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Lymphocytes T+Human Effector-Memory CD8

Four Functionally Distinct Populations of

RuferCorthesy, Estelle Devevre, Daniel E. Speiser and NathaliePittet, Cédric Touvrey, Emanuela M. Iancu, Patricia Pedro Romero, Alfred Zippelius, Isabel Kurth, Mikaël J.

http://www.jimmunol.org/content/178/7/4112doi: 10.4049/jimmunol.178.7.4112

2007; 178:4112-4119; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/178/7/4112.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Four Functionally Distinct Populations of HumanEffector-Memory CD8� T Lymphocytes1

Pedro Romero,* Alfred Zippelius,*† Isabel Kurth,‡§ Mikael J. Pittet,*¶ Cedric Touvrey,*Emanuela M. Iancu,‡ Patricia Corthesy,‡ Estelle Devevre,* Daniel E. Speiser,*and Nathalie Rufer2‡

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functionalproperties of ex vivo effector-memory (EM) CD45RA�CCR7� T lymphocytes present within the circulating CD8� T cell pool ofhealthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28expression into four subsets. EM1 (CD27�CD28�) and EM4 (CD27�CD28�) T cells express low levels of effector mediators suchas granzyme B and perforin and high levels of CD127/IL-7R�. EM1 cells also have a relatively short replicative history and displaystrong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA�CCR7�) cells. Incontrast, EM2 (CD27�CD28�) and EM3 (CD27�CD28�) cells express mediators characteristic of effector cells, whereby EM3 cellsdisplay stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiatedeffector (CD45RA�CCR7�) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss ofCCR7, CD28, and CD27 both characterize CD8� T cell differentiation. Finally, memory CD8� T cells not only include central-memory cells but also EM1 cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid andperipheral tissues, respectively. The Journal of Immunology, 2007, 178: 4112–4119.

U pon productive interaction between mature Ag-present-ing dendritic cells and specific but functionally naive Tlymphocytes, the latter undergo both clonal expansion

and differentiation into memory and effector type T cells (reviewedin Ref. 1). Although memory T cells acquire the ability to respondwith an accelerated kinetic to a second encounter with Ag, effectorT cells display functions such as lytic activity against Ag-express-ing target cells and production of cytokines such as IFN-� that aremeasurable in short-term assays (2). Major efforts have been madein recent years to understand T cell differentiation pathways. Animportant task has been to define molecular markers that readilyidentify and isolate T cells sharing discrete stages of differentia-tion. The introduction of multimers of MHC/Ag peptide, that bindstably to specific TCR on the surface of T cells, has made it pos-sible to carry out these types of analyses at the Ag-specific T celllevel (3–6). Nonetheless, this endeavor remains challenging due tothe relatively low numbers of single Ag-specific T cells that can be

retrieved from immune individuals and to the apparent complexityof the T cell differentiation process (7–13).

Four major subsets of human CD8� T lymphocytes have beendelineated with the help of two cell surface markers, the high m.w.isoform of the common lymphocyte Ag CD45RA and the chemo-kine receptor CCR7 (14, 15). Although the relationship betweenthe phosphatase activity of the former and T cell differentiationremains ill-defined, the CCR7 is involved in the molecular cascadeleading to lymphocyte recirculation from peripheral blood to sec-ondary lymphoid tissues (reviewed in Ref. 16). Thus, CCR7� na-ive and central-memory (CM)3 T cells are characterized by theability to repeatedly circulate into lymph nodes and eventuallyencounter Ag presented by incoming CCR7� mature dendriticcells. In contrast, effector-memory (EM) and effector T lympho-cytes down-regulate the CCR7 and appear specialized in migratingto peripheral nonlymphoid tissues. Although this two-marker pro-cedure to identify functionally distinct CD8� T cell subsets hasproven popular, increasing evidence indicates the existence ofhighly heterogeneous functional CD8� T subpopulations (7–13).For instance, five-color analysis including two additional surfaceAgs, CD27 and CD28, has proven useful in defining two additionalsubsets of pre-effector CD8� T lymphocytes (17). In the presentstudy, we uncovered additional heterogeneity among the EM sub-set by studying the functional attributes of pools of EM cells sep-arated on the basis of the various combinations of costimulatoryreceptor (i.e., CD27 and CD28) cell surface expression. We pro-pose the identification of four functionally distinct subsets of EMT cells, EM1, EM2, EM3, and EM4, and show data supporting thenotion that these populations represent T cells with progressivedifferentiation toward lymphocytes with potent cytokine and lytic

*Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lau-sanne Branch, University Hospital of Lausanne, Lausanne, Switzerland; †MedicalOncology, Department of Internal Medicine, University Hospital Zurich, Zurich,Switzerland; ‡Swiss Institute for Experimental Cancer Research, Epalinges, Switzer-land; §Columbia University Medical Center, Irving Cancer Research Center, NewYork, NY 10032; and ¶Center for Molecular Imaging Research, Massachusetts Gen-eral Hospital, Harvard Medical School, Charlestown, MA 02129

