PD-L1 driven Tolerance Protects Neurogenin3 …...PD-L1 driven Tolerance Protects...
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PD-L1 driven Tolerance Protects Neurogenin3-induced Islet Neogenesis to Reverse
Established Type 1 Diabetes in NOD Mice
Rongying Li1, Jeongkyung Lee
1, Mi-sun Kim
1, Victoria Liu
1, Mousumi Moulik
2, Haiyan Li
3, Qing
Yi3, Aini Xie
1, Wenhao Chen
1, Lina Yang
1, Yimin Li
1, Tsung Huang Tsai
1 , Kazuhiro Oka
1, 4,
Lawrence Chan1,4,5, Vijay Yechoor
1, 4,5
1Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center &
Department of Medicine, Baylor College of Medicine, Houston, TX,77030; 2Division of Cardiology,
Department of Pediatrics, University of Texas Medical School at Houston, 3Department of Cancer
Biology, Cleveland Clinic, Lerner Research Institute, Cleveland, OH, 44195; 4Division of Molecular and
Cellular Biology, Baylor College of Medicine, Houston, TX,77030; 5Correspondence should be addressed
to V. Y. ([email protected]) and L. C. ([email protected])
Abbreviated Summary: PD-L1 protects Ngn3-induced-islets to reverse T1D
Total number of words in the main text: 3893
Contact Information:
Vijay Yechoor, M.D.
Asst. Professor, Div. of Diabetes, Endocrinology & Metabolism
Depts. of Medicine and Molecular & Cellular Biology
Diabetes Research Center, Baylor College of Medicine
R612, One Baylor Plaza
Houston TX 77030
713-798-4146
713-798-8764 (fax)
Page 1 of 49 Diabetes
Diabetes Publish Ahead of Print, published online October 20, 2014
Abstract
A breakdown in self-tolerance underlies autoimmune destruction of β-cells and Type 1 diabetes.
A cure by restoring β-cell mass is limited by the availability of transplantable β-cells and the
need for chronic immunosuppression. Recent evidence indicates that inhibiting co-stimulation
via PD-1/PD-L1 pathway is central to immune tolerance. We, therefore, tested if induction of
islet neogenesis in the liver, protected by PD-L1-driven tolerance, reverses diabetes in NOD
mice. We demonstrate a robust induction of neo-islets in the liver of diabetic NOD mice by gene
transfer of Ngn3, the islet-defining factor, along with betacellulin, an islet growth factor. These
neo-islets express all the major pancreatic hormones and transcription factors. However, an
enduring restoration of glucose-stimulated insulin secretion and euglycemia occurs only when
tolerance is also induced by the targeted overexpression of PD-L1 in the neo-islets, which results
in inhibition of proliferation and increased apoptosis of infiltrating CD4+T-cells. Further analysis
revealed an inhibition of cytokine production from lymphocytes isolated from the liver but not
from the spleen of treated mice, indicating that treatment did not result in generalized
immunosuppression. This treatment strategy leads to persistence of functional neo-islets that
resist autoimmune destruction and consequently an enduring reversal of diabetes in NOD mice.
Page 2 of 49Diabetes
Restoration of functional β-cell mass to cure type 1 diabetes (T1D) has been limited by a lack of
long-lasting transplantable β-cells (1). The long term success of islet transplantation is limited by
the requirement for chronic immunosuppression, the limited donor availability and eventual graft
failure (2). Although immunosuppressive regimens have been optimized, they lead to
generalized immunosuppression, with some of the drugs themselves being β-cell toxic (3).
Targeted immunomodulation, without systemic immunosuppression, to prevent islet destruction
by autoimmunity still remains an elusive goal.
T-effector cells mediate the autoimmune destruction of β-cells in T1D, though mechanisms
underlying this loss of self-tolerance remains poorly understood. Recent work has highlighted
the central role of the Programmed Death-1/Programmed Death Ligand-1 (PD-1/PD-L1)
pathway in induction and maintenance of peripheral tolerance in autoimmune diabetes (4-9). The
engagement of PD-L1, expressed normally by β-cells, with PD-1 on T-effector cells leads to
truncation of the TCR signal by inhibiting required co-stimulation pathways and limits cytolysis
by local self-reactive T-cells (10;11), in both native and transplanted islets (12-14). In addition,
NOD transgenic mice constitutively expressing PD-L1 under the human insulin promoter (RIP)
were significantly protected from diabetes (15), attesting to the tolerogenic role of the PD1-
PDL1 pathway.
While induction of islet neogenesis is an attractive approach to the restoration of β-cell mass, it
still requires immunomodulation to prevent autoimmune destruction of the induced neo-islets.
We have demonstrated previously that delivery of the islet-lineage determining gene,
Neurogenin3 (Ngn3), with the islet growth factor gene, betacellulin (Btc), using helper-
dependent adenoviral (HDAd) vectors, induces ectopic islet neogenesis in the periportal regions
Page 3 of 49 Diabetes
of the liver that is sufficient to reverse insulin-deficient diabetes in streptozotocin (STZ)-induced
diabetic mice (16;17). However, in NOD mice, this regimen does not lead to a diabetes reversal
due to autoimmune-mediated destruction of the induced neo-islets. In this study, we demonstrate
that targeted induction of tolerance by overexpression of PD-L1 in the newly induced β-cells
promotes their long-term survival, leading to a reversal of diabetes in NOD mice, with
restoration of glucose tolerance. We show that this tolerance is due to a local reduction in
number and activation of CD4+T-cells only in the periportal regions surrounding the neo-islets.
This study demonstrates that tolerance can be conferred to Ngn3-induced islet neogenesis by
inhibition of co-stimulation with PD-L1 to effectively reverse T1D.
Methods
Animals
NOD/ShiLtJ and NOD.CB17-Prkdcscid
/J (NOD-Scid) mice (Jackson Labs) were housed under
standard conditions. All animal protocols were approved by the IACUC at Baylor College of
Medicine. Non-fasting body weight and blood glucose were monitored weekly ~9AM. The
vectors encoding Ngn3 (HDAd-Ngn3), Btc (HDAd-Btc) and RIP-PD-L1 (HDAd-PD-L1) were
generated on serotype 5, as described previously (16). Total vector dose was maintained at
7×1011
viral particles (vp) in all treatment groups: [5×1011
vpNgn3+1×1011
vpBtc+1×1011
vp
empty vector], [5×1011
vpNgn3+1×1011
vpBtc+1×1011
vpPD-L1] or [1×1011
vpPD-L1+6×1011
vp
empty vector]. HDAd were injected intravenously via tail vein within 48h after diagnosis of
diabetes (two blood glucoses between 250-500 mg/dl). A glucose tolerance test (GTT) was
performed 4 and 8 weeks after treatment, on 6hr fasted mice administered 1.5 gm/kg of D-
Page 4 of 49Diabetes
Glucose IP, and glucose and insulin were assayed at 0, 15, 30, 60, 120 min. BrdU (15µl/gm) was
injected 2hr before sacrifice.
