Induction of Tolerance to Cardiac Allografts Using Donor
Splenocytes Engineered to Display on Their Surface an
Exogenous Fas Ligand Protein1
Esma S. Yolcu,2,3*†Xiao Gu,2* Chantale Lacelle,* Hong Zhao,* Laura Bandura-Morgan,*
Nadir Askenasy,‡and Haval Shirwan3*†
The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immuno-
modulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing
FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and
clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently
engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The
i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor,
but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and
induction/expansion of CD4?CD25?FoxP3?T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance
as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite
survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display
on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential
application to regenerative medicine, transplantation, and autoimmunity. The Journal of Immunology, 2008, 181: 931–939.
end-organ failure, autoimmunity, malignancies, and congenital en-
zyme deficiencies. However, rejection of foreign grafts by immu-
nocompetent recipients presents a major hurdle for the routine ap-
plication of transplantation in the clinic (1, 2). Although general
immunosuppression is presently used to control foreign graft re-
jection, the chronic use of nonspecific immunosuppression is not
only inefficient in preventing graft rejection but also associated
with various complications, including but not limited to infections,
malignancies, and organ toxicity (1, 2). Induction of specific tol-
erance to foreign grafts has the potential to overcome these com-
plications, and as such has been the subject of intense studies since
the first successful organ transplantation in 1954 (3). Irrespective
of extensive efforts, the routine and consistent induction of trans-
plantation tolerance in the clinic remains to be realized (4).
ransplantation of foreign cellular, tissue, and organ grafts
represents an important therapeutic modality for the treat-
ment of various inherited and acquired disorders, such as
Modulation of immune responses using donor cells genetically
modified to express immunological molecules that play key roles
in the regulation of the immune system has the potential to induce
transplantation tolerance (5). However, using gene therapy to ex-
press immunomodulatory molecules has various complications,
such as safety, inefficient targeting, and low levels of expression of
the therapeutic protein (6). In particular, in settings of organ, tis-
sue, and primary cell transplantation where rapid and robust ex-
pression of immunological proteins are a prerequisite for thera-
peutic efficacy, gene therapy has severe limitations. Inasmuch as
cell surface receptor ligand interactions are critical to immune de-
cision making and these interactions do not need to be extensive in
duration (7), we sought direct display of exogenous immunological
proteins on the cell surface as a practical alternative to gene ther-
apy for immunomodulation and developed the ProtEx technology
(8). ProtEx involves: 1) generation of recombinant proteins that
contain extracellular domains of immunological ligands fused to a
modified form of core streptavidin (SA)4; 2) modification of cell
membrane with biotin; and 3) display of chimeric proteins on the
modified surfaces (8).
We tested the immunomodulatory potential of our ProtEx tech-
nology using Fas ligand (FasL) as an apoptotic molecule to spe-
cifically eliminate pathogenic lymphocytes in settings of autoim-
munity and allograft transplantation. The choice of FasL as an
immunomodulatory molecule is because of its critical role in ac-
tivation-induced cell death and tolerance to self Ags (9, 10). As
*Institute for Cellular Therapeutics and†Department of Microbiology and Immunol-
ogy, University of Louisville, Louisville, KY 40202; and‡Frankel Laboratory of
Experimental Bone Marrow Transplantation, Department of Pediatric Hematology
Oncology, Schneider Children’s Medical Center of Israel, Petach Tikvah, Israel
Received for publication November 21, 2007. Accepted for publication May 7, 2008.
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 in parts by grants from the National Institutes of Health
(R21 DK61333, R01 AI47864, R21 AI057903, and R21 HL080108 to H.S. and
E.S.Y.), the Juvenile Diabetes Research Foundation (1-2001-328 to H.S.), the Amer-
ican Diabetes Association (1-05-JF-56 to E.S.Y. and H.S.), and the Commonwealth of
Kentucky Research Challenge Trust Fund.
2E.S.Y. and X.G. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Esma Yolcu or Dr. Haval
Shirwan, Institute for Cellular Therapeutics, 570 South Preston Street, Donald Baxter
Biomedical Building, Suite 404E, University of Louisville, Louisville, KY 40202.
E-mail addresses: firstname.lastname@example.org and email@example.com
4Abbreviations used in this paper: SA, streptavidin; FasL, Fas ligand; SA-FasL,
chimeric molecule containing the extracellular functional domain of FasL lacking the
metalloproteinase cleavage sites fused to streptavidin; WF, Wistar-Furth; Treg, T
regulatory; MST, median survival time; SA-FasL LT, SA-FasL-engineered cells
treated long-term graft recipients; SA Rej, SA-engineered cell-treated animals with
acute graft rejection; Teff, T effector; BiP, Drosophila secretion signal.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
such, there has been significant interest in using FasL as an im-
munomodulatory molecule to induce transplantation tolerance
with conflicting observations (11). Although some studies reported
therapeutic effect of FasL, others demonstrated its contribution to
the pathogenesis of the disease (12–20). Mechanisms responsible
for these opposing effects are complex and may be regulated by
levels of FasL and its receptor, Fas, on target tissues and the sen-
sitivity of these tissues to FasL-mediated apoptosis, the cytokine
milieu, the tissue microenvironment, and/or the differential effects
of membrane-bound and soluble forms of FasL (reviewed in Ref.