Received for publication September 29, 2006. Accepted for publication January12, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was sponsored and supported by the Swiss National Center of Compe-tence in Research Molecular Oncology and Swiss National Science FoundationGrants 3100-068016 and 3100A0-105929. A.Z. was supported in part by the Emmy-Noether Program of the Deutsche Forschungsgemeinshaft (Zi-685/2.3) and a grantfrom the Swiss National Foundation (3200B0-103608/1).2 Address correspondence and reprint requests to Dr. Nathalie Rufer, Swiss Institutefor Experimental Cancer Research, 155 ch. des Boveresses, CH-1066 Epalinges,Switzerland. E-mail address: Nathalie.Rufer@isrec.ch

3 Abbreviations used in this paper: CM, central-memory; EM, effector-memory; sj,signal joint; TRAP, telomerase repeat amplification protocol; FISH, fluorescence insitu hybridization; HD, healthy donor; TREC, TCR excision circle.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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effector functions. Moreover, the EM1 subset is nearly identicalwith the CM one except for the lack of CCR7 cell surface expres-sion, suggesting that this subset shares functional features withmemory cells.

Materials and MethodsCell preparation and flow cytometry

Peripheral blood samples were collected from 15 healthy donors, aged22–46 years, with a normal proportion of CD8� T lymphocytes (average,26%; range, 18–34%). PBMC were obtained by density centrifugationusing Ficoll-Hypaque (Pharmacia). Our experimental procedures involvetwo steps that exclude NK cell contamination. First, CD8� T lymphocyteswere positively enriched from cryopreserved or fresh PBMCs using anti-CD8-coated magnetic microbeads (Miltenyi Biotec), a procedure that elim-inates most NK cells because they are not efficiently retained by the mag-net. Cells were stained with appropriate mAbs in PBS, 0.2% BSA, 50 �MEDTA for 20 min at 4°C and either directly analyzed or sorted into definedpopulations on a FACSVantage SE, using CellQuest software (BD Bio-sciences). Immediate reanalysis of the isolated populations revealed onaverage �95% purity, and in the case of naive T cells, over 99% purity. Ofnote, as naive T cells represent a homogeneous subset that is distinctivelyCD45RA-, CCR7-, CD27-, and CD28-positive, contaminant naive cellswere not present within sorted CM and EM1 cells in significantly highnumbers (�1%), thus allowing us to exclude bias in the TCR excisioncircle (TREC) analysis of those cells and in their estimate of proliferativehistory (data not shown). Second, FACS analysis and sorting was performedon gated CD8 bright T cells, allowing the exclusion of any residual contam-inating NK cells in the sorted populations (�2%). Intracellular content ofgranzyme B and perforin was measured in freshly isolated CD8� T lympho-cytes without previous stimulation as previously described (17). The follow-ing mAbs were purchased from BD Biosciences or BD Pharmingen:anti-CD27-FITC and -PE, anti-CD28-PE and -allophycocyanin, anti-CD8-allophycocyanin/Cy7, anti-HLA-DR-FITC, and goat anti-ratIgG-allophycocyanin. Other sources of mAbs were: Beckman Coulter(anti-CD45RA-PE-Texas Red) and Caltag Laboratories (goat anti-ratIgG-PE). Anti-CCR7 rat IgG mAb 3D12 was provided by Dr. M. Lipp(Max Delbruck Institute, Berlin, Germany). Anti-granzyme B-FITC andanti-perforin-FITC mAbs were obtained from Holzel Diagnostika andAlexis, respectively. Synthesis of PE-labeled HLA-A*0201/peptidemultimers with Melan analog peptide26 –35 (ELAGIGILTV), and allo-phycocyanin-labeled HLA-A*0201/peptide multimers with Flu matrixprotein58 – 66 (GILGFVTL), CMV pp65495–503 (NLVPMVATV), andEBV BMFL1280 –288 (GLCTLVAML), were prepared as described pre-viously (5).

cDNA amplification and five-cell RT-PCR

To avoid contamination of small populations by more abundant subsets,10 � 103 T cells of each subset were sorted by flow cytometry and five-cellaliquots of the purified subsets were then resorted directly into wells of96-V-bottom plates. The procedures for cDNA preparation, cDNA ampli-fication as well as the RT-PCR were recently described in details (17, 18).Additional primers were used in the present study: CD27, 5�-ACGTGACAGAGTGCCTTTTCG-3�; reverse-5�-TTTGCCCGTCTTGTAGCATG-3�; CD127/IL-7R�: 5�-ATCTTGGCCTGTGTGTTATGG-3�; reverse-5�-ATTCTTCTAGTTGCTGAGGAAACG-3�.

Cytolytic activity

Cytolytic activity was tested in a CD3 mAb-mediated “redirected” 51Crrelease assay. In brief, FcR-expressing P815 target cells were radiolabeledwith Na51CrO4 (PerkinElmer) for 1 h at 37°C. Sorted CD8� T subsets wereincubated with P815 target cells (103 cells/well) at varying effector-targetcell ratios in the presence or absence of 300 ng/ml anti-CD3 mAb (OKT3).After 4 h at 37°C, supernatants were collected and counted on a gammacounter. Percent lysis was calculated as (experimental release � sponta-neous release) � 100/(total release � spontaneous release).

