number of studies have proposed that naive andmemory Tregs express different homing molecules and migrate todifferent tissues in mice (31, 32). To clarify this further, we studiedthe localization of human CD45RA+ and CD45RO+ Tregs in tis-sues. We focused especially on their capacity to home to skin,which is often subject to inflammation and bone marrow, a pri-mary lymphoid organ.When we investigated the proportion of naive and memory Tregs

in the bone marrow by staining with CD25, CD4, CD45RA, andCD45RO, we found a significantly increased proportion ofCD45RA+ Tregs compared with the peripheral blood of the samedonor and a correspondingly decreased proportion of CD45RO+

Tregs (Fig. 7A). Similar results were seen when staining withFoxp3 rather than CD25 (data not shown). This correlated with theexpression of the bone marrow homing receptor CXCR4 (33),which was expressed on both CD45RO+ and CD45RA+ Tregs(Supplemental Fig. 4A) but was significantly higher on CD45RA+

FIGURE 5. Expression profile of naive Treg, naive responder, memory

Treg, and memory responder populations. A, Relative expression of donor

samples to reference for 121 significant genes. Colors indicate relative

signal intensities. The expression profile was clustered hierarchically,

clustering genes according to patterns of expression. Letter designations

represent gene clusters used for DAVID pathway association. Full gene

names and signal values are presented as additional data. B, Summation of

gene cluster expression pattern across donor cell populations.

4322 CHARACTERISTICS OF HUMAN CD45RO+ and CD45RA+ Tregs

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Tregs (CD45RO+ Tregs: mean fluorescence intensity mean 16.0 62.6, n = 11; CD45RA+ Tregs: mean 54.0 6 12.4, n = 11; p =0.0234). Furthermore, we found that proliferation of naive Tregswas similar in blood and bone marrow (bone marrow mean 4.26 1[n = 5], versus 5.06 1.2 in peripheral blood). Interestingly, memoryTregs found in the bone marrow proliferate less than those in theperipheral blood (bone marrow mean 7.2 6 0.7 [n = 5], comparedto 13.4 6 1.5 in peripheral blood) (Fig. 7B). One possible ex-planation is that naive Tregs proliferating in the bone marrowremain CD45RA+; this would suggest that bone marrow might actas a reservoir of naive Tregs.In a previous study, we showed that CD4+Foxp3+ Tregs can be

found in significant numbers in normal human skin, both beforeand after injection of Ag (18). These cutaneous CD4+Foxp3+

T cells expressed typical phenotypic and functional characteristicsof regulatory cells (17). To investigate the phenotype of Tregs inthe skin in terms of CD45RA and CD45RO expression, 5-mm skinsections were stained with Foxp3 and CD45RO and CD45RA Absand examined using two-color immunofluorescence. We foundthat a vast majority of skin resident Tregs have a memory phe-notype (95.4 6 2.6%) (Fig. 7C). This was related to differences inchemokine receptor expression because both CLA and CCR4were significantly higher in CD45RO+ compared with CD45RA+

Tregs, confirming previous reports (Supplemental Fig. 4B) (34).We sought functional data to verify that memory Tregs showed

preferential skin homing characteristics, as this had importantimplications for our previous studies, where we found higherproportions of these cells in the skin of older humans (18). Wetherefore used time-lapse phase-contrast microscopy to investigatethe physical capacity of naive and memory Tregs to migrate acrossprimary HDMECs in vitro (Fig. 7D). CD4 T cell migration wasminimal when using resting endothelium (Fig. 7D). Activation ofthe HDMECs with TNF-a and IFN-g stimulates E-selectin,ICAM-1, and VCAM-1 expression and allows efficient CD4+

T cell migration (18). In agreement with the expression of highlevels of CLA, both CD45RO+ Tresps and CD45RO+ Tregs mi-grated equally well across the activated endothelium. In contrast,both CD45RA+ Tregs and naive Tresps migrated significantly lessthan the respective memory populations. Hence, the difference inthe expression of skin homing molecules correlates well with thecapacity to cross HDMECs in vitro.

