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Advances in the understanding of acute graft-versus-host disease
Edward S. Morris1,2 and Geoffrey R. Hill2
1Department of Haematology, Royal Hallamshire Hospital, Sheffield, UK, and 2Bone Marrow Transplantation Laboratory, Queensland
Institute of Medical Research, Brisbane, Qld, Australia
Summary
Allogeneic stem cell transplantation (SCT) remains the defin-
itive immunotherapy for malignancy. However, morbidity and
mortality due to graft-vs.-host disease (GVHD) remains the
major barrier to its advancement. Emerging experimental data
highlights the immuno-modulatory roles of diverse cell
populations in GVHD, including regulatory T cells, natural
killer (NK) cells, NK T cells, cd T cells, and antigen presenting
cells (APC). Knowledge of the pathophysiology of GVHD has
driven the investigation of new rational strategies to both
prevent severe GVHD and treat steroid-refractory GVHD.
Novel cytokine inhibitors, immune-suppressant agents known
to preserve or even promote regulatory T-cell function and the
depletion of specific alloreactive T-cell sub-populations all
promise significant advances in the near future. As our
knowledge and therapeutic options expand, the ability to limit
GVHD whilst preserving anti-microbial and tumour responses
becomes a realistic prospect.
Keywords: graft-vs.-host disease, cytokines, regulatory T cells,
natural killer T cells, antigen presenting cells.
The pathophysiology of acute graft-vs.-hostdisease
Morbidity and mortality due to graft-vs.-host disease (GVHD)
remain major barriers to the advancement of allogeneic
haemopoietic stem cell transplantation (SCT). Advances in
our understanding of the pathophysiology of GVHD and
peripheral regulatory mechanisms, along with novel approa-
ches to the prophylaxis and treatment of GVHD, have
significantly advanced the field. Translation of basic mechan-
istic knowledge from animal models into clinical practice is
crucial if the rapid advancement of allogeneic transplantation
is to continue.
The pathophysiology of GVHD may be divided into three
distinct but collaborative phases (Fig 1):
1 Tissue damage attributable to previous therapy and condi-
tioning regimens;
2 Donor T-cell activation following interaction with antigen
presenting cells (APC);
3 Cytokine- and cell-mediated target tissue damage.
Conditioning related tissue damage
Tissue damage is the catalyst for the development of rapid
GVHD, and often occurs well before the transfer of the
allogeneic stem cell graft. Underlying malignancy, effects of
previous induction therapies and new cellular damage induced
by transplant conditioning regimens lead to generation of large
amounts of inflammatory cytokines that enhance early host
APC activation (Zhang et al, 2002; Ferrara et al, 2003).
Crucially, total body irradiation (TBI) induces secretion of
pro-inflammatory cytokines (including tumour necrosis factor
[TNF], interleukin [IL]-1 and IL-6) (Xun et al, 1994) and
direct epithelial damage to the gastrointestinal (GI) tract (Paris
et al, 2001). Translocation of lipopolysaccharide and other
putative bacterial-derived glycolipids across damaged intestinal
mucosa activates the innate immune system and promotes the
inflammatory cytokine cascade (Hill et al, 1997; Hill & Ferrara,
2000).
Donor T-cell activation
Recent data have demonstrated that naıve but not memory
donor T cells are capable of inducing acute GVHD (Anderson
et al, 2003; Zhang et al, 2004). This finding has reignited the
concept of specific (naıve) donor T-cell depletion with the
concept of transferring only memory T cells that contain
responses to nominal infectious antigens. The reasons for the
inability of memory T cells to induce GVHD are not yet clear
but may relate to ineffective trafficking to lymphoid organs
(due to the absence of adhesion molecules like CD62L),
restricted T-cell receptor (TCR) repertoire (i.e. a very low
precursor frequency of host reactive T cells), or combinations
of both.
Graft-vs.-host disease requires the stimulation of naıve
donor T cells by APC, and a large body of recent work has
focused on the requirement for alloantigen expression by
specific cellular compartments. Induction of CD4-dependent
Correspondence: Dr G. R. Hill, Bone Marrow Transplantation
Laboratory, Queensland Institute of Medical Research, Brisbane, Qld
4029, Australia. E-mail: [email protected]
review
ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19 doi:10.1111/j.1365-2141.2007.06510.x
GVHD requires the expression of alloantigen on APC, but not
target tissue epithelium, whereas maximal CD8-dependent
GVHD requires alloantigen to be expressed on both APC and
target tissue (Teshima et al, 2002). Although residual host APC
alone are absolutely required and sufficient for the induction
of CD8-dependent GVHD (Shlomchik et al, 1999), once
initiated GVHD may be amplified by donor-derived APC,
thought to be attributable to donor APC cross-priming
alloreactive CD8+ T cells (Matte et al, 2004). In contrast, the
efficient presentation of exogenous host antigens through the
classical major histocompatibility complex (MHC) class II-
dependent pathway means that expression of alloantigen on
donor APC alone is sufficient to induce CD4-dependent
GVHD (Anderson et al, 2005). While differences in the class I
and class II major histocompatibility antigens stimulate CD8+
and CD4+ T-cell responses (Sprent et al, 1988), the relative
efficacy of either T-cell subset in inducing GVHD following
human leucocyte antigen (HLA)-matched SCT remains
unclear, although both cell subsets are important in animal
models of GVHD directed to minor antigens (Sprent et al,
1988). Minor histocompatibility antigens (miHA) are peptides
from polymorphic cellular proteins, encoded by regions
outside the MHC and presented within class I or class II
MHC (Chao, 2004). The precise nature and importance of
individual miHA, and the potential to exclude miHA-reactive
T cells specifically, is a major focus for strategies to prevent
GVHD. Furthermore, tissue distributions of miHA may differ
and, in particular, miHA expressed only by haemopoietic
tissues represent a very attractive target for the induction of
graft-vs.-leukaemia (GVL) effects without promoting GVHD.
