`
`Mouse models of graft-versus-host
`disease*
`Pavan Reddy1 and James L.M. Ferrara1,§, 1University of Michigan Cancer
`Center, Ann Arbor, Ml 48109-5942, USA
`
`Table of Contents
`1. Introduction ............................................................................... 1
`2. Mouse models ............................................................................. 2
`3. Immunobiology ............................................................................ 3
`3.1. Phase 1: Activation of Antigen Presenting Cells (APCs) ................................... 3
`3.2. Phase 2: Donor T cell activation, differentiation and migration ............................. 5
`3.2.1. Costimulation ................................................................. 5
`3.2.2. T cell subsets .................................................................. 5
`3.2.3. Cytokines and T cell differentiation ............................................... 7
`3.2.4. Leukocyte migration ............................................................ 8
`3.3. Phase 3: Effector phase .............................................................. 8
`3.3.1. Cellular effectors .............................................................. 9
`3.3.2. Inflammatory effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
`4. Conclusion ............................................................................... 11
`5. References ............................................................................... 11
`
`1. Introduction
`
`Allogeneic hematopoietic cell transplantation (HCT) represents an important therapy for many hematological
`and some epithelial malignancies and for a spectrum of nonmalignant diseases (Appelbaum, 2001). The development
`of novel strategies such as donor leukocyte infusions (DLI), nonmyeloablative HCT and cord blood transplantation
`(CBT) have helped expand the indications for allogeneic HCT over the last several years, especially among older
`patients (Welniak et al., 2007). However, the major toxicity of allogeneic HCT, Graft-Versus-Host disease (GVHD),
`remains a lethal complication that limits its wider application (Ferrara and Reddy, 2006). Depending on when it occurs
`after HCT, GVHD can be either acute or chronic (Deeg, 2007; Weiden et al., 1979; Weiden et al., 1981; Lee, 2005).
`Acute GVHD is responsible for 15% to 40% of mortality and is the major cause of morbidity after allogeneic HCT,
`while chronic GVHD occurs in up to 50% of patients who survive three months after HCT. Mouse models have
`provided the majority of insights into the biology of this complex disease process.
`
`*Edited by Diane Mathis and Jerome Ritz. Last revised October 20, 2008. Published February 28, 2009. This chapter should be cited as: Reddy,
`P. and Ferrara, J.L.M., Mouse models of graft-versus-host disease (February 28, 2009), StemBook, ed. The Stem Cell Research Community,
`StemBook, doi/10.3824/stembook. l .36. l, http://www.stembook.org.
`Copyright: © 2009 Pavan Reddy and James L.M. Ferrara. This is an open-access article distributed under the terms of the Creative Commons
`Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`§To whom correspondence should be addressed. E-mail: ferrara@umich.edu
`
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`Mouse models of graft-versus-host disease
`
`The GVHD reaction was first noted when irradiated mice were infused with allogeneic marrow and spleen
`cells (van Bekkum and De Vries, 1967). Although mice recovered from radiation injury and marrow aplasia, they
`subsequently died with "secondary disease" (van Bekkum and De Vries, 1967), a syndrome that causes diarrhea,
`weight loss, skin changes, and liver abnormalities. This phenomenon was subsequently recognized as GVHD disease
`(GVHD). Three requirements for the developing of GVHD were formulated by Billingham (Billingham, 1966-1967).
`First, the graft must contain immunologically competent, now recognized as mature T cells. In both experimental and
`clinical allogeneic BMT, the severity of GVHD correlates with the number of transfused donor T cells (Kernan et
`al., 1986; Komgold et al., 1987). The precise nature of these cells and the mechanisms they use are now understood
`in greater detail (discussed below). Second, the recipient must be incapable of rejecting the transplanted cells (i.e.,
`immunocompromised). A patient with a normal immune system will usually reject cells from a foreign donor. In
`allogeneic BMT, the recipients are usually immunosuppressed with chemotherapy and/or radiation before stem cell
`infusion (Welniak et al., 2007). Third, the recipient must express tissue antigens that are not present in the transplant
`donor. This area has been the focus of intense research that has led to the discovery of the major histocompatibility
`complex (MHC; Petersdorf and Malkki, 2006). Human leukocyte antigens (HLA) are proteins that are the gene
`products of the MHC and that are expressed on the cell surfaces of all nucleated cells in the human body, HLA proteins
`are essential to the activation of allogeneic T cells (Petersdorf and Malkki, 2006; Krensky et al., 1990) discussed
`below. This chapter on mouse models of acute GVHD will place the immuno-biological mechanisms of Billingham 's
`postulates in perspective.
