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: geoffh@qimr.edu.au

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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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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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