fasl-transfected endothelial cells decrease the proliferative response of allogeneic pbl

Transplant Immunology 10(2002) 293–302

0966-3274/02/$ - see front matter� 2002 Elsevier Science B.V. All rights reserved.PII: S0966-3274Ž02.00073-4

FasL-transfected endothelial cells decrease the proliferative response ofallogeneic PBL

Sandrine Cappellesso, Gilles Thibault, Cyrille Hoarau, Olivier Herault, Sophie Iochmann,´Pierre Bardos, Yvon Lebranchu*

UPRES-EA 3249 ‘Cellules Hematopoıetiques, Hemostase et Greffe’, Faculte de Medecine, 2 bis boulevard Tonnelle, 37032 Tours cedex,´ ¨´ ´ ´ ´ ´France

Received 28 January 2002; accepted 22 March 2002

Abstract

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells.Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to theimmunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sitesexpress Fas ligand(FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasLcould inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasLcDNA and used to transfect an endothelial cell line(ECV304 cells). We demonstrated that FasL-transfected ECV304 cells wereeffective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelialcell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathwaysinhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells couldbe interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organtransplantation.� 2002 Elsevier Science B.V. All rights reserved.

Keywords: Apoptosis; Transplantation; Toleranceysuppression

1. Introduction

Transplantation has become the treatment of choicefor end-stage organ failure. However, because immuno-suppressive agents can be toxic to the new organ andsuppress the patient’s immune defenses, there have beenmany attempts to establish specific tolerance in the fewpast years, particularly by inducing apoptosis of allo- orxenoreactive activated T lymphocytesw1x. One promis-ing molecule for induction of tolerance is the Fas ligand(FasL or CD95L or TNFSF6 or CD178), a type IItransmembrane protein that belongs to the TNF super-family and induces apoptosis by binding to its membrane

Abbreviations: 7-AAD, 7-amino actinomycin D; DC, dendriticcells; EC, endothelial cells; FasL, Fas ligand; MLEC, mixed lympho-cyte endothelial cell culture.*Corresponding author. Tel.:q33-2-47-47-47-13; fax:q33-2-47-

47-38-02.E-mail address: [email protected](Y. Lebranchu).

receptor Fas(CD95 or TNFRSF6) w2x that is expressedon activated T cellsw3,4x.Several reports have demonstrated that immune-priv-

ileged tissues express FasL and are protected againstinflammatory responses and allogeneic rejectionw5–8x.Similarly, a relationship between graft acceptance andFasL expression has been observed in human recipientsof corneal allograftsw9x. It has also been proposed thatFasL expression by the placenta could be one mecha-nism allowing maternal immune tolerance of the fetusw10,11x.Enhanced or elevated expression of FasL on specific

tissues or cells by a transgene technique has beensuccessfully applied to rat liver allografts protected fromhost immune response by incorporating an inactivatedhemagglutinating virus conjugated to FasL plasmid-bearing liposomew12x. It has also been used to prolongrat renal allograft survival transduced by adenoviralvectors with FasL cDNAw13x. Allogeneic transplanta-

294 S. Cappellesso et al. / Transplant Immunology 10 (2002) 293–302

tion of islets of Langherans has been facilitated bycotransplantation with FasL-expressing myoblastsw14xor with testicular cellsw15x. However, islet allograftsthat have been genetically engineered to express FasLare not protected but undergo rapid destruction mediatedby infiltrating neutrophilsw16–18x. These results suggestthat the role of FasL-expression on the acceptance ofgrafted organs and tolerance induction might depend onthe organ and the gene delivery methods. However, apromising strategy for inducing tolerance to alloantigensis the use of FasL-expressing APCw19–21x.During transplantation, allograft endothelial cells

(EC) regulate transendothelial migration and graft infil-tration by activated T lymphocytes expressing Fas ontheir surface. Moreover, they could be considered to belike APC w22,23x. Indeed, they present alloantigens,provide costimulation signals and are able to induce theproliferation of allogeneic T cells in vitrow24–27x. It istherefore tempting to induce the expression of FasL onthe surface of human EC in order to reduce the responseof allogeneic T lymphocytes. In the present study weused an expression vector containing human FasL cDNAto transfect a human EC line, ECV304, and studied theproliferative response of allogeneic lymphocytes.

