Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells

Ex vivo isolation protocols differentially affect the phenotype of

human CD4+ T cells

Frederic Bernarda, Sara Jalecoa, Valerie Dardalhona, Marcos Steinberga,Hans Ysselb, Nelly Noraza, Naomi Taylora,*, Sandrina Kineta

a Institut de Genetique Moleculaire de Montpellier, UMR 5535/IFR 22, 1919 Route de Mende, F34293 Montpellier, Cedex 5, Franceb INSERM U454, Montpellier F34293, France

Received 27 May 2002; received in revised form 12 August 2002; accepted 12 September 2002

Abstract

Leukemic T cell lines have facilitated signal transduction studies but their physiological relevance is restricted. The use of

primary T lymphocytes overcomes this limitation but it has long been speculated that methodological aspects of blood

collection and the isolation procedure modify the phenotype of the cell. Here we demonstrate that several characteristics of

human peripheral T cells are affected by the selection conditions. A significantly higher induction of the chemokine receptor

CXCR4 was observed on CD4+ lymphocytes isolated by sheep red blood cell (SRBC) rosetting and CD4 MicroBeads as

compared with positively selected CD4+ cells where the antibody/bead complex was immediately detached. These latter cells

expressed CXCR4 at levels equivalent to that observed on CD4+ lymphocytes obtained by negative antibody-mediated

selection. Furthermore, CD4+ cells isolated by SRBC rosetting and CD4 MicroBeads formed aggregates upon in vitro culture.

CD4+ lymphocytes obtained by SRBC rosetting as well as those isolated following positive selection demonstrated basal

phosphorylation of the extracellular signal-regulated kinase (ERK)-2. Altogether these data suggest that certain discrepancies

concerning signal transduction in primary human T cells can be attributed to the selection conditions. Thus, it is essential to

establish the parameters influenced by the isolation protocol in order to fully interpret T cell responses to antigens, chemokines,

and cytokines.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: T lymphocyte; Purification; SRBC; Isolation; Signal transduction; Chemokine

1. Introduction

Dissection of the intracellular signaling pathways

triggered in response to T cell receptor (TCR) and

chemokine receptor engagement has been the subject

of intense investigation. TCR-induced cascades result

in the transcription of numerous genes leading to

functional T cell responses while certain chemokine

receptors are involved in the trafficking of lympho-

cytes.

Human leukemic T cell lines, such as Jurkat, have

been invaluable tools for studies designed to charac-

terize various signaling intermediates. Nevertheless, it

is clear that tumor lines have a strongly altered

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00412 -X

* Corresponding author. Tel.: +33-4-67-61-36-28; fax: +33-4-

67-04-02-31.

E-mail address: [email protected] (N. Taylor).

www.elsevier.com/locate/jim

Journal of Immunological Methods 271 (2002) 99–106

phenotype in comparison to primary T lymphocytes.

The evidence for this includes the following: (i) many

T cell lines are permanently cycling whereas freshly

isolated primary T lymphocytes are quiescent; (ii)

some T cell lines spontaneously secrete high levels

of cytokines in marked contrast with nonactivated

primary cells; (iii) the formation of lateral lipid

clusters (rafts) in the plasma membrane is dramati-

cally altered in tumor cells; and (iv) many signaling

molecules are differentially expressed in transformed

and primary cells (Peterson et al., 1998; Van Leeuwen

and Samelson, 1999; Lin and Weiss, 2001). In partic-

ular, the levels of several proteins involved in the TCR

signaling cascade are strikingly different in Jurkat

cells as compared to primary T cells; these include

the ~ subunit of the TCR, the CD4 receptor, the

phosphatases PTEN and SHIP, the adaptor protein

Grb2 and the docking protein Cas-L (Astoul et al.,

2001; Ohashi et al., 1999, and our own published

observations). Indeed, the absence of SHIP and PTEN

is associated with a high basal level of phosphoinosi-

tide 3-kinase and protein kinase B activities in Jurkat

cells (Astoul et al., 2001).

