Rag2−/− γ-chain−/− mice as hosts for human vessel transplantation and allogeneic human leukocyte reconstitution

Department of Cardiac Surgery, Friedrich-Alexander University, Erlangen-Nürnberg, Krankenhausstrasse 12, 91054 Erlangen, Germany.
Transplant Immunology (Impact Factor: 1.46). 05/2010; 23(1-2):59-64. DOI: 10.1016/j.trim.2010.04.003
Source: PubMed
ABSTRACT
Rodent models are a very helpful tool to investigate immunological mechanisms in allograft rejection. The aim of this study was to compare two different immunodeficient recipients in a humanized mouse model of arterial xenotransplantation in terms of reconstitution of the human immune system and rejection of the arterial graft.
Side branches of human mammary artery were transplanted as infrarenal aortic interposition grafts into C.B-17-SCID beige and C57BL/6-Rag2(-/-)gammac(-/-) recipients. 7days after surgery mice were reconstituted with 5x10(7) human peripheral blood mononuclear cells (hu PBMCs) and 30days after reconstitution mice were sacrificed and histologic analysis was performed. Peripheral blood and splenocytes were investigated by FACS and ELISA analysis to ensure engraftment of human CD45(+) cells.
Transplant arteriosclerosis developed in non-PBMC-reconstituted C.B-17-SCID beige mice (intimal proliferation: 36.31+/-4.37%), but significantly less in C57BL/6-Rag2(-/-) gammac(-/-) recipients (intimal proliferation: 12.26+/-5.21%). After reconstitution with 5x10(7) unfractionated human PBMCs both mouse strains showed intima proliferation 30days after reconstitution (C.B-17-SCID beige: 28.49+/-7.95% and C57BL/6-Rag2(-/-) gammac(-/-): 44.58+/-11.08%). Whereas only very few human CD45(+) cells were found in mouse blood and spleen of C.B-17-SCID beige mice, C57BL/6-Rag2(-/-) gammac(-/-) mice revealed a reliable reconstitution. In addition, levels of human IgG and IgM within the peripheral blood were markedly higher in C57BL/6-Rag2(-/-) gammac(-/-) recipients.
In this study we can show, that the use of C57BL/6-Rag2(-/-) gammac(-/-) mice may be advantageous compared to C.B-17-SCID beige recipients in a humanized mouse model of vessel transplantation.

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Rag2
/
γ-chain
/
mice as hosts for human vessel transplantation and allogeneic
human leukocyte reconstitution
Silke Abele-Ohl
a
, Martina Leis
b
, Shohreh Mahmoudian
b
, Michael Weyand
a
,
Thomas Stamminger
b,1
, Stephan M. Ensminger
a,
,1
a
Department of Cardiac Surgery, Friedrich-Alexander University, Erlangen-Nürnberg, Krankenhausstrasse 12, 91054 Erlangen, Germany
b
Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Germany
abstractarticle info
Article history:
Received 21 December 2009
Received in revised form 30 March 2010
Accepted 5 April 2010
Keywords:
Rag2
/
γc
/
mice
Transplant arteriosclerosis
SCID/beige mice
huPBMC
Background: Rodent models are a very helpful tool to investigate immunological mechanisms in allograft
rejection. The aim of this study was to compare two different immunodecient recipients in a humanized
mouse model of arterial xenotransplantation in terms of reconstitution of the human immune system and
rejection of the arterial graft.
Methods: Side branches of human mammary artery were transplanted as infrarenal aortic interposition grafts
into C.B-17SCID beige and C57BL/6Rag2
/
γc
/
recipients. 7 days after surgery mice were reconstituted
with 5×10
7
human peripheral blood mononuclear cells (hu PBMCs) and 30 days after reconstitution mice
were sacri ced and histologic analysis was performed. Peripheral blood and splenocytes were investigated
by FACS and ELISA analysis to ensure engraftment of human CD45
+
cells.
