CD26: A Negative Selection Marker for Human Treg Cells · Department of Biochemistry and Molecular...

CD26: A Negative Selection Marker for

Human Treg Cells

Francisco J. Salgado, Amparo Perez-Dıaz, Nora M. Villanueva, Olaya Lamas,Pilar Arias, Montserrat Nogueira*

� AbstractA major obstacle hampering the therapeutic application of regulatory T (Treg) cells isthe lack of suitable extracellular markers, which complicates their identification/isola-tion. Treg cells are normally isolated via CD25 (IL-2Ra) targeting, but this protein isalso expressed by activated CD41 effector T (Teff) lymphocytes. Other extracellular(positive or negative) Treg selection markers (e.g., HLA-DR, CD127) are also nonspeci-fic. CD26 is an extracellular peptidase whose high expression has been traditionallyused as an indicator of immune activation and effector functions in T cells. Now, weprovide flow cytometry data showing high levels of CD26 within CD41CD252 orCD41FoxP32/low effector T (Teff) lymphocytes, but negative or low levels (CD262/low)in Treg cells selected according to the CD41CD25high or the CD41FoxP3high phenotype.Unlike the negative marker CD127 (IL-7Ra), which is down modulated in CD41 Tefflymphocytes after TCR triggering, most of these cells upregulate CD26 and take aCD41CD251/highCD261 phenotype upon activation. In contrast, there is only aslight upregulation within Treg cells (CD41CD25highCD262/low). Thus, differencesin CD26 levels between Treg and Teff subsets are stable, and assessment of thismarker, in combination with others like CD25, FoxP3, or CD127, may be usefulduring the quantitative evaluation or the isolation of Treg cells in samples containingactivated Teff lymphocytes (e.g., from patients with autoimmune/inflammatorydiseases). ' 2012 International Society for Advancement of Cytometry

� Key termsCD26; regulatory T cells; effector T cells; flow cytometry; magnetic sorting

REGULATORY cells are included in different leukocyte populations, like CD81 (1) or

CD41 (2,3) T subsets. CD41CD25high natural Treg (nTreg) cells constitute a small

CD41 subpopulation (\5% of CD41 T lymphocytes) with a thymic origin. Numeri-

cal or functional deficit of nTreg cells is linked to autoimmune diseases such as multi-

ple sclerosis (MS), Type 1 diabetes or rheumatoid arthritis (RA) (4). In addition, they

are also important to prevent (or delay) grafts rejection or disproportionate responses

to bacterial/viral antigens (5,6). Alternative subpopulations of regulatory T cells (e.g.,

TH3 and Tr1), called ‘‘adaptive’’ or ‘‘induced’’ regulatory CD41 T cells (iTreg), are

generated in the periphery (7). Both nTreg and iTreg lymphocytes (altogether, Tregs)

limit the biological activities (e.g., proliferation, cytokine production) of adaptive

‘‘effector’’ cells, a miscellaneous group consisting of CD41 helper T cells (TH1, TH2,

TH17, TFH, TH9, TH22), CD81 cytotoxic T lymphocytes and B cells. To finely tune the

strength of effector responses, Treg cells employ various suppressor mechanisms, like

inhibitory soluble molecules (adenosine, TGFb, IL-10) or cell contact-mediated path-

ways (e.g., membrane cytokines like TGFb1 or surface molecules like CTLA-4) (8,9).

Any research aimed at controlling Treg function, either enhancing (e.g., in auto-

immune diseases) or blocking it (e.g., in cancer), will hold a great interest

(5,6,10,11). However, this kind of research faces several challenges; for example, how

to distinguish regulatory (Treg) from effector (Teff) CD41 T populations. Human

Department of Biochemistry andMolecular Biology, Faculty of Biology/CIBUS, University of Santiago deCompostela, Coru~na, Espa~na

Received 19 September 2011; RevisionReceived 18 May 2012; Accepted 24 July2012

Grant sponsor: Xunta de Galicia; Grantnumber: INCITE08PXIB200062PR (to A.P.-D.).

Additional Supporting Information may befound in the online version of this article.

Francisco J. Salgado, Nora Mart�ınez-Villanueva, Amparo Perez-D�ıaz, andOlaya Lamas did the experimental workand data analysis. Francisco J. Salgado,Pilar Arias, and Montserrat Nogueiradesigned the research, supervised thework, and wrote the article.

*Correspondence to: MontserratNogueira. Laboratorio BQ1 (2o piso)CIBUS. Departamento de Bioqu�ımica yBiolog�ıa Molecular, Universidad deSantiago de Compostela. C/Lope Gomezde Marzoa s/n (Campus vida), Santiagode Compostela, CP. 15782, Coru~na,Espa~na

Email: [email protected]

Published online 4 September 2012 inWiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.22117

© 2012 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 81A: 843�855, 2012

Treg cells constitutively express surface proteins like CD25,

CD45RO, CTLA-4, HLA-DR, or GITR (see Table 1 in Sup-

porting Information) (2,12), but these markers are neither

present in 100% of Tregs or exclusive of this cell lineage. Three

examples can illustrate this point: CD25 (IL-2Ra), FoxP3, and

CD127 (IL-7Ra).

The majority of human Treg cells strongly and constitu-

tively express CD25 (CD25high). However, conventional/effec-

tor T cells (2,12,13) and a portion of CCR71 central memory

T lymphocytes start expressing CD25 upon TCR-mediated

activation (14). Therefore, even highly pure CD41CD25high

Treg populations may contain a significant fraction of proin-

flammatory Teff cells (15). On the other hand, FoxP3 is the

most specific Treg marker (2). Despite this fact, FoxP3 still

shows a transitory and activation-dependent expression in

CD41CD252 Teff cells, which together with its intracellular

nature disqualifies this marker for Treg identification and,

especially, isolation purposes (16). CD127 is another protein

whose levels inversely correlate with FoxP3 expression in Treg

lymphocytes (17,18). Nevertheless, the mere existence of an

underlying disease (e.g., HIV infection) (19) or the in vitro

activation (20) cause an intense CD127 down modulation on

formerly CD1271 Teff cells. Other alternative markers have

arisen more recently. Markus Kleinewietfeld et al. (21) have

reported CD49d (a chain of VLA-4 integrin) as expressed in

most of IFN-c or IL-17-producing proinflammatory T cells

but reduced on Tregs, even though data from the same group

(22) as well as other researchers (23) still reflect some degree of

CD49d expression in some subsets of Treg cells. Mandapathil

et al. (24) have focused on CD39 as a positive selection marker.

This ectoenzyme catalyzes the generation of AMP from ATP,

which is necessary to produce adenosine, an important media-

tor of active suppression (22). Fifty to 90% of CD41CD391 T

lymphocytes are FoxP31 and express low levels of CD127 (24).

However, activated T cells upregulate CD39 (25), and a novel

population of human CD41CD391FoxP32 T cells that produce

IFN-c and IL-17 has been found (26). Thus, it seems that more

phenotypic studies on Treg cells are still necessary (27).

CD26 is a serine protease with dipeptidyl peptidase IV

(DPPIV) activity (28). Activated/memory T cells display a

CD26high phenotype, and TH1 cytokines like IL-12 raise the

number of CD26 molecules on T lymphocytes (28,29). Thus,

high surface levels of this protease are an indication of, at least,

TH1 effector responses (28,29). Ligation of CD26 leads to acti-

vation of signal transduction proteins (e.g., ERK, p56lck,

CD3f, ZAP-70, CARMA-1/NF-jB), cell proliferation, and

cytokine (IL-2, IFN-c) production (28,30). Both cis-interac-

tion of CD26 with CD45 (31,32) and trans-association of

CD26 (T cell) with caveolin-1 (APCs) (33) seem important

for CD26 functions. However, despite the large amount of

data supporting the costimulatory role of CD26 in T cells, an

extensive research on CD26 levels in Treg cells has not been

undertaken. The present work shows that CD26 is present on

FoxP3-expressing activated CD41 Teff cells, but reduced or

absent from Tregs. This CD262/low phenotype is stable, and

therefore useful to differentiate these two antagonistic CD41

T cell subsets.