To study signaling processes in a more physio-

logical context many groups have invested signifi-

cant time and effort isolating large numbers of

primary human lymphocytes. Nevertheless, conflict-

ing data have been reported in many of these studies.

Specifically, there are inconsistencies concerning the

surface expression and induction of the HIV core-

ceptor, CXCR4 (Bleul et al., 1997; Carroll et al.,

1997; Bermejo et al., 1998; Jourdan et al., 1998),

and the activation of signaling intermediates in dis-

tinct T cell subsets (Risdon et al., 1995; Pallier et al.,

1998; Sato et al., 1999).

Some of the discrepancies in the human T cell

biology literature may be the consequence of the

experimental conditions under which the peripheral

blood lymphocytes were isolated. Specifically, there

are a variety of available techniques currently uti-

lized for isolating CD4+ T cells, including those

based on the unique ability of T cells to bind and

form rosettes with sheep red blood cells (SRBC), as

well as those based on positive and negative anti-

body-mediated selection. Here, we show that the

phenotype of CD4+ lymphocytes with respect to

CXCR4 surface expression, overall morphology,

and basal extracellular signal-regulated kinase

(ERK)-2 phosphorylation are modified by the iso-

lation protocol.

2. Materials and methods

2.1. Purification and culture of human CD4+ T cells

Peripheral blood, obtained from healthy adult

donors, was collected in heparinized tubes after

informed consent was obtained. CD4+ cells were

isolated by one of the five selection methods detailed

below. For all isolations with the exception of the

RosetteSepk procedure (see below), mononuclear

cells were first separated over Ficoll –Hypaque

(Sigma). (1) CD4+ T cells were purified by SRBC

(BioMerieux) rosetting followed by elimination of

CD8+ T cells with aCD8 mAb-coated magnetic

beads (Dynal) as indicated by the manufacturer. (2)

Alternatively, CD4+ T cells were purified by positive

selection using aCD4 mAb-coated magnetic beads

that were immediately removed using DETACH-

aBEADR (Dynal). (3) Cells were also positively

selected using CD4 MicroBeads (Miltenyi) whereby

magnetically retained CD4+ cells were eluted in the

positively selected cell fraction as indicated by the

manufacturer. (4) CD4+ cells were selected by a

‘‘classical’’ negative selection: Ficoll, incubation with

a cocktail of mAbs (directed against CD8, CD14,

CD16, CD19, and CD56), incubation with goat anti-

mouse-coated magnetic beads (Dynal), and elimina-

tion of mAb-bound cells on a magnet. (5) Negatively

selected CD4+ T cells were also obtained by incuba-

tion of whole blood with a cocktail of monoclonal

antibodies directed against CD8, CD16, CD19,

CD36, and CD56 held in tetrameric arrays with a

murine antibody recognizing human glycophorin A

on the surface of red blood cells (RosetteSepk,

StemSep). This latter protocol allowed the unwanted

cells to be cross-linked to the RBC present in the

whole blood sample, and these cells were then

eliminated in the pellet upon Ficoll centrifugation.

The purities of all populations were monitored on a

FACScalibur (Becton Dickinson) following staining

with FITC-conjugated aCD3 and PE-conjugated

aCD4 mAbs.

For indicated experiments, isolated CD4+ T cells

were cultured in RPMI media supplemented with 1%

F. Bernard et al. / Journal of Immunological Methods 271 (2002) 99–106100

or 10% fetal calf serum. Cell morphology was

assessed on a Leica Leitz DM IL microscope.

2.2. Flow cytometry

To detect CXCR4 cell surface expression, cells

were incubated with a PE-conjugated aCXCR4

mAb (12G5; PharMingen). Background fluorescence

was measured using an immunoglobulin isotype

control mAb. Cells were incubated with mAbs for

20 min on ice and washed in PBS before FACS

analysis.

