of November 13, 2011
This information is current as
; Prepublished online 7 November 2011;
Chantale Lacelle, Kyle B. Woodward, Nadir Askenasy and
Esma S. Yolcu, Hong Zhao, Laura Bandura-Morgan,
Inducing Regulatory T Cells in Mice
Protein Establish Robust Localized Tolerance by
Pancreatic Islets Engineered with SA-FasL
is online at
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on November 13, 2011
The Journal of Immunology
Pancreatic Islets Engineered with SA-FasL Protein Establish
Robust Localized Tolerance by Inducing Regulatory T Cells
Esma S. Yolcu,*,†,1Hong Zhao,*,†,1Laura Bandura-Morgan,*,†Chantale Lacelle,*,†
Kyle B. Woodward,*,†Nadir Askenasy,‡and Haval Shirwan*,†
Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of
this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of
chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if
pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with
streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin
following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL–engineered
islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute
toxicity. Most importantly, the transplantation of SA-FasL–engineered BALB/c islet grafts in conjunction with a short course of
rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and main-
tained by CD4+CD25+Foxp3+regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established
tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of
long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming
the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface
immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the
treatment of type 1 diabetes.The Journal of Immunology, 2011, 187: 000–000.
CD4+T cells responding to a set of b cell-specific Ags (1–3).
Restoration of insulin-secreting b cell mass using allogeneic islet
transplantation has been viewed as a preferred treatment modality,
and its efficacy in restoring physiological glycemic control has
been demonstrated in clinical trials (4). However, the success of
allogeneic islet transplantation is compromised by immunological
rejection and secondary graft failure due to the continuous use
of immunosuppressive drugs to control rejection (5). Therefore,
ype 1 diabetes (T1D) is an autoimmune disease caused by
the destruction of insulin-producing b cells by a complex
set of immunological events initiated and coordinated by
novel approaches that specifically target and control destructive
auto- and alloimmune responses without continuous immuno-
suppression remain to be developed for the successful application
of allogeneic islet transplantation in the clinic.
Inasmuch as T cells play a critical role in the initiation of islet-
destructive auto- and alloreactive immune responses (6), specific
elimination of these cells or control of their function through
active regulatory mechanisms may prove effective in achieving
long-term islet allograft survival without the continuous use of
immunosuppression (7). In this context, immunomodulation with
FasL presents an attractive approach due to the critical role played
by Fas/FasL-mediated apoptosis in activation-induced cell death
(8), an important homeostatic molecular mechanism that controls
T cell responses to self-Ags (9). The immunomodulatory function
of FasL has been extensively exploited for the induction of tol-
erance to auto- and alloantigens using gene therapy (10–15). How-
ever, although gene therapy showed efficacy in some settings (10,
12–15), the controlled ectopic expression of FasL in transfected
cells and tissues is not only technically challenging, but also poses
We recently generated a chimeric form of FasL protein, strep-
tavidin (SA)-FasL, in which the extracellular domain of FasL
lacking potential metalloproteinase sites was cloned C terminus to
the core SA (16). This molecule exists as tetramers and oligomers
with potent apoptotic activity and can be displayed on the sur-
face of biotinylated cells in an efficient and rapid manner (16).
Most importantly, systemic immunomodulation with SA-FasL–
engineered donor splenocytes resulted in tolerance to cardiac
allografts (17). However, the application of this novel approach to
engineering tissues remains to be demonstrated. In this study, we
tested if pancreatic islets, instead of isolated cells, can be engi-
neered with SA-FasL protein and whether the engineered islets
*Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202;
†Department of Microbiology and Immunology, 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 49202, Israel
1E.S.Y. and H.Z. contributed equally to this work.
Received for publication October 1, 2010. Accepted for publication October 4, 2011.
This work was supported in part by Grants R21 AI057903 and R41 DK077242 from
the National Institutes of Health and Juvenile Diabetes Research Foundation (1-2001-
328); Kentucky Diabetes Research Board (KDR-PP09-23); the Commonwealth of
Kentucky Research Challenge Trust Fund; the Keck Foundation; and an American
Heart Association Grant-in-Aid (09GRNT2380136) and National Institutes of Health
Training Fellowship 5T32 HL076138-07 (to H.Z. and L.B.-M.).
Address correspondence and reprint requests to Dr. Haval Shirwan and Dr. Esma S.
Yolcu, Institute for Cellular Therapeutics, Donald Baxter Biomedical Building, Uni-
versity of Louisville, 570 South Preston Street, Suite 404E, Louisville, KY 40202.
E-mail addresses: email@example.com (H.S.) and esma.yolcu@louisville.
The online version of this article contains supplemental material.
Abbreviations used in this article: KDLN, kidney-draining lymph node; LN, lymph
node; MST, mean survival time; SA, streptavidin; T1D, type 1 diabetes; Teff, effector
T; Treg, regulatory T.
Published November 7, 2011, doi:10.4049/jimmunol.1003266
on November 13, 2011
overcome rejection and establish euglycemia following trans-
plantation into chemically diabetic allogeneic hosts. Our data
demonstrate for the first time, to our knowledge, that pancreatic
islets can be engineered with SA-FasL in a rapid and efficient
manner, and such engineered islets under transient cover of rapa-
mycin induce localized allotolerance that was initiated and main-
tained by CD4+CD25+Foxp3+regulatory T (Treg) cells homing
to the graft and graft-draining lymph nodes (LNs).