MATERIALS AND METHODS

Materials

Phytohemagglutinin (PHA; catalog no. L1668), parafor-

maldehyde (PFA; catalog no. P-6148), penicillin-streptomycin

solution (catalog no. P0781), 5(6)-Carboxyfluorescein diace-

tate N-succinimidyl ester (CFSE; catalog no. 21888-25MG-F),

and RPMI-1640 culture medium (catalog no. R4130) were

obtained from Sigma-Aldrich (Spain), fetal bovine serum

(FBS; catalog no. DE14-801F) from Biowhittaker (Lonza Iber-

ica, Spain), and Ficoll-Paque PLUS (catalog no. 17-1440-03)

from GE Healthcare (Spain). Mouse antibodies against human

molecules CD127 (CD127-PE, catalog no. 557938, and CD127

Alexa Fluor 647, catalog no. 558598; both IgG1 clone hIL-7R-

M21), CD25 (CD25-FITC, catalog no. 555431, and CD25-

APC, catalog no. 555434; both IgG1, clone M-A251), CD39

(CD39-FITC, clone TU66, catalog no. 561444), CD49d

(CD49d-APC, IgG1, clone 9F10, catalog no. 559881), FoxP3

(anti-FoxP3-PE, IgG1, clone 259D/C7, catalog no. 560046),

and IFN-c (anti-IFN-c-PE, IgG1, clone B27, catalog no.

554701), as well as the mouse isotypic control IgG1-APC

(clone MOPC-21, catalog no. 555751) and the Alexa Fluor

647 mouse IgG1 isotype control (clone MOPC-21, catalog no.

557714), were from BD Pharmingen (BD Biosciences, Spain).

Mouse antibodies against human CD4 (CD4-PerCP, IgG1,

clone SK3, catalog no. 345770) and CD26 (CD26-FITC,

IgG2a, clone L272, catalog no. 340426) were purchased from

BD Immunocytometry Systems (BD Biosciences). Mouse

mAb against human CD26 (TP1/16) was purified from a

hybridome supernatant and used pure (in combination or not

with APC goat anti-mouse Ig; catalog no. 550826, BD Phar-

mingen) or FITC-labeled (Fluorotag FITC Conjugation Kit,

catalog no. FITC1-1KT; Sigma). Murine mAb against human

CD26 clone TP1/19 (CD26-FITC or APC, IgG2b, catalog no.

26F-100T or 26A-100T) was provided by Immunostep (Sala-

manca, Spain). Isotypic controls IgG1-FITC (clone MOPC-21;

catalog no. F6397) and IgG1-PE (clone MOPC-21; catalog no.

P4685) were purchased to Sigma. Buffers used for intracellular

staining of FoxP3 and IFN-c were BD Pharmingen stain buffer

(catalog no. 554656), BD FACSTM lysing buffer (catalog no.

349202), Human FoxP3 buffer set (catalog no. 560098), BD

Cytofix/Cytoperm bufferTM Plus (catalog no. 555028), and BD

Perm/WashTM (catalog no. 554723), all from BD Biosciences.

Methods

PBMCs purification and culture conditions. Human buffy

coats from healthy donors were kindly provided by Centro de

Transfusiones de Galicia (Santiago de Compostela, Spain).

Once informed consent for the donation was obtained, blood

samples (healthy subjects) were drawn into EDTA/K2E or LH/

170 IU tubes (BD vacutainer; BD Biosciences; catalog no.

367525 and 367526, respectively) via sterile venipuncture

(Medical Service at the University of Santiago) according to

the ethics committee guidelines. Peripheral blood mononu-

clear cells (PBMCs) were isolated from either buffy coats or

anticoagulant treated blood samples using Ficoll1 density gra-

dient centrifugation, as previously described (29,32). PBMCs

ORIGINAL ARTICLE

844 CD26 as a Negative Treg Selection Marker

were either used directly or in vitro cultured (378C, 5% CO2

humidified atmosphere). RPMI-1640 growth medium was

supplemented with 10% FBS, 100 lg/ml streptomycin and 100

UI/ml penicillin (complete medium). Stimulation of PBMCs

was performed by using 1 to 2 lg/ml PHA (phytohemaggluti-

nin from Phaseolus vulgaris) for 5 days in either 6/24-well

plates or 75/150 cm2 flasks. Alternatively, PBMCs were acti-

vated with beads coated with monoclonal antibodies against

CD2/CD3/CD28 (Treg suppression inspector human, Miltenyi

Biotec, Auburn, CA; 0.5–1 bead per lymphocyte; catalog no.

130-092-909). In IFN-c assays, culture medium was supple-

mented with GolgiPlugTM (BD Biosciences; 1/1,000 dilution)

during the last 4 h of incubation before immunostaining.

Human Treg purification. Viable (>90%; trypan blue exclu-

sion assay) Treg cells were isolated from either resting or acti-

vated PBMCs by two different magnetic methods. The first

one (Dynabeads1 Regulatory CD41CD251 T cell Kit; Life-

Technologies, Spain; catalog no. 113.63D) was a ‘‘classical’’

procedure. During the first step, CD41 T cells were enriched

by negative selection by means of a cocktail of monoclonal

antibodies (Antibody Mix Human CD4; mouse IgG antibodies

against CD14, CD16a, CD16b, CD56, CDw123, CD36, CD8,

CD19 and glycophorin A) and anti-mouse IgG Abs linked to

super paramagnetic polystyrene beads (Depletion MyOneTM

Dynabeads). Afterwards, both CD41CD252 Teff and CD25high

Treg cells were purified from CD41 T cells by means of beads

coated with antibodies against CD25 (Dynabeads CD25).

Finally, magnetic beads were removed from Treg cells (DETA-

CHaBEAD1 buffer). Cell recovery was calculated as follows:

(number of cells within the Treg enriched fraction 3 Treg pu-

rity)/(starting cell number 3 starting Treg percentage).

The second approach was the Human CD41

CD127lowCD49d2 Regulatory T Cell Enrichment Kit (Stem-

Cell Technologies, Grenoble, France; catalog no. 19232). This

fully negative selection protocol uses CD127 and CD49d to

isolate the Treg population from a PBMCs suspension. We

have followed strictly the manufacturer’s manual (EasySepTM

protocol) in combination with the EasySep magnet (for stand-

ard 5 ml polystyrene tubes; catalog no. 18000). Despite this

fact, a low Treg viability was always observed.

Flow cytometry assays. All the incubations during immuno-

fluorescence protocols were performed in the dark, and

unstained cells, isotype controls, and single fluorochrome

stained cells were used to set-up the flow cytometer. In all

cases, recommended concentrations were employed for all

monoclonal antibodies. In both proliferation and immunoflu-

orescence experiments, sample acquisition (typically 20,000 to

200,000 events) was performed on a FACScalibur Flow Cyto-

metry System (BD Biosciences), a 2-laser, 4-color flow cytom-

eter used for clinical samples at the USC University Hospital

Complex (CHUS). This instrument has the following laser-

fluorochrome combinations: (1) 488 nm blue laser—fluores-

cein isothiocyanate (FITC), 5(6)-carboxyfluorescein diacetate

N-succinimidyl ester (CFSE), phycoerythrin (PE), and peridi-

nin-chlorophyll-protein complex (PerCP); (2) 635-nm red-

diode laser—allophycocyanin (APC) and AlexaFluor 647. We

used WinMDI software (Dr. Joe Trotter, The Scripps Institute,

Flow Cytometry Core Facility) for data analysis.

For CFSE-based proliferation studies, PBMCs (RPMI me-

dium, 10 3 106 cells/ml) were firstly incubated with 5 mM

CFSE for 8 min (RT) in the dark. To block the reaction, an

equal volume of FBS was added and cells were thoroughly

washed before counting. Initial cell density was 50,000 cells/

well (0.25 3 106 PBMC/ml; 96-round bottom well micro-

plates), and 2 lg/ml PHA was used as mitogen (34). Each ex-

perimental condition was tested nine times. At the end of the

incubation period (5 days), wells were pooled to generate sin-

gle triplicates. Cells were washed with PBS pH 7.4, and indir-

ectly labeled with either isotype antibody or a purified mAb

against CD26 (TP1/16) plus GAM-APC. Unlabeled (CFSE2)

cells served as negative controls, while unstimulated CFSE-la-

beled PBMCs allowed us to identify those lymphoblasts that

did not divide.