The identity and location of APC responsible for inducing
GVHD has also been the focus of intense investigation. The APC
family includes dendritic cells (DC), monocytes/macrophages
and B cells, and relative roles in the induction of GVHD have
been difficult to dissect using available reagents. Much circum-
stantial evidence suggests that residual host DC are the critical
APC subset that initiate GVHD (Zhang et al, 2002; Duffner
et al, 2004). Tissue-specific DC populations, such as Langerhans
cells (LC), may be required for the induction of organ-specific
GVHD. In murine studies, depletion of host LC before transfer
of donor T cells effectively prevents the development of skin
GVHD (Merad et al, 2004). Interestingly, turnover of host LC
appears to be dependent on donor T cells through a Fas-
dependent pathway (Merad et al, 2004), and clinical studies
have demonstrated that development of complete donor LC
chimerism is associated with prior cutaneous GVHD (Collin
et al, 2006). Conversely, host B cells seem to play little role and
can even inhibit GVHD by virtue of their ability to generate IL-
10 following TBI (Rowe et al, 2006). Further advances will
require the ability to deplete APC subsets specifically and can be
expected to move the field forward rapidly.
Although Peyer’s Patches (PP) have been shown to be
a central early site of donor T-cell activation during GVHD in
the absence of recipient conditioning (Murai et al, 2003),
recent studies have confirmed that lethal GVHD is still induced
following myeloablative SCT even in their absence (Welniak
Host tissue damage
LPS
Th1
IFN-γ
Monocyte
Donor APC
GVHD
Cytolytic pathway
Host APC
Naïve CD4+
T cell
Naïve CD8+
T cell CTL
Inflammatorypathway IL-1, IL-6,
TNF-α
IL-1, IL-6, TNF-α, LPS
Fig 1. The pathophysiology of acute graft-vs.-host disease (GVHD). Stage 1 – Tissue damage, attributable to underlying malignancy, effects of
previous induction therapies and transplant conditioning regimens results in tissue damage and generation of large amounts of inflammatory
cytokines (including TNF, IL-1 and IL-6) which enhance early host antigen presenting cell (APC) activation. Translocation of lipopolysaccharide
(LPS) and other pathogen-derived glycolipids across damaged intestinal mucosa further activates the innate immune system and enhances the
inflammatory cytokine cascade. Stage 2 – Naıve donor CD8+ T cells are stimulated by residual host APC, inducing potent cytotoxic T cell (CTL)
priming. Naıve donor CD4+ T cells are stimulated by both host APC and donor APC presenting host-derived antigens, driving Th1-biased
polarization and cytokine production, further promoting T-cell proliferation and monocyte priming. Stage 3 – Target tissue damage is induced via
direct cytotoxic T cell cytotoxicity and indirect cytokine-mediated damage.
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ª 2007 The Authors4 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19
et al, 2006). Thus, although PP are an early site of donor T-cell
activation (Beilhack et al, 2005), they, along with lymph nodes
and secondary lymphoid organs, may be redundant during
SCT, at least following myeloablative SCT across MHC barriers
(Welniak et al, 2006). Whether this is also true following HLA-
matched SCT will have important implications for the success
of strategies aimed at preventing T-cell trafficking to primary
lymphoid organs.
Following cognate interaction with activated APC, CD4+ T
cells are driven towards T-helper cell type 1 (Th1)-biased
cytokine production (Antin & Ferrara, 1992), promoting T-cell
proliferation (IL-2) and further differentiation, so that very
large amounts of pro-inflammatory cytokines are generated
[particularly interferon c (IFN-c), TNF], which induce tissue
damage in a MHC-independent fashion (Teshima et al, 2002).
In contrast, donor CD8+ T cells differentiate into efficient
cytotoxic T lymphocytes (CTL), capable of causing host tissue
damage in an MHC-I dependent fashion via their perforin,
granzyme and FasL cytotoxic pathways (Kagi et al, 1994;
Shresta et al, 1998).
Cytokine- and cell-mediated target tissue damage
The final phase of GVHD is characterised by target tissue
apoptosis induced by direct cytotoxic effector populations and
indirect cytokine-mediated damage (Fig 1). Donor effector
populations, including CD4+ T helper (Th) cells, CTL and
natural killer (NK) cells, mediate cell-dependent cytotoxicity
via the secretion of prestored perforin and granzyme or
through Fas–FasL, TNF or TNF-related apoptosis-inducing
ligand interactions (Kagi et al, 1994; Shresta et al, 1998;
Kayagaki et al, 1999a,b). Following lethal irradiation, however,
transfer of donor T cells deficient in both perforin/granzyme
and FasL still results in severe GVHD (Jiang et al, 2001).
Indeed, severe target organ GVHD is still seen following MHC
mismatched BMT when recipient mice were bone marrow
chimeras in which alloantigen were expressed on host APC but
not target epithelium (Teshima et al, 2002). In these studies,
target organ damage was prevented by the neutralisation of IL-
1 and TNF. IL-1 is a critical promoter of pro-inflammatory
immune responses, and polymorphisms in the genes encoding
the 10 members of the IL-1 family have important effects on
the incidence and severity of experimental GVHD (Cullup &
Stark, 2005; Dickinson & Charron, 2005). TNF has central
roles at several stages of GVHD, including the enhancement of
APC maturation, promotion of cellular trafficking, enhance-
ment of T-cell responses (Hill et al, 2000) and the direct
induction of tissue injury (Laster et al, 1988). Tissue injury is
further enhanced by macrophage-derived nitric oxide (Garside
et al, 1992; Hill & Ferrara, 2000), which, importantly,
significantly contributes to GVHD-induced immune suppres-
sion (Falzarano et al, 1996; Hongo et al, 2004).