Quantification of TRECs by real-time PCR

The amount of signal joint (sj) TRECs in 5–15 � 104 sorted CD8� Tsubsets was determined by real-time quantitative PCR using the ABIPRISM 7700 Sequence Detector TaqMan system (Applied Biosystems) aspreviously described (19, 20). In brief, after cell lysis in 100 mg/L pro-teinase K (Roche Diagnostics) for 2 h at 56°C followed by 15 min at 95°C,a PCR was performed in a final volume of 25 �l containing 5 �l of cellextract, 12.5 �l of TaqMan Universal Master Mix including AmpliTaqGold (Applied Biosystems), 500 nM of each primer (sj-5� forward: CA

CATCCCTTTCAACCATGCT; sj-3� reverse: GCCAGCTGCAGGGTTTAGG), and 125 nM TaqMan probe (FAM-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-TAMRA). After one cycle of 2 min at 50°C followedby an initial 10-min denaturation at 95°C, 40 cycles of 30 s at 95°C and 1min at 65°C were performed. The number of TRECs in a given sample wasestimated by comparing the cycle threshold value obtained with a standardcurve obtained from PCR performed with 10-fold serial dilutions of aninternal standard provided by Dr. D. Douek (Vaccine Research Center,National Institutes of Health, Bethesda, MD). The dilutions contained be-tween 107 and 101 copies of sjTREC and four reactions were run with eachdilution. Considering that �50,000 cells were always analyzed per subset,and that the linear range of the external standard used starts at 10 copies,our lower TREC detection limit was 10 copies/50,000 cells or 0.02% (thusvalues of �10 copies/sample were quoted as below the detection limit ofthe assay). In all PCR assays, the correlation coefficient of the standardcurve was �0.997, whereas the slope varied between �3.52 and �3.67.The TREC analysis was performed on young healthy individuals (�35years of age) because aging has been shown to inversely correlate with theTREC levels (19, 20).

Telomerase repeat amplification protocol assay

Telomerase activity was measured with the telomerase repeat amplificationprotocol (TRAP) assay using a telomerase substrate primer as describedpreviously (17). Cell extracts were obtained from 5 to 15 � 104 sortedCD8� T cell subsets. As positive control we used extracts from CD8� Tlymphocytes stimulated for 5 days with 1 �g/ml PHA (Sodiag) and 150U/ml rIL-2 in presence of 1 � 106/ml irradiated feeder cells. Extension ofthe telomerase substrate primer by telomerase was performed for 30 min at30°C in the presence of [�-32P]dGTP and the products generated wereamplified by 27 cycles of PCR at 94°C for 30 s and 60°C for 30 s using theACX-anchored return primer. One-half of the amplified products were re-solved on a 15% polyacrylamide gel and visualized by a phosphoimagingsystem.

Telomere fluorescence in situ hybridization and flow cytometry

The average length of telomere repeats at chromosome ends in individualcells was measured by fluorescence in situ hybridization (FISH) and flowcytometry as previously reported (18, 21, 22). Telomere fluorescence wascalculated by subtracting the mean fluorescence of the background control(no probe) from the mean fluorescence obtained from cells hybridized withthe telomere probe after calibration with FITC-labeled fluorescent beads(Quantum TM-24 Premixed; Bangs Laboratories) and conversion into mol-ecules of equivalent soluble fluorochrome units (MESF). The followingequation was performed to estimate the telomere length in base pair: bp �MESF � 0.495 (21, 22). Telomere length measurement was performed onin vitro-derived T cell clones by limiting dilution (23) sorted from Melan-A-, Flu-, EBV-, and CMV-specific CD8� T lymphocytes isolated from asingle healthy individual, allowing for the recovery of a sufficient numberof cells for flow FISH analysis. All T cell clones were expanded in identicalin vitro culture conditions and have undergone approximately the samemean number of population doublings. Cell extracts obtained from severalT cell clones with distinct Ag specificity were submitted to the TRAPassay. Although very low levels of telomerase activity could be detected,no significant differences among the stimulated cells were apparent (datanot shown). After a single round of mitogenic stimulation, 2 � 105 cellswere further processed by flow FISH. Because the average telomere flu-orescence from all these clones was evaluated in the same experimentaldesign, this allowed direct telomere comparison between each tested clonalcondition.

ResultsEx vivo distribution of phenotypically distinct CD8� T cellsubsets from human peripheral blood

Human CD8� T lymphocytes can be separated into four func-tionally different populations on the basis of CD45RA andCCR7 expression: naive (RA�CCR7�), CM (RA�CCR7�),EM (RA�CCR7�), and effector (EMRA; RA�CCR7�) (Fig.1A). The relationship between CD45RA/CCR7 and the expres-sion of CD27 and CD28 was further assessed within each cellsubset. Staining of peripheral blood CD8� T lymphocytes withAbs to CD45RA, CCR7, CD27, and CD28 revealed the pres-ence of nine discrete subpopulations in the blood from a rep-resentative healthy donor (Fig. 1B). Naive and CM cells uni-formly coexpressed CD27 and CD28. In contrast, as based on

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our previous report (17), the RA�CCR7� EMRA T cells weresplit into three functionally distinct subsets: pE1 (27�28�), pE2