DiscussionTregs represent a heterogeneous population of CD4+ T cells, whichare crucial in peripheral tolerance and control of immune re-sponses to pathogens (1, 3). We and others have shown that de-spite reduced thymic output, Treg numbers are maintained andactually increase with age (5–8). A number of mechanisms couldbe responsible for this, including increased proliferation of thy-mic-derived Tregs, conversion of nonregulatory to Treg pop-ulations, or existence of long-lived Tregs. To establish theimportance of the thymic-derived naive Treg for the maintenanceof the Treg pool in old humans, we investigated the proliferationof each Treg subset in subjects in the different age groups. Inaddition, we compared differences in phenotype and function of

FIGURE 6. Naive and memory Tregs have differing phenotypes but

identical suppressive capacity. A, CTLA-4 expression by CD45RO+ and

CD45RA+ regulatory and Tresps ex vivo (n = 7). B, Cumulative data

showing CD39 expression by CD45RO+ and CD45RA+ Treg and Tresp

subsets ex vivo (n = 8). C, Suppression of Tresp proliferation by CD45RO+

and CD45RA+ Tregs. Cells were isolated and added to CD252 Tresps.

Experiments were performed in triplicate. Left panel, Representative

suppression assay showing suppression of CD252 Tresps by both

CD45RO+ and CD45RA+ Tregs at a 1:1 ratio. Right panel, Cumulative

data showing percentage suppression of proliferation of responder cells by

CD45RO+ or CD45RA+ Tregs, as measured by incorporation of [3H]thy-

midine after 3 d of culture. Incorporation by cultures with CD45RA+ or

CD45RO+ Tregs was compared with incorporation by responder cells

alone, and assays were performed at Treg/Tresp ratios of 1:1, 0.25:1, and

0.1:1. D, Suppression of Tresp cytokine production by CD45RO+ or

CD45RA+ Tregs. Production of IL-2 and IFN-g by responder cells was

measured after culturing the cells alone and with added CD45RO+ or

CD45RA+ Tregs. Cells were cultured for 2 d, before addition of brefeldin

A and subsequent intracellular staining. Representative staining showing

cytokine production in the absence and presence of CD45RO+ or

CD45RA+ Tregs. Asterisks indicate significances, pp, 0.05; ppp, 0.001;

pppp , 0.0001.

The Journal of Immunology 4323

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

these two Treg subsets with a view to inform on the closeness of theirrelationship. A number of novel observations were made: 1) pro-liferation rates of naive Tregs are significantly lower than for the

memory Tregs, and naive Tregs acquire a memory phenotype as theyproliferate; 2) naive and memory Tregs show a very different geneexpression pattern; and 3) naive andmemory Tregs express differentchemokine receptors and preferentially localize in different tissues.Until recently Tregs were thought to be generated solely in the

thymus (reviewed in Refs. 2 and 3). However, because thymicoutput decreases with age, the source of these cells in later life hasbeen an issue of some debate (2, 3). We previously demonstratedthat human CD45RO+ Tregs represent a highly differentiatedpopulation’s, characterized by short telomeres, inability to upre-gulate telomerase and susceptibility to apoptosis (7). Therefore,these cells have limited capacity for self-renewal (7, 9), suggestingthat it is unlikely that the Treg pool is maintained through con-tinuous turnover of pre-existing CD45RO+ Tregs.Several studies have suggested that CD4+Foxp32 T cells can

acquire phenotypic and functional properties of Tregs in responseto specific signals, such as TGF-b and retinoic acid (35, 36), or tosubimmunogenic doses of agonist peptides (37). However, onlya fraction (∼35%) of Treg signature genes are induced, indicatingthat from the genomic standpoint converted Tregs and thymus-derived Tregs are clearly different (2, 3). Our microarray analysesof Treg and Tresp subsets (Fig. 5) suggest that naive and memoryTregs represent essentially distinct populations, a hypothesiswhich is compatible with a significant contribution of convertedTregs to the memory Treg pool. Our previous data on the closeclonal relationship between the subsets (7) and the close TCRhomology between Tregs and Tresps described by other groups(38–40) also support the hypothesis that the process of peripheralconversion from non-Tregs plays a significant role in the main-tenance of the Treg population in humans. A number of otherstudies have come to address the importance of the conversion inthe Treg pool, with different conclusions. In nonimmunized andnonlymphopenic mice, conversion does not seem to play a sig-nificant role in the shaping of the peripheral Treg repertoire (41–43). However, the situation is likely to be different in humans, whoundergo recurrent immunological challenges and have a muchlonger life span (2, 3, 9). In addition to peripheral conversion fromresponder cells, we do not rule out the possibility that the smallnumber of naive Tregs that are present in older humans is drivenby repeated divisions into the CD45RO+ Treg pool and contributeto the maintenance of these cells in adults.In this study, we investigated the proliferative potential of naive