11). FasL is initially expressed on the cell surface as a membrane-
bound type II protein that has potent apoptotic activity on Fas-
bearing cells (9). However, membrane-bound FasL is cleaved from
the cell surface by matrix metalloproteinases into a soluble form
(21), which does not have apoptotic activity and may interfere with
apoptosis by competing with the membranous form for binding to
Fas on target cells (21, 22). Furthermore, soluble FasL has che-
motactic activity on neutrophils (23, 24) that may be responsible
for the rejection of grafts ectopically expressing FasL (13, 19).
Therefore, a chimeric molecule containing the extracellular
functional domain of FasL lacking the metalloproteinase cleavage
sites fused to streptavidin (SA-FasL) was generated (8). We pre-
viously demonstrated that the direct display of SA-FasL on heart
graft vasculature resulted in the prevention of acute rejection after
transplantation into allogeneic recipients (25). However, this ap-
proach of direct manipulation of the donor graft resulted in mod-
erate prolongation of graft survival without achieving tolerance.
The lack of tolerance in this model might have been due to the
inability of the transient, local display of SA-FasL on the graft
vasculature to deplete a significant pool of alloreactive T effector
(Teff) cells. Hence, we herein tested whether recipient immuno-
modulation by systemic infusion of donor splenocytes engineered
to display SA-FasL on their surface induces tolerance to cardiac
grafts in a totally allogeneic rat strain combination. Infusion of
ACI graft recipients with Wistar-Furth (WF) donor splenocytes
displaying SA-FasL on their surface resulted in robust tolerance to
cardiac allografts. Tolerance was donor specific and maintained by
CD4?CD25?FoxP3?T regulatory (Treg) cells. The rapid and ef-
ficient display of exogenous proteins on the cell membrane, there-
fore, represents a practical and effective cell-based therapy with
potential application to autoimmunity, transplantation, and regen-
Materials and Methods
Male 8- to 12-wk-old WF (RT1u), ACI (RT1a), and Fischer (RT1lv) rats
were purchased from Harlan Sprague Dawley, and 2C mice were gener-
ously provided by Dr. J. Connolly (Washington University School of Med-
icine, St. Louis, MO). All animals were maintained under specific patho-
gen-free conditions in our barrier facility and used according to the
University of Louisville and National Institutes of Health institutional
Construction of SA-FasL and protein expression in Drosophila
The construction of the SA-FasL gene was reported previously (8). Briefly,
DNA segment encoding core streptavidin was amplified using specific
primers (forward 5?-AGATCTCATCATCACCATCACCATATCACCGG
CACC and reverse 5?-GAATTCGGAGGCGGCGGACGGCT) and total
genomic DNA from Streptomyces avidinii (American Type Culture Col-
lection) as a template for PCR. The 5?-primer was designed to include a
BglII restriction site and 6 histidine residues to allow cloning in frame with
the Drosophila secretion signal (BiP) in pMT/BiP/V5-His vector for ex-
pression in S2 cells as a secreted protein and purification using Chelating
Sepharose Fast Flow Columns. The cDNA encoding the extracellular do-
main of rat FasL without the metalloproteinase site (nucleotides 428–998)
was cloned using total RNA from Con A-activated splenocytes as a tem-
plate and specific primers (5?-primer, nucleotides 428–453; and 3?-primer,
nucleotides 977–998). These primers were engineered to include EcoRI
sites for cloning in frame with streptavidin in the pMT/BiP/V5-His vector.
The chimeric gene in pMT/BiP/V5-His vector was used to stably transfect
S2 cells (Invitrogen). Stable transfectants were induced by 1.0 mM CuSO4,
and secreted chimeric protein in culture medium was collected 1–4 days
after induction. The supernatant was either used immediately or precipi-
tated with 50% ammonium sulfate, dialyzed against PBS, and purified
using Chelating Sepharose Fast Flow Columns (Amersham). The concen-
tration of purified SA-FasL or protein in culture supernatant was deter-
mined by Western blot or ELISA using known amounts of commercially
available streptavidin as standard.
Engineering donor splenocytes to display SA-FasL on their
Spleens were harvested from WF rats and processed into single-cell sus-
pension, and RBC were lysed using buffered ammonium chloride solution.
Cells were biotinylated by incubation in 5 ?M biotin solution (Pierce) in
PBS at room temperature for 30 min. Cells were washed twice with PBS,
and then incubated with ?200 ng of SA-FasL protein per 1 ? 106cells in
PBS for 30 min by constant rocking in a cold room. After two washings,
cells were resuspended in PBS and counted as previously described (8). A
portion of the cells were stained with streptavidin-allophycocyanin, FITC-
labeled anti-FasL, or FITC-labeled anti-streptavidin Abs to assess the level
of biotinylation and SA-FasL-decoration, respectively, using flow
Splenocyte treatment and transplantation
ACI graft recipients received 13 ? 106unmodified WF donor splenocytes
by i.p. injection 6 days before heart transplantation. Graft recipients were
then treated on days ?3, ?1, ?1, ?3, and ?5 with respect to heart trans-
plantation on day 0 with the same number of unmodified (n ? 8) or SA-
FasL (n ? 23)- or SA (n ? 20)-engineered donor splenocytes. Recipients
without splenocyte treatment (n ? 10) served as control to determine the
normal rate of graft rejection. Another group of treated ACI animals (n ?
4) were transplanted with an F344 third-party heart as Ag specificity con-
trols. Intra-abdominal heterotopic heart transplantation was performed as
previously described (26). Ventricular contractions were assessed by daily
palpation, and rejection was defined as cessation of heartbeat verified by
autopsy and pathology.
T cell tracking using 2C model
A total of 20 million 2C spleen cells labeled with CFSE were injected into
the tail vein of C57BL/6.SJL mice. These animals were then immunized 1
day later by i.v. injection of 20 million allogeneic BALB/c splenocytes
either left unmodified or engineered with SA-FasL or control protein SA.