Results: Transplant arteriosclerosis developed in non-PBMC-reconstituted C.B-17SCID beige mice (intimal
proliferation: 36.34.37%), but signicantly less in C57BL/ 6Rag2
/
γc
/
recipients (intimal
proliferation: 12.26 ± 5.21%). After reconstitution with 5 × 10
7
unfractionated human PBMCs both mouse
strains showed intima proliferation 30 days after reconstitution (C.B-17SCID beige: 28.49 ± 7.95% and
C57BL/6Rag2
/
γc
/
: 44.58 ± 11.08%). Whereas only very few human CD45
+
cells were found in mouse
blood and spleen of C.B-17SCID beige mice, C57BL/6Rag2
/
γc
/
mice revealed a reliable
reconstitution. In addition, levels of human IgG and IgM within the peripheral blood were markedly higher
in C57BL/6Rag2
/
γc
/
recipients.
Conclusion: In this study we can show, that the use of C57BL/6Rag2
/
γc
/
mice may be advantageous
compared to C.B-17SCID beige recipients in a humanized mouse model of vessel transplantation.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Small animal models have been very h elpfu l to investigate
underlying immunological mechanisms in transplantation, but cer-
tain limitations are unavoidable because of species-specic differ-
ences to humans. Immunodecient mice harboring human cells or
tissues are frequently referred to as humanized mice. They are a
promising tool for studying complex mechanisms in human biology
and at least may partly overcome these species-specic differences.
Mice bearing human immune systems have been developed to
investigate immune-mediated disease pathogenesis [1,2] and alloge-
neic tissue rejection and tolerance in vivo [35].
About 20 years ago, the original SCID-hu model was developed by
McCune et al. using C.B-17SCID mice as recipients for human
hematopoietic tissues including fetal liver, bone, and/or thymus
originating from human fetuses [68].
Engrafted human hematopoietic tissue resulted in low levels of
human T and B cells that were able to produce a primary antibody
response when autologous fetal skin, serving as an additional source
of dendritic cells, was coengrafted along with thymus, bone marrow,
and lymph node [9]. Adoptive transfer of peripheral blood mononu-
clear cells (PBMC) in this same mouse strain supported low levels of
engraftment of T, B and dendritic cells [10].
Further renements of this system were achieved by introducing a
beige mutation or crossing with mice of the NOD background allowing
recipients to accept higher levels of mature human T and B cells
[11,12]. Technical advances such as the transfer of isolated hemato-
poietic stem cells (HSC) in C.B-17SCID and NODSCID mice [13,14]
and the development of more severely immunodecient mouse lines
lacking the common cytokine receptor gamma chain (γ-ch ain)
Transplant Immunology 23 (2010) 5964
Corresponding author. Tel.: +49 9131 8533590; fax: +49 9131 8532768.
E-mail address: stephan.ensminger@uk-erlangen.de (S.M. Ensminger).
1
Both senior authors equally contributed to the work.
0966-3274/$ see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.trim.2010.04.003
Contents lists available at ScienceDirect
Transplant Immunology
journal homepage: www.elsevier.com/locate/trim
Page 2
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[15,16] enhanced the applicability of humanized mice for studying the
human immune system.
Nowadays, several models using C.B-17SCID/beige immunode-
cient mouse hosts in which human memory T-cell responses to artery
graft tissue can be studied in vivo have been developed [4,1719].
Despite an essentially absent adaptive immune system, C.B-17SCID
animals are still signicantly resistant to adoptive transfer of human
peripheral blood lymphocytes (PBL). Indeed, further studies showed
that resistance to adoptive transfer of human PBL was reduced in
animals treated with antibody to asialo GM1, which depletes NK cells,
or in C.B-17SCID animals that also have the beige mutation that
impairs NK-cell-function [4,17,18,20]. The SCIDhu model is nowadays
a well established system for the study of human alloresponses in
vivo, and particularly using the technique of xenografting human
arteries into these mice, great progress in the understanding of
immune-mediated damage to vascularised transplants has been
achieved [4]. However, inherent problems of the model have left
researchers dissatised with several aspects of the experimental
system, e.g. an important shortcoming in C.B-17SCID/beige recipi-
ents is the fact that they have to be injected with fairly high human
cell numbers (up to 10
8
) and cells other than CD4
+
T cells engraft
poorly.