`
`In addition to these seminal postulates on GVH reaction, the critical requirement of immune cells from the
`donor graft for optimal leukemia/tumor elimination: a process called graft-versus-leukemia (GVL) effect, and its tight
`link with GVHD were initially made from mouse models (43). Other models such as the canine, nonhuman primate,
`and rat models also played important roles, particularly in the development of clinically used immuno-suppressants.
`Nonetheless, the presence of well-characterized in-bred strains, availability of knock-out and transgenic animals, easy
`availability of reagents, and the relative low cost have made mouse models the most utilized systems for investigating
`the mechanisms of GVH responses.
`
`2. Mouse models
`
`Mouse models of GVHD can be grouped into those in which GVHD is directed to MHC (class I, class II, or
`usually both) or to isolated multiple minor HA alone. Although multiple minor HA mismatches also exist in the former,
`their impact is usually limited relative to that induced by full MHC disparities (Reddy et al., 2008). The GVHD that
`develops in response to a full (class I and II) MHC disparity is dependent on CD4 T cells and CDS T cells provide
`additive pathology. These systems result in an inflammatory "cytokine storm," capable of inducing GVHD in target
`tissues without the requirement for cognate T cell interaction with MHC on tissue (Teshima et al., 2002). In contrast
`to CD4-dependent GVHD, CDS T cells induce GVHD primarily by their cytolytic machinery, which requires the
`TCR to engage MHC on target tissue (Reddy et al., 2008) The induction of GVHD to multiple minor HA results in
`a process where either CDS T cells, CD4 T cells, or both, depending on the strain combination (see Table 1) may
`play a role in disease. These different models have helped dissect and refine the various other complex aspects of
`GVHD (see below). It is critical from the outset to understand that although most clinical BMT recipients are MHC
`matched but minor HA disparate with the donor, there is no one single most appropriate mouse model of clinical BMT.
`Experimentally both the MHC disparate and minor HA disparate systems can also induce the full or certain specific
`aspects of the spectrum of clinically relevant GVHD while permitting the dissection of immunologic mechanisms.
`
`Most mouse models employ radiation for conditioning the recipient animals. Inbred mouse strains demonstrate
`variable sensitivity to radiation, so maximal tolerated total body irradiation (TBI) doses differ from strain to strain.
`For example, B6 are more resistant that BALB/C mice, and Fl hybrids are usually either more resistant than parental
`strain. Generally, the higher the TBI dose, the earlier and greater the intensity of the inflammatory arm of GVHD(see
`below) and BMT models utilizing low TBI doses and high donor T cell doses will result in GVHD dominated by
`later onset T cell-dependent pathology (Reddy et al., 2008). Chemotherapeutic conditioning with cyclophosphamide,
`fludarabine, and busulfan can also be delivered in mouse systems (Ferrara et al., 2005).
`
`Available mouse models (see Table 1) nicely mimic the spectrum of acute GVHD but the induction of clinically
`relevant chronic GVHD in mouse models using nonmutated inbred strains is challenging. Amongst the commonly
`utilized models, they either mimic only a few and not all of the manifestations or the kinetics of chronic GVHD. As
`such, this paucity of appropriate mouse models for chronic GVHD has resulted in a lack of significant understanding
`of the immunobiology of chronic GVHD when compared with acute GVHD. Below we briefly discuss the current
`understanding of immuno-biological mechanisms of acute GVHD derived from utilizing mouse models.