Immunofluorescence Microscopy and Immunohistochemistry
Immunofluorescence and immunostaining were performed on 5µm thick formalin-fixed paraffin-
embedded liver and pancreas sections, as previously described (16). Primary Antibodies used
and their dilutions are available on request. For assessing the T-cell numbers and their co-
localization with BrdU, TUNEL and Foxp3 in the periportal clusters, 1000-2000 cells were
counted from 30-40 clusters from 3 sections 100 µm apart from 4-5 mice from each group.
Flow Cytomentry
Mononuclear cells from the liver and spleen, 8wks after treatment (Ngn3-Btc or Ngn3-Btc+PD-
L1), were isolated as described (19) and used for flow cytometry analysis immediately or after
culture ex vivo. For ex vivo culture, cells were seeded in 96 well flat bottomed plate, coated with
anti-CD3 (4µg/ml) overnight, and cultured for 48hrs in medium containing anti-CD28 (2µg/ml).
Cells were analyzed after a 6hr incubation with 1 µl/mL Golgiplug. For in vitro stimulation and
co-incubation of CD4+ cells and β cells together, CD4
+ cells were purified from the spleen of
non-diabetic NOD mice and stimulated with anti-CD3 and anti-CD28 as above. β-cells
(Insulinoma – Ins2 β-cell line, a gift from Dr. Akio Koizumi) were infected with helper
dependent empty (HDV-empty) or PD-L1 virus (HDV-PD-L1) for 2 days. Purified CD4+
were
incubated with infected β-cells with ratio1:1 for 24 hours. Cells were analyzed after 6hr
incubation with 1 µl/mL Golgiplug. Cells were acquired on an LSR II (BD) and analyzed with
FlowJo (Tree Star, Inc.) software.
Page 5 of 49 Diabetes
Bioplex tissue cytokine assay
Lysate from the livers of mice treated for 8 weeks and pancreas from diabetic and non-diabetic
NOD mice were used at a final protein concentration of 0.5 mg/ml and assayed for cytokines
using 23 cytokine multiplexed bead-based immunoassay kits and Bio-PlexTM
Protein Array
System, using the high PMT settings and at least 100 bead count per analyte, per manufacturer’s
protocol (Bio-Rad).
Adoptive transfer
2x106
splenocytes (from Ngn3-Btc-PD-L1 or Ngn3-Btc treated groups), in 250µl of serum-free
media were injected IP into 6 week female NOD-Scid mice. 3-5 donors were used per treatment
group with 1-2 recipients per donor. 2x106
splenocytes from diabetic NOD mice were injected
into 6 week old female NOD-Scid mice or Ngn3-Btc-PD-L1 treated mice. Weekly blood glucose
was measured in the recipients to determine the diabetes transfer rate.
Skin Transplantation
Skin Transplantation was performed as described previously (20). Briefly, ear skin (1.0 cm2)
from the donor mice (8-12 weeks old, C57/BL6) was grafted onto the flank of recipient NOD
mice (Ngn3-Btc-PD-L1 or Ngn3-Btc treated mice). Grafts were scored daily until rejection
(defined as 80% of grafted tissue becoming necrotic and reduced in size).
Page 6 of 49Diabetes
Virus clearance
Helper virus, Ad2LC8cCARP – serotype 2 serotype, 1*10^11vp/mouse) was injected IV (21), to
Ngn3-Btc-PD-L1 or Ngn3-Btc mice. Mice were euthanized at day 3 or 3 weeks after injection
and liver tissue was harvested. DNA was extracted using Dneasy Blood and Tissue Kit (Qiagen)
and viral copy number analyzed by qPCR, using serotype specific primers
Statistical Methods
Student’s t-testing or ANOVA were used for significance testing. For adoptive transfer
experiment, Kaplan-Meier survival analysis using log-rank test was used in Sigma Plot. p<0.05
was considered significant.
Results
Ngn3-Btc+PD-L1 gene transfer reverses diabetes in NOD mice
We have shown before that Ngn3-Btc gene transfer using HDAd vectors leads to induction of
insulin-expressing neo-islets in the liver and reverses diabetes in streptozotocin-induced diabetic
mice (16;22). However, this did not ameliorate the hyperglycemia in overtly diabetic NOD mice,
which continued to lose weight with time (Fig. 1A-B). We, therefore, combined Ngn3-Btc with
Rat Insulin Promoter (RIP) -driven PD-L1, so that the expression of PD-L1 is limited only to
insulin-expressing cells induced in the liver of treated mice. In contrast to Ngn3-Btc or PDL-1
gene transfer alone, the combination therapy led to a rapid reversal of hyperglycemia and
promoted weight gain in over 75% of treated mice. This was sustained for at least 14 weeks, the
duration of the study (Fig. 1A-B). The treatment restored normal circulating plasma insulin (Fig.
Page 7 of 49 Diabetes
1C) with no deleterious effects on liver function (Fig. 1D-E). A glucose tolerance test (GTT)
demonstrated restoration of normal glucose tolerance and in vivo glucose-stimulated insulin
secretion (GSIS) to the levels seen in non-diabetic mice, by 4 weeks (Fig. 1F-G) and sustained at
8 weeks after treatment (Fig. 1H-I) only when treatment with Ngn3-Btc was combined with PD-
L1.
Neo-islets induced in the liver of Ngn3-Btc treated NOD mice persist only when PD-L1 is
also expressed.
Ngn3-Btc transiently induced insulin-expressing cells in the periportal regions of the liver in
overtly diabetic NOD mice (Fig. 2A), which were quickly destroyed by an immune infiltrate. In
contrast, the periportal neo-islets induced by Ngn3-Btc+PD-L1 combination therapy in the liver
are similar to pancreatic islets, in that they express the four major islet hormones that were
clearly identified by 4 weeks, and sustained when re-examined at 8 and 12 weeks, after treatment
(Fig. 2A-E and Fig. S1A). These neo-islets stained robustly both for insulin and C-peptide (Fig.
2B), indicating they produced proinsulin that was processed to mature insulin. The induction and
persistence of neo-islets was limited to the liver, as the pancreas did not display any recovery of
islets (Fig. S1B-C). Strong staining for PD-L1 was limited only to the insulin expressing neo-
islets (Fig. S2A-B).