2. Materials and methods

2.1. Antibodies

Agonist anti-Fas mAb(CH11; mouse IgM), antago-nist anti-Fas mAb(ZB4; mouse IgG1), anti-FasL mAb(4H9; hamster IgG), anti-CD2 mAb (T11.1; mouseIgG1), anti-CD11a mAb (AFOL-1; mouse IgG1),mouse IgG1 isotypic control(679.1 Mc7), PE-conjugat-ed anti-CD45 mAb(J33) and FITC-conjugated goatanti-mouse IgG F(ab9) were purchased from Beckman2

Coulter (Villepinte, France). Hamster IgG irrelevantcontrol mAb were obtained from Jackson Immuno-Research Laboratories(West Grove, PA, USA).

2.2. Cell culture

The human endothelial cell line ECV304, a kind giftof Dr Takahashi(Saimoto, Japan) w28x, and the humanJurkat T-cell line were cultured in 75-cm tissue culture2

flasks(Falcon 3024; Becton Dickinson, Pont de Claix,�

France) at 37 8C in 5% CO humidified atmosphere.2

Cells were grown in RPMI 1640 medium(Life Tech-nologies; Cergy Pontoise, France) supplemented with10% heat-inactivated FCS(ATGC; Noisy-le Grand,France), 25 mM sodium bicarbonate(ATGC), 2 mMglutamine(Bio Whittaker, Fontenay-sous-Bois, France),1 mM sodium pyruvate(Life Technologies), 50 IUymlpenicillin, 50mgyml streptomycin(ICN, Orsay, France)and 60mgyml tylocin (Life Technologies) (referred toas complete medium). ECV304 cells were removed

from culture flasks using trypsin-EDTA(0.4–1 gyl; LifeTechnologies) and resuspended in complete medium at1=10 cellsyflask. The cells were routinely subcultured6

every 3–4 days.

2.3. Isolation of human PBL

Human PBMC were isolated from heparinized periph-eral venous blood of healthy donors by Ficoll–Hypaque(Lymphoprep ; Nycomed, Oslo, Norway) density-gra-�

dient centrifugation. The mononuclear cell-rich interfacewas collected, washed three times and resuspended incomplete medium. PBMC were incubated twice for 45min at 37 8C in plastic tissue culture flasks(Falcon�

3024) to remove monocytes. Non-adherent PBL werecollected by gentle washing with RPMI 1640 andadjusted to 1=10 cellsyml. In some experiments, PBL6

were preactivated with PHA(Murex Diagnostics SA,Chatillon, France) (50 ngyml) for 3 days.ˆ

2.4. Stable transfection

The vector pBX-hFL1 containing human FasL cDNAw2x was kindly provided by Dr Nagata(Osaka, Japan).To construct an expression vector, the human FasLcDNA was cut out of pBX-hFL1 as aNot I-Hind IIIfragment and ligated into the pREP expression vector4

as previously describedw29x (Invitrogen, Leek, TheNetherlands). The resulting plasmid was designatedpREPyhFL1.4

ECV304 cells were seeded at 5=10 cells in 25 cm5 2

flasks(Falcon 3013) and grown for 24 h with complete�

medium. Cells were transfected by the CaPO method4

using the Mammalian Transfection Kit(Stratagene, La�

Jolla, CA, USA) according to the manufacturer’sinstructions. Briefly, cells were exposed overnight to atotal amount of 20mg CaPO precipitated vector4

(pREPyhFL1) in MEM medium (Life Technologies)4

with 10% FCS. The medium was then removed and thetransfected cells were rinsed twice with Earle buffersaline solution(Life Technologies) and grown withcomplete medium. At confluence, cells were seeded in6-well plates at 2=10 cellsywell (Falcon 3046) after5 �

trypsin-EDTA treatment and incubated for an additional24 h before applying Hygromycin B(Calbiochem,Meudon, France) for selection of stable transfectants.

