Download Mouse models of graft-versus-host disease: advances and limitations

PERSPECTIVE
Disease Models & Mechanisms 4, 318-333 (2011) doi:10.1242/dmm.006668
Mouse models of graft-versus-host disease:
advances and limitations
Disease Models & Mechanisms DMM
Mark A. Schroeder1 and John F. DiPersio1,*
The limiting factor for successful hematopoietic stem cell transplantation (HSCT) is graft-versus-host disease (GvHD), a
post-transplant disorder that results from immune-mediated attack of recipient tissue by donor T cells contained in the
transplant. Mouse models of GvHD have provided important insights into the pathophysiology of this disease, which
have helped to improve the success rate of HSCT in humans. The kinetics with which GvHD develops distinguishes
acute from chronic GvHD, and it is clear from studies of mouse models of GvHD (and studies of human HSCT) that the
pathophysiology of these two forms is also distinct. Mouse models also further the basic understanding of the
immunological responses involved in GvHD pathology, such as antigen recognition and presentation, the involvement
of the thymus and immune reconstitution after transplantation. In this Perspective, we provide an overview of currently
available mouse models of acute and chronic GvHD, highlighting their benefits and limitations, and discuss research
and clinical opportunities for the future.
Introduction
GvHD immunopathology in humans and mice
A major complication and limitation to the broad application of
hematopoietic stem cell transplantation (HSCT) is graft-versushost disease (GvHD), which results from immune-mediated attack
of recipient tissue by donor T cells contained in the transplant.
GvHD accounts for 15-30% of deaths that occur following
allogeneic HSCT that is carried out to treat malignant diseases,
and is a major cause of morbidity in up to 50% of transplant
recipients (Ferrara et al., 2009). This high incidence is despite
the use of aggressive immunosuppressive therapies after
transplantation, as well as allele-level human leukocyte antigen
(HLA) matching between donor and recipient. Despite recent
advances to reduce the incidence of GvHD through altering
prophylactic regimens and reducing the intensity of conditioning
prior to transplantation, effective treatments for GvHD are lacking.
Thus, accurate and reproducible experimental models of GvHD
are essential for advancing our fundamental understanding of this
disorder and for developing new treatments. In this Perspective,
we provide an overview of available mouse models of acute and
chronic GvHD, discuss their uses and limitations, and provide
guidance on selecting an appropriate model. We also briefly discuss
the graft-versus-leukemia (GVL) effect, although readers are
referred to other recent reviews for more detail on this topic
(Bleakley and Riddell, 2004; Ringden et al., 2009). We begin by
describing the clinical symptoms and immunopathology of GvHD.
In humans, GvHD manifests as acute GvHD (aGvHD), which
occurs within 100 days of transplant, or as chronic GvHD (cGvHD),
which typically develops 100 days after transplantation and can take
2-5 years to become clinically evident. This temporal distinction
does not necessarily translate to mouse models of the disease, which
can differ in time of onset and are mainly defined by their clinical
phenotype: cGvHD develops within weeks after transplantation in
most mouse models. In addition, in humans, aGvHD typically
precedes cGvHD, although in some cases cGvHD can occur
without the occurrence of clinically obvious aGvHD. One factor
that confounds the translation of findings in mouse models to
the human disease is that most patients are given
immunosuppressive therapy to prevent aGvHD, and these
medications might influence the development of cGvHD.
Interestingly, interventions to prevent aGvHD in the clinic have,
in general, not significantly decreased rates of cGvHD in humans
(Lee, 2010).
The sequence of events that lead to the development of GvHD
has largely been defined using mouse models. Early work
established that T-cell alloreactivity is the underlying cause of the
disease (Korngold and Sprent, 1978; Sprent et al., 1986). The
pathology of both acute and chronic mouse models of GvHD relies
on T-cell alloreactivity, but each form has a different phenotype
owing to differential involvement of cytotoxic (CD8+) or helper
(CD4+) T-cell subsets. Donor CD8+ T cells are activated when their
T-cell receptor (TCR) binds to recipient peptides presented in the
context of recipient class I major histocompatibility complex
(MHC) molecules (see Box 1 for a glossary of immunological
terms). T-cell activation also requires the engagement of costimulatory receptors on activated donor T cells by their cognate
ligands expressed on donor antigen-presenting cells (APCs). CD8+
T-cell cytotoxicity is mediated by perforin, granzymes and Fas
ligand, and inflammatory cytokines augment this response. Donor
CD4+ T cells are activated when their TCR binds to exogenous
1
Division of Oncology, Siteman Cancer Center, Washington University School of
Medicine, St Louis, MO 63110, USA
*Author for correspondence ([email protected])
© 2011. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons
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distributions of the work or adaptation are subject to the same Creative Commons License
terms.
318
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PERSPECTIVE
Mouse models of GvHD
Disease Models & Mechanisms DMM
Box 1. Glossary of immunological terms
Antigen cross presentation: a process by which exogenous antigens derived
from endogenous cells (such as damaged epithelium in the case of GvHD) are
taken up by APCs and presented, typically in MHC class I molecules, to CD8+ T
cells. In setting of GvHD, donor APCs cross-prime donor T cells by presenting
recipient antigens.
Antigen presentation: the process by which either self or non-self antigens
are captured, processed and presented on the surface of APCs for cognate
antigen recognition by lymphocytes.
Antigen-presenting cells (APCs): a heterogenous population of cells
(including B cells, monocytes/macrophages and dendritic cells) that present
self and non-self antigens to T cells.
Chimerism: the presence of two or more genetically distinct cell populations
(from donor and recipient) in the same organism, as occurs following HSCT;
can be measured by determining the percentage of donor cells present in the
blood or bone marrow through genetic or immunocytometry testing.
Cognate antigen recognition: recognition of antigen by T or B cells
expressing receptors specific for that antigen.
Cytokine storm: elevated levels of inflammatory mediators (such as TNF, IL-1
and IL-6) that potentiate further stimulation of immune effector cells such as T
cells and macrophages in a positive-feedback loop; can occur in GvHD or in
response to infections.
Effector T cells: mature activated T cells that produce large amounts of
cytokines (in the case of CD4+ T cells) or kill target cells via perforin and
granzymes (in the case of CD8+ T cells).
Epitope: a specific region of an antigen that is recognized by antibodies, or by
B-cell or T-cell receptors.
Epitope spreading: a normal or pathological (in the case of auto-antigens)
immune response in which epitopes distinct from the inducing epitope
become targets of the immune response. This typically occurs in the setting of
chronic inflammatory responses. APCs process and present a variety of
epitopes from the same larger antigenic peptide, resulting in an increased
repertoire of T-cell responses against multiple epitopes from the original
antigenic peptide.
Major histocompatibility complex (MHC): a cluster of highly polymorphic
genes that encode proteins involved in antigen presentation (also known as
HLA antigens in humans and H2 in mice); important for enabling the immune
system to differentiate between self and foreign tissues. MHC class I enables
presentation of mainly endogenous antigens to CD8+ T cells, whereas MHC
class II enables presentation of mainly exogenous antigens to CD4+ T cells.
Minor histocompatibility antigens (miHAs): a class of polymorphic antigens
thought to arise from polymorphisms in the genes encoding common cellular
proteins; presented by MHC class I and are often responsible for organ
rejection and GvHD in MHC-matched transplants. An example is the H-Y
antigen.
Regulatory T (TReg) cells: a subset of CD4+ T cells that is defined by
expression of the transcription factor FoxP3 and that suppresses immune
responses to a wide variety of antigens, including alloantigens and autologous
antigens.
T helper 1 (Th1) cell: a CD4+ T-cell effector subset characterized by the
production of the cytokines IL-2, IFN and IL-12. Th1 cells can activate
macrophages and B cells.
T helper 2 (Th2) cell: a CD4+ T-cell effector subset characterized by the
production of the cytokines IL-4, IL-5, IL-6 and IL-10. Th2 cells provide help to B
cells for antibody production.
T-cell alloreactivity: the T-cell response induced when a graft is performed
with tissue of the same species but with a different genotype, owing to the
recognition of foreign tissue (allograft) by the T cells.
Th17 cells: a subset of CD4+ T effector cells that expresses proinflammatory
molecules of the IL-17 family and mediates cytotoxic responses resulting in
inflammation and tissue injury.
Thymic negative selection (also known as central tolerance): the deletion
of self-reactive T cells (i.e. T cells with autoimmune potential) during their
development in the thymus.