Animals were euthanized 3 days after immunization, and their spleens and
mesenteric lymph nodes were harvested for single-cell preparation. Cells
were stained with Abs to CD45.2, Vb8.2, and CD8 and analyzed using flow
cytometry by gating on CD45.2?CD8?Vb8.2?T cells. Cell division was
assessed using FlowJo software.
Spleens were processed into single-cell suspensions, labeled with 2.5 ?M
CFSE, and resuspended in DMEM; then 50 ? 106CFSE labeled spleno-
cytes were plated on a petri dish for 45 min at 37°C to enrich lymphocytes.
After 45 min, nonadherent cells were collected, washed, and incubated
(1 ? 105cells) with irradiated (2000 cGy; 1 ? 105cells) naive ACI, WF,
or Fischer splenocytes in 96-well U-bottom titer plates in MLR medium.
Irradiated syngeneic cells and F344 third-party splenocytes were used as
controls. After 5 days, cells were stained with fluorochrome-labeled Abs
against rat CD4 and CD8 and analyzed by flow cytometry. Data were
analyzed using FlowJo (Tree Star) software. All proliferation assays were
performed in triplicates and are representative of a minimum of three an-
imals per group.
Intracellular cytokine staining
Splenocytes were resuspended in MLR medium at a concentration of 2 ?
106cells/ml and stimulated with PMA (5 ng/ml; Sigma-Aldrich) and iono-
mycin (500 ng/ml; Sigma-Aldrich) for 2 h at 37°C in a 5% CO2incubator.
Two hours later, cultures were supplemented with Golgi Plug (1 ?l/ml; BD
Biosciences) and incubated for an additional 2.5 h. Cells were then stained
with fluorescent-conjugated Abs against rat CD4, CD8, and IFN-? and
analyzed by flow cytometry as previously described (27).
932TRANSPLANTATION TOLERANCE WITH FasL-ENGINEERED DONOR CELLS
T cell phenotyping
Spleen and mesenteric lymph nodes were processed into single-cell sus-
pensions and stained with CD4-allophycocyanin, CD8-PerCP, and
CD25-PE mAbs or isotype controls at 4°C for 30 min. Cells were then
washed twice with FACS buffer and analyzed by flow cytometry. For
FoxP3 staining, cells were stained with Abs against CD4, CD8, and CD25;
washed twice with FACS buffer; fixed; and then stained for intracellular
FoxP3 according to the manufacturer’s protocol (eBiosciences).
Adoptive transfer studies
Spleens were harvested from long-term (?90 days) graft survivors or
acutely rejecting animals at rejection and processed into single-cell sus-
pensions. Naive unirradiated or 450 cGy-irradiated ACI rats received 80–
95 ? 106cells via i.v. injection 24 h after irradiation. These animals were
transplanted with WF hearts 24 h after cell transfer. Graft survival was
monitored by abdominal palpation.
In selected experiments, sorted CD25?T cells were used for adoptive
transfer experiments. Briefly, lymphocytes from rats with long-term graft
survival or acutely rejecting animals were stained with a saturating dose of
anti-rat CD25 Ab (OX-39) conjugated to PE for 30 min at 4°C. After
extensive washing to remove unbound Ab, cells were incubated with the
appropriate amount of anti-PE microbeads (Miltenyi Biotec). Magnetic
separation was performed according to the manufacturer’s instructions.
Positively selected CD25?and lymphocytes depleted of this population
were stained with fluorochrome-labeled Abs to FoxP3 and CD4 and ana-
lyzed using multiparameter flow cytometry. CD4?FoxP3?and CD4?
FoxP3?populations were found to be ?90% pure.
Cardiac graft samples fixed in 10% formalin were embedded in paraffin,
sectioned, and stained with H&E for evaluation of cellular infiltration.
Elastin staining was performed using a kit from Sigma-Aldrich according
to the manufacturer’s instructions to demonstrate the vascular lesions and
myocardial fibrosis. Briefly, tissue sections were stained in hematoxylin-
iodine-ferric chloride solution and differentiated by use of a dilute ferric
chloride solution. Van Gieson solution was used as a counterstain to stain
collagen fibers red and myocardium yellow. Chronic rejection was assessed
by light microscopy. The severity of chronic rejection was graded accord-
ing to the percentage luminal occlusion by intimal thickening as previously
Data were analyzed using Student’s t test, the nonparametric Mann-Whit-
ney U test, Kaplan-Meier’s log-rank test, and Tarone-Ware tests as appro-
priate. A p value of ?0.05 was considered significant. Statistical analysis
was performed using SPSS 13.0 software.