2.5. mRNA isolation and RT-PCR

Total mRNA was isolated from 1=10 transfected6

cells using the Dynabeads mRNA Direct kit(Dynal�

France SA, Compiegne, France) according to the man-`ufacturer’s instructions and as described before by ourgroup w30x. The total mRNA isolated was reverse-transcribed in a final reaction volume of 100ml contain-ing incubation buffer for AMV-reverse transcriptase(50

295S. Cappellesso et al. / Transplant Immunology 10 (2002) 293–302

mM Tris–HCl, 8 mM MgCl , 30 mM KCl, 1 mM DTT,2

pH 8.5), 500mM of each dNTP and 1.25mM of oligo(dT) (Eurobio, Les Ulis, France). mRNA and primers20

were denatured at 658C for 5 min and cooled on icebefore adding 25 units of RNase inhibitor(Boehringer-Mannheim, Meylan, France) and 25 units of AMV-reverse transcriptase. After incubation for 1 h at 428C,the enzyme was inactivated at 958C for 5 min.PCR was performed from 10 cells in a total reaction5

volume of 50ml containing Taq buffer(10 mM Tris–HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 mMMgCl 200 mM of each dNTP, 1mM of each reverse2,

and forward primer specific for FasLw31x or GAPDHw30x (Eurobio), and 0.5 units of Taq DNA polymerase(Promega, Charbonnieres, France). cDNA was amplified`by 40 cycles of denaturation at 948C, annealing at 548C and extension at 728C each for 30 s. Samples ofproducts were analyzed by electrophoresis through a1.6% agarose gel in Tris borate EDTA buffer(89 mMTris–HCl, 89 mM borate acid, 2 mM EDTA) (Euro-medex, Souffelweyersheim, France) containing 1mgyml ethidium bromide(Sigma, Saint Quentin Fallavier,France). wx174 DNA digested withHinc II was usedas a molecular weight marker(Eurobio).

2.6. Apoptosis assay

Jurkat T cells or PHA-preactivated PBL were appliedto transfected ECV304 cells or untransfected cells at aratio of 1:1 in 24-well plates(Falcon 3047) and�

incubated for 4 or 16 h at 378C in a final volume of 1ml of complete medium with or without mAbs. Cellswere harvested by trypsin-EDTA treatment, and washedtwice in PBS. In order to study the apoptotic status ofthe CD45 cell fraction, we used an original flowq

cytometry method previously described by our groupw32x. Cells (5=10 ) were stained with an optimal5

concentration of anti-CD45-PE mAb for 30 min at 48C, then washed twice with PBS. The cell pellets wereresuspended with Ca binding buffer(Beckman Coul-2q

ter) then 1 mg of 7-amino actinomycin D(7-AAD)(Sigma) and 125 ng of FITC-labeled annexin V(Beck-man Coulter) were added, followed by incubation onice for 10 min in the dark. Analyses were performedimmediately after incubation by flow cytometry using aFACStar flow cytometer(Becton Dickinson). Weplus

used a CD45 mAb to eliminate the residual EC andtarget only the lymphocyte cell fraction. 7-AAD wasused as exclusion dye to detect late apoptotic andnecrotic cells, and annexin V to recognize the apoptoticcell fraction. All fluorescence parameters were simulta-neously collected on the cell preparation to characterizenecrotic and late apoptotic cells(7-AAD annexin V-q

FITC cells), early apoptotic cells(7-AAD annexinq y

V-FITC cells) and viable cells(7-AAD annexin V-q y

FITC cells) in the subset of CD45-PE cells.y q

2.7. Mixed lymphocyte endothelial cell culture (MLEC)

MLEC was performed as previously describedw33x.Briefly, EC were seeded in 96-well tissue culture plates(Falcon 3072) to obtain confluent monolayers�

(3=10 cellsywell) and then irradiated(30 Gy).4

Responding cells(PBL) were added at 10 cellsywell5

with PHA (50 ngyml) in a final volume of 200ml.Cocultures were performed over 3 days at 378C in ahumidified 5% CO incubator. Cells were pulsed with 12

mCi (3.6=10 Bq) tritiated thymidine(Amersham, Les4

Ulis, France) for 18 h before they were collected onfilter discs using an automated harvester(Filtermate�

196, Packard) and counted in ab-counter(TopCount ,�

Packard). Results were expressed in cpm as mean"S.E.of triplicate wells. In costimulatory blocking experi-ments, anti-CD2 and anti-CD11a mAbs were addedduring the 3 days’ MLEC and the percentage of inhibi-tion of PBL proliferation was calculated using theformula:

w(Isotypic control cpmxyinterest mAb cpm)yIsotypic control cpm=100.