The periportal neo-islets also expressed transcripts of the different islet hormones (Fig. 3A). At 4
weeks, consistent with the data shown in Fig. 2, Ngn3-Btc, either alone or combined with PD-L1
induced many of these hormones. However, by 8 weeks after treatment, the level of expression
of these hormones was significantly higher in the mice that were treated with Ngn3-Btc+PD-L1,
Page 8 of 49Diabetes
in parallel with the persistence and expansion of the neo-islets when PD-L1 was added to Ngn3-
Btc (Fig. 3A and S3A). A similar pattern of gene expression was observed with the islet
transcription factors (Fig. 3B, Fig. S3B) and in the progressive increase of the protein levels of
Pdx-1 and Nkx6.1, transcription factors critical to the normal development and function of β-
cells (Fig. 3C-D). Interestingly, expression of Ngn3 itself and many of its immediate downstream
target genes, such as Neurod1, persisted in mice treated only with Ngn3-Btc, despite the auto-
immune mediated destruction of insulin-expressing cells. This observation supports the
hypothesis that the Ngn3-Btc is effective in inducing the transcription factors required for neo-
islet development but PD-L1 is required, only to allow their persistence in the face of an
autoimmune attack. These neo-islets also expressed hepatic oval cell markers OC2-1D11 (23)
and A6 (Fig. S4A-B), consistent with our previous study that these neo-islets arose from
reprogramming of hepatic oval cells (16).
Targeted PD-L1 expression in ‘neo-islets’ leads to a decrease in CD4+ T-cells locally in and
around neo-islets
To elucidate mechanisms underlying PD-L1 induced tolerance of these neo-islets in the liver, we
quantified the number of T-cells and their subsets in the liver sections from mice treated with
Ngn3-Btc, with and without PD-L1. Immunostaining revealed that CD3+ T-cells were
significantly reduced in and around the induced neo-islets, in the periportal regions of the livers
of Ngn3-Btc+PD-L1 treated mice (Fig. 4A-B). Interestingly, this was due to a decrease in the
CD4+T-cells, but not CD8+ T-cells. We then tested if neo-islet-restricted expression of PD-L1
had an effect on T-cells beyond the periportal areas. When single cell suspensions of the whole
Page 9 of 49 Diabetes
liver were assessed by flow cytometry, the total number of CD4+ and CD8+T-cells was not
different in the two groups treated with Ngn3-Btc with or without PD-L1 (Fig. S5A-D), in
contrast to that observed only in the periportal areas surrounding the neo-islets (Fig. 4A-B). This
suggests that expression of insulin promoter-driven PD-L1 resulted only in local
immunomodulation in the periportal regions.
Targeted PD-L1 expression in ‘neo-islets’ inhibits proliferation and promotes apoptosis of
local CD4+ T-cells by a Foxp3-independent mechanism
To determine the mechanism of the decrease in local CD4+T-cells in Ngn3-Btc+PD-L1 treated
mice, we tested the rate of proliferation and apoptosis of the infiltrating T-cells in periportal
regions of the liver. BrdU incorporation, indicative of active proliferation, was significantly
lower in CD3+ T-cells (Fig. 4C-D) but not in CD8+ T-cells (Fig. 4C-D), consistent with the
reduction only in numbers of CD4+T-cells. Furthermore, an increase in apoptosis was detected
by TUNEL staining in CD3+ T-cells infiltrating the neo-islets in Ngn3-Btc+PD-L1 treated mice,
as compared to the controls that received only Ngn3-Btc (Fig. 4E-F).
Since, Foxp3+ T-regulatory (Treg) cells have been invoked as mediating tolerance induction by
PD-L1 (24;25), we measured the number and expression of Foxp3+ cells in these mice.
Surprisingly, the number of Foxp3+ cells in the whole liver, assessed by flow cytometry, was not
different between the mice that received PD-L1 and those that did not (Fig. S6A-B), a finding
that was corroborated by western blotting of whole liver lysate for Foxp3 protein (Fig. S6C-D).
Furthermore, the number of Foxp3+ cells among the infiltrating periportal T-cells also was not
different between the two treatment groups (Fig. S6E). This data excluded Foxp3+ Tregs as
Page 10 of 49Diabetes
playing a major role in the induction and maintenance of peripheral tolerance to the neo-islets
with Ngn3-Btc+PD-L1 treatment.
Inactivation of infiltrating T-cells in the liver results from targeted PD-L1 expression in
neo-islets
We then investigated if PD-L1 expressed on the neo-islets was also leading to local T-cells
inactivation. To test this, we assessed, by flow cytometry, the expression of cytokines in T-cells
isolated from mice 8 weeks after treatment and stimulated ex vivo using anti-CD3 and anti-
CD28. CD4+T-cells expressing IFN-γ (7.38%) or TNF-α (6.63%) were significantly decreased
in the livers of mice that received Ngn3-Btc+PD-L1 (Fig. 5A-B), as compared to mice that
received Ngn3-Btc but no PD-L1 (16.4% and 13.1% respectively). This led us to conclude that
addition of PD-L1 to the neo-islet inducing Ngn3-Btc regimen is sufficient to decrease the
activation of the liver T-cells in the periportal ‘fighting’ zone. In parallel experiments we also
show that this degree of CD4+ activation is similar to that seen in the liver of untreated diabetic
NOD mice (Fig. 5C-D). This was also consistent with a significantly lower TNF-α protein on
western blotting of Ngn3-Btc+PD-L1 treated mouse liver lysates, as compared controls that
received only Ngn3+Btc (Fig. 5E).
We then explored the molecular mechanisms underlying this local T-cell inactivation by PD-L1
expression on neo-islets. PD-L1 mediated co-stimulation inhibition occurs by inhibiting the
TCR-MHC-II/co-stimulation pathways. However, the neo-islets do not express MHC-classII
antigens (Fig. S7A). This raised the possibility that a simultaneous spatio-temporal TCR-
engagement may not be required for PD-L1 mediated inhibition of co-stimulation. To test this,
Page 11 of 49 Diabetes
we stimulated isolated CD4+T-cells, with anti-CD3 and anti-CD28 antibodies to activate the
TCR/co-stimulation pathways. When these stimulated CD4+T-cells were subsequently co-
incubated with PD-L1 overexpressing mouse insulinoma cells, there was a significant decrease
in IFN-γ and TNF-α positive cells as compared to controls (Fig. 5E-G). This demonstrated that
PD-L1 mediated T-cell inactivation can be spatio-temporally separated from TCR-MHC-II
engagement. We also identified MHC-II expressing B-cells infiltrating the neo-islet that may be
providing the stimulus to activate the TCR signaling cascade in these infiltrating CD4+ T-cells
(Fig. S7B-C).