Therefore, it was the aim of the present study to directly compare
C.B-17SCID/beige animals with C57BL/6Rag2
/
γc
/
mice lack-
ing T, B and NK cells [21] as recipients of human arterial transplants
reconstituted with human PBMCs from the peripheral blood of adult
donors and evaluate if this modication improves the experimental
humanized mouse system of arterial transplantation. Here we can
show, that C57BL/6Rag2
/
γc
/
mice offer technical advantages
over C.B-17SCID/beige recipients as they much more readily accept
human blood PBMCs, show improved reconstitution rates with lower
levels of human PBMCs and more reliably reject human arterial
transplants. In addition, vascular lesions were not detected in C57BL/
6Rag2
/
γc
/
recipients in the absence of cell transfer. Therefore,
the use of C57BL/6Rag2
/
γc
/
mice may be advantageous
compared to C.B-17SCID/beige recipients in a humanized mouse
model.
2. Methods and materials
2.1. Animals
Original breeding pairs for C.B-17SCID beige as well as C57BL/6
Rag2
/
γc
/
mice were obtained from Charles River (Sulzbach,
Germany) and Taconic (Germantown, N.Y., USA). Mice were aged
between 6 12 weeks at the time of experimental use and were bred
and maintained at the central animal facility of the University of
Erlangen-Nuernberg (FranzPenzoldtZentrum) in isolated ventilat-
ed cages with sterilized food, water and bedding. All experimental
animals were treated in accordance with institutional and state
guidelines. In addition, mice were maintained on trimethoprim
(8 mg/kg) and sulfamethoxazole (40 mg/kg) in their drinking water.
2.2. Measurement of mouse and human immunoglobulin
The le vel of mouse immunoglobulin (mu Ig and IgM) was
quantied by ELISA technique at 4 weeks of age as described in a
previous publication [22]. Mice with immunoglobulin levels superior
to 1 µg/ml were considered leaky and were not used for experi-
ments. Human immunoglobulin (IgM and IgG) was measured by an
enzyme linked immunosorbent assay (ELISA). Blood samples were
taken from the mouse retroorbital plexus and collected in serum
microtainers (BD Microtainer SST, Schubert, Wackersdorf, Germany).
Briey, ELISA plates (NUNC MaxiSorp plates) were coated with
a polyclonal rabbit anti-human IgG + I gM antibody (Jackson
ImmunoSearch Laboratories, Suffolk, UK). After blocking of unspecic
binding capacities by incubation with 5% fetal calf serum in phosphate
buffered saline, dilutions of mouse sera were applied together with
serial dilutions of control sera with known immunoglobulin contents.
After extensive washing, bound human antibodies were detected via a
peroxidase-conjugated anti-human IgG+ IgM antibody (Dianova,
Hamburg, Germany) together with TMB peroxidase substrate
(Medac, Wedel, Germany). Human immunoglobulin concentrations
in mouse sera were calculated from standard curves derived from the
samples with known immunoglobulin contents.
2.3. Abdominal transplantation of human mammary artery segments
The procedure wasperformed using the technique initially described
by Lorber et al. [23]. Briey, size-matched segments of side branches
from the human mammary artery obtained during routine CABG-
surgery were transplanted as infrarenal aortic interposition grafts into
the abdominal cavity of SCID/beige and Rag2
/
γc
/
mice. A proximal
and distal end to end anastomosis was performed with single
interrupted sutures using 11-0 monolament nylon suture. Before
closure of the abdomen, hemostasis and blood ow through the
transplanted arterial segment were conrmed and warm sterile saline
was administered into the abdomen.