2.8. Flow cytometry assay

To detect cell-surface expression of Fas, unstimulatedPBL or PHA-preactivated PBL were washed in PBSand 5=10 cells were incubated with an optimal con-5

centration of mouse IgG1 irrelevant control or ZB4 anti-Fas mAb for 30 min at 48C. After washing in PBS,cells were incubated for an additional 30 min at 48Cwith FITC-conjugated goat anti-mouse IgG F(ab9) .2Cells were then washed in PBS and fixed in 0.5 ml of0.5% paraformaldehyde PBS solution. To detect Fas-expression on PBL from MLEC, coculture was per-formed for 3 days as described below in six-well platesin the same ratio as used for 96-well plates, and theECV304 monolayer was washed twice with PBS tocollect PBL. After staining with anti-Fas mAb or isotyp-ic control and FITC-conjugated secondary mAb, PBLwere washed with PBS and incubated for 30 min at 48C with an optimal concentration of mouse IgG1 tosaturate non-specific sites. Cells were then washed inPBS, incubated for an additional 30 min at 48C withan anti-CD45-PE mAb to eliminate Fas expression onresidual ECV304 cells and fixed with paraformaldehydesolution. Cell surface molecule expression was thenanalyzed by flow cytometry using a FACStar flowplus

cytometer.

2.9. Cytotoxicity assays

Transfected ECV304 cells or untransfected cells wereseeded at 4=10 cellsywell in 96-well plates and main-4

tained in complete medium for 7–8 h before the test.


Fig. 1. FasL mRNA detection by RT-PCR assay in ECV304 and ECV-FasL cells. Total mRNA was isolated from 10 untransfected ECV3045

cells (lanes 2 and 5) and FasL-transfected ECV304 cells(lanes 3 and6). RT-PCR for GAPDH(lanes 1, 2, 3) and FasL(lanes 4, 5, 6) wasperformed with cDNA of cells or with distilled water as negativecontrol (lanes 1 and 4) as described in Section 2.

PBL target cell pellets(8=10 cells) were labeled for6

90 min with 0.1 mCi of Na CrO(DuPont-NEN, Les512 4

Ulis, France), then washed three times in RPMI 1640supplemented with 10% FCS, resuspended in 3 ml ofthe same medium and incubated for 1 h at 378C toallow spontaneous release of Cr. Cells were then51

washed twice in complete medium and applied totransfected ECV304 cells or untransfected cells at aratio of 1:1 in a final volume of 200ml. After 16 hincubation at 378C, 50ml of supernatants was collectedfrom each well and counted in ab-counter(TopCount , Packard, Rungis, France). Spontaneous�

Cr-release(spont. cpm) was measured in wells con-51

taining target cells in the presence of wild ECV304cells. Maximum Cr-release(max. cpm) was deter-51

mined by adding 100ml of 1% Triton X-100 (Sigma)to wells containing labeled PBL only. Each assay wasset up in triplicate and the results were expressed as thepercentage of Cr-release and calculated using the51

formula:

experimental cpmyspont. cpm51% of Cr releasesmax. cpmyspont. cpm

=100

In experiments in which mAbs were used to blockthe Fas–FasL pathway, effector cells and target cellswere incubated with blocking anti-FasL mAbs(4H9),antagonist anti-Fas mAbs(ZB4) or isotypic controlmAbs (2 mgyml each). We verified that mAbs were notcytotoxic for target cells in our experiments.