To comprehensively test the effect of the targeted expression of PD-L1 in the neo-islet on the
liver cytokine profile, we assessed 23 cytokines in whole liver lysate, using Bio-Plex suspension
array system. Many cytokines involved in the pathogenesis of diabetes in NOD mice, including
IL-1β, TNF-α, ΙL−10 and IL-17, were down regulated in the liver, when PD-L1 was added to
Ngn3-Btc (Fig. 6A). Interestingly, when compared with the results from that of diabetic NOD
mouse pancreas (Fig. 6B), there was a remarkable inverse correlation, in that most of the
cytokines that were increased in the NOD diabetic pancreas were decreased in the liver of mice
that were treated with Ngn3-Btc+PD-L1, indicating that mechanisms that destroy β-cells in the
pancreas have been specifically attenuated by PD-L1 and supports this as a mechanism for the
tolerance induction by PD-L1 in this setting.
Ngn3-Btc-PD-L1 treatment is not associated with systemic immunosuppression
We then assessed if systemic immunosuppression could have contributed to the observed
tolerance in Ngn3-Btc+PD-L1 treated mice. To assess this we isolated lymphocytes from the
Page 12 of 49Diabetes
spleen of mice treated with Ngn3-Btc, with and without PD-L1 and compared the expression of
TNF-α and IFN-γ upon TCR-dependent stimulation with anti-CD3 and anti-CD28 . Splenocytes
from mice treated with Ngn3-Btc with or without PD-L1 exhibited similar response upon
stimulation, in terms of IFN-γ and TNF-α expression (Fig. 7A-C). Other controls for this
experiment performed are shown in Fig. S8A-D. We also performed skin allografts from
C57Bl6 mice into NOD-diabetic mice that had received Ngn3-Btc with or without PD-L1. Ngn3-
Btc+PD-L1 mice rejected the skin graft at the same rate and time course as the controls (data not
shown) that did not receive PD-L1, confirming that generalized immunosuppression was not
induced by Ngn3-Btc+PD-L1 therapy. Having, thus, excluded systemic immunosuppression as a
mechanism for the tolerance to neo-islets in Ngn3-Btc+PD-L1, we then tested to see if the
immune system in the liver could clear other antigens. We injected mice treated with Ngn3-Btc
with or without PD-L1 with an adenovirus (of a serotype that was different from the original
vectors) and assessed viral clearance rate after 3 weeks. This experiment indicated that both
groups had similar viral clearance rates (Fig. S9) indicating that PD-L1 expression limited to the
neo-islets in the liver did not impair the ability of the liver to clear other antigens.
Ngn3-Btc+PD-L1 induced neo-islets resist destruction by adoptively transferred
diabetogenic splenocytes
To exclude the possibility that a loss of diabetogenicity of the T-cells in mice that received PD-
L1 underlies the tolerance, we performed adoptive transfer of splenocytes from the Ngn3-
Btc+PD-L1 and Ngn3-Btc treated mice into NOD-Scid recipients. Splenocytes from Ngn3-
Btc+PD-L1 treated donor mice not only were able to robustly transfer diabetes (Fig. 7D),
Page 13 of 49 Diabetes
indicating that there was no reduction in the diabetogenicity of the splenocytes and no peripheral
tolerance to islet antigens in the donor Ngn3-Btc+PD-L1 mice. Indeed, they actually led to a
significantly earlier onset of diabetes in the recipients as compared to control NOD-Scid mice
that received splenocytes from Ngn3-Btc treated donors, which may be a reflection of the
persistent islet antigenic presence in the Ngn3-Btc+PD-L1. We then did the converse experiment
wherein we performed adoptive transfer using diabetogenic splenocytes from newly diabetic
NOD mice and transferred them to NOD-Scid mice (controls) and to euglycemic Ngn3-Btc+PD-
L1 treated NOD-diabetic mice. The Ngn3-Btc+PD-L1 mice remained completely resistant to
diabetes up to 15 weeks of the study, while all the NOD-Scid recipients became diabetic by 7
weeks (Fig. 7E). These experiments demonstrate that Ngn3-Btc-+PD-L1 treatment induced a
local tolerance in the periportal regions despite the presence of diabetogenic T-cells in the body
leading to the persistence of the neo-islets and a long-term reversal of diabetes in NOD mice.
Discussion
While there have been innumerable studies that have been reported to decrease the incidence of
diabetes in NOD mice (26), there have been only a few successful interventions that reverse
diabetes after disease onset. Most notable success has been achieved by targeting the TCR
complex by anti-CD3 antibodies (27-30) and anti-thymocyte globulin (31-34), though
generalized immunosuppression limits the translational applicability of these approaches. Other
targeted therapies including antigen-specific approaches by using DC-expanded islet specific
Tregs (35), IL-2 therapy expanded Tregs (36), have had some success in restoring euglycemia,
though an enduring response was rarely seen and never with a high efficacy. Recently, mixed
hematopoietic chimerism achieved by anti-CD3/anti-CD8 conditioning regimen with bone
Page 14 of 49Diabetes
marrow transplantation to inhibit autoimmunity reversed diabetes in 60% of NOD mice when
combined with 2 months of daily injections of EGF and Gastrin to induce β-cell formation (37).
In this study, we tested if engineered neo-islets that specifically resist T-cell targeted destruction
offer a cure for diabetic NOD mice. With targeted expression of PD-L1 in the Ngn3-Btc induced
insulin-expressing cells, we demonstrate that this approach is sufficient to reverse diabetes by
persistent functional neo-islets in the liver. In data not shown, these treated mice remain
euglycemic for prolonged periods up to a year without any untoward effects such as liver
dysfunction, tumors or hypoglycemia. We show that this occurs due to PD-L1 mediated
reduction in activated T-cells locally around the neo-islets, without systemic
immunosuppression. Our data is consistent with previous studies demonstrating that the
expression of PD-L1 induces tolerance by a reduction in the number of infiltrating T-cells, both
by inducing apoptosis while inhibiting proliferation of T-cells (38).