Materials and Methods
Mice and recombinant proteins
C57BL/6 (CD45.2; H-2b), C57BL/10 (CD45.2; H-2b), C57BL/6
Foxp3EGFP, and BALB/c (H-2d) mice were purchased from The Jackson
Laboratory. Congenic C57BL/6.SJL (CD45.1; H-2b) and TCR-transgenic
OT-I (CD8+T cell) mice on the rag22/2background were purchased from
Taconic Farms (Germantown, NY) and bred in our specific pathogen-free
animal housing facility at the University of Louisville using protocols
approved by the Institutional Animal Care and Use Committee. Recom-
binant SA, human SA-CD40L, and rat SA-FasL proteins were produced in
our laboratory using the Drosophila DES expression system (Invitrogen) as
previously described (16, 18).
Pancreatic islet isolation and engineering with SA-FasL
Pancreatic islets were harvested from 8–12-wk-old BALB/c mice under
anesthesia using a standard protocol as previously described (16). Islets
were engineered by first incubating in 5 mM EZ-Link Sulfo-NHS-LC-
Biotin solution (Thermo Scientific) in PBS at room temperature for 30
min followed by extensive washing to remove free biotin. Biotinylated
islets were then incubated in PBS containing SA-FasL or SA-CD40L
proteins (200 ng protein/450–550 islets/200 ml PBS) or equal molar of
SA as a control at room temperature for 30 min.
After several washes, islets were cultured in vitro for various days,
stained first with anti–SA-FITC Ab (Vector Laboratories) to detect SA-
FasL and SA-CD40L, washed several times in PBS, and then stained with
SA-allophycocyanin (BD Biosciences) to visualize biotin. Z-stack analysis
was performed to measure fluorescence intensity of SA-FasL on engi-
neered islets using LAS AF software on Leica TCS SP5 confocal mi-
croscopy (Leica Microsystems). Unmodified or SA-engineered islets were
used as controls for background staining.
Diabetes was induced in C57BL/6 mice by i.v. injection of streptozotocin
(200 mg/kg) and confirmed by two consecutive blood glucose readings
.300 mg/dl. Pancreatic islets were harvested, cultured overnight, and then
engineered with SA, SA-CD40L, or SA-FasL proteins. These islets were
then immediately transplanted under the kidney capsule of diabetic mice
(450–550 islets/mouse). Unless otherwise indicated, graft recipients were
injected i.p. with 0.2 mg/kg rapamycin (LC Company) starting on the day
of transplantation daily for 15 d. Animals were monitored for diabetes, and
those with two consecutive daily measurements of $250 mg/dl blood
glucose level were considered diabetic and confirmation of graft failure.
Assessing localized tolerance
To investigate the nature of observed tolerance, we performed two sets of
experiments on long-term (.100 d) graft acceptors. In the first set, uni-
lateral nephrectomy was performed to remove the SA-FasL–engineered
islet graft. After confirmation of hyperglycemia, a second set of unmodi-
fied islet graft was transplanted under the contralateral kidney capsule. To
eliminate the effect of surgery associated inflammation on graft rejection,
in a second model, unmodified donor islets were transplanted under the
contralateral kidney capsule 40 d prior to surgical removal of the kidney
harboring SA-FasL–engineered islet graft.
CD4+CD25+Foxp3+Treg cell analysis
Lymphocytes from peripheral LNs, spleen, and kidney-draining LNs
(KDLNs) of various treatment groups were harvested, stained with Abs to
mouse CD4-allophycocyanin and CD25-PE (BD Pharmingen) molecules,
washed with PBS, and permeabilized/fixed overnight at 4˚C using a Per-
meabilization/Fixation kit from eBioscience. The FcgII/III receptors were
blocked using 2.4G2 Ab, followed by staining with anti–Foxp3-FITC Ab
according to the manufacturer’s protocol (eBioscience). The cells were run
on the FACSCalibur (BD Biosciences), and the data were analyzed by
FlowJo software (Tree Star).
Snap-frozen sections of islet grafts were incubated in a blocking solution
(0.5% Triton X-100, 0.1% BSA, 5% goat serum, and 1:400 FcgII/III re-
ceptor block). Guinea pig anti-insulin Ab (DakoCytomation) and a rat anti-
mouse CD4 mAb (BD Pharmingen) were used to detect islets and CD4+
T cells, respectively. Staining of these Abs was visualized using secondary
Abs conjugated with Alexa Fluor-647 (CD4) and Alexa Fluor-555 (insulin)
followed by direct staining with FITC-conjugated rat anti-Foxp3 Ab
(eBioscience) to visualize Treg cells. Hoechst (Molecular Probes) was
used to stain the nucleus of the cell. Fluorescent images were obtained
using Leica TCS SP5 confocal microscopy (Leica Microsystems) under
320 original magnification. H&E staining was performed on formalin-
fixed and paraffin-embedded kidney tissue blocks as previously de-
CD25+T cell depletion
Selected groups of mice were injected i.p. with 300 mg purified anti-CD25
Ab/animal (PC61; Bio X Cell) to deplete CD25+T cells on days 14 or 100
posttransplantation of SA-FasL–engineered allogeneic islets. Depletion
was confirmed using the 7D4 Ab recognizing a different epitope of CD25
than PC61 Ab (19) to stain PBLs harvested at various times post-PC61
treatment. Groups of mice were also injected i.p. with 300 mg/animal
isotype Ab or NK1.1 Ab (clone PK136; Bio X Cell) on day 14 post-
transplantation as controls.
CD4+CD25+Foxp3+Treg cell adoptive transfer assay
CD4+CD25+T cells were sorted from KDLNs or spleens of long-term SA-
FasL–engineered islet graft acceptors, SA-engineered islet graft rejectors,
and naive C57BL/6 mice using Abs against CD4-FITC and CD25-PE in
flow cytometry. CD4+CD252effector T (Teff) cells from naive C57BL/6
mice spleen were sorted using flow cytometry. The purity of cells was
.95%. One million Teff cells were adoptively transferred i.v. into chem-
ically diabetic OT-I mice 1 d prior to the transplantation of unmodified
BALB/c islets either alone or with 2,500–10,000 CD4+CD25+Treg cells.