The display of SA-FasL on the surface of splenocytes does not
alter the distribution of endogenous cell membrane proteins
The transplant experiments were preceded by a series of prelimi-
nary studies to test whether the display of SA-FasL on the surface
of splenocytes interferes with the normal distribution of endoge-
nous cell surface proteins, including molecules involved in Ag
presentation. WF rat splenocytes were labeled with biotin (5 ?M)
and engineered with SA-FasL (?200 ng/106cells). The levels of
biotin and SA-FasL on the cell surface were assessed by fluoro-
chrome-conjugated SA and an Ab against SA or FasL, respec-
tively, using flow cytometry. Almost all targeted cells were posi-
tive for the cell surface biotin and SA-FasL (Fig. 1A). Biotinylated
and SA-FasL-engineered cells were then analyzed for the presence
of a series of endogenous cell surface molecules, such as CD3,
CD4, CD8, class I MHC, and CD80 molecules, involved in the
immune response using their respective Abs in flow cytometry. As
shown in Fig. 1B, neither biotinylation nor engineering with SA-
FasL significantly altered the cell surface expression patterns of
these molecules. Taken together, these data demonstrate that pri-
mary cells, such as splenocytes, can be engineered to display on
their surface SA-FasL in a rapid and efficient manner without sig-
nificantly altering the cell surface distribution of endogenous
Systemic immunomodulation with SA-FasL-engineered donor
splenocytes induces allograft tolerance
T cells become sensitive to Fas/FasL-mediated apoptosis follow-
ing activation, several rounds of proliferation, and re-encounter
with the Ag (10). We therefore pretreated ACI recipients of WF
donor heart allografts with 13 ? 106unmodified donor splenocytes
i.p. to mobilize the alloreactive T cell pool and sensitize them to
Fas/FasL-mediated apoptosis. These animals were then injected
cytes does not alter the distribution of endogenous cell
surface proteins. A, Engineering WF rat splenocytes
with SA-FasL protein. Splenocytes were modified with
biotin (5 ?M) and engineered with chimeric SA-FasL
(?200 ng/106cells). The levels of cell surface biotiny-
lation and display of SA-FasL were assessed using SA-
allophycocyanin (left), anti-SA-FITC Ab (middle) and
anti-FasL-FITC Ab (right) in flow cytometry. B, The
presence of endogenous cell surface proteins was as-
sessed using fluorochrome-conjugated mAbs to the in-
dicated molecules in flow cytometry. Shaded curve, Iso-
type control; solid line, unmodified splenocytes; dashed
line, SA-FasL-engineered splenocytes.
Display of SA-FasL on donor spleno-
933The Journal of Immunology
with several doses of SA-FasL-engineered donor splenocytes pre-
and post-cardiac allograft transplantation as shown in Fig. 2A. Sev-
enty percent of ACI rats treated with SA-FasL-modified donor
splenocytes accepted WF heart grafts over the 100-day observation
period, whereas the remaining 30% rejected their allografts in a
median survival time (MST) of 12 ? 1.2 (Fig. 2B). In contrast, all
untreated animals (n ? 10) rejected their grafts in a MST of 9 ?
0.4 days (Fig. 2B). Treatment with splenocytes engineered with
control SA protein (n ? 20) resulted in only ?25% graft survival,
which was similar to that achieved using unmodified splenocytes
(n ? 8). The induced tolerance was donor specific given that ACI
rats (n ? 4) treated with SA-FasL-engineered WF splenocytes re-
jected F344 third-party heart allografts in a normal tempo (MST
8 ? 0 days; Fig. 2B). Importantly, long-term grafts (100 days) in
the SA-FasL treatment group had no inflammatory infiltrates and
showed minimal neointimal proliferation (Fig. 2Ca,b). In marked
contrast, grafts that survived long-term (100 days) as a result of
immunomodulation with SA-engineered donor splenocytes had in-
flammatory infiltrates and showed significant incidences and levels
of neointimal proliferation (Fig. 2Cc,d).
To rigorously test the ability of donor splenocytes to induce
tolerance in a clinically relevant setting, where the heart and
splenocytes are simultaneously available from cadaveric donors,
the treatment commenced posttransplantation (Fig. 2D). Inasmuch
as allogeneic heart grafts are efficient in activating alloantigen-
specific T cells and as such sensitizing them to Fas-mediated ap-
optosis (29, 30), all infusions contained donor splenocytes engi-
neered with SA-FasL. As shown in Fig. 2E, ?62% of ACI
recipients (n ? 8) treated with SA-FasL-engineered WF spleno-
cytes did not reject WF heart allografts, whereas all animals (n ?
6) treated with control SA-engineered splenocytes rejected their
grafts in a MST of 11 ? 1.2 days. Taken together, these data
demonstrate that systemic immunomodulation with SA-FasL-en-
gineered splenocytes was effective in inducing tolerance to cardiac
allografts without the use of immunosuppression.
Systemic immunomodulation with SA-FasL-engineered
allogeneic splenocytes results in reduced proliferation and
accumulation of alloreactive T cells
To determine whether systemic infusion of SA-FasL-engineered
splenocytes physically eliminates alloreactive cells in the host, we
took advantage of the TCR-transgenic 2C mouse model the CD8?
T cells of which are specific for H-2Ldclass I alloantigen (31).
C57BL/6 (CD45.1) mice were adoptively transferred with con-
genic CFSE-labeled 2C T cells (C57BL/6; CD45.2) followed by
immunization with BALB/c (H-2d) splenocytes engineered with
SA-FasL or SA control protein. Three days later, splenocytes and
lymph node cells from the immunized animals were harvested and
analyzed using flow cytometry for the proliferation of 2C cells.
Immunization with SA-engineered BALB/c splenocytes resulted
in robust proliferation (more than eight cycles) of 2C cells and
accumulation of increasing numbers of daughter cells per gener-
ation (Fig. 3A). In marked contrast, mice immunized with SA-
FasL-engineered splenocytes had moderate proliferation (?4 cy-
cles) of 2C cells. Importantly, there was a steady decrease in the
number of daughter cells per generation plausibly due to apoptosis.
This notion is consistent with published literature that naive T cells
require few cycles of proliferation in response to Ag before be-
coming sensitive to Fas/FasL-mediated apoptosis (10).