While Foxp3+ Tregs have also been shown to mediate the tolerance induction by PD-L1 in some
settings (25), our data indicates that the locally induced tolerance to the neo-islets is Foxp3-
independent. This is also consistent with other studies that reveal that systemic development of
Tregs may not occur when manipulation of immune responses is done in an organ-specific or
limited to a microenvironment (39-41). While the liver microenvironment has been suggested to
be more tolerogenic than other tissues (42), it may have a contributory but not a determinant role
in this study as the neo-islets induced in the control mice that received Ngn3-Btc were rapidly
destroyed and the expression of PD-L1 on the neo-islets was required for their continued
survival. The persistent diabetogenicity of splenocytes in the mice that expressed PD-L1 on the
neo-islets and the resistance of these neo-islets to destruction by extraneous diabetogenic
splenocytes demonstrate the critical tolerogenic role played by modulation of the
Page 15 of 49 Diabetes
microenvironment surrounding the neo-islets without perturbing the systemic immune system.
Our experiments also demonstrate that it is not required for the PD-L1 expression to be on the
same cell that carries the MHC-II to engage the TCR on the CD4+ cells.
There have been some paradoxical observations in transgenic models wherein PD-L1 was
overexpressed in β-cells in different genetic backgrounds – overexpression in a B6 background
led to an increase in allograft rejection and induction of autoimmune diabetes (43), whereas in
the NOD background there has been a clear protection from diabetes (15). However, the
preponderance of evidence in NOD mice points to a significant inhibition of T-cell response and
protection with the activation of the PD/PD-L1 pathway in both transgenic and transplant models
(12;15;44;45). These studies are consistent with our observation that expression of PD-L1 in
target cells leads to a decrease in activation of T-cells, and an inhibition of their proliferation.
One other study, using a first-generation adenoviral vector-mediated gene transfer of Pdx-1 to
the liver, reported induction of insulin expression in hepatocytes with a decrease in
hyperglycemia in less than half of the mice treated (46). A decreased diabetogenicity of the
splenocytes from treated mice was reported as the reason for the tolerance, though the
mechanism of this decreased diabetogenicity was not defined. In contrast to this, splenocytes
from the treated mice in our study displayed increased diabetogenicity in destroying recipient
pancreatic islets in adoptive transfer experiments. This is likely to be secondary to the persistent
antigen exposure from the neo-islets that would perpetuate diabetogenic lymphocytes in the
body.
A recent study demonstrated the cell-intrinsic requirement of PD-1 expression on islet-reactive
CD4+T-cells in diabetes induction (45;47). This is consistent with a model wherein the PD-L1
Page 16 of 49Diabetes
expressed on the neo-islets engages the PD-1 on the infiltrating T-cells to inactivate them for
tolerance induction. However, other studies have identified the important role of PD-L1 binding
to B7-1 mediating T-cell inhibition (48;49), specifically in diabetogenesis in NOD mice (50).
The current study was not designed to address this question and future experiments will have to
address whether the binding of PD-L1 to PD-1 or B7-1 mediates the tolerance induction seen in
this study.
With the regimen of Ngn3-Btc+PD-L1, 24% of the mice did not respond to tolerance induction
and remained diabetic and displayed significant ‘insulitis’ and loss of insulin-positive ‘neo-
islets’, similar to mice that did not receive PD-L1. It is likely that other pathways that are not
regulated by the inhibition of co-stimulation by PD-L1 exist in these mice and unraveling the
mechanisms that underlie the resistance to tolerance induction will require further study.
In summary, we demonstrate that Ngn3-Btc gene transfer is adequate to induce ectopic islet
neogenesis in the liver of diabetic NOD-mice and reverse diabetes when combined with targeted
PD-L1 expression to inhibit the co-stimulatory pathway selectively in T-cells infiltrating the
newly induced β-cells. To our knowledge, this is the first report of a successful enduring
reversal of diabetes that was achieved in a majority of overtly diabetic NOD mice by inducing
islet neogenesis with concurrent β-cell specific tolerance without systemic immunosuppression.
This treatment strategy may be an attractive and viable approach to β-cell replacement therapy of
autoimmune type 1 diabetes.
Page 17 of 49 Diabetes
Acknowledgements
The work was supported by grants from the NIH: R03 DK089061-01 (VY); NIH: K08 DK068391 (VY);
NIH: R56 DK089061 (VY); the Diabetes Research Center-(DRC - P30DK079638) P&F grant
(VY); NIH: K08HL091176 (MM), Juvenile Diabetes Research Foundation: JDRF Award # 5-2006-
134 (VY), JDRF Award #46-2010-752 (LC), the Betty Rutherford Chair from St. Luke’s Episcopal
Hospital and the Charles and Barbara Close Foundation (LC). We thank Dr. Shixia Huang and Myra
Custorio for assistance with the Bioplex cytokine assay. We thank Dr. Marcus Grompe for generously
providing the OC2-1D11 antibody for oval cells. There are no potential conflicts of interest relevant to
this article. R.L.,Q.Y.,M.M.,W.C.,K.O.,L.C.,V.Y. designed the study and analyzed the data.
R.L.,J.L.,M.K.,V.L.,A.X.,L.Y.,Y.L.,T.H.T., H.L. performed all the experiments. V.Y. supervised the
project and is the guarantor of this work and, as such, had full access to all the data in the study and
takes responsibility for the integrity of the data and the accuracy of the data analysis.
Page 18 of 49Diabetes
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Page 26 of 49Diabetes
Figure Legends
Fig. 1: Ngn3-Btc+PD-L1 reverses hyperglycemia and restores glucose tolerance. (A, B) Blood
glucose and body weight in diabetic NOD mice. Ngn3-Btc+PD-L1 group (n=16), n=4-7 for the
other groups. (C) Non-fasting plasma insulin at indicated time points. (n=4-8). (D) Plasma
aspartate aminotransferase (AST) and (E) alanine aminotransferase (ALT) at the indicated time
points. n=4-6. (F-I) Plasma glucose and insulin during an IP-GTT at 4 weeks (F, G) and 8 weeks
(H, I). Non-diabetic (green); PD-L1 only (blue); Ngn3-Btc (purple); Ngn3-Btc+PD-L1 (orange).
All values are mean±SEM; * p≤0.05.
Fig. 2: Ngn3-Btc+PD-L1 induces neo-islets in the periportal regions of the liver. (A-E)
Representative sections of Ngn3-Btc+PD-L1 treated diabetic NOD mouse liver stained by
immunohistochemistry for (A) insulin; (B) C-peptide; (C) glucagon; (D) somatostatin (SST); (E)
pancreatic polypeptide (PP) at indicated time points after diabetes onset or treatment Scale bar
represents 20µm. PV-Portal vein. Of note, periportal cluster of cells are only occasionally seen in
PD-L1 group and are shown in panels A&B to contrast their staining with that of the Ngn3-Btc
and Ngn3-Btc+PD-L1 groups. Most of the periporatal areas in the PD-L1 group are similar to
those shown in panels C-E.
