Hepatobiliary Pancreat Dis IntVol 11No 5 October 152012 www.hbpdint.

com
Author Afliations: Department of Physiology, Xiangya Medical
School, Central South University, Changsha 410078, China (Ji M); Cell
Transplantation and Gene Therapy Institute, Third Xiangya Hospital,
Central South University, Changsha 410013, China (Ji M); Center
for Transplant and Renal Research, Westmead Millennium Institute,
Westmead Hospital, Westmead, NSW, Sydney 2145, Australia (Ji M, Jin
X, Phillips P and Yi S)
Corresponding Author: Shounan Yi, MD, Center for Transplant and Renal
Research, Westmead Millennium Institute, Westmead Hospital, Westmead,
NSW, Sydney 2145, Australia (Tel: 61-2-98457365; Fax: 61-2-98937440;
Email: shounan_yi@wsahs.nsw.gov.au)
2012, Hepatobiliary Pancreat Dis Int. All rights reserved.
doi: 10.1016/S1499-3872(12)60213-6
BACKGROUND: A major barrier to the clinical application
of xenotransplantation as a treatment option for patients
is T cell-mediated rejection. Studies based on experimental
rodent models of xenograft tolerance or rejection in vivo have
provided useful information about the role of T cell immune
response in xenotransplantation. However not all observations
seen in rodents faithfully recapitulate the human situation.
This study aimed to establish a humanized mouse model of
xenotransplantation, which mimics xenograft rejection in the
context of the human immune system.
METHODS: NOD-SCID IL2r
-/-
mice were transplanted
with neonatal porcine islet cell clusters (NICC) followed by
reconstitution of human peripheral blood mononuclear cells
(PBMC). Human leukocyte engraftment and islet xenograft
rejection were conrmed by ow cytometric and histological
analyses.
RESULTS: In the absence of human PBMC, porcine NICC
transplanted into NOD-SCID IL2r
-/-
mice revealed excellent
graft integrity and endocrine function. Human PBMC
demonstrated a high level of engraftment in NOD-SCID IL2r
-/-

mice. Reconstitution of NICC recipient NOD-SCID IL2r
-/-

mice with human PBMC led to the rapid destruction of NICC
xenografts in a PBMC number-dependent manner.
CONCLUSIONS: Human PBMC-reconstituted NOD-SCID IL2r
-/-

mice provide an ideal model to study human immune responses
in xenotransplantation. Studies based on this humanized
mouse model will provide insight for improving the outcomes
of clinical xenotransplantation.
(Hepatobiliary Pancreat Dis Int 2012;11:494-498)
KEY WORDS: humanized mice;
xenotransplantation;
neonatal porcine islet cell cluster;
xenorejection;
tolerance
Introduction
X
enotransplantation has immense potential as an
alternative source for clinical transplantation.
However T cell-mediated rejection remains
a barrier to its clinical application.
[1, 2]
Small-animal
models of xenograft tolerance or loss due to rejection are
important tools for studies on this issue.
[3, 4]
Despite the
promising results achieved in different mouse models,
comparable and efcient translation to the clinic has
been difcult.
[5]
It is therefore important to develop an
animal model that can faithfully represent xenograft
rejection in a setting that is based upon and functionally
similar to the human immune system. Immunodecient
mice reconstituted with human immune cells could
help achieve this goal.
NOD-SCID IL2r
-/-
mice are gaining popularity
as an immunodecient host and have been shown
to support high levels of human hematopoietic stem
cell and peripheral blood mononuclear cell (PBMC)
engraftments.
[3, 4, 6]
This human immune system-
engrafted mouse model has been used to study
hematopoiesis, development and function of the immune
system, infectious diseases, and autoimmunity as well as
cancer.
[4, 7, 8]
In this study, we demonstrated that neonatal
porcine islet cell clusters (NICC) transplanted into
NOD-SCID IL2r
-/-
mice had excellent engraftment with
normal endocrine function. Reconstitution of recipient
mice with human PBMC achieved a high level of human
leukocyte engraftment and consequently induced rapid
destruction of the transplanted porcine islet graft. Our
study demonstrated a new human-mouse chimeric
model to study the role of human immune cells in
xenograft rejection in vivo.
A humanized mouse model to study human
immune response in xenotransplantation
Ming Ji, Xi Jin, Peta Phillips and Shounan Yi
Sydney, Australia
Original Article / Transplantation
A new human-mouse chimeric model to study xenorejection
Hepatobiliary Pancreat Dis IntVol 11No 5 October 152012 www.hbpdint.com
Methods
Animals
Newborn pigs 1 to 3 days old supplied by an approved
research animal supply unit were used for the isolation
of NICC. NOD-SCID IL2r
-/-
mice were housed under
specic pathogen-free conditions in the Animal Care
Department at Westmead Hospital (Westmead, NSW,
Australia). Mice (aged 6 to 8 weeks) were used for
porcine islet transplantation. The animal study was
approved by the Sydney West Local Health District
Animal Ethics Committee.
Porcine islet isolation and transplantation
NICC were isolated from the pancreas of 1-3 days old
piglets and cultured for 6 days as described previously.
[9]