2.3. Cell stimulation and immunoblotting

Cells were resuspended at 1�107 cells/ml, and

stimulated for 5 min at 37 jC with an aCD3 mAb (2.5

Ag/ml) (kindly provided by D. Cantrell, ICRF) fol-

lowed by cross-linking with an amouse F(abV)2 frag-

ment (10 Ag/ml). Cells were lysed in a 1% NP40 lysis

buffer, resolved on SDS-PAGE gels, and transferred

electrophoretically to nitrocellulose membranes as

reported (Noraz et al., 2000). Membranes were blotted

with a pAb recognizing the phosphorylated forms of

Erk1/2 (Cell Signaling Technology) and an aZAP-70

mAb (kindly provided by A. Weiss, UCSF). Immu-

noreactive proteins were visualized using enhanced

chemiluminescence.

3. Results and discussion

3.1. Purity and recovery of ex vivo isolated CD4+ T

cells

To analyze the effects of various isolation methods

on the characteristics of CD4+ T cells, these lympho-

cytes were isolated by one of five methods: (1) rosett-

ing with sheep red blood cells (SRBC) followed by

negative selection of the CD4+ T cell population using

aCD8 mAb-coated immunomagnetic beads; (2) pos-

itive selection using aCD4-coated immunomagnetic

beads followed by removal of the antibody/bead com-

plex from the cell surface with DETACHaBEAD

(Dynal); (3) positive selection using CD4 MicroBeads

(Miltenyi) which are biodegradable and therefore do

not require removal; (4) ‘‘classical’’ negative selection

in which PBMC are incubated with a cocktail of

monoclonal antibodies coupled to goat anti-mouse-

coated magnetic beads (Dynal) and antibody-coupled

cells are depleted on a magnetic field; and (5) negative

selection using tetrameric antibody complexes in

which one antibody recognizes a surface antigen on

an unwanted cell and the other recognizes glycophorin

A on the surface of the red blood cells that are pelleted

during the Ficoll centrifugation (RosetteSepk, Stem-

Sep). To minimize donor-to-donor variability, all com-

parisons were performed concurrently with blood

obtained from a single donor and all isolation protocols

and experimental manipulations were repeated with

two to six different donors. The purity of the CD4+ T

cell population obtained by either positive selection

method was 98–99% (Dynal orMyltenyi) whereas that

obtained by SRBC rosetting and RosetteSepk nega-

tive selection ranged between 79–91% and 87–95%,

respectively (Table 1). CD4+ cell isolations using

‘‘classical’’ negative selection resulted in significantly

lower purities (mean of 75%, range of 67–83%). Thus,

in subsequent experiments performed with negatively

selected CD4+ cells, lymphocytes were isolated solely

using the RosetteSepk method.

CD4+ T cell yields were equivalent using both

positive selection methods, SRBC rosetting, and

‘‘classical’’ negative selection, but were consistently

at least 1.5-fold higher upon negative selection by the

RosetteSep method ( p < 0.02) (Table 1). This differ-

ence was probably due to the decreased numbers of

manipulations and centrifugations performed in the

Table 1

Purity and yield of CD3 +CD4+ cells following their isolation from

whole blood

Type of selection CD3+CD4+

cell purity

Cell yield

(� 106)

Positive selection

(Dynal)

99.2%

(98–99.5)

8.3

(7–10)

MicroBead selection

(Miltenyi)

98.4%

(98–99)

5.8

(5–6)

SRBC rosetting 85.0%

(78–91)

8.6

(7–10)

Negative selection

(RosetteSepk)

89.9%

(87–95)

14.7

(14–16)

Negative selection 75.6%

(67–83)

8.0

(6–10)

CD4+ lymphocytes were isolated by the methods indicated above in

two to six independent experiments (from 33� 106 to 42� 106

PBMC). The range of values obtained is noted in parentheses.