Disease Models & Mechanisms
peptides presented in the context of class II MHC molecules on
recipient APCs. CD4+ T-cell activation results in the development
of a T-helper (Th)1 inflammatory response [marked by the
production of interferon- (IFN), interleukin-12 (IL-12) and IL2] or a Th2 response (marked by the production of IL-4, IL-5, IL6 and IL-10). In mice, cGvHD mainly involves a Th2 response,
whereas aGvHD is mainly Th1 biased.
Cellular isolates used for HSCT in mice contain, among other
cells, stem cells and T cells. Within 24 hours after transplantation,
T cells traffic to secondary lymphoid organs (Panoskaltsis-Mortari
et al., 2004; Beilhack et al., 2005). This initial migration of donor
T cells and their interaction with recipient APCs is believed to play
a crucial role in alloantigen priming and donor T-cell activation
prior to egress to primary target organs (Kim et al., 2003; Beilhack
et al., 2005). Activated donor T cells traffic and cause cytotoxicity
in the gut, skin, liver, lung, thymus and lymph nodes. This unique
tissue restriction is observed both in mouse models of GvHD and
in human patients suffering from GvHD. Chemokines and adhesion
molecules also play a crucial role in T-cell trafficking (Wysocki et
al., 2005).
aGvHD immunopathology
aGvHD involves the direct cytotoxic effects of donor T cells on
recipient tissues, the activation of APCs and an inflammatory
cascade known as a ‘cytokine storm’, which involves the production
of cytokines, including IL-1, IL-6, IL-12, IFN and tumor necrosis
factor- (TNF). IFN has diverse roles, regulating immune
suppression as well as cytotoxicity (for a review, see Lu and Waller,
2009). The impact of IFN on aGvHD is temporally related: IFN
can have immunosuppressive effects if present immediately after
transplant but inflammatory effects if it is added later (Brok et al.,
1998). This inflammatory cascade contributes to a systemic
syndrome of weight loss, diarrhea, skin changes and high mortality.
Key events involved in the development of aGvHD in mice are
illustrated in Fig. 1. First, priming of the immune response
establishes a milieu for enhanced T-cell activation and expansion,
and occurs as a direct result of ‘conditioning’. Conditioning
suppresses the recipient’s immune system and is applied to allow
engraftment of donor hematopoietic stem cells. It can be achieved
with cytotoxic agents such as irradiation or chemotherapy, or with
immunosuppressive agents such as anti-thymocyte globulin or
drugs that target recipient T cells, such as fludarabine. The most
common method of conditioning in mouse models is total body
irradiation, which ablates the recipient’s bone marrow, allowing
donor stem cell engraftment and preventing rejection of the graft
by limiting the proliferation of recipient T cells in response to donor
cells. Subsequent activation of donor T cells depends on cognate
antigen recognition. T cells are also activated by non-cognate
stimulation by cytokines such as IL-1 and TNF that are released
during priming by many different cell types (Ferrara et al., 1993).
Subsequent expansion of donor T cells and release of cytokines
such as IFN leads to the activation of additional effector cell types,
including neutrophils, monocytes and natural killer (NK) cells,
which can result in further donor T-cell proliferation independent
of their interactions with cognate antigen (Teshima et al., 2002).
In addition, proinflammatory macrophages are activated, which can
release IL-1, TNF and nitric oxide (Nestel et al., 1992; Cooke et
al., 1998).
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PERSPECTIVE
Mouse models of GvHD
A
Immune priming
Inflammatory
cytokines
Damage
XRT
Chemotherapy
Damage to host tissues (skin, gut, liver)
B
TNFα
IL-1
IL-6
T-cell activation and co-stimulation
Lymph nodes and target organs
Recipient
APC
Donor
APC
Endogenous
antigen
APC maturation
and antigen
presentation
MH
MHC II
CD8+
T cell
C
cGvHD immunopathology
Exogenous
antigen
MHC I
CD4+
T cell
Alloreactive T-cell expansion
Disease Models & Mechanisms DMM
Lymph nodes
T-cell
expansion
D
Trafficking
Skin, gut, liver, lung
CD4+ or
CD8+ T cell
Trafficking
Blood
ood and lymph
lymp
E
Tissue injury and destruction
Skin, gut, liver, lung
Tissue damage
Tissue-resident
DC or
macrophage
TNFα
IL-1
IFNγ
TNFα
IL-1
IFNγ
Perforin/granzyme
Fas/Fas-ligand
Host cell
Fig. 1. Steps to aGvHD in mice. The progression of events occurring in the
development of aGvHD in the mouse is illustrated. The five crucial steps of
immune priming (A), activation (B), T-cell expansion (C), T-cell trafficking (D)
and host tissue injury (E) are outlined. DC, dendritic cell; XRT, radiation
conditioning. See main text for full details.
The final step leading to end organ damage in aGvHD is T-cellmediated tissue toxicity, which involves soluble mediators,
including TNF, perforin, granzymes, Fas and Fas ligand (Baker
et al., 1996; Braun et al., 1996; Graubert et al., 1997; Jiang et al.,
2001; Maeda et al., 2005). In addition, T helper 17 (Th17) cells are
a subset of T cells that have recently been shown to contribute to
this final cytotoxic phase and can cause increased inflammation
and cytotoxicity when supplemented in the graft in an
experimental setting. A recent report demonstrated that Th17 cells
were sufficient but not necessary to cause GvHD (Iclozan et al.,
320
2009). Surprisingly, however, some studies have reported that their
absence from the graft can also worsen GvHD (Yi et al., 2008;
Carlson et al., 2009; Kappel et al., 2009). It is hoped that future
studies will resolve this discrepancy to clarify the role of Th17 cells
in GvHD pathology. A crucial counterpart to Th17 cells are
regulatory T (TReg) cells, which have been shown to suppress a
wide range of immune responses, including alloreactive and
autoreactive T-cell responses (Edinger et al., 2003; Sakaguchi et
al., 2006).
Unlike aGvHD, cGvHD typically presents as an autoimmune-like
syndrome. As well as effects mediated by T cells, cGvHD involves
B-cell stimulation, autoantibody production and systemic fibrosis.
Mouse models of cGvHD are associated with prolonged survival
compared with mouse models of aGvHD. Additional clinical
characteristics depend on the mouse model and can include
splenomegaly and lymphadenopathy resulting from B-cell
expansion, glomerulonephritis, biliary cirrhosis and scleroderma.
The immunopathology underlying cGvHD is less well
understood than that of aGvHD, in part because of the limitations
of currently available mouse models. Unlike aGvHD, there is no
unifying theory for the development of cGvHD, and the
pathological mechanisms differ depending on the model system.
Mouse models of cGvHD typically involve three main pathological
mechanisms: autoantibody production, pro-fibrotic pathways and
defective thymic function. Here, we describe the main events that
underlie the phenotype, although models exploring the three main
mechanisms will be discussed in detail later.
A summary of our current knowledge of the presumed sequence
of events leading to end organ damage in cGvHD is outlined in
Fig. 2. First, unlike in aGvHD, immune priming in cGvHD models
is not dependent on tissue damage from conditioning: compared
with aGvHD models, the initial inflammatory milieu is reduced
or absent in cGvHD models, which involve reduced levels of
radiation or no conditioning at all. The outcome of transplantation
is mixed T-cell chimerism in most models. Thymic damage can
result in dysfunctional negative selection of autoreactive T cells,
and there can be decreased TReg cell number or function. In
addition, donor and recipient B cells recognize and process
extracellular antigens for presentation in the context of MHC. All
of these processes can contribute to the initiation of cGvHD.