Then NICC were pooled and 5000 were transplanted
under the renal capsule of both kidneys of NOD-SCID
IL2r
-/-
mice.
Human PBMC isolation
Blood was collected from healthy volunteers
and PBMC isolated using Ficoll-Paque (Amersham
Biosciences, Uppsala, Sweden) and density gradient
centrifugation. The study was approved by the Sydney
West Local Health District Human Ethics Committee.
Adoptive transfer of human PBMC
Human PBMC were suspended in phosphate buffered
saline (PBS) and injected into mice intravenously (i.v.)
at doses of 110
6
, 510
6
or 1010
6
. Peripheral blood,
spleen and NICC grafts were collected from recipient
mice at predetermined time points after human PBMC
reconstitution to assess human leukocyte engraftment
and NICC graft survival. Graft rejection was dened
as little or no intact graft with a heavy lymphocytic
inltrate on histological examination.
[10]
Flow cytometry
Single-cell suspensions from mouse spleen or
peripheral blood were prepared using red blood cell lysis
buffer (eBiosciences, San Diego, CA, USA) according
to the manufacturer's protocol. Phycoerythrin (PE)-
conjugated antibody specic for human antigen CD45
was used for ow cytometric analysis of human leukocyte
engraftment. In brief, spleen cells or PBMC were
incubated with anti-human CD45 or PE-conjugated
mouse IgG1 isotype control (BD, San Jose, CA, USA)
for 30 minutes at 4 . Labeled cells were washed and
resuspended in PBS containing 0.1% sodium azide and 1%
fetal calf serum (FCS). All samples were analyzed on an
LSRII ow cytometer (BD). FlowJo Software (Windows
version 7.1; Tree Star, Ashland, USA) was used for analysis.
Reconstitution was considered successful if 0.1% human
CD45
+
cells were present in the peripheral blood and 1%
in the spleen four weeks after reconstitution.
[11, 12]
Immunohistochemistry
Grafts were removed from NICC recipients at
necropsy at the indicated time points. Six-micron
frozen sections were cut and used for histological and
immunohistochemical examination of graft survival,
porcine endocrine secreting cells and graft-inltrating
human leukocytes. Graft survival was analyzed by
hematoxylin and eosin (HE) staining as described
previously.
[13]
For analysis of endocrine secreting cells,
the sections were incubated with guinea-pig anti-porcine
insulin (Dako Laboratories, Mississauga, Ontario,
Canada) for 60 minutes at room temperature, followed
by horseradish peroxidase (HRP)-conjugated goat anti-
guinea-pig antibody (Dako). For analysis of human cells
inltrating the graft, the sections were incubated with
rabbit anti-human CD45 antibody (Abcam, Cambridge,
UK), followed by HRP-conjugated goat anti-rabbit
antibody (Dako). After antibody binding, the sections
were detected with diaminobenzidine (Dako) and
counterstained with hematoxylin.
Statistical analysis
Data were expressed as meanSD. Comparisons
of two means were analyzed using Student's t test, and
those involving multiple groups were evaluated using
one-way analyses of variance with Tukey's multiple
comparison test (JMP, version 4.02, SAC, USA). P<0.05
was considered statistically signicant.
Results
Engraftment of porcine NICC in NOD-SCID IL2r
-/-