Interferon c has important effects at a variety of stages
during the pathogenesis of acute GVHD, although at the
present time, its roles are incompletely understood. Early non-
irradiation based studies using IFN-c deficient (IFN-c)/))
mice as donors demonstrated a delay in GVHD mortality in
the absence of IFN-c (Ellison et al, 1998). This was associated
with a T-helper cell type 2 (Th2)-biased cytokine profile
(Ellison et al, 2002) and impairment of CTL function (Puliaev
et al, 2004). Surprisingly, however, following TBI-based
experimental transplantation, administration of exogenous
IFN-c was associated with reduced experimental GVHD (Brok
et al, 1993, 1997) and GVHD was increased in recipients of
IFN-c)/)grafts (Murphy et al, 1998; Yang et al, 1998). The
mechanism responsible for the apparently contradictory effects
of IFN-c remains unclear at this time.
Although the central role of HLA matching in haemopoietic
SCT is well established, many recipients of fully-matched
sibling allografts still develop GVHD. It has become apparent
that other genetic systems affect the development of GVHD.
Although miHA contribute to alloreactivity and GVHD, genes
controlling inflammatory processes, such as cytokines, chem-
okines and their receptors, can modulate GVHD. Gene
polymorphisms affecting IL-1 (Cullup & Stark, 2005), IL-6
(Fishman et al, 1998), IL-10 (Cooke & Ferrara, 2003; Lin et al,
2003), TNF (Cavet et al, 1999), transforming growth factor b(TGF-b) (Hattori et al, 2002) and IFN-c (Cayabyab et al,
1994) have all been implicated in the incidence and severity of
GVHD, both in experimental models and immuno-genetic
analysis of retrospective clinical data [recently reviewed
(Dickinson & Charron, 2005)].
Diagnostic difficulties
A major recurring difficulty for transplant physicians is the
ability to differentiate immune-mediated GVHD damage from
tissue injury induced by other processes (Atkinson et al, 1988).
The definitive diagnostic procedure remains the demonstra-
tion of GVHD in biopsy specimens from affected organs,
although this is associated with inherent delays due to
difficulties in obtaining specimens, processing of samples and
examination by an experienced histopathologist. Even when
appropriate specimens are available histological diagnosis may
still be difficult, and there may be significant overlap in the
pathological appearances of acute GVHD and damage attrib-
utable to direct radiation damage, drugs, infectious agents and
microangiopathy. Although novel diagnostic techniques, such
as serum proteomic analysis (Kaiser et al, 2004; Srinivasan
et al, 2006), may allow accurate and rapid diagnosis of GVHD
in the future, at the present time differentiation of GVHD
from other pathological processes frequently still rests on the
input of experienced transplant clinicians (Deeg & Antin,
2006).
The emerging roles of novel cell populations inGVHD
The fine control of alloreactivity following SCT reflects
a complex set of interactions between the innate and adaptive
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ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19 5
immune systems including NK T cells (NKT cells), NK cells
and cd T cells. Alloreactivity may be further modulated by
regulatory cell populations including regulatory T cells (Treg),
regulatory APC populations and perhaps, mesenchymal stem
cells.
NKT cells
In the late 1980s, a number of groups independently reported
the existence of a distinct subset of ab T cells in mice which
secreted large amounts of potentially immunoregulatory cytok-
ines, including IFN-c, IL-4 and TNF (Budd et al, 1987; Ceredig
et al, 1987; Fowlkes et al, 1987). The population was subse-
quently shown to express the C-type lectin, NK1.1, previously
thought to be restricted to NK cells (Sykes, 1990; Arase et al,
1993), and is now known to include a diverse variety of
functionally distinct cell types, collectively referred to as NKT
cells. NKT cells are dependent on the presentation of hydro-
phobic glycolipid molecules by the MHC class-I like molecule,
CD1d, for positive selection within the thymus and subsequent
activation in the periphery. Type 1 NKT cells (also referred to as
invariant or iNKT cells) are further characterised by their
specific recognition of the synthetic glycolipid, a-galactosylcer-
amide (a-GalCer), and may therefore be identified via their
specificity for a-GalCer-loaded CD1d-tetramer. Type 1 NKT
cells express highly conserved TCRs consisting of an invariant
TCR a chain and a limited selection of TCR b chains due to
marked skewing of Vb gene usage. In mice, both CD4+ and
double negative (DN; CD4) CD8)) subsets are recognised,
existing in different proportions in different tissues (Eberl et al,
1999). Type 1 NKT cell recognition of glycolipid antigens
characteristically leads to rapid production of immuno-mod-
ulatory cytokines, particularly IFN-c and IL-4 (Crowe et al,
2003). The functional significance of different subsets is
becoming clear, and it appears that CD4+ type 1 NKT cells
produce both Th1 and Th2 cytokines, whereas DN NKT cells
produce predominantly Th1 cytokines (Lee et al, 2002) and may
have more important roles in tumour surveillance (Crowe et al,
2005; Morris et al, 2005). Type 2 NKT cells have been less well
characterised, largely because they cannot be specifically iden-
tified using conventional technologies (Godfrey et al, 2004a).
The immuno-modulatory effects of NKT cells following
allogeneic transplantation are critically dependent on whether
donor or residual host NKT cells predominate. The Strober
group has demonstrated that specific enrichment of host NKT
cells by conditioning with total lymphoid irradiation and anti-
lymphocyte globulin significantly abrogates GVHD in both
preclinical (Lan et al, 2001, 2003) and clinical studies (Lowsky
et al, 2005) and this appears dependent on the ability of NKT
cells to generate IL-4 (Lan et al, 2001; Higuchi et al, 2002).