3. Results

3.1. Specific expression of FasL by transfected ECV304cells

The human endothelial cell line ECV304 was trans-fected with human FasL cDNA subcloned in an expres-sion vector (pREP) containing a Hygromycin4

B-resistant gene. After selection with this antibiotic,several clones of transfected cells were isolated andFasL mRNA was detected by RT-PCR. RT-PCR prod-ucts (Fig. 1) were obtained as a single band of 345 bpfor FasL-transfected ECV304 cells(Fig. 1, lane 6). Thisspecific signal was not found in untransfected ECV304cells (Fig. 1, lane 5). The expected single band of 265bp for GAPDH mRNA expression was analyzed as acontrol (Fig. 1, lanes 2 and 3). One clone was chosenfor subsequent experiments and named ECV-FasL. Sol-uble FasL has been detected by an ELISA test in thesupernatants of this cell culture(data not shown).

3.2. Induction of Jurkat T cell apoptosis by FasL-transfected ECV304 cells

To test the efficacy of FasL protein expressed onECV304 cells, we used the Fas-positive Jurkat T lym-

phoma cell line, which is sensitive to Fas-mediatedkilling.After 4 h coculture, the cells were collected and

stained with CD45 PE mAb, annexin V-FITC and 7-AAD, as described in Section 2. Fig. 2 shows that FasL-transfected ECV304 cells induced a high level of JurkatT cell apoptosis(Fig. 2d) compared with a very lowlevel in the presence of wild type ECV304 cells(Fig.2a). Similarly, the addition of the CH11 agonist anti-Fas mAb(Fig. 2c) induced a dramatic increase in JurkatT cell apoptosis but the addition of the ZB4 antagonistanti-Fas mAb(Fig. 2b) did not. As expected, addingthe blocking ZB4 anti-Fas or 4H9 anti-FasL mAbsduring coculture with ECV-FasL greatly reduced apop-tosis of Jurkat T cells(from 39%(Fig. 2d) to 18% and12%, respectively(Fig. 2f,h) whereas isotypic controlmAbs had no blocking effects(Fig. 2e,g), demonstratingthat Jurkat T cell apoptosis is dependent on the Fas–FasL pathway. It should be noted that in all cases weobserved a small percentage of necrotic cells correspond-ing to the natural mortality of Jurkat T cells in thisexperiment.

3.3. Inhibition of allogeneic lymphocyte proliferation byFasL-transfected ECV304 cells

We then determined the effect of FasL transfectionon allogeneic PBL proliferative response using a MLECassay. In this model, human monocyte-depleted PBLwere incubated on irradiated confluent EC monolayersin the presence of PHA(50 ngyml) and both PHA andEC were necessary to induce T cell proliferation. When


Fig. 2. FasL-transfected ECV304 cells induced Jurkat T cell apoptosis. FACS analysis of Jurkat T cells after 4-h culture with ECV304 cells alone(a), in presence of ZB4 antagonist anti-Fas mAb(b) or in presence of CH11 agonist anti-Fas mAb(c) or with ECV-FasL cells alone(d). Cellswere triple stained with PE-conjugated anti-CD45 mAb, FITC-conjugated annexin V and 7-AAD. The results represent double staining(annexinV-FITC vs. 7-AAD) of CD45 positive cells. Four hours’ culture was performed with ECV-FasL cells alone(d) or in presence of blocking ZB4anti-Fas mAb(f), 4H9 anti-FasL mAb(h) or isotype irrelevant mAbs, mouse IgG1(e) and hamster IgG(g), respectively. One experimentrepresentative of three.

PBL were incubated with either EC or PHA alone, noproliferation was observed(data not shown). As shownin Fig. 3a, the maximal proliferative response wasobserved following 3 days’ coculture with both trans-fected and untransfected ECV304 cells. However, PBL

proliferation was decreased in the presence of ECV-FasL cells, especially after 3 days’ coculture. Thisdecrease was observed with the PBL of all donors tested(mean inhibition of proliferation 37"15%; ns9; datanot shown).