Tregs for the first time. We found that the ratio of CD45RA+ (naive)to CD45RO+ (memory) Tregs decreased significantly with ageconfirming previous reports (12, 13). Interestingly, a large pro-portion of CD45RA+ Tregs in adults express CD31, a markerpreviously shown to be lost on activation and suggested to beexpressed by RTEs (24, 44), suggesting some “new” Tregs mayenter the periphery throughout life. However, there was a sharpdecline in the number of CD31+CD45RA+ Tregs with increasingage, confirming that only a very small population of RTE Tregsare present in older humans (mean percentage of Tregs with RTEphenotype in donors . 70 y, 1.37 6 0.26%, n = 10). NaiveCD45RA+ Tregs proliferate significantly less than the CD45RO+

Tregs. The lower proliferation rate may be explained by the factthat on division they become CD45RO+ and disappear from theCD45RA+ pool. This is in agreement with a recent study byMiyara et al. (22) showing that there are two subsets of Tregs—resting and activated corresponding to naive and memory Tregs,respectively. In that study, resting Tregs become activated Tregsand then disappear quickly through apoptosis in agreement withour observation that memory Tregs represent a rapidly dividingpopulation that is prone to apoptosis (7, 22). Furthermore, basedon tracking of specific clonotypes, it was demonstrated that

FIGURE 7. Migratory potential of naive and memory Tregs. A, Bone

marrow samples from healthy donors were analyzed by flow cytometry and

compared with PBMCs from the same donors. Paired scatter plots are

shown displaying percentage of CD25hi expressing CD45RA (left panel)

or CD45RO (right panel) in blood and bone marrow samples from the

same donors. B, Cumulative data showing ex vivo Ki67 expression by

CD45RA+ and CD45RO+Foxp3+ and Foxp3- subsets from the peripheral

blood and bone marrow of healthy donors (n = 6). C, Double immuno-

fluorescence staining of representative biopsies from normal skin;

CD45RO+ (green) and Foxp3+ (red) cells in a perivascular lymphocytic

infiltrate. Foxp3+ cells are indicated by an arrow. Images were acquired

using the appropriate filters on a Leica DMLB microscope with a 340

objective, in conjunction with a Cool SNAP-Pro cf Monochrome Media

Cybernetics camera and ImagePro PLUS 6.2 software. D, Cumulative data

showing migration of Treg and Tresp subsets through stimulated and un-

stimulated endothelium in vitro (n = 4). Data were obtained using a Zeiss

Axiovert 200M light microscope and Hamamatsu Orca-ER digital camera,

in conjunction with SimplePCI software, to generate time-lapse videos of

T cell movement in a live-cell chamber set at 37˚C and 5% CO2.

4324 CHARACTERISTICS OF HUMAN CD45RO+ and CD45RA+ Tregs

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

CD45RA+ Tregs and CD45RO+ activated Tregs represent distinctdifferentiation stages of the same T cell (22). However, the impactof age on this process was not addressed.It is not clear how big the contribution of CD45RA+ Tregs to the

maintenance of the total Treg pool is during aging. We are cur-rently unable to distinguish between those cells within the mem-ory Treg population that are recently converted from the naivepool and those that have been derived from responder cells. Fur-thermore, a lack of phenotypic markers that distinguish betweennatural and induced CD45RO+ Tregs makes it difficult to ascertainthe relative contribution of these two mechanisms for Tregmaintenance (3). In an attempt to further address this conundrum,we compared gene expression patterns for naive and memoryTregs, as well as naive and memory responders. The data showvery distinct expression patterns between naive Tregs and all othersubsets, again in agreement with the study by Miyara et al. (22).However, on the basis of our previous data, we suggest that thisindicates that memory Tregs and thymically derived naive Tregsarise from different sources.Finally, naive and memory Tregs express different chemokine