Consistent with this, the administration of a-GalCer to
recipients in a mouse model of allogeneic SCT following
sublethal irradiation significantly reduced GVHD (Morecki
et al, 2004), due to residual host NKT cell-dependent Th2
polarisation of donor T cells (Hashimoto et al, 2005). The host
NKT cell subset responsible for this effect has not been
characterised. Conversely, donor DN NKT cells within the
stem cell graft are associated with increased GVHD severity but
significantly augment GVL activity (Morris et al, 2005). We
recently reported that stem cell mobilisation with potent
granulocyte colony-stimulating factor (G-CSF) analogues can
modulate the NKT cell compartment, with dramatic effects on
both GVHD and GVL responses (discussed below). Thus NKT
cells represent an important new cell subset that may be
modulated to improve GVHD and GVL outcomes.
NK cells
Natural killer cells are a unique lymphocyte population that
participate in complex bi-directional interactions with APC
and are central to the co-ordinated induction of innate and
primary adaptive immunity (Degli-Esposti & Smyth, 2005).
Although heterogeneous, two broad functional subsets can be
determined in humans: CD56bright NK cells, which produce
large amounts of immuno-modulatory cytokines (including
IL-2, IL-12 and IL-18), and CD56dim NK cells, which are highly
cytotoxic (mediated predominantly via the perforin pathway)
(Cooper et al, 2001). NK-cell reactivity is tightly controlled
through a balance between activating and inhibitory receptors
(Lanier, 2005). Lysis of autologous targets is physiologically
prevented through NK-cell expression of inhibitory killer cell
immunoglobulin-like receptors (KIR) that recognise self class I
MHC molecules. Normal NK-cell development dictates that
NK cells tolerate healthy autologous cells due to the interaction
of the particular inhibitory KIR receptor with autologous HLA
class I ligands (Valiante et al, 1997).
Natural killer cell cytotoxicity contributes directly to the
cellular effector stage of GVHD (Hill & Ferrara, 2000; Ferrara
& Yanik, 2005), but recent data suggest that following haplo-
identical SCT NK cell alloreactivity may in fact be beneficial.
Bi-directional T-cell alloreactivity would clearly be predicted to
represent a major barrier to haplo-mismatched transplants due
to prevention of engraftment (host-vs.-graft alloreactivity) or
conversely, overwhelming GVHD. The use of high-dose,
vigorously T cell depleted grafts in conjunction with highly
immuno-suppressive conditioning regimens may overcome
these barriers (Dey & Spitzer, 2006). Adequate T-cell depletion
of grafts is critical and may be achieved through negative
selection and T-cell depletion or positive CD34 or CD133 stem
cell selection (see below) (Bitan et al, 2005; Lang et al, 2005).
The optimal method ensuring maximal T-cell depletion
without excessive loss of progenitor cells remains to be
determined. In clinical studies to date engraftment rates have
been impressive (>90%), and the incidence of GVHD has been
low with persistence of GVL effects, at least against myeloid
malignancies (Aversa et al, 1998). In this specific setting,
donor NK cells recognise the absence of donor class I on
recipient APC and leukaemia cells and eliminate both,
reducing GVHD whilst mediating GVL respectively (Ruggeri
et al, 2002). Importantly, since NK cells are not dependent on
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stimulation by host APC to respond to host tissues, they retain
the capacity to induce GVL reactions even after host APC have
been cleared (Ruggeri et al, 1999).
Regulatory T cells
Central tolerance is classically attributed to clonal deletion of
self-reactive T cells in the thymus upon interaction with self-
antigens. However, since not all self-antigens gain access to the
thymus, central tolerance cannot be complete and the induc-
tion of tolerance in the periphery is critical for prevention of
autoimmunity and maintenance of immune homeostasis. Treg
were originally identified as a CD4+ CD25+ population of ab T
cells that permitted the acceptance of allogeneic skin grafts
(Sakaguchi et al, 1995), and may be characterised by their
expression of FoxP3 (Fontenot et al, 2005a). In mouse models
of allogeneic SCT, depletion of Treg within grafts accelerates
GVHD (Cohen et al, 2002; Hoffmann et al, 2002; Taylor et al,
2002), whereas adoptive transfer of large numbers of donor-
derived Treg effectively prevents GVHD (Cohen et al, 2002;
Hoffmann et al, 2002). Although suppression of proliferation
and function of conventional T cells might be predicted to
impair cellular GVL effects, a number of studies have demon-
strated the persistence of immune-mediated GVL effects
(Edinger et al, 2003; Jones et al, 2003; Trenado et al, 2003).
The mechanisms for maintenance of GVL effects remain to be
clearly defined, but since GVHD is immunosuppressive in its
own right (Krenger et al, 1996; Ferrara et al, 1997) it is
plausible that suppression of GVHD by Treg, allows residual
cellular effectors (including T cells and NK cells) to mount
effective GVL responses. Importantly, the absolute numbers of
alloreactive T cells may have critical effects, with relatively small
numbers being sufficient to mount a GVL effect, but larger
numbers of T cells driving robust alloreactive responses and
GVHD (Edinger et al, 2003). Nguyen et al (2006) have recently
demonstrated that infusion of conventional and Treg subsets at
almost physiological doses (10:1) prevented dramatic early
proliferation of effector T cells, but allowed the persistence of
sufficient numbers to mount an effective GVL response.
Stem cell mobilisation with G-CSF limits the ability of
T cells to induce GVHD on a per cell basis (Morris et al, 2006)
and enhanced Treg activity has been demonstrated in patients
following G-CSF administration (Rutella et al, 2002). Studies
from our laboratory have recently demonstrated that protec-
tion from GVHD may be provided by stem cell mobilisation
with the pegylated form of G-CSF (peg-G-CSF) (Morris et al,
2004). Donor T cells from peg-G-CSF treated donors had
impaired proliferation in response to alloantigen stimulation
and inhibited the alloreactivity of donor T cells following SCT
in an IL-10 dependent manner (Morris et al, 2004). Import-
antly, despite reducing GVHD, GVL effects were enhanced due
to expansion of donor NKT cells with enhanced functional
activity following SCT (Morris et al, 2005).