Fig. 3. Inhibition of allogeneic lymphocyte proliferation by ECV-FasL cells. Proliferation of hPBL in the presence of ECV304(j) or ECV-FasLcells (d) for 1–6 days’ incubation(a) or for 3 days’ incubation in the presence of blocking mAbs: anti-Fas(ZB4, mouse IgG1), anti-FasL(4H9,hamster IgG), mouse IgG1(isotypic control) or hamster IgG(isotypic control) (b). Human PBL(10 cellsywell) were added in triplicate to5

irradiated confluent ECV304 or ECV-FasL monolayers(3=10 cellsywell) in the presence of PHA(50 ngyml). At 18 h prior to termination of4

culture, wells were pulsed with tritiated thymidine(3.6=10 Bqywell). The proliferation of hPBL was measured by thymidine uptake, quantitated4

on a liquid scintillationb-counter and expressed in cpm as mean"S.E. of triplicate determination in one experiment representative of four(a)and five experiments(b).

Fig. 4. Cell surface expression of Fas on stimulated and unstimulated hPBL. Unstimulated PBL and PBL stimulated by PHA for 3 days werestained with a mouse IgG1 isotype irrelevant mAb(dotted histograms) or ZB4 anti-Fas mAb(solid line histograms) followed by FITC-conjugatedgoat anti-mouse IgG F(ab9) , and analyzed by flow cytometry. PBL were removed from MLEC for 3 days in the presence of ECV304 cells as2

described in Section 2, stained in the same conditions and then secondary stained with PE-conjugated anti-CD45 mAb. The results represent Fas-staining of CD45-positive cells. Fluorescence intensity is displayed on thex-axis (in log scale) and cell number on they-axis.

We first verified that the transfection of ECV304 cellsdid not alter the two principal costimulation pathways.Monoclonal Abs against CD2 or thea chain of LFA-1(CD11a) were therefore added during the 3 days’MLEC. Blocking of CD2yLFA-3 and LFA-1yICAM-1pathways inhibited PBL proliferation in the presence ofECV-FasL cells in the same way as in the presence ofwild type ECV304 monolayers(data not shown).To confirm that ECV-FasL cells reduced the lympho-

cyte proliferative response by a FasyFasL pathway,blocking anti-Fas or anti-FasL mAbs were added duringthe 3 days’ MLEC with ECV-FasL cells. As expected,an increase in lymphocyte proliferation was observed(Fig. 3b) leading to a proliferation level similar to thatobserved with untransfected ECV304 cells.

3.4. Apoptosis and cytotoxicity of allogeneic lympho-cytes by FasL-transfected ECV304 cells

In order to determine the role of ECV-FasL cells inthe inhibition of PBL-proliferation, the efficiency ofFasL expressed on ECV304 cells to induce apoptosis ofPBL was tested. We stimulated PBL with a suboptimaldose of PHA for 3 days, as for MLEC conditions, torender these cells sensitive to FasL and then verifiedthat Fas expression was induced as effectively as duringMLEC. Fig. 4 shows that a small fraction of fresh PBLexpressed a low level of Fas whereas 100% of PBLexpressed Fas after 72 h of stimulation by PHA aloneor during MLEC with ECV304 cells.


Fig. 5. FasL-transfected ECV304 cells induced PHA-preactivated hPBL apoptosis. Human PBL were stimulated by PHA(50 ngyml) for 3 daysbefore 16-h co-culture with ECV304 cells without mAbs(a), with ZB4 antagonist anti-Fas mAb(b), with CH11 agonist anti-Fas mAb(c) orwith FasL-transfected ECV304 cells(d). The results represent double staining(annexin V-FITC vs. 7-AAD) of CD45 positive cells. One exper-iment is representative of three.

Fig. 6. ECV-FasL cells induced PHA-preactivated hPBL cytotoxicityby the Fas-FasL pathway. Human PBL were preactivated for 3 daysby PHA and labeled with Cr before 16-h co-culture with confluent51

ECV304-FasL monolayers. The cells were cocultured in the presenceof 2 mgyml of anti-FasL mAb(4H9, hamster IgG), anti-Fas mAb(ZB4, mouse IgG1) or isotypic control mAb hamster IgG and mouseIgG1. The result represents the percentage of specific lysis and isexpressed as mean"S.E. of triplicate determinations. Data are fromone experiment representative of three.