Second, unlike in aGvHD, T-cell activation is not dependent on
recipient APCs, but is a mix of cognate antigen recognition and
cross-priming of donor T cells. CD4+ cells are necessary and
sufficient to cause cGvHD in mouse models, whereas cytotoxic
CD8+ T cells are not required, in contrast to aGvHD (Rolink and
Gleichmann, 1983; Rolink et al., 1983; Zhang et al., 2006). Recipient
B cells are activated by minor antigens and Th2 cytokines, and
mature to present processed antigens in the context of MHC to
donor CD4+ T cells, which in turn co-stimulate B cells to produce
autoantibodies and additional stimulatory cytokines (Morris et al.,
1990a; Shimabukuro-Vornhagen et al., 2009). Third, T and B cells
clonally expand in response to antigenic stimulation and costimulation, and form memory and effector cells. In addition,
epitope spreading can occur, resulting in oligoclonal T-cell
expansion. Fourth, clonally expanded T and B cells can traffic to
target organ sites and mediate damage through cytokine-mediated
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PERSPECTIVE
Mouse models of GvHD
A
Immune priming
Thymic damage
Decreased number
of TReg cells
B-cell priming
Antigen
Antibody
Thymus
B
Recipient and
donor B cells
Endogenous
antigen
Donor
APC
Exogenous
antigen
MHC I
Mouse models of aGvHD
B cell
MHC
MH II
CD8+
T cell
Disease Models & Mechanisms DMM
B cell
T-cell and
an B-cell activation and co-stimulation
Recipient
APC
C
MHC II
TReg cells
Autoreactive
T cell
+
Th2
cytokines
(IL-4, IL-10)
CD4+
T cell
T-cell and B-cell expansion and autoantibody production
Antigen-stimulated
clonal T-cell expansion
Epitope spreading
CD4+ or
CD8+ T cells
B-cell clonal
expansion
Autoantibodies
D
Trafficking
Skin, gut, liver, lung, salivary glands, oral mucosa
Autoantibodies
B cell
d and lymph
Blood
Autoreactive
T cell
Autoactivated
T cell
Target organs
E
Tissue injury, inflammation and fibrosis
Autoreactive
T cell
Cytokines
(IL-13,
IL-4, IL-10)
Antigen
recognition
Cytokine
receptor
Fibroblasts
+
Direct
cytotoxicity
TGFβ
Collagen
Macrophage
Host cell
+
B cell
Autoantibodies
PDGF
receptor
Fig. 2. Steps to cGvHD in mice. The progression of events occurring in the
development of cGvHD in the mouse is illustrated. The five steps illustrate
immune priming (A), activation of T and B cells (B), T-cell expansion and B-cell
autoantibody production (C), trafficking to sites of tissue damage (D), and end
organ damage through chronic inflammation and fibrosis (E). See main text
for full details.
Disease Models & Mechanisms
fibrosis and chronic inflammation. Additionally, bone-marrowderived T cells inappropriately selected in a dysfunctional thymus,
or through dysfunctional peripheral tolerance mechanisms, traffic
to target organs, can be activated by self antigens and perpetuate
the Th2 response. Finally, tissue injury results from activated T
and B cells that release inflammatory and fibrosing cytokines, such
as IL-13, IL-10 and IL-4, and activated macrophages producing
transforming growth factor- (TGF) stimulate collagen
production from fibroblasts, collectively leading to a sclerodermalike syndrome. Autoantibodies against double-stranded DNA,
single-stranded DNA and chromatin can be deposited at the site
of target organs, leading to immune-complex glomerulonephritis
or causing activation of the platelet derived growth factor receptor,
leading to fibrosis (Svegliati et al., 2007).
Most mouse models of aGvHD (summarized in Table 1) involve
the transplantation of T-cell-depleted bone marrow supplemented
with varying numbers and phenotypic classes of donor lymphocytes
(either splenocytes or lymph node T cells) into lethally irradiated
recipients. The bone marrow provides donor stem cells that allow
hematopoietic reconstitution after transplant; T-cell depletion is
carried out to control the dose and type of immune cells that are
delivered.
The discovery of the MHC and minor histocompatibility antigens
(miHAs) has been integral to advancing the field of HSCT.
Differences in MHC antigens are largely responsible for immunemediated rejection of foreign tissues and cells; however, even in an
MHC-matched setting, differences in miHAs can cause grafts to
be slowly rejected. Unlike humans, inbred mouse strains are closely
related with respect to MHC antigens (note that class I MHC is
referred to as H2 in mice). Although two strains of mice might
share the same H2 (i.e. they are MHC-I-matched), differences in
miHAs can result in GvHD in the context of experimental HSCT.
MHC-mismatched and miHA-mismatched models are discussed
below. In addition, we describe more recently developed models
involving the xenotransplantation of human cells into
immunodeficient mice.
The severity of aGvHD depends on several factors. First, the dose
and type of T-cell subsets (i.e. CD4+, CD8+ or TReg cells) that are
transferred with donor bone marrow can affect the severity of the
disease (Sprent et al., 1988; Korngold, 1992; Edinger et al., 2003).
Second, irradiation dose is proportional to the degree of tissue
damage and the subsequent cytokine storm, and thus is directly
proportional to aGvHD-related mortality in the mouse (Hill et al.,
1997; Schwarte and Hoffmann, 2005; Schwarte et al., 2007). In
addition, the dose of irradiation that is myeloablative varies by
mouse strain: inbred strains are generally more susceptible to
radiation than their F1 progeny, and different strains show
susceptibility differences (e.g. BALB/c>C3H>C57Bl/6). Third,
genetic disparities play a major role in disease. For example, even
mice that are MHC matched can vary by miHAs that influence
aGvHD severity. Fourth, variation in environmental pathogens
between labs and in mice from different suppliers can affect GvHD
(Nestel et al., 1992). Therefore, the same model of aGvHD can vary
between investigators because of differences in radiation dose and
delivery rate, differences in pathogens between animal facilities,
and differences in T-cell dose and sources of T cells.
321
322
C57/Bl6 (H2b)
B6C3F1 (H2k/b)
B6D2F1 (H2b/d)
B6AF1 (H2b/a)
B10.BR (H2k)
B6.C-H2bm1 (bm1)
(H2b background with
mutation at MHC I)
B6.C-H2bm12 (bm12)
(H2b background with
mutation at MHC II)
C3H/HeJ (H2k)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
DBA/2 (H2d)
B10.D2 (H2d)
Table 1 continued on next page.
CBA or BALB.K (H2k)
B10.BR (H2k)
miHA mismatched
BALB/c (H2d)
Recipient strain
C57/Bl6 (H2b)
Donor strain
MHC mismatched
Table 1. Mouse models of aGvHD
800 cGy
750 cGy
950 cGy or sublethal
XRT
950 cGy or sublethal
XRT
750-900 cGy
+/– Cytoxan
120 mg/kg
No XRT
No XRT or 1100-1300
cGy
600-1050 cGy
900 cGy
900 cGy
Conditioning
regimen
miHA mismatches
miHA mismatches
MHC II mismatch
MHC I mismatch
Mismatched for
MHC I, MHC II and
miHAs
Mismatched for
MHC I, MHC II and
miHAs
Mismatched for
MHC I, MHC II and
miHAs
Mismatched for
MHCI, MHCII and
miHAs
Mismatched for
MHCI, MHCII and
miHAs
Mismatched for
MHCI, MHCII and
miHAs
Genetics
Systemic disease; lethal
70 106 spleen cells
and 1 107 TCD BM
4 106 TCD BM cells
Systemic disease; lethal
and 1 106 T cells, or
by day 80
purified CD4+ or
CD8+ T cells
CD4+ (minor
contribution by
CD8+)
Systemic disease if CD4+
T cells infused with
death by 20-40 days
4 106 TCD BM and 15 106 CD4+ T cells
8 106 TCD BM cells
Systemic disease;
and 1 107 whole
symptoms include tail
splenocytes or
scaling, ear erosions,
5 106 CD4+ or CD8+
diarrhea, hunching
T cells
Systemic disease if CD8+
T cells infused with
death by 30-80 days
Korngold and Sprent, 1987;
Korngold, 1992
Korngold and Sprent, 1987;
Korngold, 1992; Cooke et
al., 1996; Kaplan et al.,
2004; Fanning et al., 2009
Rolink et al., 1983; Sprent
et al., 1986
Rolink et al., 1983; Sprent
et al., 1986
Vallera et al., 1981; Blazar
et al., 1991; PanoskaltsisMortari et al., 1998
2.5 105 to 2 107 TCD Systemic disease; lethal
BM cells and 5depending on T-cell
25 106 spleen cells
dose and source;
or 0.5-1 106 lymph
symptoms by 14-28
node cells
days
4 106 TCD BM cells
and 1.5–7.5 106
CD8+ T cells
Ghayur et al., 1988; Nestel
et al., 1992; Gendelman
et al., 2004
Pickel and Hoffmann, 1977;
Via et al., 1996a; Krenger
et al., 2000a; Hildebrandt
et al., 2004; Blaser et al.,
2005; Niculescu et al.,
2005
Kanamaru et al., 1984;
Fowler et al., 1996
Blazar et al., 1991
van Leeuwen et al., 2002
Reference example(s)
Systemic disease; lethal
depending on T-cell
dose
Whole lymphoid cells
(2-6 107)
Systemic disease; lethal
by 30 days if XRT used;
symptoms in 30-50
days if no XRT
Systemic disease by 1030 days; lethal
2 107 BM cells and 525 106 spleen cells
or 0.1-1 106 lymph
node cells
Whole splenocytes: 110 107 cells if no
XRT;
2-5 106 purified T
cells with XRT
Systemic disease by 1021 days; lethal
Outcome
Splenic T cells (0.52 106) and TCD
donor BM cells
Cell type and dose
CD8+
CD4+
CD8+
CD4+ and CD8+
CD4+ and CD8+
CD4+ and CD8+
CD4+ and CD8+
CD4+ and CD8+
CD4+ and CD8+
Main T-cell type
contributing to
phenotype
Disease Models & Mechanisms DMM
PERSPECTIVE
Mouse models of GvHD
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Disease Models & Mechanisms
BALB.b (H2b)
BALB.b (H2 b)
B10.D2 (H2d)
B10 (H2b)
C57/Bl6 (H2b)
DBA/2 (H2d)
NOD/SCID 2m-null
Human peripheral
blood T cells:
naive or
CD3/CD28 bead
expanded
Mismatched for
MHC I, MHC II and
miHAs
Mismatched for
MHC I, MHC II and
miHAs
Mistmatched for
MHC I, MHC II and
miHAs
miHA mismatches
miHA mismatches
miHA mismatches
miHA mismatches
Genetics
BM, bone marrow; cGy, centigray; RO, retro-orbital injection; TCD, T-cell depleted; XRT, radiation conditioning.