Although standard GVHD prophylaxis includes IL-2 block-
ade by calcineurin inhibitors (see below), Treg suppressor
function is dependent on both IL-2 signalling (Fontenot et al,
2005b) and TCR ligation (Thornton et al, 2004). In murine
studies, Zeiser et al (2006) demonstrated that culture of Treg
with cyclosporine (CSA) suppressed Treg function in vivo and
in vitro (determined by protection from GVHD). Conversely,
alternative immune suppressants, such as sirolimus and
mycophenolate mofetil (MMF), did not impair Treg function.
Clearly the nature of immune suppression may have significant
effects on the functional activity of Treg, and future clinical
trials examining combinations of agents that spare Treg
function are needed.
cd T cells
Another novel T-cell population, cd T cells, further contribute
to the bridging of innate and adaptive immune responses
(Hayday & Tigelaar, 2003). Although cd T cells undergo
somatic DNA rearrangement to form their TCR (Asarnow
et al, 1988), they recognise both protein and non-protein
moieties in the absence of MHC (and therefore do not require
professional APC for activation) and react to patterns associ-
ated with tissue injury or pathogen invasion (Chien &
Bonneville, 2006). Epithelial-based cd T cells release pro-
inflammatory cytokines (including IFN-c and IL-4) and
chemokines that augment mucosal immunity early in the
immune response (Carding & Egan, 2002). Following an
effective immune response, cd T cells may promote its
termination through the production of anti-inflammatory
cytokines, such as IL-10 (Hayday & Tigelaar, 2003). Intrigu-
ingly, although cd T cells are rare in peripheral blood and
lymph nodes, they are seen in significantly larger numbers in
the skin and GI tract, both of which are targets of GVHD.
Using murine models, T cells from donors transgenically
modified to express high levels of cd heterotrimers facilitated
engraftment of MHC mismatched bone marrow grafts, albeit at
the expense of increased GVHD severity (Blazar et al, 1996).
Drobyski et al (2000) examined the immuno-modulatory
effects of transfer of ex vivo IL-2 activated cd T cells on GVHD
in a model of donor lymphocyte infusion (DLI) following fully
MHC mismatched BMT. Transfer of cd T cells and ab T cells at
day 0 significantly increased GVHD when compared with
recipients of ab T cells alone. Maeda et al (2005a) examined the
role of recipient cd T cells following fully MHC mismatched
BMT. The presence of recipient cd T cells was associated with
increased GVHD severity, demonstrated to be the result of
enhanced host APC activation and alloantigen presentation. In
contrast, recipient cd T cells do not appear to influence chronic
GVHD directed to miHA (Anderson et al, 2004).
Regulatory APC
Peripheral blood contains two distinct populations of DCs:
conventional DCs (cDC) and plasmacytoid DCs (pDC), both
of which are myeloid in origin at steady state (MacDonald
et al, 2005a). cDC are CD11chi, CD13+ and CD33+ and require
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the presence of granulocyte-macrophage CSF (GM-CSF) for
survival (Caux et al, 1992; Young et al, 1995). Furthermore,
cDC express high surface levels of co-stimulatory molecules,
secrete large amounts of cytokines (including IL-12) when
stimulated with TNF or CD40L, and can initiate primary
T cell-dependent immune responses biased towards Th1
cytokine profiles (Macatonia et al, 1995; Cella et al, 1996).
Several recent studies, however, have suggested a role for cDC
in the development of peripheral tolerance through the
induction of Treg populations. The ability of cDC to induce
immunity or tolerance is critically linked to maturation, RelB
activity, and CD40 expression (Dhodapkar et al, 2001; Martin
et al, 2003). Immature cDC generated from murine bone
marrow induced T-cell unresponsiveness in vitro and pro-
longed cardiac allograft survival in vivo (Lutz et al, 2000).
Immature cDC have also been shown to induce CD4+ Treg
in vitro and CD8+ Treg in vivo, both of which produce high
levels of IL-10 and low levels of IFN-c (Jonuleit et al, 2000;
Dhodapkar et al, 2001). Sato et al (2003) described the
generation of regulatory host type DC, by differentiation in
the presence of IL-10, that induce transplant tolerance via the
induction of regulatory CD4+CD25+ T cells. These regulatory
DC were required to express host MHC to promote tolerance,
demonstrating a requirement for direct antigen presentation.
Plasmacytoid DC may be defined as HLA-DR+, CD11c),
CD4+and IL-3Ra+ and are dependent on IL-3 (but not GM-
CSF) for survival and differentiation (Grouard et al, 1997;
Kohrgruber et al, 1999). pDC preferentially express a distinct
profile of toll-like receptors (TLR7 and TLR9) which respond to
single stranded RNA and DNA viruses with rapid production of
type 1 IFN (IFN-a and IFN-b) (Asselin-Paturel & Trinchieri,
2005). pDC may promote Th2-biased T-cell responses (Short-
man & Liu, 2002) and can also induce Treg (Wakkach et al,
2003), a process that occurs predominantly within lymph nodes
(Ochando et al, 2006). Haemopoietic stem cell mobilisation is
known to augment pDC numbers within the blood, and this
has been mooted as a major pathway of immune modulation by
G-CSF (Kared et al, 2005). The adoptive transfer of splenic-
derived cDC or pDC from mobilised donor mice failed to
confer protection from GVHD (MacDonald et al, 2005b).
However, it is possible that more immature marrow- or blood-
derived pDC will have more tolerogenic properties and this will
require further study. We have demonstrated that stem cell
mobilisation expands a novel granulocyte–monocyte precursor
population (termed GM cells) (MacDonald et al, 2005b) that
promote transplant tolerance and prevents GVHD by MHC
class II-restricted generation of IL-10-secreting, antigen-speci-
fic Treg. These effects are all enhanced by mobilisation with
newer potent G-CSF analogues and their effects in large clinical
trials are awaited.