receptors and therefore localize to different tissues. In particular, wefind that expression of the skin homingmolecules,CLAandCCR4, isrestricted to circulating memory Tregs, and these cells can beidentified in both normal and inflamed skin (17, 34). The localizationof Tregs in the skinmay be essential as the skin is constantly exposedto numerous antigenic challenges from the environment. It is ofinterest that the proportion of Tregs in the skin increases with ageand can contribute to reduced immune responses observed in elderlyindividuals (18). Although we were not able to examine this, otherhuman tissues, such as gut and lung, also reportedly contain sig-nificant numbers of Tregs (45, 46). From the available, data it ap-pears that Tregs in these peripheral tissues also display a memoryphenotype (31, 32, 47). An intriguing observation was that CD45RATregs express significantly more CXCR4 and accumulate prefer-entially in the bonemarrow. The equivalent accumulation is not seenin the naive responders, although they also express relatively highlevels of CXCR4. The significance of this observation is unclear, butit is known that the bone marrow can serve as a secondary lymphoidorgan (48). It is possible therefore that the bone marrow may rep-resent a nichewhere naive Tregs accumulate tomature, survive, and/or undergo homeostatic proliferation while maintaining their naivephenotype.In conclusion, our studies re-enforce the hypothesis that the

human Treg pool is heterogeneous and contains cells that may arisefrom the thymus and also from Tresps in the periphery. Further-more, the source of cells that seed this pool changes with age and inolder humans where the majority of these cells may be derived fromTresps (7, 40, 49). The differences in function and homing po-tential of distinct Treg subsets may relate to the specialized role ofthese cells, in particular tissue microenvironments. It is not clearat present whether this may have implications for reduced immunefunction that is associated with aging; however, we showed re-cently that the reduced cutaneous responses of older humans tochallenge with recall Ags may be due in part to the presence ofhigher numbers of Tregs in their skin (18). It is well known thatolder individuals respond poorly to vaccination and have morefrequent and more severe infections (50). It would be important toclarify whether the age-associated increase in Tregs contributes tothis alteration in immunity.

AcknowledgmentsWe are grateful to all the volunteers who took part in this study. We thank

Prof. John Greenwood and Prof. Nigel Klein for assistance and advice on

microscopic analyses and Dr. Joanne Masters for expert cell sorting.

DisclosuresThe authors have no financial conflicts of interest.

References1. Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cells

and immune tolerance. Cell 133: 775–787.2. Curotto de Lafaille, M. A., and J. J. Lafaille. 2009. Natural and adaptive foxp3+

regulatory T cells: more of the same or a division of labor? Immunity 30: 626–635.3. Feuerer, M., J. A. Hill, D. Mathis, and C. Benoist. 2009. Foxp3+ regulatory T cells:

differentiation, specification, subphenotypes. Nat. Immunol. 10: 689–695.4. Douek, D. C., R. D. McFarland, P. H. Keiser, E. A. Gage, J. M. Massey,

B. F. Haynes, M. A. Polis, A. T. Haase, M. B. Feinberg, J. L. Sullivan, et al.1998. Changes in thymic function with age and during the treatment of HIVinfection. Nature 396: 690–695.

5. Lages,C.S., I. Suffia,P.A.Velilla,B.Huang,G.Warshaw,D.A.Hildeman,Y.Belkaid,and C. Chougnet. 2008. Functional regulatory T cells accumulate in aged hosts andpromote chronic infectious disease reactivation. J. Immunol. 181: 1835–1848.

6. Sharma, S., A. L. Dominguez, and J. Lustgarten. 2006. High accumulation of Tregulatory cells prevents the activation of immune responses in aged animals. J.Immunol. 177: 8348–8355.

7. Vukmanovic-Stejic, M., Y. Zhang, J. E. Cook, J. M. Fletcher, A. McQuaid,J. E. Masters, M. H. Rustin, L. S. Taams, P. C. Beverley, D. C. Macallan, andA. N. Akbar. 2006. Human CD4+ CD25hiFoxp3+ regulatory T cells are derived byrapid turnover of memory populations in vivo. J. Clin. Invest. 116: 2423–2433.