2.4. Purication of human PBMCs
Human buffy coats were obtained from the regional blood bank
(Blood bank Suhl, Institute for Transfusion Medicine, Germany). Cells
were tested for a variety of pathogens, including HIV, HCV and CMV to
exclude infections. Immediately upon receipt, mononuclear cells were
puried by Ficoll density centrifugation using Lymphoprep (Axis-
Shield, Oslo, Norway) as described by the manufacturer. After
quantication under Trypan blue exclusion a dened number of
cells (5× 10
7
) was resuspended in endotoxin-free phosphate buffered
saline (volume of 300 µl) and used for intraperitoneal (i.p.) injection
of mice.
2.5. Determination of human lymphocytes
The success of lymphocyte engraftment was assessed by ow
cytometry 30 days after PB MC ad ministr atio n. F or this, eithe r
peripheral blood was collected by puncture of the retroorbital plexus
using EDTA-containing 1.5 ml tubes. Alternatively, spleen cells were
harvested from sacriced animals. Flow cytometry was performed on
a FACScalibur ow cytometer according to standard protocols. The
dire ctly labelled monoc lonal antibodies (allophycocyanin [APC],
uorescein-isothiocyanate [FITC], or phycoerythrin [PE] conjugated)
were specic for human CD45 or murine CD45. Directly-labelled
isotype-matched monoclonal antibodies were used as controls. All
antibodies were purchased from Becton Dickinson (BD Biosciences,
Heidelberg, Germany). Two-colour uorescence was used to measure
the percentage of circulating human and murine lymphocytes. Human
PBMC engraftment was considered successful when a distinct
population of human CD45
+
cells was measured, consisting of more
than 0.1% of the total circulating leukocyte population concerning
mouse blood samples or more than 0.5% concerning mouse spleen cell
samples.
2.6. Morphometric analysis of the mammary artery graft
Mammary artery grafts were removed under anaesthesia on day 30
after transplantation. Grafts were perfused with saline and were ash
frozen in OCT medium (Tissue-Tek®, Sakura, Netherlands) in liquid
nitrogen for morphometric analysis of 7 µm cryostat sections. Five
cross-sections from different regions of each graft harvested at day 30
were stained with Elastin/van Gieson and analysed by two independent
examiners (SME and SA) blinded to the experimental conditions using a
60 S. Abele-Ohl et al. / Transplant Immunology 23 (2010) 5964
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conventional light microscope (original magnication ×200). For the
morphometric analysis of the degree of intimal thickening the area
within the lumen and the internal elastic lamina was circumscribed
manually. A digitised image of each section was captured and measured
as previously described [24]. All image analysis was carried out using
ANAlysi Image Analysis software (Olympus, Germany).
2.7. Statistical analysis
Results are given as the mean per group±SEM, which was derived
from the mean per graft. The data were analysed using a two-tailed
unpaired Student's test. A p-value of b 0.05 was considered as
signicant.
3. Results
3.1. SCID/beige recipients reject mammary artery grafts in the absence of human PBMCs
In a rst set of experiments age matched (68 weeks) C.B17SCID/beige and
C57BL/6-Rag2
/
γc
/
received an abdominal human mammary artery graft and no
reconstitution of human PBMCs. Prior to the experiment, C.B17SCID/beige recipients
were screened for leakyness and respective animals were excluded from further
experimental use. Interestingly, C.B17SCID/beige recipients showed prominent intima
proliferation even in the absence of human PBMCs (Intima p roliferation: 36.31 ±
4.37%) (Fig.1A). In contrast, C57BL/6Rag2
/
γc
/
mice undergoing the same
procedure demonstrated almost no signs of vascular lesions in the absence of human
PBMCs (Intima prolifer ation: 12.26 ±5.21%) (Fig.1B). After reconstitution with
5×10
7
human PBMCs both experim ental groups developed signi cant levels of
transplant arteriosclerosis (Fig. 1C, D).