Mesenchymal stem cells
Mesenchymal stem cells (MSC) are multipotential non-
haemopoietic progenitor cells, resident within the bone
marrow, that are able to differentiate into the various lineages
of the mesenchyme (including chondrocytes, myocytes and
neurons) (Pittenger et al, 2002). MSC are characterised by the
absence of haemopoietic markers (CD45) and CD34)) and the
expression of specific patterns of adhesion molecules (inclu-
ding CD106+ and CD105+). MSC have been shown to suppress
primary and secondary in vitro mixed lymphocyte reactions
(MLR) in an indoleamine 2,3-dioxygenase (IDO)-dependent
fashion (Meisel et al, 2004) and, in murine models, delay skin
graft rejection across MHC barriers (Bartholomew et al, 2002),
possibly via contact-dependent induction of regulatory APC
populations (Beyth et al, 2005). Lazarus et al (2005) demon-
strated the feasibility of administering bone marrow-derived,
ex vivo expanded MSC to 56 recipients of HLA-identical
sibling allografts following myeloablative conditioning. Grade
II to IV GVHD was seen in 28% of recipients and chronic
GVHD in 61% of patients surviving beyond day 100. Further
randomised clinical studies are needed to confirm the efficacy
of MSC in modulating GVHD.
Therapeutic strategies against acute GVHD
Prophylactic immune-suppression remains central to the
management of GVHD in clinical practice. The cornerstones
of GVHD prophylaxis remain the combination of methotrex-
ate (MTX) and calcineurin inhibitors (Chao & Chen, 2006),
although the optimal calcineurin inhibitor (CSA or tacroli-
mus) remains the focus of debate (Ratanatharathorn et al,
1998; Nash et al, 2000; Hiraoka et al, 2001; Yanada et al,
2004). Novel strategies will ultimately aim to potently inhibit
GVHD with relative preservation of anti-microbial immunity
and immunological anti-tumour effects.
Preventative strategies
IL-2 inhibition: novel calcineurin inhibitors Sirolimus (also
known as Rapamycin) is a novel macrolide which has received
recent attention because of its combination of
immunosuppressive, anti-fungal, anti-viral and anti-
neoplastic properties (Cutler & Antin, 2004). Excitingly, this
agent does not appear to inhibit Treg function in vivo (Zeiser
et al, 2006) and may even augment activity due to TGF-bdependent induction of Treg (Battaglia et al, 2005). Although
structurally related to other calcineurin inhibitors, it has
a novel mode of action and appears to exert some of its
immunosuppressive properties via inhibition of DC activity
through interference with antigen uptake (Monti et al, 2003),
cellular maturation (Hackstein et al, 2003), intracellular
signalling (Chiang et al, 2002) and apoptosis induction
(Woltman et al, 2003). Cutler and Antin (2004) described
the outcomes of three clinical trials using Sirolimus as
prophylaxis against GVHD after allogeneic SCT. They
reported excellent GVHD control even when mismatched
and unrelated donors were used, and reduced mortality
associated with transplantation due to a reduction in the
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ª 2007 The Authors8 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19
necessity to omit or reduce doses of prophylactic MTX.
Although rates of cytomegalovirus reactivation and invasive
fungal infection were low, concern was noted regarding
increased rates of thrombotic microangiopathy. Randomised
clinical studies examining the combination of Sirolimus with a
variety of immunosuppressive reagents are eagerly awaited.
IL-2 inhibition: monoclonal antibodies Dacluzimab (Zenapax),
basliximab (Simulect) and inolimomab are monoclonal
antibodies that bind to the IL-2Ra receptor (CD25) and
inhibit IL-2 signalling. These molecules, as expected, all have
activity in the SCT setting although they are limited by their
high cost. Lee et al (2004) examined the utility of first-line
therapy of acute GVHD with steroids and dacluzimab in a
randomised study. The study was stopped early due to
increased relapse and GVHD mortality in the dacluzimab
arm. Other studies in the setting of steroid refractory GVHD
have yielded conflicting results (Massenkeil et al, 2002; Bay
et al, 2005; Wolff et al, 2005; Teachey et al, 2006), but have
generally not been superior to other approaches. Perhaps the
real advantage of these agents is their lack of toxicity (save
immune suppression), which potentially makes them
ideal agents in the setting of calcineurin-related toxicity
[e.g. renal failure, thrombotic thrombocytopenic purpura
(TTP)] to provide effective IL-2 inhibition until such time
as calcineurin inhibitors can be reinstituted (Wolff et al,
2006).
T-cell depletion. CD52 is expressed on human T and B
cells, monocytes/macrophages and DC. Campath-1H
(alemtuzumab) is a humanised rat monoclonal anti-CD52
antibody that can be used intravenously for in vivo T-cell
depletion or added directly to the stem cell graft for in vitro
purging (Chakrabarti et al, 2004). The use of Campath in vivo
following unrelated donor myeloablative transplantation or as
part of non-myeloablative regimens is associated with durable
engraftment and a low incidence of GVHD, but at the expense
of a significant delay in immune reconstitution (Kottaridis
et al, 2001; Chakrabarti et al, 2002) and the approach of
general T-cell depletion significantly impairs the GVL effect
(Gale & Horowitz, 1990; Horowitz et al, 1990).
Pan T-cell depletion can be refined with highly specific
depletion of alloreactive T-cell subpopulations. Following
in vitro stimulation in MLR, alloreactive T cells can be
identified by the expression of surface activation markers
(including CD25 and CD69), proliferation or preferential
retention of photoactive dyes. Alloreactive cells can be
eliminated using immunotoxins (Montagna et al, 1999),
immuno-magnetic separation (van Dijk et al, 1999; Koh et al,
1999; Davies et al, 2004), flow cytometric cell sorting (Godfrey
et al, 2004b) or photodynamic purging (Martins et al, 2004).
These techniques aim to eliminate host reactive T cells and
allow transfer of the remainder of T cells to provide immunity
to microbial antigens, and early clinical results are encouraging
(Amrolia et al, 2006).