8. Gregg, R., C. M. Smith, F. J. Clark, D. Dunnion, N. Khan, R. Chakraverty,L. Nayak, and P. A. Moss. 2005. The number of human peripheral blood CD4+

CD25high regulatory T cells increases with age. Clin. Exp. Immunol. 140: 540–546.9. Akbar, A. N., M. Vukmanovic-Stejic, L. S. Taams, and D. C. Macallan. 2007.

The dynamic co-evolution of memory and regulatory CD4+ T cells in the pe-riphery. Nat. Rev. Immunol. 7: 231–237.

10. Taams, L. S., J. Smith, M. H. Rustin, M. Salmon, L. W. Poulter, and A. N. Akbar.2001. Human anergic/suppressive CD4+CD25+T cells: a highly differentiatedand apoptosis-prone population. Eur. J. Immunol. 31: 1122–1131.

11. Akbar, A. N., L. Terry, A. Timms, P. C. Beverley, and G. Janossy. 1988. Loss ofCD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol.140: 2171–2178.

12. Valmori, D., A. Merlo, N. E. Souleimanian, C. S. Hesdorffer, and M. Ayyoub.2005. A peripheral circulating compartment of natural naive CD4 Tregs. J. Clin.Invest. 115: 1953–1962.

13. Seddiki, N., B. Santner-Nanan, S. G. Tangye, S. I. Alexander, M. Solomon,S. Lee, R. Nanan, and B. Fazekas de Saint Groth. 2006. Persistence of naiveCD45RA+ regulatory T cells in adult life. Blood 107: 2830–2838.

14. Wing, K., A. Ekmark, H. Karlsson, A. Rudin, and E. Suri-Payer. 2002. Char-acterization of human CD25+CD4+ T cells in thymus, cord and adult blood.Immunology 106: 190–199.

15. Hoffmann, P., R. Eder, T. J. Boeld, K. Doser, B. Piseshka, R. Andreesen, andM. Edinger. 2006. Only the CD45RA+ subpopulation of CD4+CD25high T cellsgives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood108: 4260–4267.

16. Haas, J., B. Fritzsching, P. Trubswetter, M. Korporal, L. Milkova, B. Fritz,D. Vobis, P. H. Krammer, E. Suri-Payer, and B. Wildemann. 2007. Prevalence ofnewly generated naive regulatory T cells (Treg) is critical for Treg suppressivefunction and determines Treg dysfunction in multiple sclerosis. J. Immunol. 179:1322–1330.

17. Vukmanovic-Stejic, M., E. Agius, N. Booth, P. J. Dunne, K. E. Lacy, J. R. Reed,T. O. Sobande, S. Kissane, M. Salmon, M. H. Rustin, and A. N. Akbar. 2008.The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneousantigen-specific memory response in vivo. J. Clin. Invest. 118: 3639–3650.

18. Agius, E., K. E. Lacy, M. Vukmanovic-Stejic, A. L. Jagger, A. P. Papageorgiou,S. Hall, J. R. Reed, S. J. Curnow, J. Fuentes-Duculan, C. D. Buckley, et al. 2009.Decreased TNF-a synthesis by macrophages restricts cutaneous im-munosurveillance by memory CD4+ T cells during aging. J. Exp. Med. 206:1929–1940.

19. Montaner, D., J. Tarraga, J. Huerta-Cepas, J. Burguet, J. M. Vaquerizas,L. Conde, P. Minguez, J. Vera, S. Mukherjee, J. Valls, et al. 2006. Next station inmicroarray data analysis: GEPAS. Nucleic Acids Res. 34: W486–W491.

20. Huang da, W., B. T. Sherman, and R. A. Lempicki. 2009. Systematic and in-tegrative analysis of large gene lists using DAVID bioinformatics resources. Nat.Protoc. 4: 44–57.

21. Hoffmann, P., T. J. Boeld, R. Eder, J. Huehn, S. Floess, G. Wieczorek, S. Olek,W. Dietmaier, R. Andreesen, and M. Edinger. 2009. Loss of FOXP3 expressionin natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stim-ulation. Eur. J. Immunol. 39: 1088–1097.