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`Mouse models of graft-versus-host disease
`
`Donor
`
`Host
`
`GVHD targets
`
`T cell dependence
`
`Acute GVHD Models
`B6
`B6
`BALB/c
`B6
`B6
`C3H.SW
`B6
`B10.D2
`DBA/2
`BIO.BR
`Chronic GVHD Models
`B10.D2
`LP/J
`DBA/2
`B6
`BALB/c
`
`(B6 x DBA/2)Fl
`BALB/c
`B6
`bmI
`bm 12
`B6
`BALB/b
`DBA/2
`B10.D2
`
`CBA .
`
`BALB/c
`B6
`B6D2Fl
`(B6 x DBA/c)Fl
`BALB/c x A)Fl
`
`I, Il, mHAs
`I, Il, mHAs
`I, Il, mHAs
`I
`II
`mHAs
`mHAs
`mHAs
`mHAs
`mHAs
`
`mHAs
`mHAs
`I, Il, mHAs
`I, II, mHAs
`I, II, mHAs
`
`CD4 +/orCD8
`CD4 +/orCD8
`CD4 +/orCD8
`CD8
`CD4
`CD8
`CD4
`CD4
`CD8
`CD8
`
`CD4
`CD4
`CD4
`CD4
`CD4
`
`Table 1. Mouse models of BMT.
`Donor and host strains used in common BMT models, usual total body irradiation (TBI) doses (delivered)
`in two split doses on a single day at < 150 cGy/min), target GVHD antigens-MHC class I (I), or minor
`HA (mHA), T cell dependence of subsequent GVHD (CD4 and/or CD8). Source: Biol Blood Marrow
`Transplantation 14:129-135(2008) PMID SJ083-8791(07)00551-4.
`
`3. lmmunobiology
`
`It is helpful to remember two important principles when considering the pathophysiology of acute GVHD.
`First, acute GVHD reflects exaggerated, but normal inflammatory mechanisms that occur in a setting where they
`are undesirable. The donor lymphocytes that have been infused into the recipient function appropriately, given the
`foreign environment they encounter. Second, donor lymphocytes encounter tissues in the recipient that have often
`been profoundly damaged. The effects of the underlying disease, prior infections, and the intensity of conditioning
`regimen all result in substantial changes not only in the immune cells, but also in the endothelial and epithelial cells.
`Thus, the allogeneic donor cells rapidly encounter not only a foreign environment, but one that has been altered to
`promote the activation and proliferation of inflammatory cells. Thus, the pathophysiology of acute GVHD may be
`considered a distortion of the normal inflammatory cellular responses (Reddy and Ferrara 2003). The development
`and evolution of acute GVHD can be conceptualized in three sequential phases (see Figure 1) to provide a unified
`perspective on the complex cellular interactions and inflammatory cascades that lead to acute GVHD: (1) activation
`of the antigen-presenting cells (APCs; 2) donor T cell activation, differentiation and migration and (3) effector phase
`(Reddy and Ferrara 2003).
`
`3.1. Phase 1: Activation of Antigen Presenting Cells (APCs)
`
`The earliest phase of acute GVHD is set into motion by the profound damage caused by the underlying disease
`and its treatment or infections that might be further exacerbated by the BMT conditioning regimens of variable intensity
`which include total body irradiation (TBI and/or chemotherapy) that are administered even before the infusion of donor
`cells (Clift et al., 1990; Gale eta!., 1987; Hill and Ferrara, 2000; Paris et al., 2001; Xun et al., 1994). This first step results
`in activating the APCs. Specifically, damaged host tissues respond with multiple changes, including the secretion of
`proinflammatory cytokines, such as TNF-a and IL-1, described as the "cytokine storm" (Hill and Ferrara, 2000; Xun
`et al., 1994; Hill et al., 1997). Such changes increase expression of adhesion molecules, costimulatory molecules,
`MHC antigens and chemokines gradients that alert the residual host and the infused donor immune cells (Hill and
`Ferrara, 2000). These "danger signals" activate host APCs (Matzinger, 2002; Shlomchik et al., 1999). Damage to the
`gastrointestinal (GI) tract from the conditioning is particularly important in this process because it allows for systemic
`translocation of immuno-stimulatory microbial products such as lipopolysaccaride (LPS) that further enhance the
`activation of host APCs and the secondary lymphoid tissue in the GI tract is likely the initial site of interaction
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`Mouse models of graft-versus-host disease
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`GVHD
`pathology
`
`(I) Recipient c:ondltloning
`tissue damage
`
`Host
`
`IL-1
`IL-6
`
`Small
`intestine
`
`(II) Donor
`T cell activation
`
`Figure 1. T hree phases of GVHD immuno-biology.