Perhaps most exciting is the potential to specifically deplete
naıve T cells from the stem cell graft (on the basis of CD45RA
or CD62L) in order to retain immunity to microbial antigens
within the memory T-cell compartment. This approach lends
itself to available large scale separation techniques and thus
clinical trials of this approach are eagerly awaited. The
approach is likely, however, to eliminate effective GVL and
preclinical studies are awaited to document the potential GVL
effects retained in central and effector memory T-cell popu-
lations. If, as might be expected, these memory T cells retain
minimal GVL effects, subsequent DLI-based approaches will be
required in the clinic. Nevertheless, this remains the most
promising new approach for improving transplant outcome
for some time and was born from innovative preclinical studies
(Anderson et al, 2003; Zhang et al, 2004).
Stem cell selection. An alternative approach to T-cell depletion
is the implicit exclusion of alloreactive populations via the
specific positive selection of haemopoietic stem cells.
A number of groups have demonstrated excellent rates of
engraftment and low incidence of severe GVHD following
transplantation with CD34+ selected grafts, typically using
immune-adsorption techniques (Finke et al, 1996; Matsuda
et al, 1998; Lang et al, 2003, 2004a), although high rates of
GVHD were observed in some early studies (Bensinger et al,
1996). Furthermore, Cao et al (2001) reported the effects of
CD34-enrichment via density-gradient separation. Although
the incidence of acute GVHD was low (26%), there was
a surprisingly high rate of non-relapse related mortality (62%
by day 180) including infection (fungal and viral), veno-
occlusive disease, TTP and idiopathic pneumonia syndrome.
Further studies examining immune reconstitution and anti-
tumour effects are awaited, both of which would be predicted
to be compromised by this approach.
CD133 is a recently described glycoprotein (Yin et al, 1997)
reported to identify a CD34) stem cell population with
repopulating capacity, likely to be the precursor population of
CD34+ cells (Bhatia et al, 1998). Lang et al (2004b) recently
reported the feasibility of transplantation using magnetically
selected CD34+ and CD133+ grafts in a paediatric population.
All patients engrafted and although CD133-selection was
associated with a lower depletion of T cells when compared
with CD34+ selection, no GVHD greater than grade 2 was seen.
Bitan et al (2005) reported the successful use of CD133-
selected stem cell grafts in patients undergoing haplotype
mismatched transplantation. Again, all patients engrafted and
no patients developed acute GVHD although overall mortality
was high.
An alternative strategy involves a combined approach
utilising CD34+ selection in combination with addition of
defined doses of purified CD3+ T cells at the time of transplant
(Bunin et al, 2006) or planned DLI (Goerner et al, 2003).
Although initial reports are encouraging, with acceptable rates
of GVHD and apparent persistence of GVL effects, further data
is required.
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ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19 9
FTY720. FTY720 is a novel class of immuno-modulatory agent
that is agonistic for the sphingosine 1 phosphate receptor and
thereby inhibits lymphocyte migration from secondary
lymphoid tissues (Chiba et al, 2006). Synergism with other
agents, including CSA and tacrolimus (Furukawa et al, 2000),
makes it a very attractive agent for the therapy of GVHD.
Preclinical studies have demonstrated that FTY720 potently
inhibits the initiation of epithelial GVHD, but importantly
GVL effects within the lympho-haemopoietic system were
preserved (Kim et al, 2003). Further investigation and clinical
translation is eagerly awaited.
Keratinocyte growth factor. Keratinocyte growth factor (KGF)
is a member of the fibroblast growth factor family with
specificity for epithelial tissues expressing its receptors,
including the skin, GI tract and liver (Housley et al, 1994;
Pierce et al, 1994). Administration of KGF reduced GVHD
mortality, whilst retaining GVL effects, in preclinical models
(Panoskaltsis-Mortari et al, 1998; Krijanovski et al, 1999;
Ellison et al, 2004). Interestingly, the prevention of GVHD
by KGF is not attributable to facilitation of repair of
conditioning-induced tissue injury (Panoskaltsis-Mortari
et al, 2000), but is associated with preservation of
thymopoiesis (Rossi et al, 2002), prevention of gut injury
(Panoskaltsis-Mortari et al, 1998; Krijanovski et al, 1999;
Ellison et al, 2004) and relative preservation of peripheral
regulatory mechanisms (Min et al, 2002). KGF has significant
utility for the prevention of chemo- and radio-therapy induced
mucositis (Spielberger et al, 2004), and the effects of KGF on
the incidence and severity of GVHD following allogeneic
transplantation are currently under investigation. Although
other interventions, such as the administration of IL-7 may
also promote thymic T-cell generation (Snyder et al, 2006),
reports of increased GVHD severity in preclinical models
(Sinha et al, 2002) suggests that caution must be exercised.
Treatment strategies
Therapy with high dose methylprednisone remains the gold-
standard therapy for the treatment of acute GVHD developing
despite prophylaxis. One of the largest studies examining
primary therapy of acute GVHD prior to 2000 reported
a disappointing complete response rate of 35% and overall
responses of 55% (MacMillan et al, 2002). Additional thera-
peutic strategies for incomplete responders have included the
addition of pharmacological immune suppressants, such as
MMF, antibody therapies, such as polyclonal antithymocyte
globulin (ATG) or monoclonal TNF inhibitors, and novel
procedures, such as extracorporeal photochemotherapy (ECP).
Antithymocyte globulin. A number of studies have examined
the efficacy of pan T-cell ablation with polyclonal ATG. Van
Lint et al (2006) have recently reported the results of
a randomised study in which all patients with acute GVHD
were initially treated with methylprednisone 2 mg/kg/d. At
day +5 non-responders (n ¼ 61) were randomised to
receive either methylprednisone 5 mg/kg/d alone or
methylprednisone 5 mg/kg/d plus ATG (1Æ25 mg/kg for 5
doses on alternate days). The authors concluded that
although the response rate following the addition of ATG
was increased, this was at the expense of increased transplant-
related mortality, predominantly attributable to infection.