For the multicolor extracellular staining of unfractionated

leukocytes, either isotype (isotype-FITC, isotype-PE, isotype-

APC) or specific (CD26-TP1/19-FITC, CD127-PE, CD4-

PerCP, or CD25-APC) monoclonal antibodies were added to

test tubes. Hundred microliters of EDTA anticoagulated whole

blood was mixed with antibodies by gentle vortexing and left

for 30 min (RT). Afterwards, erythrocytes were lysated (2 ml

13 BD FACS lysing solution/test tube; 15 min, RT), and sam-

ples centrifuged (200g, 5 min, RT), washed with 2 ml of PBS

pH 7.4, 1% FBS, 0.1% sodium azide (washing solution), and

fixed with 2 ml ice-cold 2% PFA in PBS pH 7.4 (30 min, RT).

Finally, cells were washed again and resuspended in 1 ml of

washing solution.

During the simultaneous detection of extracellular (CD4,

CD25, CD26, CD49d, CD39) and intracellular (FoxP3) pro-

teins in either resting or PHA-activated PBMCs, samples were

washed with BD Pharmingen Stain Buffer, counted (hemocyt-

ometer) and adjusted to 10 3 106 cells/ml. For extracellular

labeling, adequate volumes of isotype (isotype-FITC and iso-

type-APC) or specific antibodies (CD26-FITC TP1/16, CD4-

PerCP, and either CD25-APC or CD49d-APC) were placed

inside flow cytometry tubes and 1 3 106 cells (100 ll) added.

In some experiments (Fig. 5), samples were simultaneously

stained with CD49d-APC and CD127 Alexa Fluor 647 (same

fluorescence channel). After incubation (20 min, RT), cells

were washed (2 ml of BD Pharmingen Stain Buffer per tube)

and centrifuged at 250g 10 min RT. Then, cell pellet was fixed

with 13 Human FoxP3 buffer A (2 ml/tube; 10 min at RT),

centrifuged again (500g, 5 min, RT) and the wash step

repeated. Fixed and extracellularly labeled cells were permeab-

ilized with 13 Human FoxP3 buffer C (0.5 ml/tube) for 30

min at RT. After washing, permeabilized cells were incubated

(30 min, RT) with isotype-PE or anti-FoxP3-PE. Cells were

washed again, centrifuged (500g, 5 min, RT) and fixed (1 ml,

2% PFA) during 30 min at RT. Finally, leukocytes were resus-

pended in 1 ml of BD Pharmingen Stain Buffer per tube.

We used a similar protocol to evaluate the percentage

of IFN-c-producing T lymphoblasts. In brief, GolgiPlugTM

ORIGINAL ARTICLE

Cytometry Part A � 81A: 843�855, 2012 845

treated lymphoblasts were also extracellularly labeled with

diverse combinations of isotypic (FITC- and APC-labeled) or

specific (CD26-FITC TP1/16, CD25-FITC, CD4-PerCP, and

CD49d-APC or CD25-APC) mAbs. Once washed, cells were

fixed and permeabilized (20 min, 48C) with 250 ll of BD

Cytofix/Cytoperm buffer, washed two times with 1 ml of 13

BD Perm/WashTM and incubated with anti-IFN-c-PE or iso-

type-PE in 50 ll of 1X BD Perm/Wash (30 min, 48C). Finally,

leukocytes were washed twice (1 ml/each, 1X BD Perm/Wash)

and resuspended in BD Pharmingen Stain Buffer (1 ml).

In Vitro Suppression Assays

Human PBMCs were activated with 2 lg/ml PHA for 5

days to generate T lymphoblasts. To favor Treg cell prolifera-

tion (2- to 10-fold), culture medium was also supplemented

(day 3) with 200 IU/ml IL-2 (Peprotech). CD41CD25high

and CD41CD25highCD262/low Treg subsets were purified

from T lymphoblasts (Dynabeads1 Regulatory CD41CD251

T cell Kit; Life-Technologies, Spain) and tested for suppres-

sion in co-cultures with autologous CFSE-labeled PBMCs as

responder cells (1 3 105/well; 250 ll/well). The suppression

assay was performed in round-bottom 96-well plates with a

dilution series ranging from 1:1 to 4:1 of responder cells:Treg

cells. To induce proliferation, responder cells were stimulated

with beads coated with mAbs against CD2/CD3/CD28 (Treg

suppression inspector human, Miltenyi Biotec, Auburn, CA;

1 bead per cell) in the presence of 200 IU/mL IL-2 for 4 to 5

days. Additionally, responder cells and Treg cells were cul-

tured separately, either with or without beads/IL-2. Measure-

ments were always carried out in triplicate. Potency of

suppression was calculated at 1:1 ratio as [1-(proliferation

of Treg:responders coculture/proliferation of responders

alone)].

Statistics

P values were calculated by the nonparametric Mann–

Whitney U-test using IBM SPSS statistics 19 software. P values

=0.05 were considered to be significant.

RESULTS

As previously reported (29), resting lymphocytes express

low levels of CD26. However, CD26 is upregulated during cell

activation (plateau phase at 4–6 days; Fig. 1A), and CD26

levels are positively correlated with cell size (forward scatter/

FSC measurements) (Fig. 1A). CD26 up regulation takes place

especially amongst CD4 T-cells (Fig. 1B), and actively dividing

T lymphocytes display higher numbers of CD26 molecules as

they progress through new division rounds (Fig. 1C, 5-days

PHA blasts, [90% enriched in CD31 T cells; see CD26 geo-

mean values plotted against the number of cell divisions in

line graph). Moreover, as the percentage of IFN-c1 secreting

CD41 T cells that also stained for CD26 after 5 days of stimu-

lation with PHA is very high (81.5 � 15.7%; n 5 5 donors),

this extracellular peptidase allows tracing IFN-c-producing

TH1 cells. Therefore, CD26 can be used, at least, as an activa-

tion/TH1 marker.

Treg cells display an ‘‘activated-like’’ (CD41CD25high

CD45RO1) phenotype. Consequently, we set out to determine

whether Treg cells at the steady-state expressed CD26. About

5.3 � 2.2% (n 5 10; data not shown) of resting CD41 T lym-

phocytes are CD251 (2,12), but only those displaying the

highest expression of CD25 (CD25high, �1–2% CD41 cells)

are actual Treg cells (Fig. 2) (2). In contrast, CD41 popula-

tions with intermediate levels of CD25 (CD25low) contain a

mixture of Teff and immature Treg lymphocytes (2). This fact,

together with the continuous expression of CD25, makes the

boundary for CD25high Treg populations not exempt of arbi-

trariness (10). However, we observed that CD41CD25high Treg

lymphocytes (R2 gate) were the subset with the lowest per-

centage of positive cells and the most reduced molecular den-

sity for both CD26 and CD127 antigens (Fig. 2). In contrast,

high levels of CD127 and CD26 were found amongst

CD41CD252 Teff cells (R4 gate), whereas CD41 lymphocytes

expressing intermediate densities of CD25 (R3 gate) consisted

of regulatory (CD262/lowCD1272/low) and effector

(CD261CD1271) populations (more clearly observed for

CD127). We confirmed these results with three different CD26

mAbs clones (TP1/19, TP1/16, and L272), pointing out that

this result does not depend on a particular epitope. Thus,

when CD26 levels were measured with the TP1/16 mAb

(�20% stronger staining than the TP1/19 mAb in Fig. 2), the

following results were obtained: 68.3 � 12.2% of CD41CD25high

T cells, 76.9 � 11% of CD41CD251, and 91.4 � 5.3% of

CD41CD252 T cells were CD261 (n 5 10). In addition, intra-

cellular staining of CD41CD25high Treg cells also revealed low

levels of CD26; i.e., there is not internal pool (not shown).

