Dendritic Cells Genetically Engineered to Express Fas Ligand
Induce Donor-Specific Hyporesponsiveness and Prolong
Wei-Ping Min,* Reginald Gorczynski,* Xu-Yan Huang,* Michelle Kushida,†Peter Kim,†
Masayuki Obataki,†Ji Lei,* Rakesh M. Suri,* and Mark S. Cattral2*
Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic
cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these
interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules
and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the
immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this
study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas?cells. FasL-trans-
fected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hypore-
sponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that
transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our find-
ings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of
organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune
responses. The Journal of Immunology, 2000, 164: 161–167.
nized, recent evidence suggests that DC are also capable of induc-
ing donor-specific hyporesponsiveness (2). It is not yet clear
whether these apparently opposing functions of DC reflect distinct
subpopulations of DC (3, 4), or alternatively, distinct functions
expressed at unique stages in the developmental cycle of the same
cell (5). Understanding the nature of the heterogeneity of DC func-
tion would promote the development of DC-based immunotherapy
for the treatment of many diseases, including the induction of tol-
erance in transplantation and in autoimmune disorders.
Genetic modification of DC with genes encoding immunoregu-
latory molecules is an alternative approach for artificial generation
of tolerogenic DC. Indeed, recent reports suggest that transfection
of DC with IL-10 and TGF-? can increase their tolerogenic potential
(6, 7). Several attributes make DC ideal vehicles for the delivery of
such molecules. They are potent activators of naive T cells, a function
related to their Ag-processing capacity and to high levels of expres-
sion of MHC and costimulatory molecules. In addition, they have
unique migratory capability, enabling them to move from peripheral
endritic cells (DC)3are APC that play a critical role in
the initiation of immune responses (1). Although the po-
tent immunostimulatory capacity of DC is well recog-
tissues to secondary lymphoid organs, where they interact with T and
B cells (8, 9). The cognate recognition of DC and T cells provides the
theoretic opportunity of these immunomodulatory molecules to influ-
ence the immune response in an Ag-specific manner.
One molecule that may enhance the tolerance-inducing capacity of
DC is Fas ligand (FasL), a type II integral membrane protein that
belongs to the TNF superfamily (10). Engagement of Fas by FasL
initiates a signaling cascade that leads to apoptotic cell death of Fas-
bearing cells. Apoptosis induced by Fas/FasL interactions is thought
T cell homeostasis and lymphocyte-mediated cytotoxicity. FasL is
expressed in immunoprivileged organs, including the eye and testis,
where it has been proposed to contribute to their tolerogenic milieu
and paucity of infiltrating inflammatory cells (11–13). There is evi-
dence that FasL constitutively expressed on splenic DC and bone
bone marrow cells was shown to be dependent on the expression of
FasL on the infused cells (16). In the studies described below, we
investigate the immunomodulatory effect of DC transduced to express
high levels of FasL in vitro and in vivo.
Materials and Methods
Male C57BL/6 (H-2b), BALB/c (H-2d), and C3H (H-2k) mice were pur-
chased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6-lpr/lpr
mice were purchased originally from The Jackson Laboratory and bred in
our animal facility. All mice were used at 8–12 wk of age.
Generation of bone marrow-derived DC
DC were generated from bone marrow progenitor cells, as described by
Inaba et al. (17) and modified by Suri et al.4Briefly, bone marrow cells
*Department of Surgery and Multiorgan Transplant Program, Toronto Hospital Re-
search Institute, University of Toronto, Toronto, Ontario, Canada; and†Hospital for
Sick Children, University of Toronto, Toronto, Ontario, Canada
Received for publication August 10, 1999. Accepted for publication October
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Physician Services Foundation, Juvenile Diabetes
Foundation International, and the Multiorgan Transplantation Programme.
2Address correspondence and reprint requests to Dr. Mark S. Cattral, The Toronto
Hospital, NU10-145, 621 University Avenue, Toronto, Ontario, Canada, M5G 2C4.
E-mail address: email@example.com
3Abbreviations used in this paper: DC, dendritic cell; DNFB, dinitrofluorobenzene;
FasL, Fas ligand.
4R. M. Suri, M. B. Lutz, A. L. J. Ogilvie, S. Robner, M. Nimi, N. Kukutsch, G.