`
`Target cell
`apoptosis
`
`{Ill) Effecto
`
`between activated APCs and donor T cells (Hill and Ferrara, 2000; Paris et al., 2001 ; Cooke et al. , 1998; Murai
`et al. , 2003). This scenario accords with the observation that an increased risk of GVHD is associated with intensive
`conditioning regimens that cause extensive injury to epithelial and endothelial surfaces with a subsequent release of
`inflammatory cytokines, and increases the expression of cell surface adhesion mol ecules (Hill and Ferrara, 2000; Paris
`et al. , 2001). The relationship among conditioning intensity, inflammatory cytokine, and GVHD severity has been
`supported by elegant murine studies (Paris et al. , 2001; Hill et al., 1997). Furthermore, the observations from these
`experimental studi es have led to two recent clinical innovations to reduce clinical acute GVHD: (a) reduced-intensity
`conditioning to decrease the damage to host ti ssues and, thus, limit activation of host APC and (b) KIR mismatches
`between donor and rec ipients to eliminate the host APCs by the alloreactive NK cells (Slavin, 2000; Velardi et al.,
`2002). However, reduced intensity conditioning also causes substantial GVHD. This suggests that in out-bred species
`that are exposed to infectious agents and in some parent into Fl mouse models , tissue stress and inflammation not
`caused by conditioning regimen are also sufficient to prime and induce a GVH response.
`
`Host type APCs that are present and have been primed by conditioning are critical for the induction of this
`phase; recent ev idence suggests that donor type APCs exacerbate GVHD, but in certain experimental models donor
`type APC chimeras also induce GVHD (Teshima et al. , 2002; Shlomchik et al., 1999; Jones et al. , 2003; Reddy et al.,
`2005). In clinical situations, if donor type APCs are present in sufficient quantity and have been appropriately primed,
`they too might play a role in the initiation and exacerbation of GVHD (Arpinati et al., 2000; Auffermann-Gretzinger
`et al. , 2002; MacDonald et al. , 2005 ). Amongst the cells with antigen-presenting capability, DCs are the most potent
`and play an important role in the induction of GVHD (Banchereau and Steinman, 1998). Experimental data suggest
`that GVHD can be regulated by qualitatively or quantitatively modulating distinct DC subsets (Chorny et al., 2006;
`Duffner et al. , 2004; Macdonald et al. , 2007; Paraiso et al. , 2007; Sato et al., 2003). In one clinical study persistence
`of host DC after day 100 correlated with the severity of acute GVHD while elimination of host DCs was associated
`with reduced severity of acute GVHD (Auffermann-Gretzinger et al. , 2002). The allo-stimulatory capacity of mature
`monocyte derived DCs (mDCs) after reduced-intensity transplants was lower for up to six months compared to the
`mDCs from myeloablative transplant recipients, thu s suggesting a role for host DCs and the reduction in "danger
`signals" secondary to less intense conditioning in acute GVHD (Nachbaur et al. , 2003). Nonetheless thi s concept of
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`Mouse models of graft-versus-host disease
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`enhanced host APC activation explains a number of clinical observations, such as increased risks of acute GVHD
`associated with advanced stage malignancy, conditioning intensity and histories of viral infections. This has been
`further suggested by recent NOD2, MBL and TLR4 polymorphism studies in humans (Holler, 2006; Rocha, 2002;
`Lorenz et al., 2001).