FoxP3 is still the preferable marker to accurately identify

the Treg subset, even though the existence of nonregulatory

FoxP3low T cells in normal individuals also precludes the use

of FoxP3 as a sole marker for Treg cells (2). For that reason,

cell size (FSC), CD25, CD127, and especially, CD26 levels,

were evaluated in resting CD41FoxP3high (R2), CD41FoxP3low

(R3), and CD41FoxP32 (R4) T cells from healthy individuals

(Fig. 3A). Our results clearly showed that, compared with

CD41FoxP32/low lymphocytes (R3 and R4), CD41FoxP3high

Treg cells (R2) were mainly CD25high leukocytes with an interme-

diate size and a dim expression of CD4, CD127, and CD26

(82.2–85.2% vs. 46.5% for CD127; 82.9–85.2% vs. 39.4% for

CD26). Thus, our findings suggest that CD262/low levels are

linked to Treg cells in resting lymphocyte populations, while high

CD26 levels can be found in CD41 subsets with effector func-

tions, which is in line with data from Mandapathil et al. (24).

However, both CD25 and CD26 antigens also reflect the

cell activation status. Consequently, we wanted to exclude the

possibility of blurry limits between different T helper subsets

for CD26 levels because of cell activation. Thus, data in resting

PBMCs were compared with those obtained after subjecting

the same cells to 5 days of in vitro polyclonal activation (Fig

3B). As expected, the nonspecific mitogenic stimuli (PHA)

caused an intense FoxP3 up regulation in both CD41 and

CD81 T cells (16), as well as augmented levels of CD25 within

the CD41FoxP32/low (R3 and R4) and, especially, the

CD41FoxP3high Treg subset (R2 gate) (14). On the contrary,

ORIGINAL ARTICLE


the percentage of CD1271 cells (but not the fluorescence in-

tensity value) was diminished (Fig. 3B) in all CD41 T subsets

(R2-R4) (19,20). Therefore, our data support the notion that

there is not a clear boundary between Treg and Teff lympho-

cytes under activating conditions when selection is exclusively

based on extracellular markers such as CD25 or CD127.

Figure 1. CD26 is an activation marker. (A) Human PBMCs, cultured and activated with 2 lg/ml PHA for 5 days in 24-well plates (0.25 3 106

cells/ml), were harvested and immunostained with CD4(SK3)-PerCP and CD26(TP1/16)-FITC. During flow cytometry data acquisition

(20,000 events) and analysis, lymphocytes were gated (R1; not shown) according to their size (FSC) and complexity (SSC), three subsets

differentiated based on cell size (small, medium, big), and CD26 levels detected in each one of these subpopulations. The discontinuous

line indicates the CD26 expression in resting lymphocytes (day 0). (B) CD26 levels in CD41 and CD42 populations from resting (day 0) and

PHA-activated lymphocytes (day 5). (C) CFSE-labeled PBMCs, seeded at 0.25 3 106 cells/ml in 96-round well microplates, were either kept

unstimulated (upper left dot plot) or activated during 5 days with 2 lg/ml PHA (upper right dot plot). Then, cells were indirectly labeledwith isotype control antibody or mAb against CD26 (TP1/16) plus GAM-APC, and CD26 expression (percentage and geomean) plotted

against the number of cell divisions of gated lymphocytes (FSC vs. SSC plot; R1 gate, not shown). Results in (A), (B) and (C) belong to rep-

resentative experiments. Data in (A) and (C) are shown as mean � standard deviation (SD) of triplicate measurements.

ORIGINAL ARTICLE


In clear contrast, steady state levels of CD26 were only

slightly upregulated (see fluorescence intensity data) by in

vitro stimulation in all CD41 T subsets (Fig 3B), which likely

reflects that augmented levels of CD26 are mainly confined to

the CD42 cell subset (Fig. 1B). More important, different

levels of CD26 expression between FoxP32/low (Teff) and Fox-

P3high (Treg) subsets were kept or even magnified after in vitro

activation. Similar data were obtained with CD2/CD3/CD28

beads, or when the TH1-polarizing cytokine IL-12 was used as

costimulus (not shown). Thus, our results collectively demon-

strate that CD26 could be used as additional criterion, in con-

junction with CD25 (or FoxP3) and CD127, to distinguish

Teff from Treg in both resting and activated populations.

Moreover, Supporting Information Figure S1 provides an

example where either CD4, CD25, CD26, or cell size (FSC)

were used with FoxP3 to segregate both T subsets. As shown,

the boundary between both FoxP32/low and FoxP3high cells

was blurred in resting CD41 T lymphocytes, when just CD4

and FoxP3 levels were considered, whereas the most polarized

FoxP3 levels in activated CD41 T cells allowed a better discri-

mination. However, the use of complementary Treg features

(e.g., CD25high, CD262/low, or FSCintermediate), in conjunction

with CD4 and FoxP3, significantly improved the selection of

this subset.

Other currently used Treg selection markers are CD49d (achain of the VLA-4 integrin) (21) and CD39 (22). Expression of

CD25 and CD26 antigens was compared with CD49d levels on

Treg cells (CD41FoxP3high; R2) and Teff lymphocytes

(FoxP32/low; R3) under resting (not shown) and activating con-

ditions (Fig. 4A). We found marked differences for CD25 and

CD26 antigens, but not for CD49d. Consequently, our results

fit with previously published data reporting the presence

of CD49dhigh subsets among Treg cells (22,23). Of note, CD26

also was better than CD49d as negative selection criterion to

Figure 2. Resting CD41CD25high Treg cells display a low or null expression of CD26. Human whole blood was directly labeled with the fol-

lowing fluorescent monoclonal antibodies: CD26(TP1/19)-FITC, CD127(hIL-7R-M21)-PE, CD4(SK3)-PerCP, and CD25(M-A251)-APC. Subse-

quently, erythrocytes were lysated and samples washed and fixed before acquisition (200,000 events). During data analysis, lymphocytes

were gated (R1) according to their forward (FSC) and side (SSC) scatter properties, and three subsets (R2, R3, and R4) selected based on

CD4 and CD25 levels. In each one, extracellular expression of both CD26 and CD127 markers was measured as percentage of positive cells

(%) and mean fluorescence intensity (geometric mean, Gm). CV, coefficient of variation. Md, median. One representative experiment out

of 10 performed is shown.

ORIGINAL ARTICLE


discriminate IFN-c-producing cells within the CD41CD25high

compartment in PHA-activated PBMCs (Supporting Information

Fig. S2).

Unlike CD49d, CD39 is a marker with a clearly divergent

expression pattern for Treg (R2; �72% CD391) and Teff (R3;

�3% CD391) cells cultured under activating conditions

(Fig. 4B), but not at the steady state (data not shown; Treg,

�3% CD391; Teff �0.1% CD391). Moreover, CD39 is linked

to adenosine production (22), an immunosuppressive molecule

whose accumulation is also favored by the CD262/low phenotype

of activated CD391 Treg cells (Fig 4B), since the CD26 protease

is the main adenosine deaminase (ADA) binding protein (28).

Figure 3. Phenotypic stability of CD41CD25highCD262/low Treg cells upon activation. PBMCs were isolated by Ficoll density gradient

separation, and either directly labeled (resting, day 0) (A) or activated in complete medium supplemented with 1 lg/ml PHA before immu-nofluorescent labeling with antibodies (activated, 5 days) (B). In both cases, cells were initially incubated with the following mAbs: CD26-

FITC (clone TP1/16), CD4-PerCP (clone SK3), and either CD25-APC (clone M-A251) or CD127-Alexa Fluor 647 (clone hIL-7R-M21). Then, cells

were fixed/permeabilized and stained with anti-FoxP3-PE (clone 259D/C7). Background values were set with the corresponding isotype

mAbs. During data analysis (WinMDI), the lymphocyte population was selected according to its cell size (FSC) and complexity (SSC) (R1).

Three subpopulations of CD41 T cells were identified as a function of its FoxP3 expression (R2, CD41FoxP3high Treg; R3, CD41FoxP3low;

R4, CD41FoxP32), and the level of CD25, CD127, and CD26 as well as the cell size (FSC) quantified afterwards. Numbers on top of histo-

grams represent the percentage of positive cells for each marker (%) as well as the corresponding intensity of fluorescence (geomean; in

brackets). Data belong to one representative experiment out of four performed.