Mice were monitored for graft survival by assessing blood glucose levels
on a regular basis.
Assessing apoptosis of islet-infiltrating T cells
BALB/c islets engineered with SA-FasL or SA proteins were transplanted
under the kidney capsule of streptozotocin diabetic C57BL/6 Foxp3EGFP
mice subjected to daily rapamycin (0.2 mg/kg) treatment starting on the
day of transplantation. Three days posttransplantation, mice were eutha-
nized, and grafted islets were dissected with fine forceps from the kidney
capsule and mechanically dispersed into single cells. Cells were stained
with fluorochrome-conjugated Abs against CD3, CD4, CD8, CD25, Gr-1,
and CD11b molecules and Annexin V, run on a BD LSR II flow cytometer
(BD Biosciences), and analyzed using Diva software (BD Biosciences).
Assessing chemotactic function of SA-FasL for neutrophils
C57BL/6.SJL (CD45.1+) splenocytes were engineered with SA-FasL (40
ng SA-FasL protein/106cells) or equal molar of SA protein (20 ng SA
protein/106cells), and 5–10 3 106of the engineered cells were injected
i.p. into C57BL/6 (CD45.2+) mice. LPS (Sigma-Aldrich) injected i.p. at 10
mg/mouse served as positive control. Animals were terminated at various
times (17–44 h) postinjection, and peritoneal lavage was aspirated under
aseptic conditions. Lavage cells were stained with Abs against CD3,
CD19, Gr-1, CD11b, CD45.1, CD45.2, and F4/80 molecules. Percentages
of lavage neutrophils (CD11b+Gr-1high) were assessed by gating on re-
cipient (CD45.2+) cells using an LSR II and Diva software (BD Bio-
The t test and ANOVA were used to determine significance between two
and multiple groups, respectively. Graft survival was assessed using the
Kaplan-Meier method and the log-rank test. Data are expressed as mean 6
SD. The p values ,0.05 were considered significant. Statistical analysis
was performed using SPSS 13.0 software (SPSS).
Pancreatic islets engineered with SA-FasL protein ex vivo
establish euglycemia in diabetic syngeneic host
Various conditions were used to optimize the rapid and efficient
display of SA-FasL on the surface of mouse pancreatic islets
2SA-FasL–ENGINEERED ISLET GRAFTS INDUCE LOCALIZED TOLERANCE
on November 13, 2011
ex vivo without compromising their survival and function. After
testing various doses of biotin and SA-FasL, concentrations of 5
mM EZ-Link Sulfo-NHS-LC-Biotin and 200 ng SA-FasL/450–
550 islets were found to be optimum for islet engineering (Fig.
1A). Z-stack analysis demonstrated intense staining for both biotin
and SA-FasL on the external surface of islets, with the inner core
showing only moderate staining (Fig. 1B). Flow cytometric anal-
ysis of mechanically dispersed SA-FasL–engineered pancreatic
islets further confirmed the presence of both biotin and SA-FasL
on the majority of islet cells (Fig. 1C). Under these conditions,
SA-FasL did not have any toxic effect on islets, and the protein
persisted on the surface of islets for .1 wk ex vivo (Fig. 1D).
Importantly, transplantation of SA-FasL–engineered islet grafts
into chemically diabetic syngeneic C57BL/10 mice resulted in
normalized blood glucose levels and survival in all mice over a
100-d observation period (Fig. 1E). Collectively, these results dem-
onstrate that pancreatic islets can be engineered with SA-FasL
protein in a rapid and efficient manner without compromising
their function for establishing glucose homeostasis in diabetic
hosts in the absence of detectable acute toxicity.
SA-FasL–engineered islet grafts survive indefinitely in
allogeneic hosts treated with a short course of rapamycin
We next tested if the SA-FasL protein on islet grafts is effective in
preventing rejection in allogeneic hosts in the absence of immu-
nosuppression. SA-FasL–engineered BALB/c islets, although they
showed significantly (p = 0.001) prolonged survival in the ab-
sence of any immunosuppression in chemically diabetic allogeneic
C57BL/6 mice as compared with unmodified or SA protein-engi-
neered islets, only a moderate percentage (∼18%) of grafts sur-
vived over the 100-d observation period (Fig. 2A). To improve
long-term survival, SA-FasL–engineered islets were transplanted
in conjunction with a short course of rapamycin treatment. The
rationale for using rapamycin was 2-fold. First, rapamycin has
been shown to cause apoptosis of alloreactive T cells (20, 21) and
as such may serve to physically eliminate circulating alloreactive
T cells evading apoptosis induced by SA-FasL displayed on islet
grafts. Second, rapamycin has also been shown in various settings
to induce CD4+CD25+Foxp3+Treg cells (22, 23). All SA-FasL–
engineered islet grafts (n = 45) survived indefinitely and nor-
malized blood glucose levels in chemically diabetic allogeneic
C57BL/6 mice treated with 0.2 mg/kg rapamycin daily for 15
doses starting on the day of transplant (Fig. 2B). In marked con-
trast, all control islets engineered with SA (n = 10) or human SA-
CD40L (n = 5) protein that does not interact with mouse CD40
receptor (18) underwent acute rejection within 30 d. Taken to-
gether, these data demonstrate that SA-FasL alone is effective in
prolonging allogeneic islet graft survival with a modest effect on
long-term survival and that, in combination with a short course of
rapamycin treatment, SA-FasL is effective in inducing tolerance in
all islet graft recipients. Therefore, we used rapamycin as a com-
ponent of our immunomodulation protocol for the rest of the
Robust tolerance induced by SA-FasL–engineered islet grafts is
islet specific and localized
To test if euglycemia is maintained by the transplanted SA-FasL–
engineered allogeneic islet grafts, the kidney harboring the grafted
islets was surgically removed 100 d posttransplantation. All hosts
developed hyperglycemia within 3 d (n = 5). These mice were then
transplanted with a second set of unmanipulated donor allogeneic
islet grafts under the remaining kidney capsule. All islet grafts
were rejected in acute fashion (n = 5; mean survival time [MST] =
18 6 7.3 d; Fig. 3A). To test that surgery-associated trauma/
inflammation or temporary lack of donor Ags in the mice with
unilateral nephrectomy was not the cause of secondary islet graft
rejection, another group of long-term survivors (n = 4) were first
transplanted with a second set of unmanipulated allogeneic islets
under the contralateral kidney capsule and then nephrectomized
40 d later to remove the kidney harboring the primary graft. All
mice maintained euglycemia until the removal of the kidney har-
boring the primary islet grafts, which then resulted in the devel-
opment of hyperglycemia within 3 d (Fig. 3A). These data
demonstrate that the secondary unmanipulated grafts are rejected
without an effect on the survival of the primary grafts, thereby
demonstrating the localized nature of tolerance.