250-300 cGy
350 cGy
BALB/cA-RAG2–/– IL2r
Human PBMCs
–/–
No XRT or
200-250 cGy
NOD/SCID IL2rγ-null
(NSG)
820 cGy
850–1000 cGy
775 cGy
Sub-lethal 600-750
cGy or
Fractionated 1000
cGy
Conditioning
regimen
Human PBMCs
Xenotransplantation models
BALB/c (H2d)
Recipient strain
B10.D2 (H2d)
Donor strain
miHA mismatched
Table 1. Continued
–
CD4+ and CD8+
CD4+
CD8+
Berger et al., 1994; Zhang
et al., 2005
Kaplan et al., 2004
Korngold and Sprent, 1987;
Eyrich et al., 2005
Reference example(s)
Systemic disease
Systemic disease
1 107 human T cells
RO injection
Death from GvHD in 3050 days
5-30 106
1 107 or
5-10 106
Nervi et al., 2007
van Rijn et al., 2003
Ito et al., 2009; King et al.,
2009
4 106 TCD BM cells
Minimal systemic disease Korngold and Sprent, 1987
and 1 106 T cells, or
with whole spleen cells
but increased with
purified CD4+ or
+
purified CD8+ T cells;
CD8 T cells
up to 50% mortality at
day 25
Systemic disease
5 106 TCD BM cells
and 2 106 T cells
CD4+
Systemic disease; no
cutaneous
involvement
8 106 TCD BM cells
and 107
unfractionated
spleen cells or
1.4 106 CD4+ or
6 105 CD8+ T cells
CD4+ (with some
contribution from
CD8+)
Systemic disease
Outcome
1 107 BM cells and
1 108 whole
splenocytes
Cell type and dose
CD4 (minor
contribution by
CD8+)
Main T-cell type
contributing to
phenotype
Disease Models & Mechanisms DMM
Mouse models of GvHD
PERSPECTIVE
323
PERSPECTIVE
Disease Models & Mechanisms DMM
MHC-mismatched models
The most commonly studied MHC-mismatched model of aGvHD
is C57/Bl6 (H2b) r BALB/c (H2d) (transplantation of cellular
isolates from C57/Bl6 (H2b) donors into BALB/c (H2d) recipients).
MHC-mismatched models of aGvHD also contain a number of
miHA mismatches, although the impact of miHA mismatches on
the pathophysiology of the clinical phenotype observed is limited.
All models incorporate myeloablative radiation conditioning either
as a single dose or, when radiation doses of more than 1100 cGy
are delivered, as a fractionated dose separated by 3-8 hours, which
decreases gut toxicity. Most MHC-mismatched models are
dependent on both CD8+ and CD4+ T cells. To dissect the
mechanisms by which these T-cell subsets induce aGvHD,
transgenic mice that have a mutant MHC I (which is involved in
CD8+ T-cell activation) [B6.C-H2bm1 (bm1)] or MHC II (which is
involved in CD4+ T-cell activation) [B6.C-H2bm12 (bm12)] have
been used. The H2bm1 and H2bm12 transgenic mouse models have
been important in dissecting the interaction of T cells with
recipient and donor APCs (Sprent et al., 1990). CD8+ T cells induce
aGvHD by engagement of the TCR and release of perforin,
granzymes and Fas ligand (Via et al., 1996b; Graubert et al., 1997;
Maeda et al., 2005). Only recipient APCs can stimulate CD8+ T
cells in CD8+ T-cell-dependent models (Shlomchik et al., 1999).
Conversely, either recipient or donor APCs can stimulate CD4+
T-cell responses (Anderson et al., 2005). TNF mediates the
cytotoxic effects of alloreactive CD4+ T cells. The target of these
cytotoxic T-cell responses is recipient epithelium, although the
epithelium is not required to present antigen to T cells (Teshima
et al., 2002).
Another MHC-mismatched model of aGvHD is the ‘parent-toF1’ model. Parent-to-F1 transplants have been studied using either
lethal irradiation regimens or no irradiation conditioning. The latter
model allows the study of aGvHD without the effects of radiation,
thus mimicking the situation in humans that undergo nonmyeloablative or ‘reduced intensity conditioning’ regimens. The
cells transplanted into these models are either whole splenocytes
(in the non-irradiated models) or purified T cells with T-celldepleted bone marrow (in the irradiated models). The development
of aGvHD in these models depends on differences in class I and
class II MHC antigens, and involves the effects of CD4+ and CD8+
T cells (Hakim et al., 1991). The characteristics of aGvHD differ
in the irradiated and non-irradiated parent-to-F1 model: whereas
the irradiated model exhibits weight loss and skin problems, the
non-irradiated model has relatively limited skin pathology or
weight loss, and aGvHD mainly manifests as immune deficiency
and mixed chimerism, with lower mortality. Importantly, not all
parental donor strains cause aGvHD. Some can cause cGvHD (e.g.
DBA/2 r B6D2F1), possibly because of the natural bias of some
strains to mount either a Th1- or Th2-cell response (De Wit et al.,
1993; Nikolic et al., 2000; Tawara et al., 2008).
miHA-mismatched models
miHA-mismatched models of aGvHD represent human HSCT
more closely than MHC-mismatched models, because MHCmismatched transplants in humans are not commonly performed.
Similar to the human setting, miHA-mismatched models exhibit
less morbidity than MHC-mismatched models, but still result in
lethal aGvHD. An example of a miHA-mismatched transplant
324
Mouse models of GvHD
model is B10.D2 (H2d) r BALB/c or DBA/2 (H2d). The aGvHD
that occurs in this model is primarily dependent on CD4+ T cells
and less on CD8+ T cells (Korngold and Sprent, 1987). However,
transplants between congenic strains illustrate that differences in
MHC (which means that alloantigens are presented differently in
vivo) influence the type and specificity of the T-cell response
underlying the GvHD phenotype (Kaplan et al., 2004). For example,
in other miHA-mismatched transplant models, such as C3H.SW
r Bl6 (H2b) and DBA/2 r B10.D2 (H2d), GvHD depends primarily
on CD8+ T cells.
Xenogeneic transplant models
These models have been developed to generate a system whereby
human T-cell-mediated aGvHD can be studied and manipulated
in vivo. However, transplanting across species (xenotransplantation)
requires significant immunosuppression to prevent graft rejection,
because the immune response to xenografts is typically much more
robust than to allografts. Initial attempts to xenotransplant human
cells into mice were conducted using non-obese diabetic severe
combined immunodeficiency (NOD/SCID) mice and resulted in a
1-20% rate of engraftment (Mosier et al., 1988; Hoffmann-Fezer et
al., 1993; Christianson et al., 1997). Low engraftment was due to
the fact that NOD/SCID mice lack functional T and B cells but
retain NK cell function and can therefore respond to foreign
antigens. Subsequently, better engraftment of xenotransplanted
cells was obtained using RAG2-deficient and IL-2-receptor-deficient mice (BALB/cA-RAG2–/– IL2r–/–), which lack functional
T, B and NK cells (van Rijn et al., 2003). Our group has used the
NOD/SCID 2-microglobulin-null mouse (Christianson et al.,
1997; Nervi et al., 2007) to image the trafficking of xenotransplanted
human T cells in vivo. Using this system, we demonstrated that
retro-orbital injection of human T cells into sublethally irradiated
NOD/SCID 2m-null recipients resulted in consistent engraftment
and expansion of human T cells, the trafficking of T cells to target
organs including the lung, skin, liver and kidney, and clinical
symptoms consistent with severe aGvHD. T cells could be detected
1 hour after retro-orbital injection and trafficked to secondary
lymphoid organs, resulting in lethal GvHD; by contrast, intravenous
injection of T cells caused them to traffic first to the lungs, and
was associated with the development of less severe aGvHD and
less morbidity and mortality (Nervi et al., 2007).