`
`Other professional APCs such as monocytes/macrophages or semi-professional APCs might also play a role
`in this phase. For example, recent data suggests that host type B cells might play a regulatory role under certain
`contexts (Rowe, 2006). Also host or donor type nonhematopoietic stem cells, such as mesenchymal stem cells or
`stromal cells when acting as APCs have been shown to reduce T cell allogeneic responses, although the mechanism
`for such inhibition remains unclear. The relative contributions of various APCs, professional or otherwise, remain to
`be elucidated.
`
`The other aspects of the innate immune system such as complement activation, PMNs, and defensins remain
`poorly understood and they too might play a role in enhancing or regulating the induction and propagation of GVHD.
`In this regard, a recent study suggests that target tissue inflammation might account for the unique organ specificity of
`acute GVHD (Chakraverty, 2006).
`
`3.2. Phase 2: Donor T cell activation, differentiation and migration
`
`The infused donor T cells interact with the primed APCs and initiate the second phase of acute GVHD. This
`phase includes antigen presentation by primed APCs, the subsequent activation, proliferation, differentiation and
`migration of alloreactive donor T cells.
`
`After allogeneic HSC transplants, both host- and donor-derived APCs are present in secondary lymphoid organs
`(Beilhack et al., 2005; Korngold and Sprent, 1980). The T cell receptor (TCR) of the donor T cells can recognize
`alloantigens either on host APCs (direct presentation) or donor APCs (indirect presentation; Lechler et al., 2001;
`Shlomchik, 2003). In direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC
`molecules or allogeneic MHC molecules without peptide (Lechler et al., 2001; Sayegh and Carpenter, 1996). During
`indirect presentation, T cells respond to the peptide generated by degradation of the allogeneic MHC molecules
`presented on self-MHC (Sayegh and Carpenter, 1996). An experimental study demonstrated that APCs derived from
`the host, rather than from the donor, are critical in inducing GVHD across MiHA mismatch (Shlomchik, 2003). Recent
`data suggest that presenting distinct target antigens by the host and donor type APCs might play a differential role in
`mediating target organ damage (Shlomchik, 2003; Anderson et al., 2005; Kaplan et al., 2004). In humans, most cases
`of acute GVHD developed when both host DCs and donor dendritic cells (DCs) were present in peripheral blood after
`BMT (Auffermann-Gretzinger et al., 2002).
`
`3.2. 1. Costimulation
`
`The interaction of donor lymphocyte TCR with the host allo-peptide presented on the MHC of APCs alone is insufficient
`to induce T cell activation (Appleman and Boussiotis, 2003). Both TCR ligation and costimulation via a "second"
`signal through interaction between the T cell costimulatory molecules and their ligands on APCs are required to achieve
`T proliferation, differentiation and survival (Sharpe and Freeman, 2002). The danger signals generated in phase 1
`augment these interactions and significant progress has been made on the nature and impact of these "second" signals
`(Bromley et al., 2001; Dustin, 2001). Costimulatory pathways are now known to deliver both positive and negative
`signals and molecules from two major families; the B7 family and the TNF receptor (TNFR) family play pivotal
`roles in GVHD (Greenwald et al., 2005). Interrupting the second signal by blockade of various positive costimulatory
`molecules (CD28, ICOS, CD40, CD30, 4-lBB and OX40) reduces acute GVHD in several murine models while
`antagonism of the inhibitory signals (PD-1 and CTLA-4) exacerbates the severity of acute GVHD (Welniak et al.,
`2007; Blazar et al., 1994; Blazar et al., 1995; Blazar et al., 1997; Blazar et al., 2001; Blazar et al., 2003; Blazar et
`al., 2003). The various T cell and APC costimulatory molecules and the impact on acute GVHD are summarized in
`Table 2. The specific context and the hierarchy in which each of these signals play a dominant role in the modulation
`of GVHD remain to be determined.
`
`3.2.2. T cell subsets
`
`T cells consist of several subsets whose responses differ based on antigenic stimuli, activation thresholds and effector
`functions. The alloantigen composition of the host determines which donor T cell subsets proliferate and differentiate.