22. Miyara, M., Y. Yoshioka, A. Kitoh, T. Shima, K. Wing, A. Niwa, C. Parizot,C. Taflin, T. Heike, D. Valeyre, et al. 2009. Functional delineation and differ-entiation dynamics of human CD4+ T cells expressing the FoxP3 transcriptionfactor. Immunity 30: 899–911.

23. Liu, W., A. L. Putnam, Z. Xu-Yu, G. L. Szot, M. R. Lee, S. Zhu, P. A. Gottlieb,P. Kapranov, T. R. Gingeras, B. Fazekas de St Groth, et al. 2006. CD127 ex-pression inversely correlates with FoxP3 and suppressive function of humanCD4+ T reg cells. J. Exp. Med. 203: 1701–1711.

24. Kimmig, S., G. K. Przybylski, C. A. Schmidt, K. Laurisch, B. Mowes,A. Radbruch, and A. Thiel. 2002. Two subsets of naive T helper cells withdistinct T cell receptor excision circle content in human adult peripheral blood. J.Exp. Med. 195: 789–794.

The Journal of Immunology 4325

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

25. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau,and B. L. Salomon. 2003. Continuous activation of autoreactive CD4+CD25+

regulatory T cells in the steady state. J. Exp. Med. 198: 737–746.26. Tang, A. L., J. R. Teijaro, M. N. Njau, S. S. Chandran, A. Azimzadeh,

S. G. Nadler, D. M. Rothstein, and D. L. Farber. 2008. CTLA4 expression is anindicator and regulator of steady-state CD4+FoxP3+ T cell homeostasis. J. Im-munol. 181: 1806–1813.

27. Read, S., V. Malmstrom, and F. Powrie. 2000. Cytotoxic T lymphocyte-associ-ated antigen 4 plays an essential role in the function of CD25+CD4+regulatorycells that control intestinal inflammation. J. Exp. Med. 192: 295–302.

28. Wing, K., Y. Onishi, P. Prieto-Martin, T. Yamaguchi, M. Miyara, Z. Fehervari,T. Nomura, and S. Sakaguchi. 2008. CTLA-4 control over Foxp3+ regulatoryT cell function. Science 322: 271–275.

29. Borsellino, G., M. Kleinewietfeld, D. Di Mitri, A. Sternjak, A. Diamantini,R. Giometto, S. Hopner, D. Centonze, G. Bernardi, M. L. Dell’Acqua, et al.2007. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis ofextracellular ATP and immune suppression. Blood 110: 1225–1232.

30. Deaglio, S., K. M. Dwyer, W. Gao, D. Friedman, A. Usheva, A. Erat, J. F. Chen,K. Enjyoji, J. Linden, M. Oukka, et al. 2007. Adenosine generation catalyzed byCD39 and CD73 expressed on regulatory T cells mediates immune suppression.J. Exp. Med. 204: 1257–1265.

31. Huehn, J., K. Siegmund, J. C. Lehmann, C. Siewert, U. Haubold, M. Feuerer,G. F. Debes, J. Lauber, O. Frey, G. K. Przybylski, et al. 2004. Developmentalstage, phenotype, and migration distinguish naive- and effector/memory-likeCD4+ regulatory T cells. J. Exp. Med. 199: 303–313.

32. Wei, S., I. Kryczek, and W. Zou. 2006. Regulatory T-cell compartmentalizationand trafficking. Blood 108: 426–431.

33. Zou, L., B. Barnett, H. Safah, V. F. Larussa, M. Evdemon-Hogan, P. Mottram,S. Wei, O. David, T. J. Curiel, and W. Zou. 2004. Bone marrow is a reservoir forCD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals.Cancer Res. 64: 8451–8455.

34. Hirahara, K., L. Liu, R. A. Clark, K. Yamanaka, R. C. Fuhlbrigge, andT. S. Kupper. 2006. The majority of human peripheral blood CD4+CD25high

Foxp3+ regulatory T cells bear functional skin-homing receptors. J. Immunol.177: 4488–4494.

35. Coombes, J. L., K. R. Siddiqui, C. V. Arancibia-Carcamo, J. Hall, C. M. Sun,Y. Belkaid, and F. Powrie. 2007. A functionally specialized population of mu-cosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-b and retinoicacid-dependent mechanism. J. Exp. Med. 204: 1757–1764.