mice
We at rst investigated if porcine NICC grafts could
survive and function in NOD-SCID IL2r
-/-
mice.
In all 6 recipient mice that received 5000 porcine
NICC, the grafts survived beyond 60 days. HE
staining of porcine islets transplanted under the renal
capsule of both kidneys of NOD-SCID IL2r
-/-
mice
demonstrated staining of intact islet grafts 60 days after
transplantation (Fig. 1A). Staining with anti-human
CD45 antibody did not stain any human leukocyte
inltration either in the graft or areas surrounding it (Fig.
1B). Porcine insulin staining was performed to conrm
the endocrine function of the porcine NICC in the
transplanted mice as described previously,
[14, 15]
showing
intact islets with positive staining for insulin (Fig. 1C).
Hepatobiliary & Pancreatic Diseases International
Hepatobiliary Pancreat Dis IntVol 11No 5 October 152012 www.hbpdint.com
Cell number-dependent human PBMC engraftment
in NOD-SCID IL2r
-/-
mice
It has been shown that NOD-SCID IL2r
-/-
mice,
which are decient in T, B, and natural killer (NK)
cells
[16-20]
support high-level engraftment of human
hematopoietic stem cells
[4, 20, 21]
and human PBMC.
[3, 6]

The number of human CD45
+
cells in the spleen of
NOD-SCID IL2r
-/-
mice reaches a plateau three weeks
after PBMC injection.
[3]
NOD-SCID IL2r
-/-
mice
transplanted with NICC xenografts were reconstituted
with 110
6
, 510
6
or 1010
6
human PBMC. Human
PBMC engraftment was determined by ow cytometry
(Fig. 2). Human PBMC engraftment was dened as >1%
splenic human leukocyte chimerism.
[3, 11, 12]
Human
CD45
+
cells were detected in the blood and spleen of
recipient mice four weeks after PBMC reconstitution.
The results showed that the level of PBMC engraftment
in the recipient mice was cell number-dependent.
Increasing the number of injected PBMC increased the
level of human cell engraftment. Moreover, the level of
PBMC engraftment was higher in the spleen than in the
blood of mice transplanted with islet xenografts (Fig. 2).
Porcine islet xenograft rejection in NOD-SCID IL2r
-/-

mice
NOD-SCID IL2r
-/-
mice are considered capable
of rejecting human islet allografts when engrafted
with allogeneic human PBMC.
[3]
We then investigated
whether porcine islet xenograft rejection would be
induced in the same immunodecient mice by adoptive
human PBMC transfer. NOD-SCID IL2r
-/-
mice were
transplanted with porcine NICC prior to reconstitution
with human PBMC at 110
6
, 510
6
, or 1010
6
. Islet
xenograft survival was monitored at different time
points by histological examination until rejection was
observed, when no intact grafts were detected by HE or
insulin staining. Islet xenograft rejection was detected
at day 28 after PBMC reconstitution at 1010
6
(Fig. 3).
However, the PBMC-induced rejection was delayed at
lower doses, showing intact islet xenografts detected
with HE staining in mice receiving 110
6
or 510
6

human PBMC at day 28 after PBMC reconstitution (Fig.
3A, B) compared to mice reconstituted with 1010
6

PBMC (Fig. 3C). Sixty days after PBMC reconstitution,
islet xenografts were rejected completely in all mice
tested (Fig. 3G, H) with no insulin-positive cells
detected in the grafts (Fig. 3D-F, I, J).
Porcine islet xenograft rejection results from human
PBMC reconstitution
Fig. 1. Representative immunohistochemical sections of graft samples
from mice receiving NICC alone 60 days after transplantation. These
mice did not receive human PBMC (original magnication 100).
HE (A), human CD45 (B) and porcine insulin (C) stains.
Fig. 2. Flow cytometric analysis of the percentage of human
leukcocyte engraftment in the spleen and peripheral blood of NOD-
SCID IL2r
-/-
mice 28 days after human PBMC reconstitution (n=
6/cell dose).
Fig. 3. Representative histological and immunohistochemical
sections of NICC xenografts from mice receiving human PBMC.
The sections of grafts from mice 28 days after human PBMC
transfer were stained with HE (A-C) and for porcine insulin (D-F);
HE (G, H) and porcine insulin (I, J) in grafts from mice 60 days
after human PBMC reconstitution (original magnication 100).
A new human-mouse chimeric model to study xenorejection
Hepatobiliary Pancreat Dis IntVol 11No 5 October 152012 www.hbpdint.com
Staining with anti-human CD45 antibody was used
to determine cell inltration either in the graft or areas
surrounding it in mice reconstituted with human PBMC.
In the absence of human PBMC, the NICC-transplanted
mice did not reject their grafts and no graft-inltrating
human CD45
+
cells were detected (Fig. 1B), whereas a
large number of graft-inltrating human CD45
+
cells
were detected in recipient mice 28 days after human
PBMC reconstitution (Fig. 4). This result showed that
islet xenograft rejection was the direct consequence
of adoptive human PBMC transfer. This was unlikely
caused by xenogeneic graft-versus-host disease (GVHD)
which has been reported in NOD-SCID IL2r
-/-
mice
4 to 5 weeks after reconstitution with 210
7
human
PBMC,
[3]
as evidenced by no signs and symptoms of
GVHD such as hunched posture, rufed fur, diarrhea,
cachexia and human cell inltration in kidney and liver
as assessed by weight (data not shown) development in
any NICC-recipient NOD-SCID IL2r
-/-
mice at day 28
after PBMC reconstitution. Thus, this suggests specic
xenograft rejection resulting from the human anti-pig
xenogeneic response but not GVHD.
Discussion
There are ever increasing shortages of organs and tissues
from deceased human donors for clinical organ and
cell transplantation. Xenotransplantation has immense
potential as a solution to this critical shortage. However,
the T cell-mediated xenoimmune response often results
in damage and ultimate destruction of the xenograft.
[1, 2]