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`Mouse models of graft-versus-host disease
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`T cell costimulation
`
`Tcell
`
`APC
`
`Adhesion
`
`Recognition
`
`Costimulation
`
`ICAMs
`LEA-1
`CD2 (LEA-2)
`TCR/CD4
`TCR/CD8
`CD28
`CD152 (CTLA-4)
`ICOS
`PD-1
`Unknown
`CD 154 (CD4OL)
`CD134 (0X40)
`CD137 (4-IBB
`HVEM
`
`LEA-1
`ICAM~
`LFA-3
`NIIIC hi
`Mi-Ice 1
`CD80/86
`CD80/86
`B7H/B7RP-1
`PD-Ll, PD-L2
`B7-H3
`CD40
`CD13L(OX40L)
`CD137L (4-lIBBL
`LIGHT
`
`Table 2. T-cell-APC Interactions that Regulate GVHD.
`
`• CD4+ and CD8+ cells
`
`CD4 and CD8 proteins are coreceptors for constant portions of MHC class II and class I molecules, respectively
`(Csencsits and Bishop, 2003). Therefore, MHC class I (HLA-A, -B, -C) differences stimulate CD8+ T cells and MHC
`class II (HLA-DR -DP, -DQ) differences stimulate CD4+T cells (Csencsits and Bishop, 2003; Ferrara et al., 1993;
`Komgold and Sprent, 1982; Komgold and Sprent, 1985; Komgold and Sprent, 1987). But clinical trials of CD4+ or
`CDS+ depletion have been inconclusive (Wu and Ritz, 2006). This may not be surprising because GVHD is induced
`by MiHAs in the majority of HLA-identical BMT, which are peptides derived from polymorphic cellular proteins that
`are presented by MHC molecules (Goulmy, 2006). Because the manner of protein processing depends on genes of the
`MHC, two siblings will have many different peptides in the MHC groove (Goulmy, 2006). Thus, in the majority of
`HLA-identical BMT, acute GVHD may be induced by either or both CD4+ and CDS+ subsets in response to minor
`histocompatibility antigens (Wu and Ritz, 2006). The peptide repertoire for class I or class II MHC remains unknown
`and likely to be distinct between one individual to the next (Spierings et al., 2006). But it is plausible that only a few
`of these many peptides might behave as immunodominant "major minor" antigens that can potentially induce GVHD.
`In any event, such antigens remain to be identified and validated in large patient populations.
`
`Central deletion by establishment of stable mixed hematopoietic chimeric state is an effective way to eliminate
`continued thymic production of both CD4+ and CDS+ alloreactive T cells and thus reduce GVHD (Sykes, 2001;
`Wekerle et al., 1998; Wekerle et al., 2000). In contrast peripheral mechanisms to induce tolerance of CD4+ and CDS+
`Tcells appears to be distinct (Wells et al., 1999; Wells et al., 2001). The T cell apoptosis pathways by which peripheral
`deletion occurs can be broadly categorized into activation-induced cell death (AICD) and passive cell death (PCD;
`Lechler et al., 2003). Experimental data suggests that deletional tolerance by AICD is operative via the Fas (for CD4+)
`or TNFR (CDS+) pathways in Thl cells and when there is a higher frequency of alloreactive cells (Combadiere et al.,
`1998; Min et al., 2004; Siegel et al., 2000; Zhang et al., 1997; Zheng et al., 1995). PCD or "death by neglect" is due to
`rapid downregulation of Bcl-2 and appears to be critical in non-irradiated, but not after irradiated BMT (Drobyski et
`al., 2002). Thus, distinct mechanisms of tolerance induced by apoptosis have a dominant role depending on the T cell
`subsets, the conditioning regimens and the histocompatibility differences. Nonetheless strategies aimed at selective
`elimination of donor T cells in vivo after HCT, either by targeting a suicide gene to the allo-T cells or by photodynamic
`cell purging appear promising in reducing experimental acute GVHD (Bondanza et al., 2006; Bonini et al., 1997;
`Bordignon et al., 1995; Chen et al., 2002; Chen et al., 2002; Drobyski and Gendelman, 2002).