Fig. 3: Neo-islets express islet hormones and transcription factors. RT-qPCR showing the
expression of (A) islet hormones and (B) transcription factors involved in islet development, at 8
weeks. n=4-5, all values are mean±SEM; * p≤0.05. (C-D) Representative sections of liver
stained for Pdx-1 and Nkx 6.1 in different groups. Scale bar represents 20µm. PV-Portal vein.
Page 27 of 49 Diabetes
Fig. 4: Ngn3-Btc+PD-L1 treatment reduces the local number of CD4+ T-cells infiltrating the
peri-portal neo-islet clusters. (A) Representative sections in control and Ngn3-Btc+PD-L1
treated NOD mice liver stained for CD3, CD4 and CD8. (B) CD3, CD4 and CD8 positive cells
represented as a percent of all cells only in periportal clusters. (C) Representative sections in the
liver of control and Ngn3-Btc+PD-L1 treated NOD mice co-stained for Brdu with CD3, CD4 or
CD8. (D) Percent of BrdU positive T-cells in the peri-portal clusters. (E) Representative sections
in the liver of control and Ngn3-Btc+PD-L1 treated NOD mice co-stained for TUNEL and CD3.
(F) Percent of TUNEL positive T-cells in peri-portal clusters. All values are mean±SEM;
*p≤0.05. n=4-5 for each group. Scale bar represents 50µm.
Fig. 5: PD-L1 protects neo-islets by inactivating T-cells. (A-B) IFN-γ and TNF-α positive CD4+
T-cells isolated from livers of treated mice after ex vivo stimulation with anti CD3 and anti
CD28 on FACS analysis. Representative dot plot from one set of mice is shown in (A) and
quantification from 3-5 separate mice in (B). (C) IFN-γ and TNF-α positive CD4+ T-cells
isolated from liver or spleen of non-diabetic or diabetic mice after ex vivo stimulation with anti-
CD3 and anti-CD28 on FACS analysis. Representative dot plot from one set of mice is shown in
(C) and quantification from 3-4 separate mice in (D). Control shown is unstimulated CD4+ T-
cells from the spleen of Ngn3-Btc treated (A) or untreated diabetic (C) mice. (E) Western
blotting of whole liver lysate for TNF-α at 8 weeks after treatment. All values are mean ± SEM;
* p≤0.05, # p≤0.06.
Fig. 6: (A-C) IFN-γ and TNF-α protein was assessed in isolated CD4+ T-cells purified from the
spleen of non-diabetic NOD mice that were first stimulated with anti-CD3 and anti-CD28 for 2
Page 28 of 49Diabetes
days and then co-incubated with HDAd-empty or HDAd-PD-L1 virus infected β-cells
(Insulinoma - Ins2 cells) for 24 hrs. Representative dot plot of one set of experiments is shown in
(A-B) and quantification from 4 separate experiments in (C). CD4-empty: stimulated CD4+ cells
co-incubated with β-cells infected with HDAd-empty; CD4-PD-L1: stimulated CD4+ cells co-
incubated with β-cells infected with HDAd-empty PD-L1. Control indicates unstimulated CD4+
cells. All values are mean ± SEM; * p≤0.05.
Fig. 7: The levels of 23 cytokines in the livers (A) of Ngn3-Btc and Ngn3-Btc+PD-L1 treated
NOD mice or in the pancreas (B) of diabetic NOD mice (5-6 weeks after diabetes onset) and
non-diabetic NOD mice (27 weeks old) analyzed by Bioplex tissue cytokine assay. N=3-5/group.
All values are mean±SEM. * p≤0.05; #p value between 0.05-0.06.
Fig. 8: No peripheral immunosuppression in the mice treated with Ngn3-Btc+PD-L1. (A-C)
IFN-γ and TNF-α positive CD4+ T-cells isolated from spleens of treated mice after ex vivo
stimulation with anti CD3 and anti CD28 on FACS analysis. Representative dot plot from one set
of mice is shown in (A-B) and quantification from 3-5 separate mice in (C). (D) Diabetes
induction in NOD-Scid mice after adoptive transfer of splenocytes from Ngn3-Btc-PD-L1 or
controls. (E) Diabetes induction in Ngn3-Btc-PD-L1 or control mice after adoptive transfer of
splenocytes from newly diabetic NOD mice. n=3-5 group. All values are mean±SEM; * p≤0.05.
Page 29 of 49 Diabetes
15
18
21
24
27
-3 -1 1 3 5 7 9 11 13 15
Bo
dy
Wei
gh
t(g
m)
0
0.5
1
0 30 60 90 120
Pla
sma
Insu
lin (
ng
/ml)
Time (min)
****
0
200
400
600
800
-3 -1 1 3 5 7 9 11 13 15
Blo
od
Glu
cose
(mg
/dl) PD-L1
Ngn3+BtcNgn3+Btc-PD-L1
Weeks after Treatment
A BHDAd
Fig. 1
0
200
400
600
800
1000
0 30 60 90 120
Pla
sma
Glu
cose
(mg
/dl)
Non-diabeticPD-L1Ngn3+BtcNgn3+Btc+PD-L1
D
**
* *
Weeks after Treatment
HDAd
F
*
Time (min)
*******
***********
Time (min)
0
0.5
1
1.5
0 30 60 90 120
Pla
sma
Insu
lin (
ng
/ml)
*
**
**
0
25
50
75
100
0 4 8 12
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sma
AS
T (
IU/L
)
0
25
50
75
100
0 4 8 12P
lasm
a A
LT
(IU
/L)
G
I
*
Weeks after Treatment Weeks after Treatment
E
*
4 weeks4 weeks
0
200
400
600
800
1000
1200
0 30 60 90 120
Pla
sma
Glu
cose
(m
g/d
l)
Time(min)
Ngn3+BtcNgn3+Btc+PD-L1
****
H
*
8 weeks8 weeks
C
0
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2
3
Basal 2 4 6 12Pla
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** **
0 4 8 12Basal
Page 30 of 49Diabetes