As expected, PHA-preactivated PBL apoptosis wasvery low during 16 h coculture with wild type ECV304cells (Fig. 5a). The addition of the CH11 agonist anti-Fas mAb(Fig. 5C) (but not of the ZB4 anti-Fas mAb(Fig. 5b) induced increased PBL-apoptosis. Interesting-ly, increased apoptosis was observed in the same pro-portions after coculture with ECV-FasL(Fig. 5d),indicating that the transfected cells had the same capac-ity to induce PHA-preactivated PBL apoptosis as theagonist anti-Fas mAb.We used a cytotoxicity test to confirm this result and

very little cytotoxicity occurred(below 5%; data notshown) with resting PBL after 16 hours’ coculture withuntransfected or FasL-transfected ECV304 cells. Cr-51

release of PHA-preactivated PBL was then performedand, despite interindividual variations, ECV-FasL cellsinduced more than 10% cytotoxicity of PHA-preactivat-ed PBL in 6 experiments of 7(mean 16.6"8.0% ofCr release; data not shown). Cytotoxicity induced by51

FasL-expressing ECV304 cells against PHA-preactivat-ed PBL was totally abolished by the addition of mAbsdirected against either FasL or Fas(Fig. 6).Thus, the transfection of FasL on the EC surface

induced inhibition of allogeneic T cell-proliferation that


was accompanied by T-cell apoptosis in a FasL-depend-ent manner.

4. Discussion

This study was undertaken to investigate the effectsof FasL expression on the proliferative response of PBLto EC in a human allogeneic model. Apoptosis ofallogeneic T cells has been observed with FasL-trans-fected APC such as murine macrophagesw19x anddendritic cellsw21x. Nevertheless, Matsue failed to blockthe induction of immune response to alloantigens witha FasL-transfected macrophage cell linew20x. Theseresults suggest that the effects of FasL-expression onthe allogeneic response may depend on the cells usedto express FasL. Graft EC regulate graft infiltration byallogeneic activated T cells. Moreover, they are able toinduce the proliferation of allogeneic T cells in vitrosuch as DCw24–27x. Therefore, EC expressing highmembrane levels of FasL could induce specific apoptosisof alloantigen-activated T cells. Transfection of EC toexpress FasL seems to be possible as these cells areresistant to Fas-mediated apoptosis despite Fas-expres-sion. Indeed, EC remains viable in the presence of theagonist anti-Fas mAb, even when the expression of thisreceptor is upregulated by treatment with interferong,whereas macrophages do notw34x. EC are also resistantto cell death when FasL is highly overexpressed on thecell surface by adenovirus-mediated gene transfer, incontrast to other cell types such as vascular smoothmuscle w35x or islet b cells w17x that express Fas andare sensitive to FasL-induced apoptosis. This may bedue to different capacities to regulate anti-apoptoticmolecules that may protect them from fratricidal orsuicidal induction of apoptosis. Although it has beenreported that FasL expression on a graft can lead toneutrophil infiltration and rapid destruction of the organw16–18x, it has never been reported that FasL-overex-pressing EC led to organ destruction by a FasyFasLpathway. On the contrary, the low level of FasL-expres-sion on HUVEC, which has recently been demonstrated,seems to be of physiological importance because FasLis downregulated by TNFa suggesting that leukocyteadherence and transendothelial migration during inflam-matory responses takes place by controlling FasL-expression of ECw36,37x. The authors demonstratedthat HUVEC can induce slight apoptosis of adherentleukocytesw38x and this might mean that EC engineeredto express FasL could affect activated lymphocytes invivo. It therefore seems more interesting to transducegraft endothelium, instead of inducing FasL-transfectedDC, to insure local production of FasL and inducealloreactive T cell apoptosis in the graft.We showed that ECV304 cells could be successfully

transduced to express human FasL and that the transfec-tion revealed a very stable expression of FasL during