3.2. Rag2
/
γc
/
recipients show higher levels of circulating CD45
+
cells after
reconstitution with human PBMCs
Initial reconstitution experiments with different doses of human PBMCs
revealed, that after transfer of 5 ×10
7
PBMCs stable reconstitution levels were seen
in C57BL/6Rag2
/
γc
/
recipients and no signs of GVHD like weight loss, bad fur
structure or cha nges in s kin , liver and gut could be detected (data not shown). There-
fore, on day 7 after implantation of the mammary artery graft, C.B17SCID/bei ge and
C57BL/6Rag2
/
γc
/
recipients were recons tituted with 5 ×10
7
hu PBMCs.
30 days after i.p. PBMC-reconstitution almost no human CD45
+
cell-chimerism
was detectable in the blood (0.13 ±0.48% human CD45
+
cells) or spleen (0.36±
0.87% human CD45
+
cells) of SCID/beige recipients (Fig. 2). C57BL/6Rag2
/
γc
/
mice of the experimental group reconstituted with human PBMCs showed 3.13 ±
1.74% human CD45
+
cells in the blood and 12.23 ± 6.63% human CD45
+
cells in the
spleen 30 days after reconstitution ( Fig. 2). (* =pb 0.05 vs. reconstituted C.B17
SCID/beige; ** = p b 0.05 vs. reconstituted C.B17SCID/beige (n = 5 )).These results
were paralleled by levels of human IgG and IgM of 108±22 µg/ml in C.B17SCID/
beige recipients and 748 ±27 µg/ml in C57BL/6Rag2
/
γc
/
recipients (Fig. 3).
(*= p b 0.05 vs. reconstitut ed C.B17SCID/beige/(n = 5)).
3.3. Rag2
/
γc
/
recipients display more stable rejection responses of the human
vascular graft
The read out and therefore most important aspect of this experimental model is a
reliable rejection response of the human vascular mammary artery graft after
reconstitution with human PBMCs. Overall complication rates of this study were 9.2%
(SCID) and 8.5% (RAG-2), the most frequent complication in both groups being
thrombosis of the aortic graft (n=4) and one late death of the recipient due to other
causes. None of the recipient-mice showed any kind of haemorrhage or major
postoperative bleeding. Again, there was a striking difference between the two
recipient mouse strains. Whereas mammary artery grafts recovered 30 days after
reconstitution with 5 × 10
7
human PBMCs from C57BL/6Rag2
/
γc
/
recipients
showed signicant amounts of intimal proliferation (44.58± 11.08% luminal occlusion/
n= 5), C.B17SCID/beige mice showed signicantly less intima proliferation after
reconstitution with equal levels of human PBMCs (28.49 ± 7.95% luminal occlusion/
n=5) (Fig. 1C, D, Fig. 4). In addition, variations among individual experiments within
the respective experimental group were much smaller in C57BL/6Rag2
/
γc
/
recipients as compared to C.B17SCID/beige recipients.
4. Discussion
In this study, we compared the engraftment of human PBMCs in C.
B-17SCID/beige and C57BL/6Rag2
/
γc
/
mice and their rejec-
tion response to a human artery graft. To allow direct comparisons in
these two mouse strains, a number of critical parameters were held
constant throughout the study, including age at the time of
engraftment and an identical number of input cells from each PBMC
source in a given experiment. On many occasions, cells from identical
buffy-coats were injected on the same day into mice of the two
different strains. Here we can show that (1) after reconstitution with
unfractionated human PBMCs C57BL/6Rag2
/
γc
/
mice revealed
Fig. 1. For the morphometric analysis of the degree of intimal thickening Miller's Elastin van Gieson stained sections were used. Areas within the lumen and the internal elastic
lamina were circumscribed manually and measured. Human artery grafts recovered from C.B17SCID/beige recipients on day 30 after transplantation displayed vascular lesions even
in the absence of human PBMCs (A), whereas C57BL/6Rag2
/
γc
/
recipients did not show signicant lesions (B). Panel (C) shows a human artery graft recovered from a C.B17
SCID/beige recipient (n = 5) reconstituted with 5 ×10
7
human PBMCs on day 30 after reconstitution; C57BL/6Rag2
/
γc
/
recipients (n = 5) reconstituted with the same amount
of human PBMCs showed signicant more intima proliferation (D) (original magnication ×200).