The most permissive mouse model for human stem-cell and Tcell engraftment was derived by back-crossing mice carrying a
homozygous deletion of the common -chain of the IL-2 receptor
onto a NOD/SCID background [referred to as NOD/SCID IL2rnull (NSG) mice]. NSG mice lack T, B and NK cells, and also have
reduced function of macrophages and dendritic cells.
Transplantation of human peripheral blood mononuclear cells
(PBMCs) causes an aGvHD-like syndrome that results in death by
20-50 days, and is manifested by overt human T-cell infiltration of
mouse skin, liver, intestine, lungs and kidneys (Ito et al., 2009).
Xenogeneic transplants of human PBMCs into immunodeficient
mice require human APCs to process mouse antigens and present
them in the presence of class II MHC. Because T-cell recognition
of MHC molecules is restricted by species, human TCRs do not
recognize mouse MHC, making this a primarily CD4+ T-celldependent model, at least in the case of NOD/SCID (Lucas et al.,
1990).
dmm.biologists.org
PERSPECTIVE
Mouse models of GvHD
Disease Models & Mechanisms DMM
Antigen-specific transgenic TCR models
A major limitation of all MHC- and miHA-mismatched transplant
models of aGvHD is that it is difficult to determine the specific
alloantigen that the responding T cells recognize. In addition, the
cytokine storm induced by conditioning can lead to antigenindependent bystander activation of T cells. One solution to this
problem has been to use mouse strains that have T cells expressing
TCRs of defined specificity (TCR-transgenic mice); these strains
were generated initially to study thymic selection, but have more
recently been useful for understanding T-cell alloresponses in
GvHD. The specificity of the T cells in a TCR-transgenic mouse
is generally restricted to a single peptide epitope that is either
constitutively expressed by the transplanted allogeneic APCs or
is administered as an exogenous antigen to stimulate a T-cell
response. Examples of TCR-transgenic mice include H-Y, TEa,
2C, TS1, D011.10 and OT-II (Kisielow et al., 1988; Sha et al., 1988;
Murphy et al., 1990; Grubin et al., 1997; Barnden et al., 1998;
Wilkinson et al., 2009). These mice have been used to investigate
several aspects of GvHD, including the role of antigen affinity
(Yu et al., 2006), antigen-induced T-cell activation and
proliferation, TReg cell function (Albert et al., 2005; Damdinsuren
et al., 2010) and, more recently, cross presentation of antigens
(Wang et al., 2009).
resulting in the sclerodermatous changes (Hamilton, 1987;
Korngold and Sprent, 1987). In addition, other cellular mediators
have also been implicated: macrophages are responsible for TGF
production, whereas other cell types such as eosinophils and mast
cells have been implicated in the pathogenesis of fibrosis in target
organs. Mediators secreted by eosinophils and mast cells can cause
fibroblast proliferation and collagen production (Levi-Schaffer and
Weg, 1997). Two examples of sclerodermatous models are B10.D2
r BALB/c and LP/J r Bl6, both of which require total body
irradiation conditioning (Jaffee and Claman, 1983; DeClerck et al.,
1986). The dose of radiation can also affect the severity of disease
(Jaffee and Claman, 1983).
The sclerodermatous cGvHD models provide important
insights into the pathogenesis of fibrosis that occurs in cGvHD;
however, there are a number of limitations of these models. First,
the fibrosis in human cGvHD can be systemic or pleiotropic,
unlike mouse models, which mainly involve the skin, suggesting
other mechanisms might be at play in the development of systemic
sclerosis. Second, the time course of development of skin changes
is less rapid in humans than in mouse models. Finally, these mouse
models fail to reproduce the full clinical spectrum seen in
humans, which can involve target organs including the intestine,
lungs, liver, salivary glands, eyes, oral mucosa, muscles, tendons
and nerves.
Mouse models of cGvHD
Although the clinical phenotype and kinetics of cGvHD are distinct
from aGvHD, with fibrosis and chronic inflammation
predominating in a manner resembling autoimmune disease, most
of the same organs are affected. There are five factors that can
contribute to the pathology of cGvHD in humans and mouse
models after HSCT: cytokine-mediated fibrosis, defective negative
selection of T cells, deficiency of TReg cells, genetic polymorphisms
and dysregulated B-cell responses. However, no mouse model
developed thus far recapitulates all of these characteristics or exactly
mimics what is seen in human cGvHD.
Current mouse models of cGvHD (summarized in Table 2) can
be broadly divided into sclerodermatous (pro-fibrotic) models,
autoantibody-mediated (lupus-like) models and a more recently
reported model in which thymic function is defective (Sakoda et
al., 2007; Chu and Gress, 2008). The distinctions in the phenotypes
of models of cGvHD and aGvHD result from differences in the
factors underlying the pathogenesis of each model.
Sclerodermatous models
Sclerodermatous (pro-fibrotic) cGvHD models are characterized
by fibrotic changes in the dermis, which can involve the lung, liver
and salivary glands (Jaffee and Claman, 1983; Claman et al., 1985;
Howell et al., 1989; McCormick et al., 1999; Levy et al., 2000).
Transplantation results in full donor chimerism, and the resulting
phenotype resembles scleroderma in humans, which occurs in up
to 15% of patients with cGvHD (Skert et al., 2006). However, fibrosis
in the mouse models begins within 30 days of transplantation,
which is atypical of human cGvHD, which occurs months to years
after transplantation.
These models are MHC matched but differ in numerous miHAs.
The pathology is dependent on CD4+ T cells that release Th2
cytokines, which can stimulate other cells to release fibrosing
cytokines (such as IL-13 and TGF) (for a review, see Wynn, 2004),
Disease Models & Mechanisms
Autoantibody-mediated models
Autoantibody-mediated (lupus-like) cGvHD models, which exhibit
a pathology similar to that of lupus, are manifested by nephritis,
biliary cirrhosis, salivary gland fibrosis, lymphadenopathy,
splenomegaly, autoantibody production and, to a lesser extent, skin
pathology. These models are MHC mismatched and classically
involve parent-to-F1 transplants that result in mixed chimerism.
The most commonly studied autoantibody model is the DBA/2 r
B6D2F1 model. This model results in expansion of recipient B cells,
leading to lymphadenopathy, splenomegally and autoantibody
production. Both MHC I and II are mismatched in these models,
but the pathology is dependent on donor CD4+ T cells (Rolink and
Gleichmann, 1983; Rolink et al., 1983; Hakim et al., 1991) and is
associated with the production of Th2 cytokines. Manipulating the
cytokine balance by skewing the CD4+ T-cell response to a Th1
phenotype, by exposure to IL-12, causes a shift to a more aGvHDlike phenotype (Via et al., 1994). Importantly, the choice of parental
donor determines the phenotypic outcome. For example, if C57/Bl6
is the T-cell donor for a B6D2F1 recipient, an aGvHD phenotype
results. This might be due to naturally high proportions of cytotoxic
CD8+ T cells in the C57/Bl6 mouse strain (Via et al., 1987).
Unlike models of aGvHD, which depend primarily on donor T
cells, pathology in autoantibody models of cGvHD depends on
recipient B cells (Morris et al., 1990a). Specifically, the recipient B
cells interact with donor T cells (Morris et al., 1990b) via cell-surface
MHC and are also stimulated by cytokines, including IL-4 and IL10 (De Wit et al., 1993; Durie et al., 1994; Via et al., 1996a; Kim et
al., 2008). Recipient B cells act as potent APCs that stimulate donor
T cells, perpetuating the cycle that leads to autoantibody
production.
Mouse autoantibody cGvHD models are similar to cGvHD that
occurs in humans in that they also involve the production of
autoantibodies, although there are a number of distinctions. First,
325
326
Recipient strain
C57/Bl6 (H2b)
BALB/c Rag2–/– (H2d )
LP/J (H2b)
B10.D2
(H2d)
None
900-1100 cGy
700-900 cGy
Conditioning
regimen
MHC I
mismatched
MHC II
mismatched
650 cGy
No
conditioning
None or 900
cGy
BALB/c (H2d)
(C57/Bl6 B6.CH2bm1)F1 (H2b
background with
mutation at MHC
I)
(C57/Bl6 B6.CH2bm12)F1
(H2b background
with mutation at
MHC II)
DBA/2 (H2d)
C57/Bl6 (H2b)
C57/Bl6 (H2b)
C3H/HeN (H2k)
Mismatched for
MHC I, MHC II
and miHAs
BM-derived CD4+
and CD8+
CD4+
CD8+
–
CD4+
CD4+
CD4+
Abbreviations as for Table 1, plus: B6, C57/Bl6; B6D2F1, (C57/Bl6 x DBA/2)F1; ND, not determined.