Schuler, and J. M. Austyn. 1999. Stably immature dendritic cells induce T cell un-
responsiveness in vitro and prolong allograft survival in vivo. Submitted for publication.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00
were flushed from femurs and tibias of C57BL/6 mice, washed, and cul-
tured in 6-well plates (2 ? 106/ml) in 4 ml RPMI 1640 containing rGM-
CSF (10 ng/ml; Peprotech, Rocky Hill, NJ) and mouse rIL-4 (10 ng/ml;
Peprotech). All media and additives were documented to be free of LPS
contamination (18). Nonadherent granulocytes were removed after 48 h of
culture, and fresh media added every 48 h. By day 4 to 6 of culture,
proliferating clusters of cells with typical dendritic morphology were seen,
and by day 7 to 9 more than 90% of the cells expressed the DC cell surface
marker DEC-205. The proportion of cells staining for T (CD3) and B
(B220) lymphocytes was consistently ?3%.
pBK-CMV phagemid vector (2 ?g) containing full-length human FasL
cDNA or empty control vector was incubated with 8 ?l of Lipofectin (Life
Technologies, Gaithersburg, MD) in a volume of 100 ?l of PBS at room
temperature for 45 min. This mixture was added to 7-day cultured DC in
a final volume of 1 ml of serum-free medium. After 4-h incubation at 37°C
with 5% CO2, the cells were washed and cultured in RPMI 1640 with 10%
FCS for 48 h.
Phenotypic analysis of DC was performed at day 9 (2 days after gene
transfection) of culture using an EPICS XL-MCL Cell Analysis System
(Coulter, Miami, FL). The following mAbs were purchased from Cedar-
lane Laboratories (Hornby, Ontario, Canada), unless otherwise indicated,
and used for staining cells as primary mAbs: anti-DEC205 (clone NLDC-
145), anti-mouse H-2b, anti mouse I-Eb, and anti-mouse CD40 (PharMin-
gen, San Diego, CA). The secondary mAbs used were FITC-conjugated
anti-rat IgG2a (Caltag Laboratories) or FITC-conjugated anti-mouse IgG2a
(PharMingen). The following mAbs, purchased from PharMingen, were
used directly as FITC-conjugated mAb: anti-mouse CD80 (B7-1), anti-
mouse CD86 (B7-2), anti-mouse CD3?, anti-mouse CD4, anti-mouse CD8,
anti-mouse Mac1, and anti-mouse B220. FasL-transfected and control DC
were stained with anti-human FasL mAb (MBL, Nagoya, Japan) or isotype
control, followed by secondary PE-conjugated anti-hamster IgG (Cedar-
Total RNA was extracted from DC (1 ? 107) 48 h after transfection with
FasL or the empty control vector, with TRIzol reagent (Life Technologies),
as per the manufacturer’s instructions. First strand cDNA was synthesized
using an RNA PCR kit (Life Technologies) with the supplied oligo(dT)16
One microliter of the reverse-transcription reaction product was used for
the subsequent PCR reaction. The sequence of the human FasL primers,
which generated a 293-bp fragment of human FasL, was: sense, 5?-AAT
AGGCCACCCCAGTCCA-3?; antisense, 5?-CCCCTCCATCATCACCA
GA-3. The sequence of the mouse ?-actin primers was: sense, 5?-AGGCA
TCCTGACCCTGAAGTAC-3?; antisense, 5?-TCTTCATGAGGTAGTCT
GTCAG-3?. The samples were denaturated for 1 min at 94°C, annealed for
1 min at 53°C, and extended for 1 min at 72°C, for a total of 35 cycles. The
PCR products were subjected to electrophoresis on 1.5% agarose gel con-
taining ethidium bromide and visualized by UV illumination. ?-actin was
used as an internal control for RNA integrity.
DNA fragmentation (JAM assay)
Jurkat cells (1 ? 104) were labeled with 5 ?Ci/ml [3H]thymidine for 4 h
at 37°C (19) and seeded in triplicate in U-bottom 96-well tissue culture
plates as target cells. They were incubated with the indicated ratio of FasL-
or control-transfected DC in a total volume of 200 ?l/well for 18 h. Un-
fragmented high m.w. DNA was harvested onto glass fiber filters and
counted in a Beckman scintillation counter. Data are expressed as percent-
age of DNA fragmentation: 100 ? [(1 ? cpm in experimental group)/(cpm
of unstimulated targets alone)].