61S. Abele-Ohl et al. / Transplant Immunology 23 (2010) 5964
Page 4
Author's personal copy
a more reliable reconstitution as determined by the presence of
human CD45
+
cells within the peripheral blood and spleen compared
to C.B-17SCID/beige mice. In addition (2) levels of human IgG and
IgM within the peripheral blood were markedly higher in C57BL/6
Rag2
/
γc
/
recipients. Furthermore, (3) transplant arteriosclero-
sis after reconstitution with human PBMCs was more pronounced and
reliable in C57BL/6Rag2
/
γc
/
recipients as compared to C.B-17
SCID/beige mice, which also demonstrated vascular lesions in the
absence of reconstitution with human PBMCs.
The initial human artery transplantation model in a humanized
mouse using C.B-17SCID/beige mice as recipients was developed by
Lorber et al. [23] to overcome species specic differences between
mice and humans, such as differences in cell signalling e.g. in the
expression of MHC molecules. Rodent endothelial cells express mainly
MHC II whereas human endothelial cells express MHC I and MHC II
[25,26]. In addition, there are structural differences between mouse
and human vessels, e.g. the integrity of the human vascular media in
clinical heart transplantation is mostly preserved, whereas vascular
smooth muscle cells (VSMCs) of the mouse media disappear during
the rejection response [27]. These problems seemed to have been
overcome by implanting a human artery into a humanized mouse
recipient and indeed the authors managed to demonstrate that the
Fig. 2. (A and B) FACS analysis for human lymphocyte detection within the peripheral blood and mouse spleen after reconstitution with human PBMCs i.p. into the respective C.B17
SCID/beige (A) or C57BL/6Rag2
/
γc
/
recipients (B). Results are given as percentage of human leukocytes of total circulating leukocytes (CD45). The positive control was
peripheral human blood (A) or a mixture of human blood and mouse blood (B). (C) The diagram shows average amounts of human CD45
+
cells detected by FACS analysis in the
respective experimental groups. There were low numbers of detected human cells with blood and spleen of C.B17SCID/beige recipients, whereas C57BL/6Rag2
/
γc
/
recipients showed signicant numbers of human cell reconstitution within blood and spleen.
Fig. 3. Human IgG and IgM showed much higher concentrations within the peripheral
blood of C57BL/6-Rag2
/
γc
/
recipients compared to C.B17-SCID/beige.
62 S. Abele-Ohl et al. / Transplant Immunology 23 (2010) 5964
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Author's personal copy
vessel injury was specic to the human arterial segments and was
mediated by the human PBMCs [23]. However, results of the present
study show that after successful reconstitution, rejection responses of
human arterial grafts implanted into C.B-17SCID/beige recipients
were very variable and on a signicantly lower level as compared to
C57BL/6Rag2
/
γc
/
mice, when identical numbers of human
PBMCs were injected. One aspect that may account for this difference
is the deciency of the interleukin-2 receptor (IL-2R) γ-chain locus
that is required for high-afnity binding of the receptors for IL-2, IL-4,
IL-7, IL-9, IL-15, and IL-21 and is essential for their signalling and
function [2]. This mutation renders RAG2
/
mice more decient in
NK cells, as well as causing deciencies in T- and B-cell development
and function and may therefore provide a critical environment for
enhanced human PBMC development in C57BL/6Rag2
/
γc
/
mice as demonstrated in this report. However, defects in NK-cell
function conferred by the beige mutation of the C.B-17SCID
recipients did not result in similar levels of reconstitution in the
recipients of human vessel transplants. In addition, although C.B-17
SCID/beige mice were screened for mu IgG and IgM before each
experiment to exclude leaky C.B-17SCID/beige mice some tend to
become leaky later on as a consequence of reactivation of single T-cell
clones. This circumstance also may have prevented better engraft-
ment of human PBMCs in C.B-17SCID/beige as compared to C57BL/
6Rag2
/
γc
/
mice.