(27�28�), and effector (27�28�) T cell subpopulation. EM Tcells also exhibited high heterogeneity with a differential ex-pression of CD27 and CD28 cell surface molecules (Fig. 1B).Thus, we identified, within the RA�CCR7� EM T compart-ment, four phenotypically separate subsets referred to as EM1

(27�28�), EM2 (27�28�), EM3 (27�28�), and EM4 (27�28�).Naive T cells exhibited the highest levels of CD27, with a pro-

gressive down-regulation of CD27 cell surface expression fromnaive, through CM and EM1, to EM2 cells (Fig. 1C). In contrast,cell surface CD28 expression levels were higher in CM, EM1, andEM4 cells than in naive cells. Finally, the EM3 subset resembles tothe effector one, because both subsets have lost CD27 and CD28coexpression. These data are in line with reports showing that invitro stimulation of CD8� T cells induces down-regulation ofCCR7 and CD27 and up-regulation of CD28 (14, 24, 25). To ob-tain an overview of the distribution of the nine CD8� T cell sub-sets defined by a different pattern of CD45RA, CCR7, CD27, andCD28 expression, we analyzed PBMC from 14 healthy individu-als, ranging in age from 22 to 46 years (Fig. 1D). Although theproportion of each T cell subset varied between donors, all ninesubpopulations were found in every single individual, with thepreferential dominance of naive, effector, and EM1 CD8� Tsubpopulations.

Progressive acquisition of granzyme B and perforin expressionand of ex vivo killing activity within EM CD8� T cell subsets

Previous studies have proposed that CD27�CD28� T cells differ-entiate through a CD27�CD28� to a CD27�CD28� stage (11,17). According to this model, CD8� T cells sequentially down-regulate CCR7, CD28, and CD27 surface expression, while up-

regulating expression of molecules that confer cytolytic activity.To investigate whether EM1, EM2, and EM3 CD8� T cell subsetscorresponding to the proposed differentiation pathway (11, 17) dif-fered in the expression of genes involved in T cell effector func-tions, we used a modified RT-PCR protocol that detects specificcDNAs after global amplification of expressed mRNAs from asfew as five cells (17, 18). As expected, all naive and most CM Tcell five-cell samples contained no detectable granzyme B, per-forin, IFN-�, or NK receptor CD94 mRNA (Fig. 2A). Interestingly,despite the loss of CCR7 expression, the gene expression profile ofEM1 T cells resembled closely to the one of CM cells. Of note, weobserved differences of IFN-� mRNA expression by EM1 T cellsthat may reflect biological variations in the proportions of IFN-�-expressing cells from the same subset among different healthy do-nors (Fig. 2A; see HD1, HD2, and HD3). In contrast, EM2 andEM3 T cell aliquots exhibited detectable levels of granzyme B,IFN-�, and CD94 transcripts. This was particularly marked forgranzyme B mRNA expression and associated with the high ex-pression level for this protein (Fig. 2B). Moreover, most of naive,CM, and EM1 cells, but almost none of the aliquots of EM3 andeffector T cells, yielded a detectable CD127-specific product, en-coding for the �-chain of the IL-7R complex. According to bothmRNA analysis (Fig. 2A) and intracellular staining (Fig. 2B), allthree EM T subsets expressed perforin, but at lower levels than inthe differentiated effector subset. When we compared the cyto-lytic activity of these distinct cell populations, using a CD3mAb-mediated redirected 51Cr release assay (Fig. 2C), wefound that both EM3 and effector T cells displayed high ex vivolytic activity, whereas CM and EM1 T cells had comparablekilling activity, which was �10 times lower than that of theeffector population.

FIGURE 1. Differential expression of CD45RA,CCR7, CD27, and CD28 cell surface molecules on totalCD8� T cells from healthy blood donors. A, CD8�

gated cells were separated into four subsets (naive, CM,EM, and effector) based on CD45RA and CCR7 label-ing. B, Each of these subsets was analyzed for CD27and CD28 coexpression and nine subpopulations ofCD8� T cells could be distinguished. A representativeexample is here depicted. C, Naive-CD27�, CM-CD27�, EM-CD27� (comprising EM1�EM2), EM-CD27� (EM3�EM4), and EMRA-CD27� T cellswere analyzed for their level of CD28 expression.Naive-CD28�, CM-CD28�, EM-CD28� (comprisingEM1�EM4), EM-CD28� (EM2�EM3), and EMRA-CD28� T cells were analyzed for their level of CD27expression. D, The distribution of the nine definedCD8� T cell subsets among 14 healthy individuals isshown as mean percentage (range).

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Progressive telomere shortening and reduction in level ofTRECs within EM CD8� T cell subsets

We next investigated the replicative history of EM1, EM2, andEM3 T cell subsets by quantifying their content of TRECs, whichare stable DNA episomes formed during TCR-� gene rearrange-ment and are diluted out with each cell division (19). In all fourhealthy individuals tested (Fig. 3A), naive cells had the highestlevel of TRECs, whereas they were below the detection limit of theassay (�0.01 TREC copies/100 cells) in the EM3 and effectorsubsets. CM and EM2 T cells contained low but detectable TRECsin all healthy individuals. Intriguingly, TRECs in EM1 cells weredetected in reduced levels in two healthy donors (HD), and not atall in the two other individuals, indicating that these cells had atleast undergone six to seven more divisions than the bulk of naivecells.