Mollee et al (2001) described a single centre experience in
which patients developing GVHD whilst on CSA, and
resistant to steroids, received therapy consisting of cessation
of CSA, continuation of steroids and addition of ATG
(15 mg/kg for 5 d) and tacrolimus (0Æ025–0Æ1 mg/kg/d).
Within 28 d of treatment, they reported responses in 70%
of patients. Within 6 weeks of therapy, however, 80% of
patients developed significant infections, often with multiple
organisms. Experience with alternative T-cell depletion
antibodies including OKT3 (anti-CD3) and ABX-CBL (anti-
CD147) have been largely disappointing (Keever-Taylor et al,
2001; Knop et al, 2005; Benekli et al, 2006; Macmillan et al,
2006).
Thus, although ATG is still the standard therapy for steroid
refractory GVHD, the control of subsequent opportunistic
infection remains one of the major barriers to improving
outcome. It is anticipated that the introduction of new highly
active anti-fungal agents and improved molecular-based viral
diagnostics and anti-viral drugs may help in this regard.
Extracorporeal photochemotherapy. Extracorporeal photo-
chemotherapy is based on the immuno-modulatory action of
UV-A irradiation on blood mononuclear cells collected by
apheresis and photosensitised by 8-methoxypsoralen (Kanold
et al, 2005). The mechanism of immune suppression is poorly
understood but may involve the re-infusion of irradiated APC
and subsequent induction of antigen-specific Treg (Maeda et al,
2005b). ECP has been investigated as a therapeutic option for
the treatment of steroid refractory acute GVHD and chronic
GVHD (Greinix et al, 2006), although positive results from
randomised, prospective studies are needed to definitively
establish efficacy.
Mycophenolate mofetil. Mycophenolate mofetil is the pro-
drug of the potent immune-suppressant mycophenolic acid, a
non-competitive reversible inhibitor of inosine
monophosphate dehydrogenase, which blocks de novo
synthesis of guanosine nucleotides (Lipsky, 1996).
Lymphocytes depend on this pathway because they do not
possess the salvage pathways of other cells. MMF has been
examined in limited trials for the treatment of acute GVHD
(Mookerjee et al, 1999; Basara et al, 2001; Baudard et al,
2002), with some efficacy. The toxicity profile of MMF,
particularly its lack of renal toxicity and cross-reactivity with
CSA, make MMF an attractive candidate, although bone
marrow suppression and reports of high rates of infection
(Baudard et al, 2002) counsel caution. Unfortunately, a
recent study demonstrated that the addition of MMF to
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ª 2007 The Authors10 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 137, 3–19
CSA did not offer any benefit over standard CSA and MTX
(Nash et al, 2005), and the main use of MMF may therefore
be in the setting of steroid-refractory GVHD (Kennedy et al,
2006).
TNF inhibition. Since TNF has a central role in the
pathogenesis of GVHD, it represents a logical target for
inhibition. Currently, there a number of clinically available
anti-TNF monoclonal antibody preparations: infliximab
(Remicaid – chimeric mouse/human anti-TNF antibody),
adalimumab (Humira – fully humanised anti-TNF antibody)
and etanercept (Embrel – recombinant human TNF receptor
type II fusion protein). It must be noted that there are
important differences between these molecules which may
translate to differences seen in clinical trials (Ehlers, 2003).
Infliximab and adalimumab bind with high affinity to both
soluble and membrane-bound TNF, and do not release TNF
once bound. They also bind complement and therefore have
the ability to lyse cells expressing surface TNF (including tissue
macrophages). In contrast, etanercept binds soluble TNF and
lymphotoxin (LT) alpha homotrimers (LTa3) but has very low
affinity for membrane-bound TNF (Scallon et al, 1995) and
does not induce cellular lysis. Furthermore, etancercept rapidly
releases bound TNF and LT, and therefore rapidly binds them
in environments where they are abundant, and rapidly releases
them where concentrations are low (Ehlers, 2003).
Couriel et al (2004) reported 21 patients with steroid-
refractory GVHD treated with infliximab. The CR rate was
impressive at 62%, but infection rates were high. Uberti et al
(2005) examined the use of combined therapy with etanercept,
methylprednisone and tacrolimus as primary therapy in 20
patients with acute GVHD. Again the response to therapy was
impressive, with 75% of patients achieving CR. Kennedy et al
(2006) recently reported a retrospective analysis of 16 patients
with refractory GVHD treated with a combination of ATG,
tacrolimus and etanercept. Again, overall response rates were
impressive at 81%, and were superior to historical data in
patients receiving ATG alone.
Thus TNF appears an attractive, logical and non-overlap-
ping target (in conjunction with T cell-directed therapy) for
the treatment of steroid-refractory GVHD. While combined
therapy with anti-T cell therapies (e.g. ATG or CD25
inhibition) and TNF inhibition in this setting has become
routine therapy in many units, prospective randomised trials
are urgently required.
Concluding remarks
Acute GVHD remains one of the major barriers to the
advancement of allogeneic haemopoietic transplantation.
Advances in our knowledge of the pathophysiology of GVHD
and mechanisms of peripheral tolerance promises significant
advances in the next few years. Further investigation of
combinations of pharmacological immune suppressants, par-
ticularly agents with the ability to spare beneficial regulatory
populations in combination with cytokine inhibition and
specific removal of alloreactive T cells, will be of great interest.
The continued high rates of infectious complications following
intensive immune suppression remain of great concern, but
aggressive anti-fungal prophylaxis and the promotion of early
reconstitution of innate and adaptive anti-microbial immune
responses should begin to redress the balance.
Acknowledgement
The authors thank Dr John Snowden for helpful discussion
during the writing of this manuscript.
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