Because of the study of human biology in vivo is severely
limited by ethical and technical constraints, small
animal models have become important investigative
tools to study the human immune system. Rodent
models have greatly expanded our knowledge of
disease processes without placing patients at risk.
[5, 22, 23]

Unfortunately, these models are still based on mice,
which do not always faithfully translate to circumstances
or clinical interventions in human diseases.
[24, 25]

Immunological studies have demonstrated differences
between the mouse and human immune systems.
[26, 27]

Therefore, there is growing need to establish more
appropriate animal models for the study of human
immune responses in xenotransplantation.
The immunodecient mouse with mutations targeted
at the IL-2 receptor common chain locus (IL2r null)
is gaining popularity as an immunodecient host and
supports the high levels of human hematopoietic stem
cell and human PBMC engraftment.
[3, 4, 6]
The reason
for the high engraftment rates might be attributed to
multiple immunological functional defects, including
dendritic cells in addition to the absence of T, B, and
NK cells.
[20, 28]
In this study, we demonstrate that
porcine NICC transplanted into NOD-SCID IL2r
-/-

mice retained excellent graft integrity and endocrine
function. A high level of human leukocyte engraftment
was achieved in human PBMC-reconstituted NOD-
SCID IL2r
-/-
mice, and the adoptive transfer to NICC-
recipient NOD-SCID IL2r
-/-
mice with human PBMC
resulted in the rapid destruction of NICC xenografts.
Taken together, our data show that the humanized
NOD-SCID IL2r
-/-
mouse is an important experimental
model for investigating the in vivo mechanisms
of human T cell-mediated porcine islet xenograft
rejection and for evaluating potential therapies (such as
transplantation tolerance induction by regulatory T cells
or immunosuppression drugs) before clinical application
of xenotransplantation without potentially putting
human subjects at risk. In addition, this humanized
mouse model can be extended to test human immune
responses to other porcine xenografts, such as cornea,
neural cells, skin or red blood cells in vivo. A better
understanding of the human immune response towards
xenografts may allow for appropriate means to prevent
xenograft rejection and thus potentially many millions
of patients might benet from xenotransplantation.
In summary, we have been able to validate a
humanized NOD-SCID IL2r
-/-
mouse model for
the study of human xenoimmunity. Intravenous
reconstitution with human PBMC, even at a low dose
(110
6
) in the absence of host preconditioning, leads
to highly reproducible, robust engraftment of human
leukocytes which consequently results in islet xenograft
rejection without development of GVHD.
Contributors: JM participated in research design, performed
the experiments, analyzed data and drafted the manuscript. YS
contributed to the research design, reviewed and wrote the
manuscript. JX and PP participated in collection and analysis of the
data. YS is the guarantor.
Funding: This work was supported by grants from the National
Health and Medical Research Council of Australia, the National
Fig. 4. Representative immunohistochemical sections of graft-
inltrating human CD45
+
cells in mice receiving human PBMC
at different doses 28 days after human PBMC transfer (original
magnication 200).
Hepatobiliary & Pancreatic Diseases International
Hepatobiliary Pancreat Dis IntVol 11No 5 October 152012 www.hbpdint.com
Natural Science Foundation of China (81271712), and Hunan
Provincial Natural Science Foundation of China (11JJ4078).
Ethical approval: The animal study was approved by the Sydney
West Local Health District Animal Ethics Committee.
Competing interest: No benets in any form have been received
or will be received from a commercial party related directly or
indirectly to the subject of this article.
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Received February 22, 2012
Accepted after revision April 25, 2012