To further characterize the relationship between the mitotichistory of cells and their differentiation status, the averagelength of telomere repeats of ex vivo-sorted naive, CM, EM1,EM2, EM3, and effector T lymphocytes from two healthy do-nors, was measured by FISH and flow cytometry (Fig. 3B).Because telomeres progressively shorten as a function of celldivision (26), telomere length is a powerful indicator of thereplicative in vivo history of lymphocytes (21, 27). We ob-served a progressive reduction in the mean telomere fluores-cence from naive through CM, EM1, EM2 to EM3 and effectorT lymphocytes, that corresponded to a telomere shortening of�4.5–5 kb. EM1 cells displayed shorter average telomerelengths than those observed in CM cells from HD1, suggestingadditional cell divisions within the former subset. In contrast, inHD2, the telomeres of both subsets exhibited similar lengths.These results confirmed the heterogeneity observed when wecharacterized the content of TRECs within EM1 cells (Fig. 3A).Importantly, our data revealed that EM3 cells had relativelyshort telomeres, within the same range than those found in

FIGURE 2. Ex vivo analysis of expression of effector mediatorswithin EM T cell subsets. A, Gene expression analysis was performedon sorted naive (RA�CCR7�27�28�), CM (RA�CCR7�27�28�),EM1 (RA�CCR7�27�28�), EM2 (RA�CCR7�27�28�), EM3

(RA�CCR7�27�28�) and effector (RA�CCR7�27�28�) CD8� T cellsusing a modified RT-PCR protocol (18). Data from three or six indepen-dent five-cell aliquots per subset, and negative (�) and positive (�) con-trols, are depicted. Comparable results were obtained in three healthy in-dividuals. For IFN-� expression, data obtained from HD1, HD2, and HD3is depicted; na, not applicable. B, The proportion of granzyme B- andperforin-positive cells among CM, EM1, EM2, EM3, and effector T cellswas determined by immunofluorescence. Note that the perforin signal islower in EM1, EM2, and EM3 than in effector cells. Data are representativeof four healthy donors. C, Ex vivo-sorted CD8� naive, CM, EM1, EM2,EM3, and effector T cells were tested in a redirected cytolytic assay against51Cr-labeled P815 target cells. None of these subsets lysed P815 cells inabsence of CD3 mAbs (lysis �10%; data not shown). Data are represen-tative of two healthy donors.

FIGURE 3. Ex vivo analysis of the replicative history and telomeraseactivity of EM T cell subsets. A, Real-time PCR quantification ofTRECs was performed on sorted naive, CM, EM1, EM2, EM3, andeffector CD8� T cells from four healthy young individuals (age range,22–35 years). �, Not detectable (sorted cell number was 5 � 104 to 105,lower quantification limit � 0.01– 0.02%). Of note, the levels of TRECsmeasured in sorted CM (0.4 0.2), EM1 (0.1 0.1), and EM2 (1 0.4) cells were not significantly different. B, Telomere fluorescenceanalysis in ex vivo-sorted naive, CM, EM1, EM2, EM3, and effectorCD8� T cells isolated from two healthy donors (HD1 and HD2). Themean telomere fluorescence (in FL1 channel) was converted to kilobaseas described in Materials and Methods; n.a., not applicable. C, Telom-erase activity in cell extracts of sorted 28�DR�, 28�DR�, naive, CM(28�DR�), CM (28�DR�), EM1 (28�DR�), EM1 (28�DR�), EM2,EM3, and effector CD8� T cells. As positive controls, we used cellextracts of in vitro-PHA-activated CD8� T cells (act. CD8�). Data arerepresentative of two healthy individuals. CD45RA; RA, CCR7; R7.

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differentiated effector cells. This agrees with our recently pub-lished results (12) showing progressive shortening of the telo-meres along T cell differentiation, so that highly differentiatedCD8�CCR7�CD27�CD57� cells displayed the shortest telo-meres, with lengths equivalent to those observed in primedCD8� T cells from the elderly (22). Altogether, these data sup-port the view that T cells evolve through extensive rounds ofdivision as they differentiate further.

A previous report (28) showed telomerase activity in ex vivo-isolated CD8� T cells that express both CD28 and the activationmarker HLA-DR. To determine which if not all of the HLA-DR-expressing T cell subsets accounted for such activity, TRAP assayswith the HLA-DR� and HLA-DR� fractions of CM and EM1 Tcells were conducted (Fig. 3C). Remarkably, we observed positivetelomerase activity selectively confined to the DR-positive frac-tions of both subsets. In contrast, naive, EM2, and EM3 T cells, thatdid not contain a sizeable fraction of HLA-DR� cells, revealed noex vivo detectable telomerase activity. Of note, telomerase activitywas already detectable when EM1 T cells had been sorted withoutdiscriminating HLA-DR� from HLA-DR� cells (data not shown).Finally, within both CM and EM1 populations, the proportion ofHLA-DR� T cells represented between 5 and 15% with a trendtoward CM cells.