Fig. 2
A
B
C
D
Insu
lin
Non-diabetic
4w
PV
PD-L14w
SS
T
PV
E
PV
PV
C-p
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de
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on
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PP
PV PV
Ngn3-Btc4w
PV
Ngn3-Btc+PD-L14w
PV
PV
PV
PV
PV
PV
PV
PV
Page 31 of 49 Diabetes
0
4
8
Ins
-1
Ins
-2
Glu
cag
on
PP
SS
T
Gh
relinRel
ativ
e m
RN
A L
evel
**
*
*
*
*
Fig. 3
A B
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6.1
Pd
x-1 PV PV
PVPV
PV
PV
PV
PVPVPV
PV
C
4w 8w 12w4w4w4wNgn3+Btc+PD-L1Non-diabetic PD-L1 Ngn3+Btc
D
8 weeks 8 weeksNgn3+Btc
Ngn3+Btc+PD-L1
Ngn3+Btc
Ngn3+Btc+PD-L1
0
5
10
15
20
Pd
x-1
Ng
n3
Isl-
1
Ne
uro
d1
Pax
4
Nk
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Pax
6
Nk
x6.1
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ativ
e m
RN
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evel
*
**
Page 32 of 49Diabetes
CD4A CD3
CD3 CD4
CD8
CD8
0
20
40
60
80
100
CD3 CD4 CD8T c
ells
in p
erip
ort
alcl
ust
ers
(%)
Ngn3+Btc
Ngn3+Btc+PD-L1
**
Ng
n3-
Btc
Ng
n3-
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+P
D-L
1Fig. 4
B In the periportal cluster
0
4
8
12
CD3 CD4 CD8
Brd
U+
amo
ng
T c
ells
in
p
erip
ort
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er (
%)
0
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2
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4
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nel
+ a
mo
ng
CD
3+ in
p
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al c
lust
ers
(%)
CD3 Brdu
CD4 Brdu
C
E
Ngn3-Btc Ngn3-Btc+PD-L1
CD3 Brdu
CD4 Brdu
CD3 Tunel
CD8 Brdu
CD8 Brdu
CD3 Tunel
**
Ng
n3-
Btc
+P
D-L
1N
gn
3-B
tc
*
Ngn3-Btc Ngn3-Btc+PD-L1
D
F
Ngn3-Btc
Ngn3-Btc+PD-L1
Ngn3-Btc
Ngn3-Btc+PD-L1
In the periportal cluster
In the periportal cluster
PV
PV
PV
PV
PV
PVPVPV
PV
PV
PV
PV
PV
PV
Page 33 of 49 Diabetes
ControlDiabetic SpleenNormal SpleenDiabetic LiverNormal Liver
0
4
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IFN-γ TNF-α
IFN
-γ+
or
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+ a
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**
#
Ngn3-Btc+PD-L1
GAPDH
TNF-α
Ngn3-Btc
ED
C
CD4
TN
F-α
IFN
-γ
Ng
n3+
Btc
Ng
n+
Btc
+P
D-L
1A
Co
ntr
ol
0
5
10
15
20
IFN-γ TNF-α
Ngn3+Btc
Ngn3+Btc+PD-L1
* *
IFN
-γ+
or
TN
F-α
+
amo
ng
CD
4+ c
ells
B
Fig. 5
Page 34 of 49Diabetes
05
1015202530
IFN-γ TNF-α
IFN
-γ+
or
TN
F-α
+ a
mo
ng
cd
4+ c
ells
CD4-Empty
CD4-PD-L1
*
*
Fig. 6
CD4-Empty CD4-PD-L1
IFN
-γT
NF
-α
CD4
A
B
Control
Page 35 of 49 Diabetes
0
0.4
0.8
1.2
Rel
ativ
e P
rote
in L
evel
Ngn3+Btc liver Ngn3+Btc+PD-L1 liver
* * * * **
* *#
#
**#
Fig. 7
0
1
2
3
4
IL-1α
IL-1β
IL-2
IL-3
IL-4
IL-5
IL-6
IL-9
IL-1
0
IL-1
2(P
40)
IL-1
2(P
70)
IL-1
3
IL-1
7
Eo
taxi
n
G-C
SF
GM
-CS
F
IFN
-γ KC
MC
P-1
MIP
-1α
MIP
-1β
RA
NT
ES
TN
F-α
Rel
ativ
e P
rote
in L
evel
Non-diabetic Pancreas Diabetic Pancreas
* * **
##
#
#
Page 36 of 49Diabetes
IFN
-γ
Ngn3+Btc
TN
F-α
CD4
Ngn3+Btc+PD‐L1
0
2
4
6
8
IFN-γ TNF-α
IFN
-γ+
or
TN
F-α
+am
on
g C
D4
+(%
)
Ngn3+BtcNgn3+Btc+PD-L1
A
B
C D
0 2 4 6 8 10 12 14 16
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Fra
ctio
n D
iab
etes
fre
e
Ngn3+Btc+PD-L1 (Donor)
Ngn3+Btc (Donor)
Weeks
p=0.022
E
0 2 4 6 8 10 12
0.0
0.2
0.4
0.6
0.8
1.0
Fra
ctio
n D
iab
etes
fre
e
Ngn3+Btc+PD-L1 (Recipient)
NOD-Scid (Recipient)
Weeks
p=0.039
Fig. 8Control
Page 37 of 49 Diabetes
Supporting Figure Legends
Fig. S1: Ngn3-Btc+PD-L1 induces neo-islets in the periportal regions of the liver. (A)
Representative sections of Ngn3-Btc+PD-L1 treated diabetic NOD mouse liver stained by
immunohistochemistry for insulin; C-peptide; glucagon; somatostatin (SST); pancreatic
polypeptide (PP) at indicated time points after treatment. Periportal neo-islets are shown
encircled by the red line. (B-C) Insulin staining in the pancreas with lower (C) and higher (D)
magnifications of a neo-islet is indicated by the red arrow in (B). Scale bar represents 20μm in
(A), 100μm in (B) and 50μm in (C). PV-Portal vein.
Fig. S2: PD-L1 and PD-L2 expression level in the liver or pancreas of Ngn3-Btc and Ngn3-
Btc+PD-L1 treated NOD mice. (A) PD-L1 staining in the liver or pancreas of Ngn3-Btc and
Ngn3-Btc+PD-L1 treated NOD mice. (B) PD-L1 and PD-L2 RNA level in the liver of Ngn3-Btc
and Ngn3-Btc+PD-L1 treated NOD mice. Scale bar represents 20μm.
Fig. S3: Neo-islets express islet hormones and transcription factors. RT-qPCR showing the
expression of (A) islet hormones and (B-C) transcription factors involved in islet development
at 4 weeks and non diabetic and diabetic NOD control mice. n=4-5, all values are mean±SEM;
* p≤0.05.
Fig. S4: Neo-islets induced by Ngn3-Btc+PD-L1 in the liver expresses the hepatic oval cell
marker. (A) A6 staining. (B) OC2-1D11 and CD3 staining in different groups. Scale bar
represents 20μm.