Bl6-H2-Abl –/–
(H2b)
Thymic dysfunction model
1300 cGy split
in two
fractions
miHA mismatches
No
conditioning
(B6xBALB/c)F1
(H2b/d)
C57/Bl6 (H2b)
Mismatched for
MHC I, MHC II
and miHAs
Mismatched for
MHC I, MHC II
and miHAs
No
conditioning
(BALB/cxA)F1 (H2d/a)
BALB/c (H2d)
Mismatched for
MHC I, MHC II
and miHAs
B6D2F1 (H2b/d)
DBA/2 (H2d)
ND
ND
Mismatched for
miHAs and MHC
H2d matched but
miHA
mismatched
CD4+
Main T-cell type
contributing to
phenotype
miHA mismatched
Genetics
No
conditioning
Autoantibody-mediated (lupus-like) models
BALB/c (H2d)
B10.D2
(H2d)
Scleroderma (pro-fibrotic models)
Donor
strain
Table 2. Mouse models of cGVHD
5 106 TCD BM cells
No conditioning:
4-5 106 spleen CD4+ T cells, 23 106 BM cells;
2 doses of cells on day 0 and 7
Conditioned:
4-5 106 CD4+ T cells and 1-2 106
TCD BM cells
Epidermal atrophy, fat loss,
follicular drop out and
dermal thickening; bile
duct loss and periportal
fibrosis in liver; salivary
gland fibrosis and
atrophy; cytopenias
Systemic disease and death
by 20-40 days
Glomerulonephritis; nonlethal in unirradiated
mice; lethal in irradiated
mice at 30-60 days
5 107 spleen cells or 108 spleen
(2/3) and lymph node (1/3) cells;
2 doses of cells on day 0 and 7
Sakoda et al., 2007
Rolink et al., 1983; Ito et al., 1992;
Brown et al., 2002
Rolink et al., 1983; Ito et al., 1992
Zhang et al., 2006
Autoantibodies; skin
scleroderma
5 107 whole spleen cells
Pals et al., 1985; Vidal et al., 1996
Via et al., 1987; Fujiwara et al., 1991;
De Wit et al., 1993; Via et al.,
1996a; Slayback et al., 2000; Kim
et al., 2006; Kim et al., 2008
Tschetter et al., 2000
Polyarthritis, scleroderma,
glomerulonephritis,
Sjorgren’s, sclorosing
cholangitis
Lupus
Sjorgren’s (Sialoadenitis);
autoantibodies
Ruzek et al., 2004
Hamilton and Parkman, 1983;
DeClerck et al., 1986
Korngold and Sprent, 1978; Jaffee
and Claman, 1983; Claman et al.,
1985; Hamilton, 1987; Korngold
and Sprent, 1987; McCormick et
al., 1999; Levy et al., 2000; Kaplan
et al., 2004; Zhou et al., 2007
Reference example(s)
Acute progressing to
chronic GvHD
6 107 whole spleen cells
8-12 107 spleen cells
1-10 107
Scleroderma; progressive
fibrosis of organs;
autoantibodies
Scleroderma occurring by
day 28
20-50 106 spleen cells and 5 107
BM
Whole spleen cells
(2-5 107)
Scleroderma; progressive
fibrosis of organs
Outcome
TCD BM cells and 1 107 whole
splenocytes or purified T cells
Cells and dose
Disease Models & Mechanisms DMM
PERSPECTIVE
Mouse models of GvHD
dmm.biologists.org
Mouse models of GvHD
nephritis in cGvHD in humans is rare. Second, the repertoire of
autoantibodies associated with cGvHD is more diverse in humans
than in mice. Finally, lymphadenopathy and splenomegaly do not
occur in the human setting.
Disease Models & Mechanisms DMM
A cGvHD model involving both autoantibodies and
scleroderma
To more accurately model cGvHD that occurs in humans, attempts
have been made to combine the features of autoantibody-mediated
and scleroderma models in a single mouse model. For example,
transfer of splenocytes from DBA/2 (H2d) mice into sublethally
irradiated BALB/c (H2d) recipients results in a lupus-like and
sclerodermatous phenotype (Zhang et al., 2006). These mice
develop proteinuria with basement membrane immune complex
deposits, and skin fibrosis with collagen I deposition; the severity
of these disorders depends on the number of donor cells
transplanted. In addition, these mice have increased levels of
autoantibodies specific for double-stranded DNA. In this case,
donor CD4+ T cells are required for the pathology, whereas donor
CD8+ T cells do not contribute. Unlike pathology in the parent-toF1 autoantibody-mediated model, which involves recipient B cells,
pathology in this model involves donor B cells. Finally, the transfer
of TReg cells rescues the cGvHD phenotype, indicating that TReg
cells are functional in treating cGvHD, as has been demonstrated
in aGvHD models. In summary, this model indicates the
importance of donor CD4+ T cells as well as B cells (from both
donor and recipient) in the development of cGvHD. However, it
does not support a role for the thymus in the pathophysiology of
cGvHD, in contrast to the model discussed below.
PERSPECTIVE
periportal fibrosis in the liver; salivary gland fibrosis and atrophy;
and cytopenias (Sakoda et al., 2007). This phenotype developed
within 6 weeks of donor cell transfer, and fewer than 20% of animals
lived for more than 100 days. Importantly, thymectomized recipient
mice abrogated the phenotype, highlighting the role of the thymus
in the pathology seen in this model. Experiments involving chimeric
mice showed that donor APCs were essential for the development
of the cGvHD phenotype in this model: donor APCs that trafficked
to the thymus after transplantation were principally responsible
for negative selection. In a series of adoptive transfer studies, it was
shown that CD4+ T cells isolated from C3H/HeN recipients of
MHC-II-deficient bone marrow that develop cGvHD caused
aGvHD when transferred to the C57/Bl6 donor strain, but caused
cGvHD when transferred to established chimeric mice created from
a C57/Bl6 r C3H/HeN transfer. This implies that donor APCs are
essential for the development of the cGvHD phenotype not only
through their role in negative selection but through peripheral
antigen presentation and T-cell stimulation.
Although the mouse model described above (H2-Ab1–/– r
C3H/HeN) is the first that reproduces multiple clinical features of
human cGvHD, it might not be clinically relevant. The model
cannot rule out the possibility that additional insult to the thymus
caused by GvHD might be the cause of dysfunctional negative
selection. It is also difficult to determine the exact kinetics of
cGvHD in this model, and whether there is a critical period of
thymic damage that is necessary to cause the resulting phenotype.
Nonetheless, this model suggests that a crucial component of
cGvHD is dysfunctional thymic selection, which may or may not
be caused by donor T-cell-induced damage of the recipient thymus
as a consequence of aGvHD.
Defective thymic function model
Given the similarity between the presentation of cGvHD and some
autoimmune diseases, as well as the fact that thymectomy of 3day-old mice results in symptoms similar to some aspects of cGvHD
(Nishizuka and Sakakura, 1969; Bonomo et al., 1995), it has long
been hypothesized that defects in thymic negative selection might
have a role in the pathogenesis of cGvHD. The thymus is a crucial
target of aGvHD (Fukushi et al., 1990; Krenger et al., 2000b; HauriHohl et al., 2007), and it is known that aGvHD results in destruction
of the normal thymic architecture (Ghayur et al., 1988), especially
Hassall’s corpuscles, which have been shown to be crucial for the
development of TReg cells (Watanabe et al., 2005). The presentation
of tissue-restricted antigens by thymic APCs and epithelial cells
could result in their direct attack by cytotoxic donor T cells.
Cytotoxic attack of these cell types, which are crucial for negative
selection, could result in impaired central tolerance and subsequent
autoreactivity (Krenger and Hollander, 2008; Mathis and Benoist,
2009).
A potential role for thymic damage in GvHD was initially
proposed by a group that demonstrated dysfunctional negative
selection of autoreactive T-cell clones in mice with aGvHD (van
den Brink et al., 2000). More recently, a transgenic mouse transplant
model was developed: T-cell-depleted bone marrow from MHCII-deficient mice on a C57/Bl6 background [H2-Ab1–/– (H2b)] were
transferred to lethally irradiated C3H/HeN (H2k) recipients.