Lack of systemic tolerance was further tested by performing
BALB/c skin allografts into C57BL/6 mice with long-term (.100
d) surviving islet allografts. All skin grafts rejected at a similar
tempo (n = 4; MST = 12.75 6 1.26 d) to skin grafts (n = 6; MST =
11.3 6 1.21 d) transplanted onto naive C57BL/6 mice (Fig. 3B).
Importantly, unlike islet grafts (Fig. 3A), skin graft rejection re-
sulted in acute rejection of long-term islet grafts within 21 d
(Fig. 3B). Taken together, these data demonstrate that the transient
display of SA-FasL on pancreatic islet grafts is effective in induc-
ing localized tolerance, which can be overcome by alloreactive
responses against donor skin, but not islet, grafts.
Localized tolerance is induced and maintained by Treg cells
Although SA-FasL–engineered islet grafts may induce localized
tolerance by clonal deletion of alloreactive T cells (17), this mech-
anism is expected not to operate when the levels of SA-FasL on
pancreatic islet grafts decline with time. Therefore, newly arising
alloreactive T cells late posttransplantation need to be controlled
by ongoing immunoregulatory mechanisms, such as Treg cells. To
SA-FasL protein on their surface, and such engineered islets established
long-term euglycemia in chemically diabetic syngeneic recipients. A,
Confocal picture of SA-FasL–engineered pancreatic islets stained first with
anti–SA-FITC Ab (green) and then with SA-allophycocyanin (red) to vi-
sualize SA-FasL and biotin, respectively. Cells positive for both molecules
appear as yellow. Original magnification 320. B, Z-stack analysis of
pancreatic islets across the line shown in A. C, The FACS profile of
mechanically dispersed total engineered islet cells stained with SA-allo-
phycocyanin and anti–SA-FITC Ab to detect biotin and SA-FasL, re-
spectively. D, In vitro persistence of biotin (red) and SA-FasL protein
(green) on engineered pancreatic islets as a function of time shown in days.
Original magnification 320. E, Transplantation of 450–550 SA-FasL–
engineered islets under the kidney capsule of chemically diabetic synge-
neic host results in long-term euglycemia. Unmodified islets are used as
positive controls. Data shown for A–D are representative of two to three
Pancreatic islets can effectively be engineered to display
The Journal of Immunology3
on November 13, 2011
investigate the role of Treg cells in localized tolerance, lymphoid
organs from long-term syngeneic and allogeneic SA-FasL–engi-
neered allogeneic islet graft acceptors and SA-engineered islet
graft rejectors were analyzed at various times posttransplantation.
There were no detectable differences in the percentages of Treg
cells in the spleen, mesenteric LNs, or KDLNs of all three groups
(Fig. 4A, 4B, and data not shown).
In view of several recent reports providing evidence for in situ
intrapancreatic immune reactions playing a critical role in the de-
velopment of T1D in NOD mice (24, 25), we analyzed islet grafts
using immunohistochemistry and confocal microscopy for the
presence of Treg cells. On day 5 posttransplant, SA-FasL–engi-
grafts (Fig. 4C, 4D). Importantly, long-term (.100 d) SA-FasL–
engineered allogeneic islet grafts also had higher numbers of Treg
cells residing in the periphery of islet grafts as compared with
syngeneic grafts (Fig. 5A, 5B) and otherwise looked similar to
syngeneic grafts with respect to insulin expression and lack of sig-
nificant levels of inflammatory infiltrates (Fig. 5C, 5D).
The critical role of Treg cells in the localized tolerance was
further confirmed by in vivo elimination of these cells using an Ab
(PC61) against the CD25 molecule. Consistent with a previous
report (26), i.p. treatment of mice with PC61 Ab (300 mg/mouse)
partially depleted Treg cells because a significant percentage
(∼6%) of CD4+T cells in the blood remained Foxp3 positive with
downregulated expression of CD25, as assessed by a second Ab
(7D4) to a different CD25 epitope (Fig. 6A). Partial depletion of
Treg cells either early (day 14 posttransplant) during the estab-
lishment of tolerance or late (day 100 posttransplant), when tol-
erance has already been established, resulted in prompt islet graft
rejection (Fig. 6B). There was a direct correlation between the
depletion of CD4+Foxp3+Treg cells and the tempo of graft re-
jection. Mice that rejected islet grafts at a rapid tempo had fewer
CD4+Foxp3+Treg cells remaining in the periphery as compared
with mice that rejected at slower tempo (data not shown). Indeed,
one out of five mice that did not reject the allograft had inefficient
depletion of CD4+Foxp3+Treg cells (29 versus 39–66% depletion
in rejecting mice). Treatment with an isotype Ab or NK1.1-
depleting Ab did not prompt graft rejection in either setting (Fig.