Arterial grafts implanted in C.B-17SCID/beige recipients dis-
played variable levels of intima proliferation after reconstitution with
similar numbers of human PBMCs. Interestingly, several grafts
demonstrated strong vascular lesions in the absence of reconstitution
with human PBMCs. In contrast, similar experiments using C57BL/6
Rag2
/
γc
/
mice showed only discrete signs of intima prolifera-
tion in the absence of human PBMCs and more constant results after
reconstitution with human PBMCs. One explanation for this difference
may be the fact that C.B-17SCID/beige mice can become leaky later in
life, as already discussed and that NK-cell function is not as impaired
as in C57BL/6Rag2
/
γc
/
mice. The signicantly higher level of
human immunoglobulins in these mice is further evidence for a better
engrafting of the hu PBMCs in this mouse strain.
Engraftment levels of human PBMCs in C.B-17SCID/beige mice
are discussed controversially in the literature [2,28]. Some groups
favour C.B-17SCID/beige mice as the strain of choice for the transfer
of a human adaptive immune system [28], others demonstrated
variable and mostly low level engraftment of human PBMCs by using
this mouse strain [2]. Results of the present study support the latter
view as we could only achieve very low levels of human PBMC
engraftment in C.B-17SCID/beige mice. Recently, it was shown by
two groups that Rag2
/
γc
/
mice supported B-cell, DC and T cell
development, following engraftment of human stem cells (HSC)
[29,30]. However, for the current report, human PBMCs were used for
reconstitution of the Rag2
/
γc
/
mice and our results also support
sufcient engraftment of T cells as shown in the peripheral blood and
spleen. In addition, signicantly higher amounts of human IgG and
IgM were detected in Rag2
/
γc
/
mice than in the C.B-17SCID/
beige mice and histologic analysis of the human artery graft revealed
signicant amounts of human CD45
+
cells (data not shown).
There are two experimental-setup-specic parameters that need
monitoring: Firstly, the fact that human mammary artery segments
display variable amounts of ordinary arteriosclerosis, at the beginning
of each experiment. These differences were taken into account by
analysing the human vessel before implantation in the respective
recipient and by comparing these results to measurements performed
after explanation of the arterial graft at the end of each experiment. In
addition it was shown in previous studies, that alloimmune-mediated
intimal injury and vascular remodelling is somewhat independent of
pre-existing coronary atherosclerosis [31]. Secondly, although experi-
ments were performed in groups, there were differences in HLA-
mismatches between the implanted human artery segments and the
respective buffy-coats used for reconstitution. As both, human artery
segments and buffy coats from single donors, were tissue typed for
each experiment, data revealed that there was an average of 3.7 HLA
mismatches between arterial grafts and PBMCs used for reconstitu-
tion in both experimental groups (data not shown).
Survival rates after surgery were on a similar level for both
experimental groups (N 85%) with thrombosis of the graft being the
most freque nt complication. However, mice from both strains
analysed occasionally became ill during adult life and required
premature euthanasia for human reasons if they failed to reconstitute
or had very low engraftment levels (b 0.1%). These mice were not used
in the functional analysis experiments.