Recipient mice developed a phenotype similar to clinical cGvHD,
with skin fibrosis characterized by epidermal atrophy, fat loss,
follicular drop out and dermal thickening; bile duct loss and
Disease Models & Mechanisms
Linking acute and chronic GvHD in a mouse model
The high incidence of cGvHD that occurs following aGvHD in
humans suggests that there is a direct causal link between these
two disease states. If the mechanisms underlying this link could be
identified, targeted interventions could be used to prevent the
development of autoimmune-like responses that characterize
cGvHD from the alloresponses that underlie aGvHD.
Zhang et al. attempted to link acute and chronic GvHD in a
mouse model (Zhang et al., 2007). Donor bone-marrow-derived
CD4+ T cells that arose from hematopoietic precursors following
transplantation and were selected (educated) in the recipient
thymus during the development of aGvHD could mediate a cGvHD
phenotype when transferred to secondary allogeneic recipients,
resulting in cGvHD affecting the skin, liver and gut. When
transferred to syngeneic secondary recipients, the same cells
resulted in a lethal aGvHD phenotype, suggesting that appropriate
positive selection had taken place in the recipient thymus but that
thymic negative selection was absent or dysfunctional. This
illustrates a possible link between the cytotoxic CD8+ T-cell
responses of aGvHD and the CD4+ T-cell-mediated responses
demonstrated in these models of cGvHD. The exact mechanism is
unknown, but these data suggest that the thymus-educated donor
CD4+ T cells that should be self-tolerant are autoreactive, suggesting
that the link between aGvHD and cGvHD is related to the targeting
to the thymus by alloreactive T cells (both CD4+ and CD8+) and
subsequent elimination or alteration of the function of thymic
cellular elements that are essential for appropriate negative
327
Disease Models & Mechanisms DMM
PERSPECTIVE
selection. The effects of these autoreactive T cells can only be
demonstrated by adoptive transfer experiments because of the
highly lethal nature of aGvHD models.
Another attempt to investigate the link between aGvHD and
cGvHD was performed using an F1 donor and F1 recipient [(B10
⫻ B10.D2)F1 (H2b/d) r (BALB.b ⫻ BALB/c)F1 (H2b/d)] transplant:
this resulted in both systemic symptoms of aGvHD and skin
manifestations of cGvHD (Kaplan et al., 2004). In this model, MHC
was matched between donor and recipient, but miHAs were
mismatched. This same group investigated transplantation between
multiple strain combinations matched at MHC loci, and
demonstrated that certain MHC haplotypes were associated with
the development of GvHD involving specific target organs, which
they suspected was due to differences in MHC affinity for antigens
found in certain target tissues. For example, immunodominant
antigens in the skin might be bound with high affinity in only certain
MHC strains. This illustrates the important role of MHC in
selecting immunodominant antigens, and how this might influence
cGvHD.
A final example of the link between alloreactive T-cell responses
of aGvHD and the autoimmune responses of cGvHD involved the
use of immunodeficient mice (Tivol et al., 2005; Chen et al., 2007).
In this case, aGvHD was initiated through a C57/Bl6 r BALB/c
lethally irradiated MHC-mismatched transplant. After 20 days,
recipient animals were sacrificed and their splenic T cells were
isolated. Transfer of these isolated T cells into unconditioned
immunodeficient (Rag–/–) C57/Bl6 recipients (i.e. the same
background as the original donor) resulted in inflammatory
infiltration of the colon and liver (Chen et al., 2007). This
autoimmune phenotype was dependent on CD4+ T cells (with a
Th1 and Th17 cytokine profile) and could be rescued by the
infusion of immunosuppressive donor TReg cells. This supports the
data of Zhang et al. described above that a CD4+ T cell – derived
either centrally (through the thymus) or peripherally (from the
initial donor T-cell inoculum) – in the context of aGvHD can
stimulate the autoimmune responses observed in cGvHD. In fact,
recent evidence suggests that mature donor-derived CD4+ T cells
have the ability to cause both alloreactive and autoreactive
responses (Zhao et al., 2010). In a series of adoptive transfer
experiments using the DBA/2 r BALB/c cGvHD model, Zhao et
al. characterized the CD4+ T cells down to single cells, and showed
that a specific TCR predominated in mice with cGvHD. These T
cells recognized antigen in the presence of either allogeneic or
autologous dendritic cells.
Considerations when selecting a mouse model of GvHD
With the large number of GvHD models now available, it can be
difficult to determine which model is appropriate for the research
question at hand. We offer in this section a few suggestions.
(1) First, decide whether you are interested in studying acute or
chronic GvHD. It is of course useful to first establish from published
literature which models have been successfully validated for testing
a given intervention.
(2) Next, determine what mechanism or T-cell population you
want to be dominant in your model for testing a specific therapeutic
intervention. For example, a drug that targets CD8+ T cells would
not be effective in a model in which CD4+ T cells mediate
pathology. Conversely, if limiting the degree of cytokine storm
328
Mouse models of GvHD
produced from conditioning is important, then miHA-mismatched
or TCR-transgenic models would be useful, because conditioning
can be reduced or eliminated.
(3) Next, consider measurement outcomes such as: severity, coat
color and the ability to track specific cell populations. If easy clinical
scoring of GvHD severity and survival are the main outcomes
measured, then a model that has consistent rapid kinetics and 100%
penetrance is desirable. In this case, the C57/Bl6 r BALB/c
transplant model could be a good choice. In general, the less the
degree of MHC mismatch, the less severe the model. If
bioluminescence imaging (BLI) will be used (see Box 2), consider
the coat color of the mouse that will be imaged, because imaging
is more difficult with mice that have dark coats (which require
shaving prior to BLI). If the tracking of donor cells in the recipient
is necessary, it is possible to use flow cytometry to track MHC
disparity, to track fluorescent donor cells using BLI, or to use
congenic mice that have allelic differences at the CD45 locus (e.g.
CD45.1 or CD45.2) to track cellular subsets from donor versus
recipient when they have a common MHC.
(4) It is also necessary to consider more complex issues such as
the production of transgenic strains. Sophisticated modeling can
allow for elegant mechanistic studies, but if you are going to use a
transgenic strain as a donor or recipient, extensive backcrossing to
a single background is essential to eliminate significant genetic
disparity that could affect results.
(5) Finally, it is crucial to personally validate all new models in
your lab. Even a basic model (such as Bl6 r BALB/c) and certainly
any novel strain combinations require careful validation to
investigate the influence of various factors – including preconditioning radiation dose, mouse strain and cleanliness of
colonies (which varies between labs) – on, for example, disease
incidence, severity and pathologic evidence of GvHD.
When working with any mouse model of GvHD, there are a
number of caveats to be aware of when interpreting and translating
experimental data, which are outlined in Box 3.
Measuring the GVL effect in GvHD models
The graft-versus-leukemia (GVL) effect, also referred to as graftversus-tumor effect, describes the alloimmune responses of donor
immune cells to residual leukemia or tumor, which are indications
for which HSCT is frequently performed as a treatment. Although
a complete review of models of GVL is beyond the scope of this
article [the reader is instead referred to reviews on the mechanisms
Box 2. Imaging pathological events during GvHD
Bioluminescent imaging (BLI) has become an important tool for tracking
transplanted cells in vivo. This technique has been reviewed recently (Negrin
and Contag, 2006). Briefly, this technique uses donor mice that are transgenic
for a luciferase-GFP (green fluorescent protein) fusion protein, which is under
the control of the constitutively active chicken -actin promoter and
cytomegalovirus enhancer [L2G85 mice (FVB background, H2q)]. The activity of
the luciferase enzyme results in light emission that can be detected in vivo in
recipient mice using a specialized device. This technique is sensitive for
visualizing 100-1000 cells per region. It has been used to study the in vivo
trafficking and proliferation of T cells (Beilhack et al., 2005), TReg cells (Edinger
et al., 2003) and other T-cell subsets, including Th17 cells (Iclozan et al., 2009).
It can also be used to measure the GVL effect by tracking the expansion or
elimination of luciferase-expressing tumor cells over time after HSCT
(Nishimura et al., 2008).