long-term culture. FasL expressed on EC was functionalin this study as it induced high apoptosis of allogeneicT cells similar to that observed with an agonist anti-FasmAb. Induction of lymphocyte apoptosis by FasL-transfected EC has already been demonstrated in axenogeneic model with bovine aortic endothelial cellsw39x. More importantly, transfection of FasL on ECV304cells partially inhibited PBL proliferation in MLECwithout affecting the main costimulation pathways(CD2and LFA-1). We and others have previously shown thatHUVEC induce high levels of allogeneic PBL prolifer-ation by CD2- and LFA-1-dependent pathwaysw22x(and data not shown). We confirmed this result with theECV304 cell line. Although a recent study revealed thatthe ECV304 cell line was cross-contaminated with thebladder cancer epithelial cell line T24y83 w40x, thesecells express the same costimulatory molecules asHUVEC and EA.hy926 cells(w41x and data not shown)and are an appropriate model for T-cell proliferationstudies as well as HUVEC. On the other hand, blockingthe FasyFasL interaction in MLEC completely restoredproliferation. Because stimulation of PBL by PHA orduring MLEC induced high levels of Fas and ECV-FasLinduced apoptosis of PHA-stimulated PBL, this stronglysuggests that the decrease in PBL proliferation is at leastpartially due to FasL-induced apoptosis of PBL.The decrease in proliferation observed with FasL-

transfected cells during MLEC was incomplete,(approx.40%) despite Fas expression on 100% of PBL, corre-sponding to the partial apoptosis of allogeneic T-lym-phocytes. This may be explained by the fact thatFas-expression is necessary to induce T-cell apoptosisby FasL, but not sufficient. Indeed, T-cells which areactivated by TCR in an antigen-specific manner or withpharmacologic agents such as PHA produce a variety ofcytokines, cell surface costimulatory molecules and anti-apoptotic molecules such as Bcl-2 family proteinsw42,43x in addition to Fas and FasL expression. It wasshown in a recent study that down-regulation of Bcl-2-expression and a low level of IL-2 production coincidedwith the onset of T-cell sensitivity to Fas-mediatedapoptosis w44x. Moreover, the authors observed thatoptimal coculture conditions induced apoptosis in onlyapproximately 40% of target cells. In our model, weobserved 20% apoptosis of PBL preactivated for 3 days.This time corresponded to maximum proliferation andperhaps to minimum PBL sensitivity to FasL-inducedapoptosis. In contrast, the inhibition of proliferation wasgreater after the third day of coculture, and in someexperiments we used PHA for 7 days to render T-cellsmore sensitive to apoptosis. In these conditions approx-imately 40% cell death occurred(data not shown)induced by FasL-expressing ECV304 cells. This incom-plete ECV-FasL-induced apoptosis suggests that theefficacy of an in vivo model would be optimized usingdrugs that favor activation-induced cell death of T cells,


such as rapamycin which, unlike anticalcineurin drugs,reduces Bcl2 expressionw45x. Moreover, it might beinteresting to use anti-CD2 andyor anti-LFA-1 mAb toinduce additional effects for blocking T cellproliferation.In summary, FasL-expressing endothelium seems to

be an interesting route by which to deliver a deathsignal to activated lymphocytes during transplantationand it is necessary to define the conditions that maximizetheir ability to influence immune responses in vivo. Itmay therefore be interesting to perfuse the organ exvivo with a recombinant adenovirus encoding the FasLgene before grafting. This strategy has recently beenused with other genes to induce tolerance in cardiacw46x and liver w47x allograft transplantation, thus reveal-ing successful outcome of a feasible and potentiallyclinically relevant system of gene delivery.

Acknowledgments

We thank Dr S. Nagata for providing the FasL cDNA.We also thank Mrs H. Aget, Mr P. Louisot, Mr S.Muller, Professor O. Le Floch and Professor J.C. Bes-nard for their valuable collaboration. We are very grate-ful to Drs A. Legrand, and D. Brand for their assistanceduring molecular biology experiments and Dr T. Avrilfor his valuable assistance. S. Cappellesso was supportedby a grant from the Fondation Merieux and Ouest´Transplant.

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fasl-transfected endothelial cells decrease the proliferative response of allogeneic pbl

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