6B), demonstrating that it is not mere depletion of a lymphocyte
population that sets off the rejection reaction. Collectively, these
data demonstrate that Treg cells play a critical role in the main-
tenance of localized tolerance.
Treg cells sorted from kidney draining LNs, but not spleens, of
long-term tolerant graft recipients prevent rejection of
unmodified donor islets in an adoptive transfer model
Although the partial depletion of Treg cells using anti-CD25 Ab
demonstrates theimportance ofthesecells inthe inducedtolerance,
it does not assess if they are the primary mechanism of tolerance.
We, therefore, tested the function of these cells in an adoptive
transfer allogeneic islet model. Given that the established tolerance
was not systemic and localized to the graft, we also tested if there
was a functional difference between Treg cells residing in the graft
draining LNs and spleens of tolerant mice. Treg cells were sorted
from the KDLNs or spleens of long-term (.100 d) SA-FasL–
engineered islet graft acceptors, SA-engineered islet graft rejec-
tors, and unmanipulated naive C57BL/6 mice. The sorted cells
were then adoptively transferred into chemically diabetic rag22/2
OT-I mice on C57BL/6 background 1 d prior to BALB/c alloge-
neic islet transplantation. Animals transplanted with unmodified
BALB/c allogeneic islets did not reject their grafts (n = 5; .100 d)
due to the specificity of the TCR in OT-I mice for the OVA Ag
(Fig. 7). However, adoptive transfer of 1 3 106CD4+CD252
survival of SA-FasL–engineered BALB/c islet grafts in C57BL/6 mice in the absence of rapamycin as compared with unmodified or islet-SA groups
(p = 0.001 versus others). B, SA-FasL–engineered islet grafts induce robust tolerance when transplanted under the transient cover of rapamycin as
compared with SA, nonfunctional human SA-CD40L–engineered, or unmodified control islet grafts (p , 0.0001). Because some animals were euthanized
for mechanistic studies at various time points, the survival curve represents: n = 45, 100 d; n = 23, 300 d; n = 18, 400 d; and n = 13, 500 d post-
SA-FasL–engineered pancreatic islets establish robust allotolerance when transplanted under the transient cover of rapamycin. A, Prolonged
is localized to the graft. A, Long-term tolerant mice reject unmodified
secondary islet grafts from the donor. C57BL/6 recipients of primary SA-
FasL–engineered islet grafts underwent unilateral nephrectomy to remove
the kidney harboring primary SA-FasL–engineered grafts 100 d post-
transplantation. After the confirmation of hyperglycemia within 3 d, these
mice were transplanted with a second set of unmodified BALB/c alloge-
neic islets under the contralateral kidney capsule. All of the secondary
grafts were rejected in a normal tempo (MST = 18 6 7.3 d; 2nd islet post-
nephrectomy). To ensure that the rejection of the second set of islets is not
due to nephrectomy-associated inflammation, a second group of long-term
SA-FasL–engineered islet graft recipients was transplanted with unma-
nipulated BALB/c islets under the contralateral kidney capsule. Removal
of the kidney harboring primary islets 40 d later resulted in hyperglycemia
in all animals (2nd islet pre-nephrectomy). B, Long-term allogeneic islet
graft survivors reject allogeneic skins from BALB/c mice in a normal
tempo (MST = 12.75 6 1.26 d) as compared with naive mice (MST = 11.3 6
1.21 d). Allogeneic skin graft rejection also results in the rejection of long-
term (100 d) allogeneic islet grafts (MST = 19.25 6 6.90 d) in a similar
tempo to naive islets (MST = 14.5 6 1.76 d).
Tolerance induced by SA-FasL–engineered allogeneic islets
4 SA-FasL–ENGINEERED ISLET GRAFTS INDUCE LOCALIZED TOLERANCE
on November 13, 2011
T cells alone from naive C57BL/6 mice resulted in acute rejection
of allogeneic islets (n = 5; MST = 29.0 6 9.2 d). Cotransfer of
2,500–10,000 Treg cells sorted from graft-draining LNs of SA-
FasL–islet graft acceptors prevented islet graft rejection induced
by 1 3 106CD4+CD252T cells (n = 6; MST .100 d). In marked
contrast, cotransfer of the same number of Treg cells sorted from
either SA-islet graft rejectors or naive C57BL/6 mice did not
prevent rejection mediated by CD4+CD252T cells (n = 4; MST =
36 6 2.3 d and n = 5; MST = 34.4 6 3.5 d, respectively; Fig. 7).
Importantly, splenic Treg cells sorted from SA-FasL–islet graft
acceptors had significantly reduced efficacy in preventing the re-
jection of unmanipulated donor islet allografts in the adoptive
transfer model as compared with Treg cells sorted from graft-
draining LNs. Indeed, only one out of six mice, which received
the highest number of Treg cells, in this group accepted the donor
graft, whereas all of the rest had rejection, but in a delayed fashion
(MST = 48 6 3.8 d) as compared with controls (Fig. 7). These
studies provide direct evidence for the role of Treg cells in the
induced tolerance and further demonstrate that graft-protective
Treg cells preferentially home to the graft-draining LNs, but not
distant lymphoid organs, such as spleen.