ORIGINAL ARTICLE


According to Kleinewietfeld et al. (21), antibodies against

CD49d and CD127 alone are sufficient to isolate ‘‘untouched’’

FoxP31 Treg cells free of contaminating CD251 Teff cells. To

determine whether the combined use of these two markers is

enough to accurately discriminate Treg cells from activated

Teff cells, ‘‘untouched’’ CD41CD127lowCD49d2 Treg lympho-

cytes were purified from resting or in vitro expanded PBMCs

using the EasySepTM Human CD41CD127lowCD49d2 Regula-

tory T Cell Enrichment Kit (StemCell Technologies). Treg cells

isolated from resting PBMCs were a homogeneous population

(see CD4 and FoxP3 levels), mostly negative/low for CD127

and CD49d (as expected) and with a homogeneous CD262/low

phenotype (see R2 and R3 gates in Fig. 5A). On the contrary,

the CD41CD127lowCD49d2 cell fraction purified from acti-

vated PBMCs was heterogeneous, whether regarding the level

of CD4 and FoxP3 molecules or according to CD26 levels (see

Figure 4. Levels of CD26, CD49d and CD39 antigens in Treg (FoxP3high) and Teff (FoxP3low) lymphocytes. PBMCs were purified and

expanded with PHA during 5 days before inmunofluorescent labeling. Cells were extracellularly stained with CD4-PerCP (cloneSK3) and

different combinations of CD26-FITC (clone TP1/16), CD39-FITC (clone TU66), CD25-APC (clone M-A251), CD26-APC (clone TP1/19), and

CD49d-APC (clone 9F10) mAbs. After that, cells were fixed/permeabilized and stained with anti-FoxP3-PE (clone 259D/C7). During analysis,

lymphocytes were gated (size vs. complexity plots; R1) and two subpopulations (R2 and R3) distinguished based on CD4 and FoxP3

expression: CD41FoxP31/high Treg cells (R2; depicted as grey color in A) and CD41FoxP3low/2 Teff lymphocytes (R3; depicted as black color

in A). Finally, either plasma membrane levels of CD25, CD26, and CD49d, each one in combination with FoxP3 (A), or both CD39 and CD26

antigens (B), were evaluated. The figure shows one representative experiment out of three performed.

ORIGINAL ARTICLE


R2 and R3 gates in Fig. 5B). This heterogeneity was caused by

a preferential isolation of a FoxP3low subset (R3), which dis-

plays a higher CD26 expression than the FoxP3high subset (R2)

(Fig. 5B). Due to the activation-dependent reduction of

CD1271 cells within the Teff subset (Fig. 3), we speculate that

this FoxP3lowCD261 phenotype (R3) may be the signature of

T cells with unstable FoxP3 expression, activated-memory

phenotype, and the capability of producing IL-2, IFN-c or

IL-17 (35,36).

According to our findings, inclusion of CD26-specific

mAbs in Treg purification protocols may help to deplete con-

taminating FoxP3low T cells and allow the isolation of highly

enriched FoxP3high cells, especially from previously expanded

‘‘bulk’’ T-cell populations or samples from inflammatory/

autoimmune diseases. Most of commercially available proto-

cols are based on the CD41CD25high phenotype. According to

our data in resting T lymphocytes, 81.9 � 11.2% (n 5 10) of

CD41CD251 T cells were CD262. Thus, unlike the use of

CD45RA to isolate ‘‘naive’’ CD41CD25high Treg cells (24–61%

of resting Treg cells) (37), the CD26 strategy should yield

higher Treg numbers (38). As a first approach, we determined

by means of a pilot flow cytometry experiment whether the

strategy of selecting the CD41CD25highCD262/low yielded

more homogeneous Treg populations (according to cell size

and FoxP3; Supporting Information Fig. S3) than the usual

CD41CD25high phenotype. We used a broader gating strategy

and a lower than usual CD25 boundary. CD41CD252 T cells

were also gated for comparison, revealing that �95% of them

(R4 gate) were FoxP32/low cells. However, FoxP3low cells

(40–60%) were also detected amongst the CD41CD25high

Treg lymphocytes (R2 1 R3 gates). In contrast, the use of a

CD41CD25highCD262 phenotype (R2 gate) to select the Treg

subset yielded a more homogeneous population, as the per-

centage of FoxP3high cells reveals (in Supporting Information

Fig. S3, 43%?61% for resting PBMCs, and 59?75% for acti-

vated PBMCs).

Given these preliminary data, both CD41CD25high Treg

and CD41CD252 Teff lymphocytes were isolated from either

freshly isolated or PHA-expanded PBMCs by means of the

Dynabeads1 Regulatory CD41CD251 T cell Kit (Life Tech-

nologies/Invitrogen). As expected, the starting percentage of

Treg cells amongst resting PBMCs was low (0.84 � 0.37%; n

5 6), recovering �86% (n 5 3) of these Treg cells as

CD41CD25high fractions with a cell purity of 87% (assessed by

CD25 expression; n 5 3). In contrast, we have detected a

slightly higher starting percentage of CD41CD25high cells

amongst PHA-activated PBMCs (3.34 � 2.08%, n 5 6). Only

68% (n 5 7) of these lymphocytes was collected within

CD41CD25high fractions. Moreover, the percentage of positive

cells for CD25 and FoxP3 was, respectively, 83.6 � 5.14% and

63.63 � 9.12% (n 5 3), which can be explained because of the

presence of activated CD251 Teff cells.

To improve the purity of Treg cells obtained from PHA-

expanded PBMCs, we partially removed CD261 cells from

CD41 T lymphocytes by means of the addition of TP1/16

mAb to the cocktail of mouse IgG mAbs during the first step.

Consequently, a significant decrease in the number of CD41

Figure 5. The combined use of CD127 and CD49d does not avoid

the presence of FoxP3lowCD26high T cells within Treg populations

isolated from activated PBMCs. Regulatory T cells were purified

from resting (A) or activated PBMCs (1 lg/ml PHA, 5 days) usingthe EasySepTM Human CD41CD127lowCD49d2 Regulatory T Cell

Enrichment Kit (StemCell Technologies). Treg cells were stained

with CD26-FITC (clone TP1/16), anti-FoxP3-PE (clone 259D/C7),

CD4-PerCP (clone SK3), and CD49d-APC/CD127-AlexaFluor 647

(Clone 9F10/hIL-7R-M21) mAbs. During analysis (A and B), Treg

lymphocytes were gated (forward vs. side scatter; R1) and two

subpopulations (R2 and R3) distinguished based on the expres-

sion of CD4 and FoxP3: CD41FoxP3high (R2) and CD41FoxP3low

(R3). Finally, both CD26 and CD127/CD49d levels were evaluated

for R2 and R3 in new density plots, reflecting that Treg lympho-

cytes purified from PBMCs (A) are a more homogeneous popula-

tion as compared with the Treg population isolated from activated

PBMCs (B). A high mortality rate has been constantly observed

for Treg cells purified with this protocol (see forward vs. side scat-

ter plot is in A and B). One representative experiment out of three

performed is shown.

ORIGINAL ARTICLE


T cells was noted: 27.39 � 8.93 ?18.67 � 5.53 (3 106) CD41

T cells, 28.84 � 5.47% reduction (n 5 7). Once purification

was completed, flow cytometric analysis revealed a low expres-

sion of CD26 in CD41CD252 Teff cells and a bimodal pattern

for the CD41CD25high Treg subset, which comprises CD262/

low and CD26high subtypes (Fig. 6A). The use of mAbs against

CD26 caused an almost full depletion of CD261 cells in

CD41CD252 Teffs, but only a partial decline amongst

CD41CD25high Tregs (�40%; Figs. 6B and 6C). However, de-

spite this incomplete CD26 depletion, the percentage of positive

cells for CD25 (83.6 � 5.14% ? 91.2 � 5.05%; n 5 3) and

FoxP3 (63.63 � 9.12% ? 75.33 � 10.91%; n 5 3) (Fig. 6C)

was raised, as well as the percentage of CD25highFoxP3high cells

(e.g., from 66.8 to 84.5% in Figs. 6A and 6B). Moreover, there

was a significant rise in the number of CD1271 Treg cells after

CD26-depletion (8.9 � 9.2% CD1271 Treg ? 29.7 � 0.8%

CD1271 Treg, n 5 2 donors), which fits with a higher

suppressive activity (Fig. 6D; 8.29 � 2.17% ? 17.98 � 1.93%).