`
`• Na'ive and Memory Subsets
`
`Several independent groups have intriguingly found that, unlike memory (CD62L -) T cells, the na'ive (CD62L +)
`T cells were alloreactive and caused acute GVHD across different donor/recipient strain combinations (Anderson et
`al., 2003; Chen et al., 2004; Maeda et al., 2007; Zhang et al., 2004). Furthermore, expression of the na'ive T cell
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`marker CD62L was also found to be critical to regulate GVHD by donor natural regulatory T cells (Ermann et al.,
`2005; Taylor et al., 2004). By contrast, another recent study demonstrated that alloreactive memory T cells and their
`precursor cells (memory stem cells) sustain and also cause robust GVHD (Zhang et al., 2005; Zhang et al., 2005).
`
`• Regulatory T cells
`
`Recent advances indicate that distinct subsets of regulatory CD4+co25+, CD4+co25-1uo+ Tr cells, yoT
`cells, DN- T cells, NK T cells and regulatory DCs control immune responses by inducing anergy or active suppression
`of alloreactive T cells (Sato et al., 2003; Blazar and Taylor, 2005; Cohen et al., 2002; Cohen and Boyer, 2006; Hoffmann
`et al., 2002; Johnson et al., 2002; Lowsky et al., 2005; Maeda et al., 2005; Roncarolo, 1997; Young et al., 2003; Zeng
`et al., 1999; Zhang et al., 2004). Several studies have demonstrated a critical role for the natural donor co4+co25+
`Foxp3+ regulatory T (Treg) cells, obtained from naive animals or generated ex-vivo, in the outcome of acute GVHD.
`Donor CD4+co25+ T cells suppressed the early expansion of alloreactive donor T cells and their capacity to induce
`acute GVHD without abrogating GVL effector function against these tumors (Edinger et al., 2003; Nguyen et al.,
`2007). co4+co25+ T cells induced/generated by immature or regulatory host type DCs and by regulatory donor
`type myeloid APCs were also able to suppress acute GVHD (Sato et al., 2003). One of the clinical studies that
`evaluated the relationship between donor CD4+co25+ cells and acute GVHD in humans after matched sibling donor
`grafts and found that, in contrast to the murine studies, donor grafts containing larger numbers of CD4+ CD25+T
`cells developed more severe acute GVHD (Stanzani et al., 2003). These data suggest that coexpression of CD4+ and
`CD25+ is insufficient because an increase in CD25+ T cells in donor grafts is associated with greater risks of acute
`GVHD after clinical HCT. Another recent study found that Foxp3 mRNA expression (considered a specific marker
`for naturally occurring CD4+CD25+Tregs) was significantly decreased in peripheral blood mononuclear cells from
`patients with acute GVHD (Miura et al., 2004; Zorn et al., 2005). But Foxp3 expression in humans, unlike mice, may
`not be specific for T cells with a regulatory phenotype (Ziegler, 2006). It is likely that the precise role of regulatory T
`cells in clinical acute GVHD will, therefore, not only depend upon identifying specific molecular markers in addition
`to Foxp3, but also on the ability for ex vivo expansion of these cells in sufficient numbers. Several clinical trials are
`underway in the United States and Europe to substantially expand these cells ex vivo and use for prevention of GVHD.
`
`Host NKl .1 + T cells are another T cell subset with suppressive functions that have also been shown to suppress
`acute GVHD in an IL-4 dependent manner. (Lowsky et al., 2005; Zeng et al., 1999; Hashimoto et al., 2005) By contrast,
`donor NKT cells were found to reduce GVHD (Morris et al., 2005; Morris et al., 2006) and enhance perforin mediated
`GVL in an IFN-y dependent manner. Recent clinical data suggests that enhancing recipient NKT cells by repeated TLI
`conditioning promoted Th2 polarization and dramatically reduced GVHD (Lowsky et al., 2005). Experimental data
`also show that activated donor NK cells can reduce GVHD through the elimination of host APCs or by secretion of
`transforming growth factor-,B (TGF-,B; Morris et al., 2006). A murine BMT study using mice lacking SH2-containing
`inositol phosphatase (SHIP), in which the NK compartment is dominated by cells that express two inhibitory receptors
`capable of binding either self or allogeneic MHC ligands, suggests that host NK cells may play a role in the initiation
`of GVHD (Wang et al., 2002).