To provide direct evidence that alloreactive T cells undergo ap-
optosis following Ag recognition in the context of FasL on donor
cells, we performed in vitro stimulation assays where BALB/c
WF-to-ACI rat model. A, Schematic diagram detailing the treatment regimen for ACI recipients. Unmodified WF splenocytes (13 ? 106) were injected i.p.
into ACI recipients 6 days before heart transplantation to mobilize alloreactive T cells and sensitize them to Fas/FasL-mediated apoptosis, followed by five
doses of SA-FasL-engineered splenocytes pre- and posttransplantation as indicated. B, Survival curve. Immunomodulation of ACI recipients with SA-
FasL-engineered WF donor splenocytes (SA-FasL-Spl) resulted in the prevention of allograft rejection in ?70% of the recipients, whereas only ?25% of
animals receiving unmodified (Spl) or SA-engineered splenocytes (SA-Spl) maintained their allograft long term. Tolerance was donor specific given that
ACI recipients treated with WF splenocytes engineered with SA-FasL promptly rejected third-party F344 heart grafts. C, Long-term allografts in the
SA-FasL treatment group showed minimal incidences and levels of neointimal proliferation. Long-term (?100 days) heart grafts from SA-FasL-engineered
(SA-FasL-Spl LT) and control SA-engineered (SA-Spl LT) donor splenocyte treatment groups were stained with H&E (a and c) and elastic/Van Geison
(b and d). Posttreatment alone (D) was also effective in inducing tolerance to the majority (?62%) of WF heart allografts (E). Data were compared using
Kaplan-Meier’s Tarone-Ware test. p ? 0.05 for SA-FasL-Spl vs all other groups.
Systemic immunomodulation using SA-FasL-engineered donor splenocytes (Spl) prevents cardiac allograft rejection in a fully mismatched
934 TRANSPLANTATION TOLERANCE WITH FasL-ENGINEERED DONOR CELLS
splenocytes engineered with SA-FasL or SA control protein were
used as stimulators for 2C cells. There was a significant reduction
in the number of proliferating 2C cells responding to SA-FasL-
engineered donor cells as compared with control (Fig. 3B, top).
The observed reduction in proliferation was due to significant ap-
optosis of responding 2C T cells (Fig. 3B, bottom).
Allograft tolerance is associated with donor-specific T cell
hypoproliferation and reduced expression of IFN-?
To further elucidate the mechanisms responsible for the observed
tolerance to WF hearts, ACI graft recipients were evaluated for
evidence of donor-specific proliferation using CFSE-based MLRs
(32). As shown in Fig. 4A, lymphocytes from SA-FasL-engineered
cells treated long-term graft recipients (SA-FasL LT) responded
poorly to donor cells but showed a normal response to third-party
cells, indicating Ag specificity. In marked contrast, lymphocytes
from SA-engineered cell-treated animals with acute graft rejection
(SA Rej) and naive control animals proliferated vigorously to both
donor and third-party cells. Moreover, tolerant rats had lower per-
centages of CD8?T cells expressing IFN-?, a signature cytokine
for graft rejection (Refs. 33 and 34) and Fig. 4B) as compared with
SA-engineered cell-treated rats with acute graft rejection (7.4 ?
3.6% vs 28.5 ? 3.0% in CD8?T cell gate; Fig. 4C). In marked
contrast to CD8?T cells, only a small percentage of CD4?T cells
expressed IFN-? in all groups irrespective to the treatment regimen
(Fig. 4C; gated on CD4?cells). There was a slight increase in the
percentage of CD4?T cells expressing IFN-? in FasL-treated an-
imals, which was not statistically significant.
Tolerance is associated with increased percentage of
peripheral CD4?CD25?FoxP3?Treg cells
Although we demonstrated that systemic immunomodulation with
SA-FasL-engineered splenocytes induces apoptosis in activated al-
loreactive T cells (Fig. 3), this effect may be short lived due to the
transient present of SA-FasL on the cell surface. Therefore, phys-
ical elimination of alloreactive T cells early in transplantation may
not be sufficient for long-term allograft survival and may require
induced peripheral immunoregulatory mechanisms. We focused on
CD4?CD25?FoxP3?Treg cells because of their capacity to in-
duce and maintain tolerance in various transplantation settings
(35). Lymphocytes were isolated from the spleen of long-term
graft recipients (?90 days) and phenotyped for CD4 and CD25
expression using flow cytometry (Fig. 5A). There was a significant
(p ? 0.05) increase in the percentage of CD4?CD25?T cells
(8.2 ? 1.3% of total lymphocytes) in long-term SA-FasL-treated
graft recipients compared with results in naive rats (5.1 ? 0.4%).
In contrast, rats with acute graft rejection had similar percentages
of CD4?CD25?T cells as naive animals whether they were
treated with SA (5.7 ? 1.5%) or SA-FasL-engineered (5.1 ?
1.2%) splenocytes (Fig. 5B). The increased percentage of
CD4?CD25?Treg cells in long-term graft survivors was further
confirmed with an Ab to the signature transcriptional factor FoxP3
(Fig. 5C; gated on CD4?T cells).
Importantly, sorted CD4?CD25?T cells from long-term
graft survivors inhibited the proliferative responses of naive
Teff cells to donor alloantigens when used at various ratios in a
CFSE-based in vitro proliferation assay (Fig. 5D). This finding
indicates that CD4?CD25?T cells from long-term graft survi-
vors are Treg cells, rather than newly activated Teff cells ex-
pressing CD25. Taken together, these data demonstrate that sys-
temic immunomodulation with SA-FasL-engineered donor
splenocytes induces long-term graft survival that is associated
with increased percentages of CD4?CD25?FoxP3?Treg cells
in the periphery.