SA-FasL–engineered pancreatic islets or splenocytes lack
chemotactic activity for neutrophils
It has been shown that FasL can cause tissue destruction by serving
as a chemotactic factor for neutrophils (27–29). We, therefore,
conducted two sets of studies to directly test if SA-FasL has such
a function. In the first set of studies, SA-FasL–engineered syn-
geneic splenocytes were injected i.p. into mice, and peritoneal
exudate cells were harvested at various time points to assess the
recruitment of neutrophils using flow cytometry. Splenocytes en-
gineered with SA served as negative control, whereas LPS was
used as positive control. We observed about the same percentages
(∼12%) of neutrophils among peritoneal exudate cells harvested
increased numbers of CD4+CD25+Foxp3+Treg cells
within the islet grafts. A, Analysis of CD4+Foxp3+
Treg cells gated on total lymphocytes harvested from
spleen, mesenteric LN, and KDLN of SA-allogeneic
islet graft rejectors and long-term (.100 d) SA-FasL–
allogeneic or syngeneic islet graft survivors. B, Tabu-
lation of the data presented in A (n = 3–5 animals/
group). C, Representative confocal images of Treg
cells in day 5 posttransplant SA-FasL– or SA-engi-
neered allogeneic islet grafts. Nuclear staining is
shown in blue. Scale bars, 50 mm. D, Tabulation of
Treg cells shown in C. The data are shown as average
of Treg cells per section and a minimum of three sec-
tions per animal with three animals per group.
Localized tolerance is associated with
periphery of the graft in the absence of significant inflammatory infiltrates. A and B, Kidney harboring islet grafts were surgically removed from recipients
of syngeneic or SA-FasL–engineered allogeneic islets on day 100 posttransplantation. Tissues were stained with Abs against CD4 (red), Foxp3 (green), and
counterstained with Hoechst (blue). Arrows indicate islet grafts. Scale bars, 100 mm. Right panels are higher magnification of areas shown by rectangles in
A and B. Scale bar, 25 mm. C and D, Tissues in A and B were stained with Abs against insulin (red) and counterstained with Hoechst (blue). Scale bar, 100
mm. H&E staining pattern for syngeneic and allogeneic islet grafts is depicted in insets. Original magnification 320.
Long-term SA-FasL–engineered allogeneic islet grafts maintain increased numbers of CD4+CD25+Foxp3+Treg cells localized within the
The Journal of Immunology5
on November 13, 2011
from mice injected with SA-FasL– and SA-engineered spleno-
cytes (Fig. 8A). In marked contrast, ∼60% of peritoneal exudate
cells were neutrophils in the LPS-positive group. In a second set of
studies, we assessed the presence of neutrophils among recipient
cells infiltrating into SA- and SA-FasL–engineered BALB/c islet
grafts transplanted into C57BL/6 mice under the cover of rapa-
mycin. SA-FasL–islet grafts had low levels of neutrophils as
compared with SA-engineered islets (Fig. 8B). Taken together,
these data demonstrate that SA-FasL displayed on splenocytes or
pancreatic islets lacks chemotactic activity for neutrophils.
We demonstrate for the first time in this study, to our knowledge,
that pancreatic islets can be engineered ex vivo in a rapid and
efficient manner to display on their surface an apoptotic form of
FasL protein, SA-FasL, and that such islets induce robust local-
ized, rather than systemic, tolerance when transplanted into fully
allogeneic hosts under transient cover of rapamycin without de-
tectable toxicity to the graft or host. Tolerance by this protocol is
initiated and sustained by Treg cells, as physical depletion of these
cells in vivo early (14 d) or late (.100 d) posttransplantation
resulted in acute graft rejection. Furthermore, sorted Treg cells
from long-term, but not rejecting, graft recipients prevented the
rejection of unmanipulated donor allogeneic islets in an adoptive
transfer model, further confirming their role in the induced tol-
Tolerance in this model is completely dependent on SA-FasL
protein and accentuated by a short course of rapamycin treat-
ment. This observation is consistent with the demonstrated role
of Fas-meditated apoptosis in lymphocytes for the regulation of
chronic immune responses and control of autoimmunity (9). Upon
activation, T cells upregulate both Fas and FasL and become sen-
sitive to autocrine and paracrine apoptosis following repeated
engagement with the challenge Ag (9, 30). Therefore, the pre-
sentation of alloantigens in the context of FasL may specifically
eliminate alloreactive T cells that upregulate the Fas receptor
and thereby become sensitive to Fas/FasL-mediated apoptosis.
Indeed, analysis of T cells infiltrating into the grafts 3 d post-
transplantation revealed higher percentages of CD4+Teff and
CD8+Teff cells undergoing apoptosis in SA-FasL–islet grafts
as compared with SA-islet grafts (Supplemental Fig. 1). These
findings are also consistent with our recent studies demonstrating
that systemic immunomodulation with SA-FasL–engineered do-
nor splenocytes induces apoptosis in alloreactive T cells, resulting
in the inhibition of primary and secondary alloreactive immune
responses (16) and induction of tolerance to cardiac allografts
(17). Our findings are also consistent with several studies by others
were collected from SA-FasL–engineered islet graft recipients before (D0) or 3 d (D3) after treatment with the PC61 Ab and typed using the 7D4 Ab
recognizing a different CD25 epitope than PC61 (n = 3). A significant percentage (∼6%) of CD4+T cells expressing Foxp3 remains after PC61 Ab
treatment. B, Depletion of Treg cells in C57BL/6 mice transplanted with SA-FasL–engineered allogeneic BALB/c islets early (day 14) or late (day 100)
posttransplantation using PC61 anti-CD25 Ab. An isotype Ab and an anti-NK1.1 Ab are used on day 14 as controls. *, **p values for NK 1.1-depleted
group or isotype control versus groups depleted with PC61 on days 14 and 100 posttransplantation, respectively.