36. Benson, M. J., K. Pino-Lagos, M. Rosemblatt, and R. J. Noelle. 2007. All-transretinoic acid mediates enhanced Treg cell growth, differentiation, and guthoming in the face of high levels of co-stimulation. J. Exp. Med. 204: 1765–1774.

37. Apostolou, I., and H. von Boehmer. 2004. In vivo instruction of suppressorcommitment in naive T cells. J. Exp. Med. 199: 1401–1408.

38. Kasow, K. A., X. Chen, J. Knowles, D. Wichlan, R. Handgretinger, andJ. M. Riberdy. 2004. Human CD4+CD25+ regulatory T cells share equallycomplex and comparable repertoires with CD4+CD25– counterparts. J. Immunol.172: 6123–6128.

39. Fazilleau, N., H. Bachelez, M. L. Gougeon, and M. Viguier. 2007. Cutting edge:size and diversity of CD4+CD25highFoxp3+ regulatory T cell repertoire in hu-mans: evidence for similarities and partial overlapping with CD4+CD25– T cells.J. Immunol. 179: 3412–3416.

40. Siewert, C., U. Lauer, S. Cording, T. Bopp, E. Schmitt, A. Hamann, andJ. Huehn. 2008. Experience-driven development: effector/memory-like aE+

Foxp3+ regulatory T cells originate from both naive T cells and naturally oc-curring naive-like regulatory T cells. J. Immunol. 180: 146–155.

41. Wong, J., D. Mathis, and C. Benoist. 2007. TCR-based lineage tracing: no ev-idence for conversion of conventional into regulatory T cells in response toa natural self-antigen in pancreatic islets. J. Exp. Med. 204: 2039–2045.

42. Hsieh, C. S., Y. Zheng, Y. Liang, J. D. Fontenot, and A. Y. Rudensky. 2006. Anintersection between the self-reactive regulatory and nonregulatory T cell re-ceptor repertoires. Nat. Immunol. 7: 401–410.

43. Pacholczyk, R., H. Ignatowicz, P. Kraj, and L. Ignatowicz. 2006. Origin andT cell receptor diversity of Foxp3+CD4+CD25+ T cells. Immunity 25: 249–259.

44. Thiel, A., J. Schmitz, S. Miltenyi, and A. Radbruch. 1997. CD45RA-expressingmemory/effector Th cells committed to production of interferon-g lack expres-sion of CD31. Immunol. Lett. 57: 189–192.

45. Singh, B., S. Read, C. Asseman, V. Malmstrom, C. Mottet, L. A. Stephens,R. Stepankova, H. Tlaskalova, and F. Powrie. 2001. Control of intestinal in-flammation by regulatory T cells. Immunol. Rev. 182: 190–200.

46. Strickland, D. H., P. A. Stumbles, G. R. Zosky, L. S. Subrata, J. A. Thomas,D. J. Turner, P. D. Sly, and P. G. Holt. 2006. Reversal of airway hyper-responsiveness by induction of airway mucosal CD4+CD25+ regulatory T cells.J. Exp. Med. 203: 2649–2660.

47. Siewert, C., A. Menning, J. Dudda, K. Siegmund, U. Lauer, S. Floess,D. J. Campbell, A. Hamann, and J. Huehn. 2007. Induction of organ-selectiveCD4+ regulatory T cell homing. Eur. J. Immunol. 37: 978–989.

48. Tripp, R. A., D. J. Topham, S. R. Watson, and P. C. Doherty. 1997. Bone marrowcan function as a lymphoid organ during a primary immune response underconditions of disrupted lymphocyte trafficking. J. Immunol. 158: 3716–3720.

49. Liang, S., P. Alard, Y. Zhao, S. Parnell, S. L. Clark, and M. M. Kosiewicz. 2005.Conversion of CD4+CD25– cells into CD4+CD25+ regulatory T cells in vivorequires B7 costimulation, but not the thymus. J. Exp. Med. 201: 127–137.

50. Yoshikawa, T. T. 1997. Perspective: aging and infectious diseases: past, present,and future. J. Infect. Dis. 176: 1053–1057.

4326 CHARACTERISTICS OF HUMAN CD45RO+ and CD45RA+ Tregs

by guest on July 16, 2018http://w

ww

.jimm

unol.org/D

ownloaded from