DISCUSSION

CD26 is an activation/memory marker capable of trans-

mitting costimulatory signals (28,30). Thus, interaction

between CD26 (T cell) and caveolin-1 (APCs) leads to NF-jB

activation and recruitment of CARMA1/Bcl10/IjB (28,33), a

complex implied in thymic development of Treg cells (39),

while blockade of this costimulatory interaction causes T cell

cycle arrest (40,41). Nevertheless, CD26 may also have inhibi-

tory functions. For example, disease severity is increased in

CD262/2 mice in certain autoimmune models (42,43), with

augmented T-cell proliferation rates (43) and production of

TH1 cytokines (IFN-c, TNF-a) (42,43) but diminished TGF-b1

synthesis (43). Indeed, several inhibitory peptides or naturally

occurring ligands of CD26/DPPIV promote the secretion of

TGF-b1 (43-45). Therefore, all these findings point to some

kind of connection between CD26 and the Treg phenotype

that deserves to be ascertained, especially because Treg lym-

phocytes, with an ‘‘activated-like’’ (e.g., CD25high, HLA-DR1)

and ‘‘memory’’ (CD45RO1) phenotype, were expected to be

CD261 (46,47) but not CD262/low cells. However, our results

are in agreement with the CD262/low profile of Treg-like leu-

kocytes in classical Hodgkin’s lymphoma (48). Moreover, they

fit with recent studies in human Treg cells (24), the latest in

April this year (49), where authors describe variable CD26

levels in different TH subsets: TH17[TH1[TH2[Treg cells.

Nevertheless, our data go beyond, as they show that this

CD262/low phenotype is stable upon in vitro T-cell expansion

and can be used during the isolation/identification of Treg

cells. In addition, this CD262/low phenotype may explain why

the TH1 cytokine IL-12 causes a strong upregulation of CD26

on ‘‘bulk’’ T-cell cultures, whereas IL-2 (CD25-ligand) induces

only a slight increase (29). Still, several questions remain

unanswered: for example, whether this CD262/low phenotype

is based on differential transcription/translation, shedding, or

exosomes/microvesicles releasing processes, whether CD26

level on Tregs depends on their tissue localization (50), or how

the various Treg/Teff cells ratios found in different diseases

correlate with levels of circulating sCD26 (51).

Researchers can only get high numbers of Treg cells by

means of tedious two-step magnetic procedures based on

CD25 (35-38). However, CD25 is also expressed by FoxP3low T

cells, a group of lymphocytes that might fit with naıve or

uncommitted Tregs but also activated Teff cells (2,37,52,53).

For that reason, more efficient technologies have been placed

on the research market. Nevertheless, these new protocols may

not be entirely effective yet, as our article points out. Thus,

negative selection antigens, like CD127 (17,18) or CD49d

(21), certainly provide an additional way of ascertaining the

right phenotype. Nevertheless, this does not seem to be suffi-

cient because of the intense reduction in CD1271 cells upon

TH-cell activation (19,20,54, our data), the presence of

CD127high subsets within activated Treg cells (50, our data), or

the small differences for CD49d between FoxP3high and

FoxP32/low cells (our data).

The limited amount of biological samples or the low

abundance of Treg cells in peripheral blood are additional pro-

blems. On this regard, several protocols have been developed

for the in vitro expansion of Treg cells (35-38), but many of

them are associated with potential risks: e.g., the in vitro out-

growth of contaminating Teff cells or the possibility of

‘‘plastic’’ Treg cells reverting to FoxP3low Teff cells (2,35,37).

To circumvent these problems, CD45RA have been promoted

as a discriminating marker to identify a subset of naive

CD41CD25high Treg cells that maintain FoxP3 expression dur-

ing in vitro culture (37). However, it remains to be determined

if there is a sufficient number of such ‘‘naive’’ Treg cells in

blood samples of patients for in vitro expansion and autolo-

gous adoptive transfer (37), especially considering the age-de-

pendent differentiation from naive to effector memory Treg

cells (55). Moreover, it is known that even CD45RAhigh Treg

cells can give rise to nonregulatory T cells when cultured in a

milieu containing high-dose IL-2 (2,35,37). In clear contrast,

our study suggests using a different strategy. This new

approach consist in the in vitro expansion of bulk lympho-

cytes before isolating the Treg subset on the basis of a more

reliable Treg/Teff differentiation marker: CD26. Thus, antibo-

dies against this peptidase could be included during the first

negative selection step of any magnetic protocol to get rid of

unwanted CD261/high Teff (TH1, TH2, TH17) lymphocytes and

achieve higher numbers of either CD41CD25high or

CD41CD39high Treg populations (our data, 24,49). Moreover,

the phenotype CD41CD127lowCD49d2CD262 may be also

enough to select Treg cells through a fast and activation-degree

independent ‘‘single-step’’ purification protocol.

Which functional explanation there might be for this

CD262/low phenotype in human Treg cells? Several ligands

have been found for CD26, as the tyrosine phosphatase

CD45RO (31,32) or adenosine deaminase (ADA). The last one

is an ectoenzyme involved in degradation of adenosine, which

is an immunosuppressive molecule (22,56). Consequently, a

low level of CD26 could alter the membrane location of

CD45RO (32) or reduce the amount of surface ADA on Treg

cells (57), which may explain either their anergic phenotype in

vitro or their net production of adenosine (22). Alternatively,

interaction of Caveolin-1 (APC) with CD26 (T-cell) leads to

ORIGINAL ARTICLE


Figure 6. The use of CD26 as a negative selection marker enhances the levels of FoxP3 and CD25 in CD41CD25high Treg populations iso-

lated from activated PBMCs. CD41CD25high Treg cells were purified from activated PBMCs (1 lg/ml PHA, 5 days) using the Dynabeads1

Regulatory CD41CD251 T Cell Kit (Life Technologies/Invitrogen). During the depletion of non-CD41 cells, the cocktail of monoclonal antibo-

dies was supplemented (B) or not (A) with CD26 (TP1/16) mAb in order to deplete CD261 cells. Isolated CD41CD25high Treg and CD41CD252

Teff cells were stained with CD26-FITC (TP1/16), anti-FoxP3-PE (259D/C7), CD4-PerCP (SK3), and CD25-APC (M-A251) mAbs. During analy-

sis, viable CD41 lymphocytes were selected (R1 and R2) and FoxP3/CD25 levels and CD26 expression evaluated. According to CD26, two

different subsets (CD262/low and CD26high) were observed within CD41CD25high Treg cells (A), which points out to the presence of contami-

nating activated Teff cells (CD26high). Reduction of this CD26high subset within the CD41CD25high Treg lymphocytes raised the percentage

of CD25/FoxP3 double positive cells (B). (C) Columns plot summarizes the percentage of reduction/increase in the fluorescence intensity of

CD26, FoxP3, and CD25 in Tregs from three different healthy donors after the partial depletion of CD261 cells (mean � standard deviation).(D) Suppression of autologous (CFSE-labeled) responder PBMCs proliferation (y-axis; % of cells that divided at least once) by coculture

with increasing numbers of either CD41CD25high (Treg) or CD41CD25highCD262/low (CD26-depleted Treg) lymphocytes isolated from

preactivated (PHA 1 IL-2; 5 days) PBMCs (x-axis). Measurements were carried out in triplicate. Significant results are indicated by single

asterisks (P value is =0.05; Mann-Whitney U-test).