Mixed leukocyte reactions
Two days after gene transfection, FasL- or control-transfected DC (1 ?
104) were treated with 50 ?g/ml of mitomycin C at 37°C for 20 min,
washed twice with RPMI, and seeded in triplicate in flat-bottom 96-well
culture plates (Corning Glass, Corning, NY) for use as stimulator cells.
Responder spleen cells (2 ? 105/well) from BALB/c mice were added to
the DC in a total volume of 200 ?l of RPMI 1640 containing 10% FCS, 50
?M 2-ME, 1 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml strep-
tomycin, and cultured in a humidified atmosphere of 5% CO2in air at
37°C. The cells were pulsed with 1 ?Ci of [3H]thymidine (Amersham,
Arlington Heights, IL) at the indicated time points, cultured an additional
16 h, and collected onto glass fiber filters; [3H]thymidine incorporation was
quantified using a Beckman scintillation counter. Results were expressed as
the mean cpm of triplicate cultures.
For MLR using C57BL/6-lpr/lpr responder splenocytes, the stimulating
DC were generated from BALB/c mice and transfected as described above.
Blockade of FasL
Soluble Fas:Fc fusion protein (Alexis, San Diego, CA), or control human
IgG (Calbiochem, San Diego, CA) was added at the beginning of MLR and
DNA fragmentation assays at the concentrations indicated, and as de-
scribed by Desbarats et al. (20).
Flow-cytometric analysis for T cell apoptosis
Quantitative determination of T cell apoptosis was analyzed by flow cy-
tometry, as described by Nicoletti et al. (21). Purified T cells were activated
with Con A (5 ?g/ml) for 72 h and collected over Ficoll-Hypaque. Viable
blasts (5 ? 106) were incubated with 5 ? 105FasL-transfected or control
DC for 24 h. Cell suspensions were centrifuged at 200 ? g for 10 min,
gently resuspended in 1 ml of hypotonic fluorochrome solution (50 ?g/ml
propidium iodide, 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA,
0.1% Triton X-100), and stored in the dark for 3–4 h before being analyzed
by flow cytometry. The apoptotic cells were quantified as the percentage of
cells with subdiploid DNA.
Donor-specific hyporesponsiveness induced by allogeneic DC
FasL- or empty control vector-transfected BALB/c DC (2 ? 106) were
injected i.p. into groups of nine C57BL/6 and four C57BL/6-lpr/lpr mice
at 3-day intervals for a total of six injections, as per Zhang et al. (22). Mice
were sacrificed 3 days after the last injection, and MLR cultures were
initiated with fresh mitomycin C-treated BALB/c spleen stimulator cells, as
described above. Paraffin sections of Formalin-fixed liver and spleen bi-
opsies from these animals were stained with hematoxylin and eosin for
Heterotopic heart transplantation with DC pretreatment
FasL- or empty control vector-transfected BALB/c DC (2 ? 106) were
injected i.p. into groups of six C57BL/6 at 3-day intervals for a total of six
injections. Within 3 days of the last injection, vascualized heterotopic heart
transplants from BALB/c mice were performed and monitored daily, as
described (23). Rejection was defined by the cessation of heartbeat.
Continuous variables were compared with Student t tests. Cardiac graft
survival curves were calculated by the Kaplan-Meier method, with differ-
ences between groups compared by the log-rank test. A p value ?0.05 was
Transfection and expression of functional FasL in DC
DC were propagated from bone marrow cells cultured with GM-
CSF and IL-4, as described in Materials and Methods. By the
seventh day of culture, cells with characteristic DC morphology
and immunophenotype (DEC-205?, MHC class II?, CD40?,
CD80?, CD862?, Mac1low, B220?, CD3?, CD4?, CD8?) were
To optimize gene delivery into DC by lipofection, we first used
the Escherichia coli ?-galactosidase gene under the control of the
CMV immediate promoter as a reporter system. The optimal DNA
(?g):Lipofectin (?l) ratio was found to be 1:4 using an incubation
time of 4 h. With these conditions, the transfection efficacy was
50–70%, and cell viability was more than 90% (data not shown).