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PERSPECTIVE
Mouse models of GvHD
Disease Models & Mechanisms DMM
Box 3. Caveats and considerations when interpreting
findings from mouse models of GvHD and translating
them to the clinic
Differences in conditioning regimens
Pre-transplant conditioning in humans mainly consists of chemotherapy
regimens, given with or without radiation, which is delivered at a rate (typically
fractionated) that differs from that used in mouse models of GvHD (usually
single dose). Conditioning results in tissue damage and a proinflammatory
reaction that might differ between mice and humans and therefore influence
the GvHD phenotype. In addition, conditioning regimens and the timing of
transplantation can impact experimental outcomes (Gendelman et al., 2004;
Mabed et al., 2005; Schwarte and Hoffmann, 2005; Mapara et al., 2006). This
can be directly related to the residual host-versus-graft (HVG) effect, which can
decrease GvHD severity if the host immune system is not adequately
suppressed. The HVG reaction is a rare complication after transplant in humans
but increases as conditioning regimen intensity is decreased.
Genetic and immunological disparities between donor and recipient
In humans, HLA typing occurs via high-resolution DNA typing, and most
transplants are matched at the allele level with respect to MHC loci, although
they usually differ at the level of miHAs that lie outside of the MHC locus. By
contrast, many mouse models of GvHD involve mismatches in both MHC
antigens and miHAs. There are also key species differences between the
mouse and human immune system that can make the direct translation of
results difficult. [These have been reviewed elsewhere (Mestas and Hughes,
2004).] Some of these differences include: involution of the thymus in humans
but not mice; expression of MHC II by human but not mouse T cells; and
differences in the proportions of circulating lymphocytes and their effector
mechanisms in the two species. Furthermore, strain-specific disparities in the
proportion of lymphocyte subsets (such as CD4+, CD8+ and TReg cells)
influence the phenotype between different mouse models of GvHD. Finally,
recent reports suggest that MHC-matched mice can carry genetic
polymorphisms outside of the MHC locus that can affect the clinical
phenotype of GvHD, which might explain observed differences in results in
different labs, even when the same strain combinations are used (Cao et al.,
2003; Fanning et al., 2009).
Source of donor immune cells
Material for HSCT in humans is now most commonly derived from mobilized
stem cell products and contains donor immune cells from the circulation. The
number, origin and type of circulating immune cells can differ depending on
the mobilization regimen used. By contrast, immune cells delivered together
with transplants in mouse models are usually isolated from spleen or lymph
nodes. It is important to consider that immune-cell populations from different
sources might have different trafficking capacities and composition (i.e.
different proportions of T-cell subsets, dendritic cells and early myeloid
progenitors), and therefore have a different influence on the GvHD phenotype.
Differences in normal flora
Enteric pathogens can play a role in the kinetics and severity of GvHD, which
targets the gut in many cases. Microorganisms express molecules that can
activate the immune system and therefore can augment the severity of
disease (Cooke et al., 1998). In line with this, changes in the environment of
the mice, which in turn affect the composition of gut flora, have been shown
to affect disease severity (Bennett et al., 1998; Gerbitz et al., 2004; Gorski et al.,
2007). Given that the gut flora of a human is markedly different from that of a
mouse kept in a pathogen-free facility, this should be taken into account when
translating experimental data.
Age
HSCT is commonly carried out in adult humans, whereas mice used to study
GvHD are typically 8- to 14-weeks old (and have a 2-year lifespan). Given that
age can influence the efficiency of immune reconstitution after transplant, as
well as susceptibility to GvHD (Ordemann et al., 2002), this factor must be
taken into account when translating experimental results.
[Adapted, with permission, from Socié and Blazar (Socié and Blazar, 2009).]
Disease Models & Mechanisms
of GVL (Bleakley and Riddell, 2004; Kolb et al., 2004) as well a recent
historical perspective on GVL (Kolb, 2008)], it is worth mentioning
that separating GvHD from the GVL effect is a long-standing
dilemma in transplantation, and is still an area of active
investigation. In fact, separating the immunopathological aspects
of GvHD from the curative effect of GVL is perhaps the most
clinically meaningful endpoint of HSCT. However, this is a
complicated issue because T cells mediate both GvHD and GVL.
Research in this area has focused on identifying the mechanisms
that might differentiate cytotoxic T-cell responses in GvHD from
cytotoxic T-cell responses in GVL, as well as identifying accessory
cells that could block GvHD while allowing or augmenting GVL.
The first evidence for GVL came from mouse transplant models
(Barnes and Loutit, 1957; Weiss et al., 1983). Initial studies in the
1950s demonstrated that a mouse leukemia could not be eliminated
by irradiation or by irradiation and syngeneic bone marrow, but
irradiation plus allogeneic bone marrow prolonged survival of mice
and eliminated leukemia from detection. However, transplanted
mice developed a wasting syndrome that later became known as
GvHD. Later efforts in the early 1980s were better able to define
the immunological basis for this GVL effect, and subsequent work
defined the importance of T cells and miHAs, as well as of innate
immune cells, such as NK cells, on the GVL effect. The models
discussed above can also be used to test GVL effects of allogeneic
cellular therapies, as well as to test treatments that are designed to
prevent GvHD while preserving the GVL effect.
It could be argued that any novel therapy designed in these
models to target GvHD should be assessed for its effect on GVL
because of the clinical implications for translation to humans. To
test the GVL effect, a labeled congenic tumor cell line (i.e.
genetically compatible with the recipient, such as the A20 cell line
for a BALB/c background) or primary tumor cells [such as mouse
acute promyelocytic leukemia cells from PML/RAR transgenic
mice (Bl6/129 background) (Westervelt et al., 2003)] can be
transduced to stably express a fluorescent marker or luciferase for
bioluminescent studies. After a lethal injection of tumor cells at
the time of allogeneic transplantation, mice that develop aGvHD
generally eliminate the genetically compatible tumor, owing to the
alloreactive response mounted by donor T cells. The ultimate goal
of interventions has been to prevent GvHD while preserving GVL.
There have been a number of recent articles demonstrating this
(Liang et al., 2008; Nishimura et al., 2008; Zheng et al., 2009). The
availability of BLI (Box 2) has improved the capacity to detect
residual leukemia after transplantation and is used as a measure
of response to treatments, together with survival and GvHD
scoring, in these models.
Conclusion
Mouse models of GvHD are crucial for advancing our
understanding of the disease in its acute and chronic forms. As our
understanding grows, it is hoped that targeted therapies will be
developed to treat GvHD and preserve GVL. Recent advances in
treatment that have evolved owing to studies of mouse models of
aGvHD include targeting mediators of the cytokine storm, such as
TNF and IL-1, with antagonists (Uberti et al., 2005; Alousi et al.,
2009; Antin et al., 2002), as well as reducing the intensity of
conditioning regimens to avoid excess inflammation. Other novel
interventions include the proteasome inhibitor bortezomib (Sun
329
Disease Models & Mechanisms DMM
PERSPECTIVE
et al., 2004; Sun et al., 2005), and the DNA methyltransferase
inhibitors decitabine and azacitidine (Choi et al., 2010). The exact
mechanism by which these agents work is not known, but they
might involve suppression of alloreactive T-cell responses and/or
an increase in TReg cell number or function following
transplantation. In addition, cellular therapies, such as depleting T
cells from the graft, or infusing TReg cells, have been developed
based on studies performed in the mouse. Finally, cGvHD models
have been instrumental in developing imatinib – a tyrosine kinase
inhibitor that targets PDGFR, which has been implicated in the
pathogenesis of fibrosis (McCormick et al., 1999; Daniels et al., 2004;
Abdollahi et al., 2005) – and rituximab, an antibody that targets
potential autoreactive B cells (Cutler et al., 2006). Areas that warrant
further study include the influence of reduced intensity
conditioning regimens (Sadeghi et al., 2008), in vivo imaging, and
the genomics and epigenomics of GvHD.
As discussed, advances in in vivo imaging techniques to monitor
trafficking of immune cells have recently been developed and will
be crucial for dissecting the temporal and spatial relationships of
donor and recipient T cells, B cells and APCs in these models
(Panoskaltsis-Mortari et al., 2004). Furthermore, recent advances
in proteomics, gene profiling and whole genome sequencing will
help to elucidate the genetic basis of GvHD, provide ways to better
match donor and recipient to prevent GvHD, and improve our
ability to predict the severity and kinetics of the disease (Baron et
al., 2007; Hori et al., 2008; Paczesny et al., 2009).
Despite the advances made through preclinical modeling of
GvHD in mice, many questions remain. Mouse models will
continue to allow for the dissection of mechanistic pathways of
GvHD through the use of informative genetic knock-out, knockin and knock-down experiments that are only possible in the mouse.
Fundamental research using mouse models of GvHD should
enhance our understanding of GvHD in humans and result in
improved and novel therapeutic approaches to minimize GvHD
while optimizing the GVL effect, as well as immune reconstitution,
following HSCT.
COMPETING INTERESTS
The authors have no relevant financial or competing interests to disclose.
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