ORIGINAL ARTICLE


the up regulation of CD86 on APCs (58). Interestingly, Treg

lymphocytes down-regulate CD80 and CD86 on APCs

through a CTLA-4-dependent (9) mechanism. CD80/CD86

molecules are differentially regulated (59) and seem to have

divergent functions (60). For example, CD86 is highly respon-

sive to proinflammatory stimuli (LPS, TNFa, IFN-c, CD40-

CD40L ligation) and shows a preference for CD28, whereas

CD80 seems rather specific for the inhibitory receptor CTLA-

4 (60,61). Therefore, if we assume a co-stimulatory role for

the CD26/caveolin-1 interaction and a subsequent CD86 (but

not CD80) upregulation on APCs (58), a CD262/low pheno-

type in Treg cells may contribute to keep low levels of proin-

flammatory CD86 molecules on APCs, leading indirectly to a

lack of response (anergy) or apoptosis in antigen-primed naıve

T cells.

In conclusion, the present data substantiate a link between

null/low levels of CD26 and immunosuppressive functions

within either CD41CD25high, CD41CD39high, or

CD41CD1272/lowCD49d2 subsets. In addition, they support

further investigation to determine whether the CD262/low phe-

notype is a reliable alternative for the delineation or purification

of human Treg cells by means of extracellular markers, which

may stimulate the arising of new protocols and the progress in

Treg field. Finally, a patent application for the data described in

this paper was done on January 26, 2010, which has recently

entered the Patent Cooperative Treaty (PCT) phase.

ACKNOWLEDGMENTS

The authors are grateful to the Medical Service at the

University of Santiago, and to Dr. Juan E. Viuela, Immunology

Service, Clinic University Hospital of Santiago (CHUS), for

technical assistance. In addition, the authors want to thank

Ana M. Carballido and Alejandro Gonzalez for their colla-

boration.

LITERATURE CITED

1. Lu L, Cantor H. Generation and regulation of CD81 regulatory T cells. Cell MolImmunol 2008;5:401–406.

2. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP31 regulatory T cells inthe human immune system. Nat Rev Immunol 2010;10:490–500.

3. Feuerer M, Hill JA, Mathis D, Benoist C. Foxp31 regulatory T cells: Differentiation,specification, subphenotypes. Nat Immunol 2009;10:689–695.

4. Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, Shimizu J, TakahashiT, Nomura T. FoxP31CD251CD41 natural regulatory T cells in dominant self-toler-ance and autoimmune disease. Immunol Rev 2006;212:8–27.

5. Boros P, Bromberg JS. Human FOXP31 regulatory T cells in transplantation. Am JTransplant 2009;9:1719–1724.

6. Joosten SA, Ottenhoff TH. Human CD4 and CD8 regulatory T cells in infectious dis-eases and vaccination. Hum Immunol 2008;69:760–770.

7. Curotto de Lafaille MA, Lafaille JJ. Natural and adaptive FoxP31 regulatory T cells:More of the same or a division of labor? Immunity 2009;30:626–635.

8. Corthay A. How do regulatory cells work? Scand J Immunol 2009;70:326–336.

9. Oderup C, Cederbom L, Makowska A, Cilio CM, Ivars F. Cytotoxic T lymphocyte anti-gen-4-dependent down-modulation of costimulatory molecules on dendritic cells inCD41CD251 regulatory T-cell-mediated suppression. Immunology 2006;118:240–249.

10. Tang Q, Bluestone JA. Regulatory T-cell physiology and application to treat autoim-munity. Immunol Rev 2006;212:217–237.

11. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer2010;127:759–767.

12. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD41CD25high regulatory cellsin human peripheral blood. J Immunol 2001;167:1245–1253.

13. Aerts NE, Dombrecht EJ, Ebo DG, Bridts CH, Stevens WJ, De Clerck LS. Activated Tcells complicate the identification of regulatory T cells in rheumatoid arthritis. CellImmunol 2008;251:109–115.

14. Jourdan P, Vendrell JP, Huguet MF, Segondy M, Bousquet J, Pene J, Yssel H. Cyto-kines and cell surface molecules independently induce CXCR4 expression on CD41CCR71 human memory T cells. J Immunol 2000;165;716–724.

15. Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. Ex vivo isolation andcharacterization of CD4(1)CD25(1) T cells with regulatory properties from humanblood. J Exp Med 2001;193:1303–1310.

16. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive humanCD41FOXP32 T cells by T cell receptor stimulation is TGFb-dependent but does notconfer a regulatory phenotype. Blood 2007;110:2983–2890.

17. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gin-geras TR, Fazekas de St Groth B, et al. CD127 expression inversely correlates withFoxP3 and suppressive function of human CD41 T reg cells. J Exp Med2006;203:1701–1711.

18. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, SolomonM, Selby W, Alexander SI, Nanan R, et al. Expression of interleukin (IL)-2 and IL-7receptors discriminates between regulatory and activated T cells. J Exp Med2006;203:1693–1700.

19. Del Pozo-Balado M del M, Leal M, Mendez-Lagares G, Pacheco YM.CD4(1)CD25(1/hi)CD127(lo) phenotype does not accurately identify regula-tory T cells in all populations of HIV-infected persons. J Infect Dis 2010;201:331–335.

20. Alves NL, van Leeuwen EM, Derks IA, van Lier RA. Differential regulation of humanIL-7 receptor alpha expression by IL-7 and TCR signaling. J Immunol2008;180:5201–5210.

21. Kleinewietfeld M, Starke M, Di Mitri D, Borsellino G, Battistini L, Rotzschke O, FalkK. CD49d provides access to ‘‘untouched’’ human FoxP31 Treg free of contaminatingeffector cells. Blood 2009;113:827–836.

22. Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R,Hopner S, Centonze D, Bernardi G, Dell’Acqua ML, et al. Expression of ectonucleo-tidase CD39 by Foxp31 Treg cells: Hydrolysis of extracellular ATP and immune sup-pression. Blood 2007;110:1225–1232.

23. Venken K, Hellings N, Thewissen M, Somers V, Hensen K, Rummens JL, Medaer R,Hupperts R, Stinissen P. Compromised CD41 CD25(high) regulatory T-cell functionin patients with relapsing-remitting multiple sclerosis is correlated with a reducedfrequency of FOXP3-positive cells and reduced FOXP3 expression at the single-celllevel. Immunology 2008;123:79–89.

24. Mandapathil M, Lang S, Gorelik E, Whiteside TL. Isolation of functional human reg-ulatory T cells (Treg) from the peripheral blood based on the CD39 expression. JImmunol Methods 2009;346:55–63.

25. Alam MS, Kurtz CC, Rowlett RM, Reuter BK, Wiznerowicz E, Das S, Linden J, CroweSE, Ernst PB. CD73 is expressed by human regulatory T helper cells and suppressesproinflammatory cytokine production and Helicobacter felis-induced gastritis inmice. J Infect Dis 2009;199:494–504.

26. Moncrieffe H, Nistala K, Kamhieh Y, Evans J, Eddaoudi A, Eaton S, Wedderburn LR.High expression of the ectonucleotidase CD39 on T cells from the inflamed site iden-tifies two distinct populations, one regulatory and one memory T cell population. JImmunol 2010;185:134–143.

27. Bluestone JA, Thomson AW, Shevach EM, Weiner HL. What does the future hold forcell based tolerogenic therapy? Nat Rev Immunol 2007;7:650–654.

28. Ohnuma K, Dang NH, Morimoto C. Revisiting an old acquaintance: CD26 and itsmolecular mechanisms in T cell function. Trends Immunol 2008;29:295–301.

29. Cordero OJ, Salgado FJ, Viuela JE, Nogueira M. Interleukin-12 enhances CD26expression and Dipeptidyl peptidase IV function on human activated lymphocytes.Immunobiology 1997;197:522–533.

30. Huhn J, Ehrlich S, Fleischer B, von Bonin A. Molecular analysis of CD26-mediatedsignal transduction in T cells. Immunol Lett 2000;72:127–132.

31. Torimoto Y, Dang NH, Vivier E, Tanaka T, Schlossman SF, Morimoto C. Coassocia-tion of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lym-phocytes. J Immunol 1991;147:2514–2517.