Transfection of DC with the FasL vector construct using the same
conditions resulted in high levels of FasL gene expression, but not
with the empty control vector, as determined by RT-PCR and
flow-cytometric analysis (Fig. 1). Forty-eight hours after transfec-
tion, ?50% of the DC stained positive for FasL (range, 29% to
68% in 20 independent experiments). Transfection with either
FasL or the control vector did not adversely affect cell viability or
the expression levels of cell surface molecules (Fig. 2).
162 Fas LIGAND GENE TRANSFECTION OF MURINE DC
The biological activity of FasL in transfected DC was confirmed
in a DNA fragmentation assay that used Fas?Jurkat cells (Fig.
3A). DC transfected with FasL induced high levels of DNA frag-
mentation as compared with those transfected with the control vec-
tor. Moreover, we found that the addition of blocking Fas-Fc ef-
fectively inhibited DNA fragmentation. This inhibition was dose
dependent and specific, as it was unaffected by the addition of
control Ig (Fig. 3B).
FasL-transfected DC down-regulate T cell responses in vitro
To assess the functional activity of transfected DC in stimulating
allogeneic T cell responses, MLR reactions were performed using
FasL- or control-transfected DC. Allogeneic T cell proliferation
was significantly decreased when incubated with FasL-DC, but not
with the control DC (Fig. 4A). The stimulatory capacity of
FasL-DC could be restored in the presence of soluble Fas-Fc, but
not control Ig, indicating that inhibition of allogeneic MLR by
FasL-DC was specific to FasL (Fig. 4B). Furthermore, these results
confirmed that DC transfected with FasL were viable and capable
of presenting alloantigen.
FasL-transfected DC do not inhibit T cell responses in Fas-
To further establish the functional relevance of Fas:FasL interac-
tions in the inhibition of the MLR response by FasL-DC, the MLR
studies were repeated with lymphocytes from C57BL/6-lpr/lpr
mice, which do not express Fas. In these experiments, DC were
propagated from BALB/c bone marrow cells and transfected as
described above. FasL-DC inhibited the proliferative response of
wild-type lymphocytes, whereas there was no inhibition of
C57BL/6-lpr/lpr lymphocytes (Fig. 5).
FasL mRNA expression in FasL-DC and human spleen by RT-PCR; bot-
tom, ?-actin mRNA expression by RT-PCR. Control, empty vector-trans-
fected DC. B, FasL expression in DC by flow-cytometric analysis. Two
days after transfection with FasL or empty control vector, DC were col-
lected and stained with anti-human FasL mAb and PE-conjugated anti-
hamster IgG, as described in Materials and Methods. Thin dotted lines
denote isotype control.
A, Expression of human FasL in murine DC. Top, Human
were transfected after 7 days of culture in GM-CSF and IL-4 with empty
control vector (top) or FasL (bottom). They were stained for DEC-205,
MHC class II, CD40, and CD86, and analyzed by flow-cytometric analysis
48 h after transfection. Thin dotted lines denote isotype controls.
Phenotypic analysis of DC after transfection with FasL. DC
[3H]thymidine and incubated with graded numbers of FasL- or empty vec-
tor-transfected DC, for 18 h. The percentage DNA fragmentation was cal-
culated as described in Materials and Methods. B, Inhibition of DNA frag-
mentation by soluble Fas:Fc. Radiolabeled Jurkat cells were mixed with
FasL- or control-transfected DC (E:T ratio 50:1), and human Fas:Fc protein
or control human IgG was added at the beginning of the cultures. Results
are expressed as mean ? SD, and are representative of three independent
experiments; ?, p ? 0.02.
DNA fragmentation assay. A, Jurkat cells were labeled with
in triplicate with 2 ?105BALB/c responder spleen cells and 1 ? 104
mitomycin-treated DC that were transfected with FasL or control vectors.
Proliferation at the indicated time points was determined by [3H]thymidine
incorporation. B, The stimulatory capacity of FasL-DC was restored with
the addition of soluble Fas:Fc, but not control human IgG, at the beginning
of a 3-day MLR. Results are expressed as mean cpm ? 103? SD, and are
representative of three different experiments; ?, p ? 0.01.