F. Bernard et al. / Journal of Immunological Methods 271 (2002) 99–106 101

latter protocol; it was the only one of the five isolation

methods where the CD4+ T cell population was

obtained immediately following Ficoll density centri-

fugation. Indeed, this isolation procedure required

approximately 1.5 h whereas the positive selection

and SRBC rosetting protocols required an average of

3 and 4.5 h, respectively.

3.2. Expression of the CXCR4 chemokine receptor in

ex vivo isolated CD4+ T cells

The CXCR4 chemokine receptor has been the

focus of many studies in recent years, in part because

of its role as a coreceptor for HIV-1 entry (Feng et al.,

1996). While many reports have focused on the levels

of CXCR4 in human T cell subsets, there are wide

discrepancies concerning the effects of activation and

ex vivo culture on its cell surface expression (Bleul et

al., 1997; Carroll et al., 1997; Bermejo et al., 1998;

Jourdan et al., 1998). As these differences may have

resulted, at least in part, from the selection protocol,

CXCR4 levels were monitored in CD4+ T cells iso-

lated by the various methods described above. Surface

CXCR4 levels did not significantly differ in CD4+ T

lymphocytes immediately following isolation, regard-

less of the selection protocol (Fig. 1A). However,

following a 24-h in vitro culture, the induction in

CXCR4 expression was 2-fold higher in T cells iso-

lated by SRBC rosetting as compared to CD4+ cells

obtained by positive Dynal or negative RosetteSepkselection methods (Fig. 1A, p < 0.02). Notably, as

previously described (Bleul et al., 1997; Bermejo et

al., 1998), we found that the variability in CXCR4

expression between individual donors was significant

(not shown). Nevertheless, the induction in CXCR4

expression was consistently augmented in T cells

isolated by SRBC rosetting in all donors. Thus, these

data suggest that the surface expression of CXCR4 on

CD4+ T cells is modulated by the SRBC isolation

protocol.

It has been suggested that positive selection using

biodegradable microbeads (50 nm) may result in less

signal to the T cell. However, in the context of cells

that are in a low metabolic state, such as freshly

isolated T lymphocytes which are almost entirely in

the G0 phase of the cell cycle (not shown), the process

of antibody/bead detachment generally requires 24–

48 h and may take up to 96 h (Miltenyi, oral commu-

Fig. 1. Surface CXCR4 expression differs as a function of the

isolation protocol. (A) Peripheral blood obtained from a single

donor was divided into equal portions and CD4+ T lymphocytes

were isolated by either negative selection with a cocktail of

tetrameric antibody complexes cross-linked to red blood cells

(RosetteSepk), positive selection using aCD4-conjugated mag-

netic beads (Dynal) followed by bead detachment, or sheep red

blood cell (SRBC) rosetting followed by negative selection of the

CD4+ T cell fraction with aCD8-mAb coated magnetic beads

(Dynal). Lymphocytes thus isolated were stained with the PE-

conjugated anti-CXCR4 mAb immediately after isolation (t0) and

following a 24-h culture in RPMI supplemented with 1% FCS (t24).

The change in mean fluorescence intensity (DMFI) between freshly

isolated and cultured lymphocytes is shown for each isolation

procedure. (B) CXCR4 expression in negatively isolated CD4+ cells

and CD4+ cells isolated using CD4 MicroBeads (Miltenyi) were

compared following a 48-h culture in RPMI supplemented with 1%

FCS. The MFI of each histogram is indicated. Filled histograms

depict staining with an isotype-matched control mAb. Histograms

are representative of data obtained in independent experiments

performed with cells obtained from two to four different donors.


nication). Indeed, the level of CXCR4 was higher on

CD4 MicroBead-isolated T cells than on the corre-

sponding negatively isolated CD4+ cell population

following an overnight culture in the presence of 1%

FCS (data not shown). This difference further

increased during the next 24 h, with a >2-fold higher

mean fluorescence intensity of CXCR4 binding in the

CD4 MicroBead-isolated cells (Fig. 1B).