`
`3.2.3. Cytokines and T cell differentiation
`
`APC and T cell activation result in rapid intracellular biochemical cascades that induce transcription of many genes
`including cytokines and their receptors. The Th 1 cytokines (IFN-y, IL-2 and TNF-a) have been implicated in the patho(cid:173)
`physiology of acute GVHD (Antin and Ferrara, 1992; Ferrara and Krenger, 1998; Liu et al., 2007; Ratanatharathom et
`al., 1998; Reddy, 2003). IL-2 production by donor T cells remains the main target of many current clinical therapeutic
`and prophylactic approaches, such as cyclosporine, tacrolimus and monoclonal antibodies (mAbs) against the IL-2 and
`its receptor to control acute GVHD (Ferrara and Krenger, 1998; Liu et al., 2007; Ratanatharathom et al., 1998; Reddy,
`2003; Ferrara, 1994). But emerging data indicate an important role for IL-2 in the generation and maintenance of
`co4+co25+ Foxp3+ Tregs, suggesting that prolonged interference with IL-2 may have an unintended consequence
`of preventing the development of long-term tolerance after allogeneic HCT (Gavin et al., 2007; Liston and Rudensky,
`2007; Zeiser et al., 2006; Zhang et al., 2005).
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`Similarly the role of other Thi cytokines IFN-y or their inducers as regulators or inducers of GVHD severity
`depends on the degree of allo-mismatch, the intensity of conditioning and the T cell subsets that are involved after
`BMT (Reddy, 2004; Sykes et al., 1999; Yang et al., 1997). Thus, although the "cytokine storm" initiated in phase 1
`and amplified by the Thi cytokines correlates with the development of acute GVHD, early Thi polarization of donor
`T cells to HCT recipients can attenuate acute GVHD suggesting that physiological and adequate amounts of Thi
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`Mouse models of graft-versus-host disease
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`cytokines are critical for GVHD induction, while inadequate production (extremely low or high) could modulate acute
`GVHD through a breakdown of negative feedback mechanisms for activated donor T cells (Reddy, 2003; Reddy, 2004;
`Reddy et al., 2001; Sykes et al., 1995). Several different cytokines that polarize donor T cells to Th2 such as IL-4,
`G-CSF, IL-18, IL-11, rapamycin and the secretion of IL-4 by NKl .1 + T cells can reduce acute GVHD (Fowler et al.,
`1994; Fowler and Gress, 2000; Hill et al., 1998; Jung et al., 2006; Krenger et al., 1995; Pan et al., 1995; Reddy et al.,
`2003). But Thi and Th2 subsets cause injury of distinct acute GVHD target tissues, and some studies failed to show
`a beneficial effect of Th2 polarization on acute GVHD (Nikolic et al., 2000). Thus the Thl/Th2 paradigm of donor
`T cells in the immuno-pathogenesis of acute GVHD has evolved over the last few years and its causal role in acute
`GVHD is complex and incompletely understood.
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`IL-10 plays a key role in suppressing immune responses and its role in regulating experimental acute GVHD
`is unclear (Blazar et al., 1998). Recent clinical data demonstrate an unequivocal association between IL-10 polymor(cid:173)
`phisms and the severity of acute GVHD (Lin et al., 2003). TGF-,B, another suppressive cytokine, was shown to suppress
`acute GVHD, but to exacerbate chronic GVHD (Banovic et al., 2005). The roles of some other cytokines, such as
`IL-7 (that promotes immune reconstitution) and IL-13, remain unclear (Alpdogan et al., 2001; Alpdogan et al., 2003;
`Gendelman et al., 2004; Sinha et al., 2002). The role for Th 17 cells, a recently described novel T cell differentiation
`in many immunological processes, is not yet known (Weaver and Murphy, 2007). In any