FasL-DC inhibit allogeneic MLR. A, Cultures were set up
163The Journal of Immunology
FasL-DC induce T cell apoptosis
To explore the mechanism by which FasL-DC inhibit MLR, we
determined their ability to induce apoptosis of activated T cells
using a quantitative fluorometric assay for hypodiploid DNA. Con
A blasts were incubated with FasL-transfected or control-trans-
fected DC for 24 h. As shown in Fig. 6, DC transfected with FasL
induced significantly higher levels of apoptosis than the control-
FasL-DC induce unresponsiveness after injection into mice
To determine whether Ag-specific inhibition of alloreactivity
would be seen after pretreatment of naive mice with FasL-DC,
C57BL/6 or C57BL/6-lpr/lpr mice received i.p. injections of 2 ?
106BALB/c-derived DC (FasL or control vector transfected) at
3-day intervals for a total of six injections. Three days after the last
injection, all mice were sacrificed and MLR cultures were initiated
using spleen cells from all individuals stimulated with BALB/c
(allogeneic) or C3H (third-party) mitomycin-treated spleen stim-
ulator cells. Control cultures used cells from nonimmunized (no
DC treatment) mice.
FasL-DC treatment induced allospecific hyporesponsiveness
during restimulation with fresh stimulators in vitro, while spleno-
cytes from mice treated with control DC showed normal secondary
responsiveness (upper panel of Fig. 7). This suppression of sec-
ondary proliferative responses by FasL-DC was abrogated in lpr
mice (lower panel of Fig. 7).
FasL-DC prolong cardiac allograft survival
The capacity of FasL-DC to induce alloantigen-specific hypore-
sponsiveness in vivo suggested that these cells might prolong al-
lograft survival. We tested this possibility with a vascularized het-
erotopic cardiac transplant model. Groups of five to six C57BL/6
mice were pretreated with i.p. injections of 2 ? 106BALB/c con-
trol- and FasL-DC at 3-day intervals for a total of six injections. As
shown in Fig. 8, mean graft survival was significantly longer in
mice pretreated with FasL-DC as compared with both untreated
controls and those pretreated with control-DC (20 ? 4 vs 10 ? 2
and 9 ? 3 days, respectively; p ? 0.01 by log-rank test).
In this study, we show that primary murine bone marrow-derived
DC can be successfully transduced to express high levels of FasL.
FasL-DC were capable of killing Fas?cells through apoptosis,
down-regulating allogeneic MLR in vitro, and inducing donor-
specific hyporesponsiveness to alloantigen in vivo. Finally, we
lpr mice. DC (1 ? 104) propagated from BALB/c BM cells were trans-
fected with FasL or control vector, treated with mitomycin C, and cocul-
tured with 5 ? 105responder splenocytes from C57BL/6-lpr/lpr or
C57BL/6 wild-type mice for 3 days. Proliferation was determined by
[3H]thymidine incorporation. Results are expressed as mean cpm ? 103?
SD, and are representative of three different experiments; ?, p ? 0.02.
FasL-DC do not inhibit proliferation of splenocytes from
propagated from C57BL/6 mice and transfected with FasL or control vec-
tor, were cocultured with BALB/c Con A blasts (1 ? 106) for 24 h. Flow-
cytometric analysis for subdiploid DNA is indicated in the region marked
M1. The results are representative of three independent experiments.
FasL-DC induce apoptosis of Con A blasts. DC (1 ? 105),
vivo. Nine C57BL/6 mice per group (upper panel) or four C57BL/6-lpr/lpr
(lower panel) mice per group received six injections of 2 ? 106BALB/c
DC transfected with FasL or control vector, or were untreated (see Mate-
rials and Methods and text for details). Spleen cells were harvested 3 days
after the last injection and stimulated in vitro with mitomycin-treated
BALB/c or C3H (third party) spleen cells for 3 days. The proliferative
response, as determined by [3H]thymidine incorporation, is shown for each
mouse. The horizontal bars represent the mean proliferative response of
each group; ?, p ? 0.001 compared with mice treated with control DC.
Data are representative of two independent experiments
FasL-DC induce donor-specific hyporesponsiveness in
164 Fas LIGAND GENE TRANSFECTION OF MURINE DC
show that these cells can prolong organ allograft survival when
administered before transplantation.