Notably, Stanciu et al. (1996) previously reported

that CD4+ T cells isolated by CD4 MicroBeads

produce significantly more IL-4 than the correspond-

ing negatively-isolated population. They hypothesized

that ligation of the CD4 molecule results in cell

activation, although there was no spontaneous T cell

proliferation or increase in surface expression of the

IL-2Ra receptor (CD25) (Stanciu et al., 1996). Our

results also indicate that the selection process results

in selective, rather than global, changes in phenotype.

While changes in CXCR4 levels were observed in

both SRBC- and CD4 MicroBead-isolated cells, other

receptor levels remained unchanged (IL-2Ra, CD69,

and CD3) as compared with cells isolated by negative

selection.

3.3. Morphology of CD4+ T cells upon in vitro

culture in the absence of exogenous stimuli

Differences in CXCR4 expression were observed

under in vitro culture conditions where no exogenous

stimuli were added, and as such the morphology of

the CD4+ lymphocytes was followed. T lymphocytes

maintained in the absence of exogenous stimuli can

survive for several days but remain small and uni-

form in size. CD4+ T cells isolated by negative

selection as well as those isolated by positive selec-

tion where the CD4 antibody was immediately

detached (Dynal) remained small and round. In

contrast, the phenotype of the CD4 MicroBead-iso-

lated CD4+ T cells and the SRBC-rosetted CD4+ T

lymphocytes were distinct. Following 48 h of cul-

ture, some of these latter cells formed aggregates

while others became elongated and ‘‘blast-like’’,

resembling T cells activated through the antigen

receptor (Fig. 2). Thus, at least a subset of these

CD4+ T cells has an altered morphology resulting

from the isolation procedure. Intriguingly, high

expression of CXCR4 and cell aggregation were

observed in the same CD4+ T cell subsets. Fig.2.CD4+Tcellmorphologyismodifiedbytheisolationprotocol.CD4+Tcellswereisolatedbyeither

negativeselection(RosetteSepk),positiveselectionwithanti-CD4-

conjugated

magnetic

beadswhichwereim

mediately

detached

(Dynal),CD4MicroBead-isolatedcellswheretheMicroBeadswerenotdetached,orSRBC

rosettingfollowed

by

elim

inationofCD8+Tcells.TheCD4+Tcellpopulationsthusisolatedwereculturedfor48hin

RPMImedia

supplementedwith10%

FCS.Lymphocyteswereobserved

under

a

Leitz

DM

ILmicroscopeandphotographed

atamagnificationof125�.Sim

ilar

morphologieswereobserved

inCD4+lymphocytesobtained

from

twoto

fourdifferentdonors.


3.4. Activation of the MAPK pathway in ex vivo-

isolated CD4+ T cells

Since the Ras-Erk pathway is integrally involved in

multiple T cell responses (Cantrell, 1996), we as-

sessed whether the basal activation state of the

MAPKs, Erk1, and Erk2 was affected by the selection

method. Importantly, there was a low level of basal

Erk2 phosphorylation in lymphocytes isolated by

positive selection and SRBC rosetting that was not

observed in the negatively selected CD4+ T cell

population (Fig. 3). All isolated CD4+ T cell popula-

tions were capable of responding to aCD3-mediated

cross-linking as demonstrated by strong induction of

Erk1 and Erk2 phosphorylation (Fig. 3). However,

under conditions of positive isolation where the large

aCD4-coated Dynal beads (4.5 Am) were not

detached, a high level of ‘‘basal’’ Erk1/Erk2 phos-

phorylation was observed and this response could not

be augmented by CD3 cross-linking (data not shown).

Thus, it appears that the selection procedure is not a

‘‘neutral’’ process in that it can modulate the basal

activation state of the cell, specifically in lymphocytes

isolated by SRBC rosetting and positive selection

methods.