32. Salgado FJ, Lojo J, Fernandez-Alonso CM, Alonso-Lebrero JL, Lluis C, Franco R,Viuela JE, Cordero OJ, Nogueira M. A role for IL-12 in the regulation of T cell plasmamembrane compartmentation. J Biol Chem 2003;278:24849–24857.

33. Ohnuma K, Uchiyama M, Yamochi T, Nishibashi K, Hosono O, Takahashi N, Kina S,Tanaka H, Lin X, Dang NH, et al. Caveolin-1 triggers T-cell activation via CD26 inassociation with CARMA1. J Biol Chem 2007;282:10117–10131.

34. Canda-Sanchez A, Salgado FJ, Perez-Dıaz A, Varela-Gonzalez C, Arias P, Nogueira M.IL-12-dependent activation of ERK1/2 in human T lymphoblasts. Immunobiology2009;214:187–196.

35. Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G, Olek S, Dietmaier W,Andreesen R, Edinger M. Loss of FOXP3 expression in natural human CD41CD251regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol 2009;39:1088–1097.

36. Earle KE, Tang Q, Zhou X, Liu W, Zhu S, Bonyhadi ML, Bluestone JA. In vitroexpanded human CD41CD251 regulatory T cells suppress effector T cell prolifera-tion. Clin Immunol 2005;115:3–9

37. Hoffmann P, Eder R, Boeld TJ, Doser K, Piseshka B, Andreesen R, Edinger M. Onlythe CD45RA1 subpopulation of CD41CD25high T cells gives rise to homogeneousregulatory T-cell lines upon in vitro expansion. Blood 2006;108:4260–4267.

38. Riley JL, June CH, Blazar BR. Human T regulatory cell therapy: Take a billion or soand call me in the morning. Immunity 2009;30:656–665.

39. Molinero LL, Yang J, Gajewski T, Abraham C, Farrar MA, Alegre ML. CARMA1 con-trols an early checkpoint in the thymic development of FoxP31 regulatory T cells. JImmunol 2009;182:6736–6743.

40. Ohnuma K, Ishii T, Iwata S, Hosono O, Kawasaki H, Uchiyama M, Tanaka H, Yamo-chi T, Dang NH, Morimoto C. G1/S cell cycle arrest provoked in human T cells byantibody to CD26. Immunology 2002;107:325–333.

41. Ohnuma K, Uchiyama M, Hatano R, Takasawa W, Endo Y, Dang NH, Morimoto C.Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion proteininduces anergy in CD41 T cells. Biochem Biophys Res Commun 2009;386:327–332.

ORIGINAL ARTICLE


42. Busso N, Wagtmann N, Herling C, Chobaz-Peclat V, Bischof-Delaloye A, So A,Grouzmann E. Circulating CD26 is negatively associated with inflammation inhuman and experimental arthritis. Am J Pathol 2005;166:433–442.

43. Preller V, Gerber A, Wrenger S, Togni M, Marguet D, Tadje J, Lendeckel U, Rocken C,Faust J, Neubert K, et al. TGF-beta1-mediated control of central nervous systeminflammation and autoimmunity through the inhibitory receptor CD26. J Immunol2007;178:4632–4640.

44. Steinbrecher A, Reinhold D, Quigley L, Gado A, Tresser N, Izikson L, Born I, Faust J,Neubert K, Martin R, et al. Targeting dipeptidyl peptidase IV (CD26) suppressesautoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo. JImmunol 2001;166:2041–2048.

45. Matteucci E, Giampietro O. Dipeptidyl peptidase-4 (CD26): knowing the functionbefore inhibiting the enzyme. Curr Med Chem 2009;16:2943–2951.

46. Skripuletz T, Schmiedl A, Schade J, Bedoui S, Glaab T, Pabst R, von Horsten S, Ste-phan M. Dose-dependent recruitment of CD251 and CD261 T cells in a novelF344 rat model of asthma. Am J Physiol Lung Cell Mol Physiol 2007;292:L1564–1571.

47. Stephens LA, Barclay AN, Mason D. Phenotypic characterization of regulatoryCD41CD251 T cells in rats. Int Immunol 2004;16,365–375.

48. Ma Y, Visser L, Blokzijl T, Harms G, Atayar C, Poppema S, van den Berg A. TheCD41CD262 T-cell population in classical Hodgkin’s lymphoma displays a distinc-tive regulatory T-cell profile. Lab Invest 2008;88:482–490.

49. Bengsch B, Seigel B, Flecken T, Wolanski J, Blum HE, Thimme R. Human Th17 cellsexpress high levels of enzymatically active dipeptidylpeptidase IV (CD26). J Immunol2012;188:5438–5447.

50. Simonetta F, Chiali A, Cordier C, Urrutia A, Girault I, Bloquet S, Tanchot C, Bour-geois C. Increased CD127 expression on activated FOXP31CD41 regulatory T cells.Eur J Immunol 2010;40:2528–2538.

51. Cordero OJ, Salgado FJ, Nogueira M. On the origin of serum CD26 and its alteredconcentration in cancer patients. Cancer Immunol Immunother 2009;58:1723–1747.

52. Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S. Het-erogeneity of natural FoxP31 T cells: A committed regulatory T-cell lineage and an

uncommitted minor population retaining plasticity. Proc Natl Acad Sci USA2009;106:1903–1908.

53. Kmieciak M, Gowda M, Graham L, Godder K, Bear HD, Marincola FM, Manjili MH.Human T cells express CD25 and Foxp3 upon activation and exhibit effector/mem-ory phenotypes without any regulatory/suppressor function. J Trans Med 2009;7:89.

54. Potter SJ, Lacabaratz C, Lambotte O, Perez-Patrigeon S, Vingert B, Sinet M, Colle JH,Urrutia A, Scott-Algara D, Boufassa F, et al. Preserved central memory and activatedeffector memory CD41 T-cell subsets in human immunodeficiency virus controllers:An ANRS EP36 study. J Virol 2007;81:13904–13915.

55. Santner-Nanan B, Seddiki N, Zhu E, Quent V, Kelleher A, Fazekas de St Groth B,Nanan R. Accelerated age-dependent transition of human regulatory T cells to effec-tor memory phenotype. Int Immunol 2008;20:375–383.

56. Pacheco R, Martinez-Navio JM, Lejeune M, Climent N, Oliva H, Gatell JM, Gallart T,Mallol J, Lluis C, Franco R. CD26, adenosine deaminase, and adenosine receptorsmediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci USA2005;102:9583–9588.

57. Cordero OJ, Salgado FJ, Fernandez-Alonso CM, Herrera C, Lluis C, Franco R,Nogueira M. Cytokines regulate membrane adenosine deaminase on human activatedlymphocytes. J Leukoc Biol 2001;70:920–930.

58. Ohnuma K, Yamochi T, Uchiyama M, Nishibashi K, Yoshikawa N, Shimizu N, IwataS, Tanaka H, Dang NH, Morimoto C. CD26 up-regulates expression of CD86 onantigen-presenting cells by means of caveolin-1. Proc Natl Acad Sci USA2004;101:14186–14191.

59. Larsen CP, Ritchie, SC, Hendrix R, Linsley PS, Hathcock KS, Hodes RJ, Lowry RP,Pearson TC. Regulation of immunostimulatory function and costimulatory molecule(B7-1 and B7-2) expression on murine dendritic cells. J Immunol 1994;152:5208–5219.

60. Rudy W, Guckel B, Siebels M, Lindauer M, Meuer SC, Moebious U. Differential func-tion of CD80- and CD86-transfected human melanoma cells in the presence of IL-12and IFN-c. Int Immunol 1997;9:853–860.

61. Zheng Y, Manzotti CN, Liu M, Burke F, Mead KI, Sansom DM. CD86 and CD80 dif-ferentially modulate the suppressive function of human regulatory T cells. J Immunol2004;172:2778–2784.

ORIGINAL ARTICLE


CD26: A Negative Selection Marker for Human Treg Cells · Department of Biochemistry and Molecular...

Documents

Transcript of CD26: A Negative Selection Marker for Human Treg Cells · Department of Biochemistry and Molecular...