Sorted CD4?CD25?FoxP3?Treg cells from tolerant animals
prevent the rejection of donor hearts in secondary naive
To provide direct evidence for the role of peripheral immunoregu-
latory mechanisms in the observed tolerance, 80–95 ? 106spleno-
cytes harvested from primary graft recipients were adoptively
transferred into a second cohort of naive rats that had been sub-
jected to 450 cGy total body irradiation 1 day earlier. All rats (n ?
6) that received splenocytes from SA-FasL-engineered cells
treated long-term primary graft survivors indefinitely accepted WF
donor heart allograft (Fig. 6A). In marked contrast, animals (n ?
4) receiving splenocytes from SA-engineered cell-treated primary
results in apoptosis of alloreactive T cells. A, C57B/6.SJL (CD45.1) mice were
adoptively transferred with 20 ? 1062C (C57B/6; CD45.2) splenocytes. One
day later, the animals were immunized with 20 ? 106BALB/c splenocytes
engineered with SA or SA-FasL. Splenocytes and mesenteric lymph node
(MLN) cells were harvested 3 days later, stained with Abs against CD45.2,
CD8, TCR V?8.2, and analyzed for proliferation in multiparameter flow cy-
tometry by gating on CD45.2?CD8?V?8.2?T cells. Data are representative
of two independent experiments conducted with two animals in each group. B,
BALB/c splenocytes engineered with SA-FasL or SA were used as stimulators
for CFSE-labeled 2C cells in a 3-day MLR assay. Cells were stained with
H-2Kdto gate out stimulators and 7-aminoactinomycin D (7-AAD) and an-
nexin V to assess dead and apoptotic cells, respectively. Data are representa-
tive of two independent experiments.
Immunomodulation with SA-FasL-engineered splenocytes
donor Ags and have reduced percentages of T cells expressing IFN-?. A,
Splenocytes from long-term (?90 days) graft survivors treated with SA-
FasL-engineered donor splenocytes (SA-FasL LT), acutely rejecting ani-
mals treated with SA-engineered splenocytes (SA Rej), or naive ACI an-
imals (naive) were labeled with CFSE and used as responder (1 ? 105
cells/well) to ACI (Auto), WF (Allo), and F344 (third party) irradiated
splenocytes (1 ? 105cells/well). After 5 days, cells were analyzed by flow
cytometry gating on live CFSE?cells. Data are representative of a minimum
of three independent MLR experiments.B and C, Long-term SA-FasL animals
have low percentages of CD8?T cells expressing IFN-?. Splenocytes were
harvested from the indicated treatment groups, activated with PMA and iono-
mycin, and subjected to intracellular IFN-? staining and flow cytometric anal-
ysis by gating on total lymphocytes (B) or CD4?or CD8?T cells (C). A
minimum of three animals per group were analyzed.
Long-term SA-FasL-treated animals are hyporesponsive to
935The Journal of Immunology
graft recipients with acute rejection rejected donor heart grafts in
a MST of 9 ? 0.4 days. We also tested the efficacy of splenocytes
from the SA-FasL treatment group that had delayed rejection in
prolonging the survival of secondary grafts. Adoptive transfer of
these cells into secondary graft recipients resulted in prolonged
survival of all grafts (MST 15 ? 10.4 days) with one of six grafts
not rejecting during the 100-day observation period. These data
suggest that SA-FasL treatment in this group had induced immu-
noregulatory cells but that these cells were not sufficient to prevent
graft rejection in primary or secondary recipients after adoptive
transfer. This notion is consistent with the reduced IFN-? secretion
in T cells from this group as compared with the SA-treatment
group (Fig. 4C).
The presence of immunoregulatory cells in primary long-term
graft survivors was further confirmed in a group of nonirradiated
secondary graft recipients. Adoptive transfer of splenocytes from
SA-engineered cell-treated ACI primary graft recipients with acute
rejection into secondary unmanipulated naive ACI recipients re-
sulted in accelerated rejection of all WF donor hearts (Fig. 6B; n ?
4, MST 9 ? 1 days). In marked contrast, all WF grafts (n ? 6)
transplanted into secondary ACI recipients adoptively transferred
with splenocytes from long-term SA-FasL-engineered cell-treated
ACI primary recipients showed prolonged survival, with 50% of
the grafts not rejecting during the 100-day observation period.
Long-term (?100 days) graft survivors rejected third-party F344
hearts in a normal tempo (n ? 4; MST 7.5 ? 0.5 days) without any
effect on the survival of primary WF grafts.
survivors treated with SA-FasL-engineered donor splenocytes (SA-FasL LT), acutely rejecting animals treated with SA-engineered splenocytes (SA Rej), or naive
ACI animals (naive) were stained with fluorochrome-labeled Abs against CD4, CD25, and FoxP3 and analyzed using multiparameter flow cytometry. A, Dot plot
pattern of splenocytes showing CD4?CD25?T cells in total lymphocyte gate. B, Tabulation of CD4?CD25?T cells for a minimum of three animals per group
in the lymphocyte gate. ?, p ? 0.05 as compared with all the other groups. C, Long-term SA-FasL animals have high percentages of CD4?T cells expressing
intracellular FoxP3 (gated on CD4?T cells). D, CD4?CD25?T cells sorted from long-term SA-FasL-splenocyte-treated graft recipients are suppressive in vitro.