Both primed EM1 (27�28�) and EM4 (27�28�) T cell subsetsexpress a similar pattern of genes and display low levels ofeffector mediators

The fourth T cell population (EM4) was analyzed in additionalexperiments, as EM4 cells could not be fully investigated in par-allel in the previously shown experiments due to technical limita-tions. To gain insight into the relationship between EM4 T cellsand the EM1 subset (as outlined in Fig. 1B), we compared theirexpression of genes involved in effector functions within fivecell-sorted samples (Fig. 4A). In both EM1 and EM4 cells, nogranzyme B mRNA transcripts were detected, whereas CD127/IL7R�, perforin, and IFN-� transcripts were found in a signif-icant proportion of these samples. Moreover, these results cor-related with the analysis of granzyme B and perforin expressionby intracellular staining for these molecules (Fig. 4B). Thus,our data indicate that despite the loss of CD27 expressionwithin the EM4 compartment, these cells seem closely related tothe EM1 T cells.

Ag-specific CD8� T lymphocytes exhibit distinct differentiationphenotypes and replicative history

Several studies have reported that Ag-specific CD8� T cells di-rected against the tumor-associated self-Ag Melan-A/MART-1 oragainst viral epitopes such as the influenza matrix protein (Flu),

BMFL1 (EBV), and pp65 (CMV) show different stages of cellulardifferentiation. We previously found that the majority of circulat-ing Melan-A-specific T cells from HLA-A2 healthy donors is phe-notypically and functionally naive despite their high frequencies(20). In contrast, influenza- and EBV-specific T cells respectivelydisplay a primed CM and EM phenotype, while CMV-specific Tlymphocytes are mostly composed of differentiated effector cells(2, 8–11, 17, 29). Here, we confirmed and extended these studiesby comparing the coexpression of CCR7/CD45RA (Fig. 5A), andof CD27/CD28 (Fig. 5B) in Melan-A-specific T cells to that ofprimed Ag-specific T cells such as EBV and CMV in healthy in-dividuals. Whereas Melan-A-specific T cells shared a homoge-neous naive-like phenotype (CCR7�CD45RA�CD27�CD28�),the EBV-specific response consisted primarily of early differ-entiated T cells (EM1, mean SD, 60 16%; n � 8). Con-sistent with our recent report (17), CMV-specific T lympho-cytes displayed the phenotype of effector CD8� T cells(mean SD, 18 7%; n � 8), but also the phenotype of EM2

(19 8%) and EM3 cells (34 14%). Interestingly, our datafurther revealed that Melan-A-specific naive T cells, as com-pared with EBV-specific T cells, exhibited the highest levels ofCD27, while they expressed lower levels of CD28 (Fig. 5C), inline with the notion that activation and priming of T cells in-volves down-regulation of CD27 and up-regulation of CD28cell surface molecules (14, 24, 25).

An important aspect, often neglected in those current studies,concerns the proliferative potential of the characterized Ag-spe-cific T cells. Therefore, we investigated the replicative history ofeach of the four above-defined Ag-specific T cells that exhibitdistinct differentiation phenotypes (Fig. 5, A and B). For this pur-pose, we measured the average length of telomeres in Melan-A/MART-1, Flu-, EBV-, and CMV-specific T cell clones isolatedfrom a single healthy individual (Fig. 5D). Strikingly, we observeda progressive reduction in mean telomere fluorescence fromMelan-A- through Flu-, to EBV- and CMV-specific lymphocytes,consistent with the observation of progressive telomere shorteningfound within differentiated CD8� T cell subsets (Fig. 3B). Due tothe relative low frequencies of the four antigenic specificitieswithin CD8� T cells, we were unable to perform the flow FISHexperiment on ex vivo-sorted T cells (see Materials and Methods).Nevertheless, these results confirmed our previous finding that Flu-specific T cells, displaying an early differentiated phenotype (CMand EM1; Fig. 5, A and B), showed significant reduced telomerelengths ex vivo compared with the naive Melan-A-specific cells(20). Collectively, our data favor the notion that Ag-specific Tcells, as they differentiate, also undergo additional rounds of invivo cell division.

FIGURE 4. Comparison of the ex vivo expression ofeffector mediators between EM1 and EM4 T cell sub-sets. A, Gene expression analysis was performed onsorted CM, EM3, EM1, and EM4 CD8� T cells by RT-PCR. Data from three or eight independent five-cell ali-quots are shown. (�), Negative; (�), positive controls.B, The proportion of granzyme B- and perforin-positivecells among CM, EM1, EM4, and effector CD8� T cellswas determined by immunofluorescence. All results arerepresentative of two healthy individuals. CD45RA;RA, CCR7; R7.