Treg cells are critical for the induction and maintenance of localized tolerance. A, Depletion of Treg cells using PC61 (anti-CD25) Ab. PBLs
rejection of unmanipulated donor islets in an adoptive transfer model.
Survival of allogeneic unmodified BALB/c islet grafts in rag22/2OT-I
mice that did not receive T cell transfer (No CD4+T cells) or adoptively
transferred with 1 3 106flow-sorted conventional CD4+T cells from naive
C57BL/6 mice alone (Naive CD4+T cells) or in combination with flow-
sorted Treg cells from KDLN (n = 3, 2.5 3 103; n = 2, 5 3 103; n = 1, 10 3
103cells) or spleens (n = 2, 2.5 3 103; n = 2, 5 3 103; n = 2 10 3 103
cells) of long-term SA-FasL–islet graft survivors (tolerant CD4+Tregs),
KDLN of SA-islet rejectors (rejector CD4+Tregs; n = 4, 5 3 103cells), or
naive C57BL/6 mice (naive CD4+Tregs; n = 1, 2.5 3 103; n = 2, 5 3 103;
n = 2, 10 3 103cells). p = 0.002, KDLN tolerant CD4+Treg versus all the
other adoptive T cell transfer groups.
Treg cells sorted from LNs of long-term islet grafts prevent
C57BL/6 (CD45.2) mice were injected i.p. with LPS or C57BL/6.SJL
(CD45.1) splenocytes engineered with SA-FasL or SA. Peritoneal lavage
cells were harvested at various times postinjection, stained with Abs to
various cell surface markers, and analyzed using flow cytometry by gating
on recipient cells (CD45.2). Data are shown for 20 h postinjection (n = 3/
group). B, Graft-infiltrating cells were harvested at 3 d posttransplantation
from SA or SA-FasL–engineered BALB/c islets transplanted under the
kidney capsule of STZ diabetic C57BL/6 mice treated with rapamycin.
Cells were stained with various cell-surface markers and analyzed using
flow cytometry (n = 3/group). Results are expressed as mean 6 SD.
SA-FasL lacks chemotactic activity for neutrophils. A,
6 SA-FasL–ENGINEERED ISLET GRAFTS INDUCE LOCALIZED TOLERANCE
on November 13, 2011
using cells (13), tissues (15), or organs (31) genetically manipu-
lated to express FasL for immunomodulation.
Contradictory data demonstrating that immunomodulation with
FasL does not prevent graft rejection have also been reported. For
example, islets and heart grafts genetically modified to express
FasL lacked protection in allogeneic hosts (11, 32). These negative
results have been attributed to the use of a cleavable isoform of
FasL and diverse functions associated with the membranous and
soluble forms of this molecule. FasL is a type II membranous
protein that is converted into a soluble form via cleavage by
matrix metalloproteinases in response to various physiologic
stimuli (33). The membranous form is noted for its ability to in-
duce apoptosis in autoreactive and alloreactive T cells, thus pro-
moting tolerance. In contrast, the soluble form may inhibit ap-
optosis, initiate inflammatory responses, and promote the active
recruitment of neutrophils, thereby accelerating disease or allo-
graft rejection (29, 34, 35). Although the separation of these
distinct functions of the soluble versus membranous forms of FasL
has been the source of great controversy, a recent study using
transgenic mice expressing either membranous or secreted forms
of FasL demonstrated that the membranous form only has apo-
ptotic activity and is responsible for the elimination of self-
reactive T cells and prevention of systemic autoimmunity (9). In
marked contrast, the soluble form had no apoptotic activity and
appeared to promote autoimmunity and tumorogenesis via non-
The SA-FasL molecule used in this study lacks potential met-
form, but rather forms stable tetramers or oligomers (16) without
any detectable chemotactic activity for neutrophils as demon-
strated in this study (Fig. 8). Given that oligomerization of the Fas
receptor on the surface of T cells is critical for the delivery
of apoptotic signals (36), SA-FasL has potent apoptotic activity
in soluble form (16) or when displayed on the cell surface (17).
Therefore, SA-FasL–engineered allogeneic cells or tissues have
the potential to effectively eliminate alloreactive T cells via apo-
ptosis, thereby initiating a cascade of regulatory events resulting
in either systemic tolerance (17) or localized tolerance as shown
in this study, with CD4+CD25+Foxp3+Treg cells serving as the
common denominator. Treg cells were shown to be relatively re-
sistant to Fas-induced apoptosis under selected experimental con-
ditions (37) and are spared by rapamycin (22). Consequently, we
observed high numbers of Treg cells homing to the islet graft.
Physical depletion of these cells either early or late posttrans-
plantation resulted in the abrogation of tolerance. The critical role
of these cells in the observed tolerance was further demonstrated
in an adoptive transfer model in which Treg cells sorted from
graft-draining LNs of tolerant, but not those from naive or SA-
islet, graft rejectors prevented the rejection of unmanipulated
donor islet grafts. Importantly, splenic Treg cells isolated from
tolerant SA-FasL–islet graft recipients were inferior to Treg cells
from graft-draining LNs in preventing rejection in the adoptive
transfer model. Taken together, these data demonstrate that graft-
protective Treg cells preferentially home to graft-draining LNs
and potentially to the graft and play a critical role in keeping in
check the pathogenic Teff cells. Although we cannot totally ex-
clude other regulatory mechanisms in addition to Treg cells in-
volved in the observed tolerance, our data are consistent with a
recent study by Kendal et al. (38), demonstrating that Treg cells
play a critical role in tolerance to skin allografts induced by
nondepleting anti-T cell Abs by preferentially residing in the graft
and keeping in check the pathogenic function of Teff cells.