A promising strategy for inducing tolerance to alloantigens is
infusion of tolerogenic DC. Previous studies have shown that im-
mature DC, which characteristically express low levels of costimu-
latory molecules such as CD80 and CD86, can promote the de-
velopment of donor-specific tolerance and prolong cardiac and
islet allograft survival (24, 25). In vitro growth conditions can be
manipulated to enhance the generation of immature DC from bone
marrow cells (26, 27); however, subsequent maturation in vivo
may limit their tolerogenic potential (25). Genetic engineering of
DC with genes encoding immunoregulatory molecules provides an
alternative method of generating tolerogenic DC that might be
more effective. The feasibility of this approach is supported by
recent studies showing that DC can be genetically modified using
retroviral and adenoviral vectors to express model tumor Ags that
promote both protective and antitumor immunity (28–30), and cy-
tokines that augment (IFN-?, IL-12) and inhibit (IL-10, TGF-?)
immune responses (6, 7, 31). Other molecules that could poten-
tially enhance the capacity of DC to promote tolerance include
CTLA4-Ig, which would block CD80 and CD86 costimulatory
pathways, and OX-2, which we and others have recently shown
down-regulates T cell responses through a unique costimulatory
pathway (32, 33).
Apoptosis induced by Fas/FasL interactions is one mechanism
implicated in peripheral T cell tolerance. Enhanced or elevated
expression of FasL on specific tissues or cells by a transgene tech-
nique is being extensively applied for gene therapy of tumors (34)
(35), rheumatoid arthritis (36), autoimmune diseases (37, 38), and
regulation of rejection in transplantation (39–41). Thus, signifi-
cant prolongation of allogeneic grafts has been achieved by di-
rectly transducing FasL gene into donor tissue or organs before
transplantation (39, 40), or by cotransplanting FasL-transfected
carrier cells (41). The studies we report suggest that DC trans-
fected with FasL are capable of down-regulating T cell responses.
This modulating effect appears to function by inducing T cell ap-
optosis via a Fas/FasL pathway, because inhibition was blocked by
a Fas:Fc fusion protein, and failed to occur in lymphocytes from
C57B6-lpr/lpr Fas mutant mice. Furthermore, we show that sys-
temic administration of these cells not only inhibits donor-specific
alloresponsiveness, but also prolongs cardiac allograft survival.
Our results are consistent with those of Zhang et al., who re-
cently showed that a FasL-transfected macrophage cell line was
capable of inducing allogeneic T cell hyporesponsiveness in vivo
(22). They provide evidence with a CD8?transgenic model that
FasL-transfected macrophages are capable of inducing rapid and
profound clonal deletion of Ag-specific T cells (22). It is interest-
ing to consider the possibility that the peritoneal macrophage cell
line used these studies, which express MAC1, F4/80, MHC class
I and II, as well as significant amounts of B7 contained popula-
tion(s) of DC (42). This would explain their finding of fluoro-
chrome-labeled cells in splenic T cell areas after i.v. injection,
which is a property characteristic of DC (43). Furthermore, it
would be valuable to investigate whether the clonal deletion sug-
gested in their study might be due to the migration of Ag-specific
clones out of the spleen (44)or down-regulation of CD8 itself (45).
Another potential mechanism that might contribute to the hypore-
sponsiveness induced by FasL-DC is polarization of Th cells to-
ward a Th2 phenotype, as Th1 cells are reported to be more sen-
sitive to FasL-mediated apoptosis than Th2 cells (46). These issues
are currently under investigation in our laboratory using highly
purified subsets of FasL-DC in an allospecific transgenic model.
Similarly, Matsue et al. have recently reported the generation of
killer DC from an immortalized DC line (47). The authors show
that peptide-pulsed FasL-expressing DC are capable of inducing
Ag-specific T cell hyporesponsiveness in delayed-type hypersen-
sitivity and contact hypersensitivity responses both prophylacti-
cally and therapeutically. The homing capacity and mechanism of
action of these cells in vivo were not addressed, however. Inter-
estingly, these killer DC were unable to block the induction of
immune responses to alloantigen or Ab responses to nominal Ag,
suggesting that there may be important differences between these
cells and those used in our and Zhang’s (22) study.
Although our study indicates that FasL-DC can prolong survival
of vascularized cardiac allografts, all grafts ultimately failed from
rejection, suggesting that complete (or lasting) depletion of allo-
reactive cells did not occur. These results are in agreement with
those of Matsue et al., who showed that hyporesponsiveness to
dinitrofluorobenzene (DNFB) following treatment with a DNFB-
pulsed FasL-DC clone was temporary and could be reversed with
subsequent DNFB resensitization (47). Ongoing studies in our lab-
oratory are being performed to determine whether the duration of
allograft survival can be extended.