4. Conclusions and perspectives

Collectively, these data demonstrate that the proto-

col used to isolate lymphocytes can bias experimental

outcome. Reports dating from the early 1980s already

suggested that lymphocytes incubated with SRBC

show increased responses in mixed lymphocyte culture

and augmented mitogenic responses to suboptimal

doses of PHA and ConA (Silva et al., 1981). Moreover,

Breitmeyer and Faustman (1989) found that the SRBC

rosetting procedure inhibits TCR-mediated calcium

flux and proliferation during the first 24–48 h post-

selection. These consequences can be attributed to the

interaction of SRBC with the T cell-specific CD2

glycoprotein, a molecule that plays a role in T cell

signaling and lymphocyte adhesion (Bierer and Hahn,

1993). However, this method of selection is still com-

monly used for lymphocyte separations in signaling

studies (Tamma et al., 1997; Pallier et al., 1998;

Kennedy et al., 1999; Cicala et al., 2000). As we now

find that CXCR4 surface levels are modified by the

isolation protocol, it is important to consider this

parameter in studies of CXCR4-induced signaling

intermediates, especially in the context of SRBC and

positively selected T cells. Notably, the effects of

SRBCmay have confounded various signaling studies,

including those concerning the IL-7 cytokine. We and

others recently reported that human T cells, isolated by

negative selection, do not proliferate in response to IL-

7 but previous studies, using SRBC-isolated cells,

reported high levels of IL-7-induced proliferation

(Armitage et al., 1990; Yip-Schneider et al., 1993;

Hassan and Reen, 1998; Dardalhon et al., 2001).

Despite potential biases related to the isolation pro-

cedure andmanipulation of blood samples (David et al.,

1998), experiments utilizing primary human Tcells are

invaluable. Transformed cell lines present tractable

genetic systems in which some signaling phenomena

can be studied but the establishment of primary lym-

phocyte models is essential for investigations of human

Fig. 3. Basal Erk2 phosphorylation in CD4+ lymphocytes isolated

by positive selection and SRBC rosetting. 1�106 CD4+ T

lymphocytes isolated by either negative selection, positive selection

with anti-CD4-conjugated magnetic beads (Dynal), or SRBC

rosetting followed by elimination of CD8+ T cells were lysed

immediately (� ) or following stimulation (+) with a cross-linked

aCD3 mAb. Cell lysates were fractionated on a 10% polyacryla-

mide gel and immunoblotted with an anti-active MAPK pAb

specifically recognizing the dually phosphorylated forms of Erk1

and Erk2. To assure that equivalent numbers of T cells were used for

each activation, the blot was reprobed with a mAb recognizing the T

cell-specific ZAP-70 protein tyrosine kinase. Immunolabelled

proteins were visualized by enhanced chemiluminescence. Data

are representative of results obtained with T cells isolated from three

different donors.


immunity. It is therefore necessary to determine the

characteristics of human T cells that are influenced by

the isolation protocol. This will facilitate the interpre-

tation of important biological experiments studying T

cell responses to antigens, chemokines, and cytokines.

Acknowledgements

We are grateful to Drs. R. Hipskind, R. Feil, and

L. Dirick for helpful discussions and to L. Swainson

for critical reading of the manuscript. This work was

supported by grants from the ANRS, AFM, March

of Dimes (MOD Grant #6-FY99-406), and ARC

(to N.T.). S.K., S.J., V.D. and M.S. are funded by

fellowships from the European community (HPMF-

CT-2000-01035), Programa PRAXIS XXI, Fundac�aopara a Ciencia e Tecnologia, Portugal (Grant PRAXIS

XXI BD/19929/99), the French Ministere de la Re-

cherche, and the Fundacion YPF, respectively. Sup-

port for N.N. and N.T. is from the MOD and

INSERM, respectively.

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Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells

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