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DiscussionCirculating naive T lymphocytes form a relatively homogeneouspopulation expressing a well-defined set of cell surface glycopro-teins and are characterized by the null expression of effector me-diators (e.g., IFN-�, granzyme B, perforin, Fas/CD95) and by highproliferative potential (e.g., long telomeres, high detectable levelsof TREC copies). During the last three decades, primed Ag-expe-rienced T lymphocytes have mostly been classified into two dis-tinct subpopulations, e.g., effector and memory cells (30). Effectorsare presumably rather short-lived, produce cytolytic effector mol-ecules and are capable of migrating to the site of infection and ofkilling target cells directly ex vivo. In contrast, memory cells arelong-lived, persist after pathogen clearance, and have increasedsurvival properties and cell division capacities. However, severallines of evidence recently challenge this simplified view of defin-ing primed T cells (reviewed in Ref. 31). First, using five-colorflow cytometry, detailed analysis of human peripheral CD8� (17)and CD4� (32) T cells allowed their distribution into six majorsubpopulations, identified by the patterns of expression ofCD45RA, CCR7, CD28, and/or CD27. Second, it has been shownthat EBV-, CMV-, and HIV-specific T cells vary in differentiationphenotype during persistent viral infections, suggesting that Ag-specific T cells present in each individual type of infection are verydifferent (11). Third, several studies in mice revealed that effector-

type T cells were required in peripheral tissue before viral chal-lenge to protect against vaccinia virus, whereas CM cells weremost potent at protecting against systemic infection with lympho-cytic choriomeningitis virus (33, 34), as they have a greater ca-pacity than EM T cells to persist in vivo (35). Finally, Roberts et al.(36) recently described that both CM and EM T cells contributedto recall responses to Sendai virus infection in the lung, with aprogressive increase in efficacy of the CM subset over time. Thesedata reinforce the notion that the protective capacity of differentsubpopulations of primed T cells (i.e., effector vs memory) mayvary depending on the nature of the challenging pathogen, and maychange substantially over time. Altogether, these and other studies(7–13) indicate that primed Ag-experienced T lymphocytes arehighly heterogeneous, varying in terms of their cell surface phe-notype, functional capacities, and history of Ag encounter.

Here, by combining the simultaneous analysis of surface mark-ers by multiparameter flow cytometry with the analysis of geneexpression and of replicative history, we show that EMCD8�CD45RA�CCR7� T cells can be further divided into fourdistinct subsets, based on differential CD27 and CD28 expressionpatterns. CD27 and CD28 are costimulatory receptors involvedrespectively in the generation of Ag-primed cells and the regula-tion of T cell activation (37, 38). EM1 (27�28�), EM2 (27�28�),EM3 (27�28�), and EM4 (27�28�) T cell subsets are all present

FIGURE 5. CD8� T cells vary inphenotype and replicative history de-pending on Ag specificity. A and B,Melan-A, Flu-, EBV-, and CMV-mul-timer� T cells were characterized exvivo by flow cytometry for their cellsurface expression of CD45RA, CCR7,CD28, and CD27. The dot plots showdouble staining for CD45RA/CCR7 (A)and CD28/CD27 (B) on Melan-A(HLA-A2), Flu (HLA-A2/Matrix pro-tein), EBV (HLA-A2/BMFL1), andCMV (HLA-A2/pp65)-specific CD8�

T lymphocytes gated using the relevantmultimers. For comparison, stainingson whole CD8� T cells (CD8�) andthe distribution of the different subsetsaccording to cell surface marker ex-pressions is depicted. Eight healthy in-dividuals were included in these analy-ses. Representative data are shown. C,Enlarged dot plots of CD28 and CD27costaining on Melan-A (multimer-allo-phycocyanin) and EBV (multimer-PE)specific CD8� T cells. Dotted lines rep-resent the reference for CD28 andCD27 positivity based on the fluores-cent signal obtained after gating on theequivalent whole CD8� T cell subset.D, Telomere fluorescence analysis wasperformed on 10 in vitro-generated Tcell clones derived from either Melan-A-, Flu-, EBV-, or CMV-specificCD8� T lymphocytes isolated from asingle healthy donor as described inMaterials and Methods. The proportionof multimer-specific T cells in CD8� Tcells is indicated. The mean telomerefluorescence (in FL1 channel) was con-verted to kilobase as described in Ma-terials and Methods.

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in the peripheral blood of healthy donors with a predominancetoward EM1 cells (Fig. 1D). Functionally, at least three subsets canbe clearly identified: EM1 (that includes the very similar EM4),EM2, and EM3. EM1 are memory-like, EM2 are intermediate withpartial effector functions and replicative history, and EM3 are ef-fector-like. Taken together, our data are in agreement with themodel according to which there is a differentiation pathway withprogressive loss of CCR7, CD28, and CD27 cell surface expres-sion concomitant with up-regulation of cytolytic capacity (11).

In particular, we show that both circulating EM2 and EM3

CD8� T cells express mediators characteristic of effector cells, butgene and protein expression profiles of EM3 T cells more closelyresemble that of effector cells. In line with this notion, EM3 cellsdisplay stronger ex vivo cytolytic activity and have experienced alarger number of cell division, similarly to differentiated effector Tlymphocytes (Figs. 2 and 3). In contrast, EM2 cells contain low butyet detectable levels of TRECs (Fig. 3A). Unless EM2 cells dif-ferentiate from CM cells without cell division, our data indicatethat these cells descend from naive T cells. Moreover, loss ofCD28 cell surface expression is associated with the acquisition ofgranzyme B expression, allowing more differentiated cells (e.g.,EM3) to kill their targets through perforin/granzymes pathways.