CFSE-labeled ACI naive splenocytes (1 ? 105) were used as responder to irradiated WF splenocytes (1 ? 105) in a 5-day proliferation assay in the presence of
varying ratios of CD4?CD25?T cells sorted from long-term graft survivors. Naive CFSE-labeled responder to CD4?CD25?T cell ratios are indicated in the right
bottom corner of each graph. Percentages of proliferating alloreactive T cells (upper left) and added (ovals) CD4?CD25?T cells (lower left) are shown for each
panel. All histograms show mean ? SD and significance was assessed using Mann-Whitney U test. APC, Allophycocyanin.
Long-term SA-FasL treated animals have high percentages of peripheral CD4?CD25?Treg cells. Splenocytes from long-term (?90 days) graft
Adoptive transfer experiments into irradiated secondary graft recipients.
Splenocytes (80–95 ? 106) harvested from long-term (?90 days; SA-
FasL) or acutely rejecting (SA or SA-FasL) recipients were transferred into
450-cGy-irradiated naive ACI animals 1 day before WF heart transplan-
tation. B, Adoptive transfer experiments into nonirradiated secondary graft
recipients. Experimental conditions are as in A. C, Sorted CD4?CD25?T
cells from long-term but not from acutely rejecting animals prevent rejec-
tion of WF hearts in naive ACI recipients. Unmanipulated naive ACI rats
were adoptively transferred 1 day before WF heart transplants with 5–9 ?
106CD4?CD25?FoxP3?T (Treg) cells sorted from SA-FasL long-term
graft survivors, 50–90 ? 106Treg-depleted splenocytes from these ani-
mals, or 5–9 ? 106CD4?CD25?FoxP3?Treg cells sorted from acutely
rejecting (SA-engineered splenocytes treated and normal controls).
Tolerance is maintained by CD4?CD25?Treg cells. A,
936 TRANSPLANTATION TOLERANCE WITH FasL-ENGINEERED DONOR CELLS
To provide direct evidence for the contribution of Treg cells in
the observed tolerance, CD4?CD25?Treg cells were positively
sorted from tolerant splenocytes using Miltenyi beads. Sorted Treg
cells (5–9 ? 106cells/recipient) as well as Treg-depleted spleno-
cytes (50–90 ? 106cells/recipient) were adoptively transferred
into naive ACI recipients of WF hearts 1 day before transplanta-
tion. All recipients (n ? 4) adoptively transferred with Treg cells
accepted their grafts (Fig. 6C). In marked contrast, all grafts in the
group receiving Treg cell-depleted splenocytes underwent rejec-
tion with a MST of 23.4 ? 3.4 days. However, this rejection time
was significantly delayed as compared with normal controls (MST
9 ? 0.4 days), suggesting either the presence of other immuno-
regulatory cells or contamination with Treg cells. Importantly,
Treg cells sorted from acutely rejecting animals (SA-splenocyte
treated or normal controls) did not prevent the rejection of donor
grafts following adoptive transfer into naive secondary recipients
under similar conditions as Treg cells sorted from FasL-treated
long-term graft survivors. Taken together, these data demonstrate
that Treg cells are critical to the observed donor-specific peripheral
tolerance achieved by systemic immunomodulation using SA-
Immunomodulation with genetically engineered cells expressing
FasL has been extensively tested for the induction of tolerance to
auto- and alloantigens with reported efficacy in various preclinical
settings (14–18, 20, 36–40). However, the translation of this ap-
proach to the clinic remains to be realized primarily due to diffi-
culties associated with efficient and rapid manipulation of donor
primary cells under clinically applicable conditions to reproduc-
ibly express FasL and safety concerns of the gene therapy. Fur-
thermore, FasL has pleiotropic effects on immune and nonimmune
cells, and as such long-term stable expression of this molecule
using gene therapy might have detrimental consequences. In the
present study, we overcame these difficulties by transient display
of a recombinant form of FasL protein with potent apoptotic ac-
tivity (25) on donor splenocytes in a rapid (?2 h) and efficient
manner (?100% of the targeted cells). Systemic immunomodula-
tion with FasL-engineered donor cells resulted in peripheral toler-
ance to cardiac allografts without the use of any additional
Although a series of studies using various APC genetically en-
gineered to express FasL for immunomodulation reported alloan-
tigen-specific immune nonresponsiveness, none of these studies
demonstrated that such nonresponsiveness eventually leads to
long-term allograft tolerance (11, 16, 37, 40). To our knowledge,
this is the first study to demonstrate that systemic immunomodu-
lation with donor cells engineered to express FasL on their surface
induces long-term peripheral tolerance to cardiac allografts in a
totally allogeneic rat strain combination. The mechanistic basis of
the induced peripheral tolerance involves physical elimination of
alloreactive cells by activation-induced cell death early after trans-
plantation and maintenance of tolerance by CD4?CD25?FoxP3?
Treg cells. The enhanced immunomodulatory effect of repeated
splenocyte infusion reported in the present communication can be
attributed to effective elimination of alloreactive immune effector
cells at remote sites from the graft, robust induction/expansion of
Treg cells, or both. T cells require Ag-specific activation and sev-
eral rounds of sensitization to acquire sensitivity to Fas/FasL-me-
diated apoptosis (10). Also, it is well-established that memory im-
mune cells reside within nonlymphoid target tissues, plausibly to
generate a rapid and effective secondary immune response to re-
current infection (41). Therefore, systemic and repeated adminis-
tration of engineered donor splenocytes may have the advantage of
tolerizing compartmentalized memory responses defined as heter-
ologous immunity that serves a major barrier for tolerance induc-
tion in the clinic (4).