Earlier reports have shown that systemic administration of anti-
Fas Abs and FasL-expressing viruses to mice causes massive he-
patocyte apoptosis and liver failure, which has been attributed to
high levels of expression of Fas on hepatocytes (48, 49). FasL also
has proinflammatory properties mediated by recruitment and acti-
vation of neutrophils (50, 51). In our studies, however, treatment
of mice with FasL-DC was remarkably well tolerated. Further-
more, histologic examination of livers from both the wild-type and
lpr/lpr-treated mice showed no evidence of hepatitis or hepatocyte
apoptosis (data no shown). Specific homing patterns of DC to sec-
ondary lymphoid organs may account for the lack of toxicity, and
are currently being studied.
Recently, Matsue et al. reported that ligation of Fas on DC by
FasL on T cells is capable of inducing DC apoptosis, and sug-
gested that this may be one mechanism by which immune re-
sponses are normally terminated (52). Thus, one caveat of trans-
fecting DC with FasL is that it might directly trigger DC apoptosis.
However, the viability of DC after transfection in our studies was
consistently greater than 90%, as determined by trypan blue ex-
clusion and annexin V staining (data not shown). Whether FasL/
Fas interactions induce DC apoptosis is most likely dependent on
a variety of factors, including DC origin (e.g., spleen vs bone mar-
row), maturation stage, and expression levels of antiapoptotic pro-
teins such as Bcl-2 and Bcl-xL(53, 54). The use of phenotypically
mature DC (MHC IIhigh, DEC205?, CD40?, CD862?) in the
cipients were pretreated i.p. with six injections of 2 ? 106BALB/c DC
transfected with FasL or control vectors, or were untreated. Within 3 days
of the final injection, BALB/c cardiac allografts were transplanted, and
monitored daily. p ? 0.01 by log-rank test. Data are representative of two
FasL-DC prolong cardiac allograft survival. C57BL/6 re-
165The Journal of Immunology
present study, which express higher levels of Bcl-2 than immature
DC, may account for their tolerability to FasL transfection. In ad-
dition, several ligand/receptor interactions have been shown to af-
fect the outcome of FasL/Fas interaction. For example, DC sur-
vival signals expressed on T cells, such as TRANCE and CD40L,
can prevent Fas-induced apoptosis (55–57).
Another potential limitation of using FasL-DC to inhibit im-
mune responses is that the level of expression of functional Fas on
naive T cells may be insufficient to trigger apoptosis. In fact, Nish-
imura et al. (58) have shown that murine T cells are resistant to
anti-Fas mAb treatments. However, recent studies by Suda et al.
(59) indicate that the membrane-bound form of FasL is capable of
killing both fresh and in vitro activated peripheral blood T cells,
whereas soluble FasL only kills the latter. The ability of FasL-DC
to induce systemic T cell hyporesponsiveness in our study may be
due, at least partly, to the use of the entire (membrane form) FasL
molecule, which would be expected to provide a potent apoptotic
A variety of gene delivery methods has been reported for trans-
fecting DC, including viral vectors (7, 60–62), electroporation
(22, 63), and gene guns (64). We have also used a replication-
deficient adenoviral vector to transfect DC with FasL, but found
that despite providing a high transfection efficiency, cell viability
was significantly less than with the Lipofectin method used in the
present study (unpublished observations). Whether this was due to
direct viral injury or to the level of FasL expression is unclear.
Other important advantages of liposomal gene transfer are the
avoidance of potential biological hazards and antigenicity, which
are associated with viral vectors.
In summary, the results of the present study suggest that trans-
fection of DC with FasL may be a practical way to suppress al-
lospecific immune responses in transplant recipients, and possibly
for the treatment of autoimmune diseases. Furthermore, our results
also highlight the potential of using DC genetically engineered to
express other immunoregulatory genes. Currently, our efforts are
directed toward optimizing gene delivery and expression in DC,
and defining the conditions that maximize their ability to modulate
in vivo immune responses.
We thank Dr. Li Zhang for helpful discussions and critical reading of the
manuscript, and Marlene Kennedy for secretarial assistance.
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167The Journal of Immunology