Another major finding in this study is that EM1 T cells, despitetheir lack of CCR7 expression, have several functional features incommon with CM cells. Both populations have a similar replica-tive history (Fig. 3), thus they have undergone more cell divisionsthan naive cells but fewer than effector cells. Remarkably, theyexpress the enzyme telomerase, which is known to be involved inmaintenance of telomere length and cell proliferation potential.Because telomerase activity was exclusively found within theHLA-DR-positive fraction of CM and EM1 T cells, our data dem-onstrate that these subsets account for the previously describedtelomerase in CD8�28�DR� T cells (28). Speiser et al. (28) alsoshowed that in vivo cycling CD8� T lymphocytes expressed HLA-DR, thus supporting the idea that telomerase expression in HLA-DR� CM and EM1 cells reflects proliferative activity while main-taining telomere lengths. This would be compatible with reducedlevels of TRECs but only progressive shortening of telomerelengths as observed in the EM1 cells (Fig. 3). It is also noteworthyto mention that induction of telomerase activity in Ag-specific ef-fector and memory CD8� T cells from mice infected with lym-phocytic choriomeningitis virus has been suggested to be impor-tant for the maintenance and longevity of the memory CD8� T cellpopulation (39). Finally, both CM and EM1 T cell subsets expresshigh levels of the IL-7R� chain (CD127) necessary to memory cellsurvival (40), but only low levels of effector molecules such asIFN-�, granzyme B, and perforin. Altogether, these results, ob-tained with cells analyzed directly ex vivo, indicate that the CMand EM1 subpopulations may comprise T cells that have beenrecently activated following antigenic challenge. Alternatively, te-lomerase-expressing T cells may consist of memory cells that ex-pand following homeostatic maintenance of T cell numbers. In-vestigations of ongoing immune responses in vivo (41) as well ason T lymphocytes isolated from lymph nodes will be useful tospecifically address these questions.

Based on these findings, it is tempting to propose that humanmemory CD8� T cells include in fact two types of functionallyequivalent populations with identical cell surface phenotypes ex-cept for the expression of the chemokine receptor CCR7. CCR7�

cells (CD8� TCM, for CM) have the ability to migrate from bloodthrough secondary lymphoid organs, like naive T cells, whereasCCR7� cells (CD8� TPM, identified here as EM1, for peripheralmemory) travel from blood to nonlymphoid tissues where they candirectly re-encounter Ag. In both cases, the memory status of both

subsets allows for rapid reactivation upon Ag challenge regardlessof tissue location.

Loss of CD27 during clonal expansion and differentiation pre-sumably leads to the emergence of differentiated T cells with amore extensive replicative history and more complete effectorfunctions than CD8�CD27� cells (Refs. 17 and 42; Figs. 2 and 3).Recent analysis on sorted CD27� HIV- and EBV-specific T cellclones followed by in vitro stimulation revealed that most ofthese cells had irreversibly lost CD27 expression (43). Yet, sortedCD27� CD8� T cells transiently down-regulated the expression ofCD27 with the majority re-expressing CD27 at the end of the pro-liferative cycle. In the present study, we identified a small butsignificant proportion of EM T cells within the circulating bloodthat had down-regulated CD27 expression while maintaining theCD28 costimulatory molecule (EM4; CD27�CD28�). Similarly toEM1 cells, this subset displays low levels of effector-mediatedmolecules, while still expressing IL-7R� (Fig. 4). One possibilityis that EM4 cells differentiate from the CM T cell pool and thatsuch cells represent a transitory subset appearing during the ex-pansion phase of a secondary immune response. Alternatively, onecannot formally exclude that these cells have emerged directlyfrom the EM1 pool and have transiently or definitively down-reg-ulated CD27 expression. Future analysis involving the carefulevaluation of their replicative history combined to their cell cyclestatus is necessary to understand the role of this subset along theCD8� T cell differentiation pathways.

Ultimately, a conclusive answer to the issues concerning thefunction of the various EM T subsets described here and theirrelationship with other subpopulations will require the in vivotracking of Ag-specific T cells in humans during the course ofinfection with viruses such as influenza, EBV, or CMV. Althoughtimely access to such clinical situations is enormously difficult,they remain attractive because they would provide two major ad-vantages from an experimental viewpoint. On one hand, infectionsby these viruses are frequent and independent of geographical lo-cation. On the other hand, there are currently well-defined MHCclass I-restricted dominant T cell epitopes and enough tools exist,including fluorescent pMHC multimers, to identify and character-ize the corresponding Ag-specific CD8� T cell responses.

AcknowledgmentsWe dedicate this work to our friend and colleague Dr. Pascal Batard whocontributed indispensably to this study and passed away abruptly. We arethankful for Drs. Immanuel Luescher and Philippe Guillaume for synthesisof multimers; Dr. Daniel Douek for providing signal joint internal standard;and Dr. Martin Lipp for the anti-CCR7 mAb. We also thank the excellenttechnical and secretarial help of Dr. Lionel Arlettaz, Pierre Zaech, Martinevan Overloop, and Severine Reynard.

DisclosuresThe authors have no financial conflict of interest.

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