In our view, induction of CD4?CD25?FoxP3?Treg cells using
SA-FasL-engineered donor splenocytes is the most significant
finding of the present study. The essential role of Treg cells in
tolerance was demonstrated by adoptive transfer experiments
where cells sorted from lymphoid organs of long-term graft ac-
ceptors transferred tolerance to naive, unmanipulated secondary
recipients (Fig. 6C). Importantly, CD4?CD25?FoxP3?T cells
sorted from acutely rejecting animals did not prevent the rejection
of donor grafts in naive secondary recipients following adoptive
transfer. This may be because the sorted CD4?CD25?FoxP3?
Treg cells were not donor specific, contaminated with newly
activated alloreactive Teff cells expressing CD25, or rat Teff cells
transiently express FoxP3 following activation as reported for hu-
man Teff cells (42). The presence of Treg cells, other than
CD4?CD25?FoxP3?Treg cells, in our model is consistent with
our observations that Treg cell-depleted total splenocytes from
SA-FasL-treated long-term graft survivors did not prevent the re-
jection of donor grafts upon adoptive transfer into secondary naive
graft recipients but caused significant prolongation as compared
with controls (Fig. 6C). These regulatory T cells may include
CD4?CD8?TCR?T cells (43), CD8?FoxP3?T cells (44), and/or
newly described CD8?CD45RClowT cells expressing IFN-? (45).
These cell types are also implicated in allotransplantation achieved
using donor-specific transfusion. For example, infusion of alloge-
neic donor splenocytes into graft recipients was shown to generate
CD4?CD8?TCR?Treg cells that used FasL to physically elimi-
nate alloreactive T cells for tolerance induction (43). Furthermore,
tolerance to skin allografts disparate for H-Y Ag has recently been
shown to require FasL on donor cells used for infusion and Fas in
graft recipients (46). Therefore, the Fas/FasL system may serve as
a common denominator of mechanisms responsible for tolerance
achieved by immunomodulation of graft recipients using donor-
specific transfusion in form of donor lymphocytes.
We envision three possible mechanisms for the induction/ex-
pansion of Treg cells by SA-FasL-engineered splenocytes. First,
FasL interaction with Fas up-regulated on the surface of Treg cells
in response to donor Ags may transduce a mitogenic signal, lead-
ing to their expansion. Although Fas signaling was shown to be
involved in the physiological process of T effector cell activation
under selected conditions (47, 48), it remains to be determined
whether such a function also applies to Treg cells. Second, Treg
cells may be less sensitive to Fas-mediated apoptosis as compared
with T effector cells. Immunomodulation with SA-FasL-engi-
neered splenocytes may preferentially eliminate T effector cells,
thereby tipping the balance toward the expansion of Treg cells.
However, the sensitivity of Treg cells to Fas/FasL-mediated apo-
ptosis has been the subject of few studies with conflicting obser-
vations. Although some studies reported that Treg cells are more
sensitive to FasL-mediated apoptosis than T effector cells (49, 50),
we (51) and others indicated the exact opposite (52). These con-
flicting observations may be due to the study designs, such as lack
of comparative analysis of Treg and T effector cell sensitivity to
FasL-mediated apoptosis in the course of an immune response to
Ags under inflammatory conditions. Third, Teff cells undergoing
apoptosis may give rise to the generation of adaptive Treg cells or
expansion of naturally occurring Treg cells. Consistent with this
notion are the observations that apoptotic lymphocytes release two
cytokines, IL-10 and TGF-?, that play important roles in the gen-
eration and function of Treg cells (53). Furthermore, apoptotic
bodies from dying cells were shown to contribute to the generation
937 The Journal of Immunology
of Treg cells through mechanisms that involved TGF-? and mac-
rophages (54). Further studies are needed to establish mechanisms
that are responsible for the generation of Treg cells in our model
and whether these cells are of adaptive or natural type.
The immunomodulatory approach presented here has several
attractive features with direct relevance to the clinic. First, it al-
lows for rapid, efficient, and transient display of exogenous im-
munomodulatory proteins on the cell membrane with immediate
function without a time lag required for gene transfer-based ex-
pression. Second, the transient display of immunomodulatory pro-
teins with pleiotropic effects may minimize the potential undesired
effects arising from their stable and long-term expression achieved
by gene therapy. Third, several proteins with synergistic functions
may be simultaneously displayed on the cell surface to maximize
their therapeutic efficacy (R. K. Sharma and H. Shirwan, unpub-
lished data). In particular, our approach may be incorporated into
cell-based immunomodulation approaches, such as donor-specific
leukocyte transfusion or hemopoietic stem cell transplantation, for
tolerance induction in the clinic. Infusion of unmodified donor
leukocytes or bone marrow cells into graft recipients for the pur-
pose of immunomodulation has been extensively tested with ob-
served beneficial effects in various preclinical and clinical settings
(55, 56). The presence of SA-FasL on donor leukocytes and bone
marrow cells may further improve their therapeutic efficacy by
eliminating alloreactive pathogenic lymphocytes and/or inducing
peripheral immunoregulatory mechanisms as shown by the present
study. In conclusion, this protein display approach possesses the
simplicity, safety, and efficacy required to make it a clinically rel-
evant and practical alternative to gene therapy for immunomodu-
lation with broad application to cell-based therapies, regenerative
medicine, autoimmunity, and transplantation.
We are grateful to Dr. Suzanne Ildstad for proofreading the manuscript and
to Orlando Grimany for technical assistance.
The ProtEx technology described in this article is licensed out from the
University of Louisville by ApoImmune (Louisville, KY), for which
H.S. serves as Chief Scientific Officer, and H.S. and E.S.Y. have significant
equity interest in the Company.
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939The Journal of Immunology