Page 38 of 49Diabetes
Fig. S5: There is no difference in the number of CD4 + and CD8+ cells in the whole liver of
Ngn3-Btc and Ngn3-Btc+PD-L1 treated mice. Representative dot plot of CD4+ (A) and CD8+
(C) T cells among CD3+ T cells in whole liver treated with Ngn3-Btc or Ngn3-Btc+PD-L1. (B,
D) Percentage of CD4+ (D) and CD8+ (F) among CD3+ T cells in whole liver. All values are
mean±SEM; * p≤0.05. n=4-5 for each group. Scale bar represents 50μm.
Fig. S6: There is no difference in the number of Foxp3+ in the whole liver or in the periportal
regions of the liver of Ngn3-Btc and Ngn3-Btc+PD-L1 treated mice. (A) Representative dot plot
of Foxp3+ among CD4+ T cells in whole liver treated with Ngn3-Btc or Ngn3-Btc+PD-L1. (B)
Percentage of Foxp3+ among CD4+ T cells in whole liver. (C-D) Western Blot from the whole
liver also shows no change of Foxp3 expression in the liver of different treated groups. (E) The
quantitation for counting the number of Foxp3+ among CD4+ T-cells in the periportal clusters in
different treated groups N=4-5/group. All values are mean±SEM.
Fig. S7: Neo-islets do not express MHCII antigens. (A) MHCII staining does not co-localize
with c-peptide staining in neo-islets. (B) B220 (B cell marker) positive cells and CD4 positive
cells are both present infiltrating the periportal neo-islet. (C) MHCII is expressed in the B220
positive B lymphocytes in the periportal area. Scale bar represents 50μm.
.
Fig. S8: TNF-α and IFN-γ production in CD4 lymphocytes in different tissues of recent onset
diabetic NOD mice. Representative dot plot and quantitation of IFN-γ production (A,C) and
TNF-α (B,D) in the spleen, pancreas, pancreas lymph node and mesentery lymph node of
Page 39 of 49 Diabetes
diabetic NOD mice. Control shown is unstimulated CD4+ T-cells from the spleen of untreated
NOD mice. C & D represent the quantification from 3 separate mice in the group.
Fig. S9: Virus clearance shows no immunosuppression in the mice treated with Ngn3-Btc+PD-
L1. (A) 1*10^11 helper virus was iv injected to Ngn3-Btc and Ngn3-Btc+PD-L1 treated mice for
3d and 3w. Q-PCR shows relative virus clearance in different mice. N=2-3/group.
Page 40 of 49Diabetes
8 weeks
PV
PV
PV
PV
PV
PV
PV
PV
PV
12 weeks
PV
A Insulin Insulin B
Insu
lin
G
luca
go
n
SS
T
PP
C
-pep
tid
e Fig. S1
C
No
n-d
iab
etic
PD
-L1
Ng
n3-
Btc
Ng
n3-
Btc
+P
D-L
1
Ngn3-Btc+PD-L1
4 weeksPancreas
Liver
Page 41 of 49 Diabetes
Liv
erP
ancr
eas
Ngn3-Btc-PD-L1Ngn3-Btc
0
1
2
3
4
PD-L1 PD-L2
Rel
ativ
e m
RN
A L
evel Ngn3+Btc
Ngn3+Btc+PD-L1
*B
A
Fig. S2
Page 42 of 49Diabetes
0
1
2
3
4
Pd
x-1
Isl-
1
Neu
rod
1
Pax
4
Nkx
2.2
Pax
6
Nkx
6.1
Rel
ativ
e m
RN
A L
evel
non diabetic
diabetic
Ngn3+Btc
0
4
8
12
16
20In
s-1
Ins-
2
Glu
cag
on
PP
SS
T
Gh
relin
Rel
ativ
e m
RN
A
Lev
el
non diabetic
diabetic
Ngn3+Btc
Ngn3+Btc+PD-L1
A
B
*
*
*
C
0
100
200
300
400
Ngn3
Rla
tive
mR
NA
Lev
el
non diabeticdiabeticNgn3+BtcNgn3+Btc+PD-L1
Fig S3
Page 43 of 49 Diabetes
Ngn3-Btc+PD-L1Ngn3-BtcNo primary antibody
Non-diabetic PD-L1 Ngn3-Btc Ngn3-Btc+PD-L1
A6
A
PV
PV
PV
PV
Fig. S4
B
OC
2-1D
11 C
D3
PV
PV PV
Page 44 of 49Diabetes
CD
8C
D4
CD3
0
20
40
60
CD
4+ a
mo
ng
C
D3+
in
wh
ole
live
r (%
)
0
20
40C
D8+
am
on
g
CD
3+
in w
ho
le li
ver
(%
)
Ngn3-Btc Ngn3-Btc+PD-L1A
C
Ngn3-Btc Ngn3-Btc+PD-L1
B
D
Ngn3-Btc Ngn3-Btc+PD-L1
In the whole liverFig. S5
Page 45 of 49 Diabetes
0
5
10
15
20
FO
XP
3+ a
mo
ng
CD
4+in
w
ho
le li
ver
(%
)
A
E
Fig. S6
0
5
10
15
FO
XP
3 am
on
g C
D4+
in
p
erip
ort
alcl
ust
ers
(%)
Ngn3-Btc+PD-L1
Ngn3-Btc
Ngn3-Btc+PD-
L1
Ngn3Btc
Ngn3-Btc+PD-L1
C
GAPDH
FOXP3
Ngn3+Btc 0
0.8
1.6
FO
XP
3 R
elat
ive
Lev
elNgn3-
Btc+PD-L1Ngn3-Btc
B
D
CD4
FO
XP
3
Ngn3-Btc Ngn3-Btc+PD-L1
In the periportal cluster
In the whole liver
Page 46 of 49Diabetes
C‐peptide MHCII MergeDAPI
DAPI
DAPI
CD19
CD19MHCII
CD4 Merge
Merge
Fig. S7
Page 47 of 49 Diabetes
Fig. S8IF
N-γ
TNF-α
CD4
Spleen Pancreas PLN MLN
A
B
C D
0
0.2
0.4
0.6
0.8
1
Spleen Pancreas PLN MLN
IFN
-γ a
mom
g C
D4
0
0.4
0.8
1.2
1.6
Spleen Pancreas PLN MLN
TNF-α
amon
g C
D4
Control
Page 48 of 49Diabetes
0
20
40
60
80
100
120
Ngn3-Btc Ngn3-Btc-PD-L1
Rel
ativ
e vi
rus
clea
r ra
te
in 3
wee
ks (
%) 3d 3w
Fig. S9
Page 49 of 49 Diabetes