SA-FasL may contribute to the generation/expansion of Treg
cells by two distinct mechanisms: 1) preferential elimination of
activated alloreactive Teff cells due to their enhanced sensitivity to
FasL-mediated apoptosis and as such tilting the balance toward
Treg cells (37); or 2) generation of induced Treg cells through
mechanisms involving apoptosis (39). Unlike the human Treg cells
that are not only resistant to Fas/FasL-mediated apoptosis, but
also use FasL as an effector molecule to induce apoptosis in Teff
cells as a means of immune suppression (40, 41), the preferential
sensitivity of mouse Treg cells over the Teff cells to Fas/FasL-
mediated apoptosis has been the subject of significant controversy.
A series of studies in the autoimmunity and cancer settings has
demonstrated that Treg cells are more sensitive than Teff cells to
Fas/FasL-mediated apoptosis (42–44). For example, immunomo-
dulation with FasL protein was recently reported to selectively
deplete Treg cells from tumor (43). Similarly, the efficacy of
immunomodulation with IL-12 was shown to be dependent on
CD8+T cells expressing FasL and eliminating Treg cells within
the tumor via Fas/FasL-mediated apoptosis (42). In marked con-
trast, freshly isolated CD4+CD25+Treg cells were shown to be
less sensitive to Fas-mediated apoptosis as compared with CD4+
CD252T cells in response to CD3 stimulation in vitro (45).
However, this differential sensitivity to Fas/FasL-mediated apo-
ptosis could be reversed in coculture experiments depending on the
Treg/Teff cell ratios that were regulated by IL-2. Consistent with
this study, we recently demonstrated relative resistance of NOD
Treg cells to SA-FasL–mediated apoptosis under inflammatory
conditions as compared with Teff cells (37, 46, 47). Indeed, the
direct display of SA-FasL on the surface of Treg cells endowed
them with better regulatory activity by inducing apoptosis in Teff
cells via SA-FasL/Fas interaction (46). In marked contrast, the
direct display of SA-FasL on NOD Teff cells resulted in their
accelerated apoptosis and reduced onset and incidence of diabetes
in an adoptive transfer model (47). Consistent with these studies,
we observed higher percentages of CD4+Teff and CD8+Teff cells
undergoing apoptosis in SA-FasL–islet grafts as compared with
SA-islet group. In marked contrast, CD4+Treg cells appeared to
be resistant to SA-FasL–induced apoptosis (Supplemental Fig. 1).
Although the molecular nature of these opposing observations is
not known and remains to be elucidated, they indicate the complex
nature of Fas/FasL-mediated homeostasis of Treg and Teff cells
under normal physiological conditions and disease settings.
Alternative and/or additive to the sparing of Treg cells from Fas-
mediated death, SA-FasL–engineered islet grafts may facilitate de
novo generation of induced Treg cells via a cascade of immuno-
regulatory mechanism orchestrated by apoptosis of Teff cells. Con-
sistent with notion is a recent study demonstrating that immu-
nomodulation with anti-CD3 Ab results in apoptosis of T cells,
digestion of apoptotic bodies by phagocytes, and their secretion
of TGF-b that converts Ag-specific Teff cells into Treg cells (39).
Importantly, Treg cells harvested from SA-FasL–engineered, but
not SA-control, islet graft recipients prevented rejection of un-
modified second set donor islet grafts in an adoptive transfer
model, suggesting that these cells may represent the induced Treg
cells that show Ag specificity.
The tolerogenic effect of SA-FasL was greatly enhanced by
a short course of rapamycin treatment. Rapamycin may work in
synergy with SA-FasL to enhance tolerance by eliminating Teff
cells (20) and contributing to the generation of Treg cells either
by sparing natural Treg cells (22) or actively contributing to the
conversion of Teff cells into induced Treg cells through the reg-
ulation of the mammalian target of rapamycin (23). Important in
this context is a recent study demonstrating that in the presence
of rapamycin TCR/IL-2 signaling resulted in the upregulation of
antiapoptotic Bcl-2 family members in Treg cells and their relative
resistance to apoptosis (48). In marked contrast, under the same
The Journal of Immunology7
on November 13, 2011
conditions, rapamycin downregulated the expression of Bcl-2 fam-
ily members while increasing the expression of proapoptotic Bax
in T conventional cells, leading to their increased sensitivity to ap-
Genetic manipulation of pancreatic islets ex vivo to express
immunoregulatory molecules for long-term survival in allogeneic
hosts represents an attractive immunomodulatory approach that
may spare the recipient from harmful immunosuppressive treat-
ments. However, introduction of foreign DNA into the graft is not
only technically challenging but also has safety concerns, and
continuous expression of potent immunomodulatory molecules,
such as FasL, on the graft may have long-term complications.
Therefore, our approach of engineering donor grafts ex vivo to
FasL, in a rapid, effective, and transient manner represents a novel
means of immunomodulation with therapeutic implications in
cancer, autoimmune disease, and transplantation.
We thankO. Grimany for technical help with SA-FasL, SA-CD40L, and SA
protein expression and purification and Drs. Suzanne T. Ildstad, Michele
Kosiewicz, and Huang-Ge Zhang for critical reading of the manuscript.
The SA-FasL protein and ProtEx technology described in this article are
licensed from the University of Louisville by ApoVax, Inc., Louisville,
KY, for which H.S. serves as a Member of the Board and Chief Scientific
Officer, and H.S. and E.S.Y. have significant equity interest in the com-
pany. The other authors have no financial conflicts of interest.
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