Optimizing humanized mouse models to investigate underlying
mechanisms of chronic allograft rejection is paramount to a more
comprehensiv e understanding of this complex disease pathogenesis and
progression. The current study reports a comparison of two different
recipient mouse strains in a mouse model of human artery transplan-
tation. We can show for the rst time, that C57BL/6Rag2
/
γc
/
mice
offer technical advantages over C.B-17SCID/beige recipients as they
much more readily accept human blood PBMCs, show improved
reconstitution rates with lower levels of human PBMCs and more
reliably reject human arterial transplants. In addition, signicant vascular
lesions were not detected in C57BL/6Rag2
/
γc
/
recipients in the
absence of cell transfer. Therefore, the use of C57BL/6Rag2
/
γc
/
mice may be advantageous compared to C.B-17SCID/beige recipients in
a humanized mouse model.
Acknowledgements
The authors would like to thank Mr Johannes Roesch and Mrs.
Regina Müller for expert technical assistance. We would also like to
thank Professor Stephan von Hoersten and the staff of the animal
facility of the University of Erlangen-Nürnberg for their expert care of
animals used for this study.
This work was supported by grants from the IZKF of the University
of Erlangen-Nürnberg, the GRK1071 and the ADUMED-Foundation.
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  • Source
    • "These mice did not become leaky, as they do not involve the SCID mutation. Rag2 −/− y c −/− mice provided a more constant model, with only some discrete intima changes preceding the huPBL injection compared to SCID/bg [59,61,68] . However, a limitation of the hu-PBL- SCID model is that from all injected human cells only T-cells and a few B-cells survive. "
    [Show abstract] [Hide abstract] ABSTRACT: The interest in the use of humanized mouse models for research topics like Graft versus Host Disease (GvHD), allograft studies and other studies to the human immune system is growing. The design of these models is still improving and enables even more complicated studies to these topics. For researchers it can be difficult to choose the best option from the current pool of available models. The decision will depend on which hypothesis needs to be tested, in which field of interest, and therefore 'the best model' will differ from one to another. In this review, we provide a guide to the most common available humanized mouse models, with regards to different mouse strains, transplantation material, transplantation techniques, pre- and post-conditioning and references to advantages and disadvantages. Also, an evaluation of experiences with humanized mouse models in studies on GvHD and allograft rejection is provided.
    Full-text · Article · Feb 2014 · Transplantation reviews (Orlando, Fla.)
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    [Show abstract] [Hide abstract] ABSTRACT: Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.
    Full-text · Article · Nov 2011 · Arteriosclerosis Thrombosis and Vascular Biology
  • [Show abstract] [Hide abstract] ABSTRACT: BACKGROUND: Characteristics of vascular prostheses are similar to the natural vessels and it has high histocompatibility after transplantation. But endothelial regeneration is the key for vascular prosthesis unblocked eternally. OBJECTIVE: To investigate the histocompatibility of a novel small-diameter bioartificial vascular graft at different periods postoperatively and the histopathologic changes following intimal regeneration of the vessel wall. METHODS: A dog model of artificial vascular grafts implanted end-to-end by a continuous suture in the common carotid artery was established. RESULTS AND CONCLUSION: Light microscope results showed that at week 12 after graft, endothelial cells were sparsely distributed throughout the neointima surface near the stoma; at month 6 after graft, endothelial cells were presented on the whole surface of the patent graft lumen; 1.5 years after graft, lumens of graft vessels were unobstructed, and parts of endomembrane tissues were found chronic inflammation. Electron microscope results showed that at week 12 after graft, new vascular endothelial cells were in fusiform shape and arranged regularly; besides, the endothelial cells spread from the stoma to the middle of the graft vessel. After graft for 6 months, the endothelial cells spread from the stoma to the middle of the graft vessel. Endothelial cell colony in the middle of the graft vessel showed leaping and flake-like growth, and the cells were arranged more compactly; moreover, their shapes were more like the endothelial cells of mature vessels. After graft for 1.5 years, the whole vessel wall was covered by tightly arranged endothelial cells, and parts of endomembrane tissues revealed chronic inflammation. These findings suggest that the novel small-diameter bioartificial vascular graft has many advantages, for example, the newborn intima forms early and completely, and endangium can be well reconstructed; in addition, it has good biocompatibility and stability.
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