Mouse models of graft-versus-host disease€¦ · In addition to these seminal postulates on GVH...

Mouse models of graft-versus-hostdisease∗

Pavan Reddy1 and James L.M. Ferrara1,§, 1University of Michigan CancerCenter, Ann Arbor, MI 48109-5942, USA

Table of Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Mouse models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Immunobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1. Phase 1: Activation of Antigen Presenting Cells (APCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2. Phase 2: Donor T cell activation, differentiation and migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3.2.1. Costimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2.2. T cell subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2.3. Cytokines and T cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.4. Leukocyte migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3.3. Phase 3: Effector phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.3.1. Cellular effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.3.2. Inflammatory effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction

Allogeneic hematopoietic cell transplantation (HCT) represents an important therapy for many hematologicaland some epithelial malignancies and for a spectrum of nonmalignant diseases (Appelbaum, 2001). The developmentof 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 olderpatients (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 occursafter 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 haveprovided 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.1.36.1, http://www.stembook.org.

Copyright: C© 2009 Pavan Reddy and James L.M. Ferrara. This is an open-access article distributed under the terms of the Creative CommonsAttribution 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: [email protected]

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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 spleencells (van Bekkum and De Vries, 1967). Although mice recovered from radiation injury and marrow aplasia, theysubsequently 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 andclinical allogeneic BMT, the severity of GVHD correlates with the number of transfused donor T cells (Kernan etal., 1986; Korngold et al., 1987). The precise nature of these cells and the mechanisms they use are now understoodin 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. Inallogeneic BMT, the recipients are usually immunosuppressed with chemotherapy and/or radiation before stem cellinfusion (Welniak et al., 2007). Third, the recipient must express tissue antigens that are not present in the transplantdonor. This area has been the focus of intense research that has led to the discovery of the major histocompatibilitycomplex (MHC; Petersdorf and Malkki, 2006). Human leukocyte antigens (HLA) are proteins that are the geneproducts of the MHC and that are expressed on the cell surfaces of all nucleated cells in the human body, HLA proteinsare essential to the activation of allogeneic T cells (Petersdorf and Malkki, 2006; Krensky et al., 1990) discussedbelow. This chapter on mouse models of acute GVHD will place the immuno-biological mechanisms of Billingham’spostulates in perspective.

In addition to these seminal postulates on GVH reaction, the critical requirement of immune cells from thedonor graft for optimal leukemia/tumor elimination: a process called graft-versus-leukemia (GVL) effect, and its tightlink 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, easyavailability of reagents, and the relative low cost have made mouse models the most utilized systems for investigatingthe 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, orusually 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 thatdevelops in response to a full (class I and II) MHC disparity is dependent on CD4 T cells and CD8 T cells provideadditive pathology. These systems result in an inflammatory “cytokine storm,” capable of inducing GVHD in targettissues without the requirement for cognate T cell interaction with MHC on tissue (Teshima et al., 2002). In contrastto CD4-dependent GVHD, CD8 T cells induce GVHD primarily by their cytolytic machinery, which requires theTCR to engage MHC on target tissue (Reddy et al., 2008) The induction of GVHD to multiple minor HA results ina process where either CD8 T cells, CD4 T cells, or both, depending on the strain combination (see Table 1) mayplay a role in disease. These different models have helped dissect and refine the various other complex aspects ofGVHD (see below). It is critical from the outset to understand that although most clinical BMT recipients are MHCmatched 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 specificaspects 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 demonstratevariable 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 F1 hybrids are usually either more resistant than parentalstrain. Generally, the higher the TBI dose, the earlier and greater the intensity of the inflammatory arm of GVHD(seebelow) and BMT models utilizing low TBI doses and high donor T cell doses will result in GVHD dominated bylater 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 clinicallyrelevant chronic GVHD in mouse models using nonmutated inbred strains is challenging. Amongst the commonlyutilized models, they either mimic only a few and not all of the manifestations or the kinetics of chronic GVHD. Assuch, this paucity of appropriate mouse models for chronic GVHD has resulted in a lack of significant understandingof the immunobiology of chronic GVHD when compared with acute GVHD. Below we briefly discuss the currentunderstanding of immuno-biological mechanisms of acute GVHD derived from utilizing mouse models.

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Donor Host GVHD targets T cell dependence

Acute GVHD ModelsB6 (B6 × DBA/2)F1 I, II, mHAs CD4 +/or CD8B6 BALB/c I, II, mHAs CD4 +/or CD8BALB/c B6 I, II, mHAs CD4 +/or CD8B6 bm I I CD8B6 bm 12 II CD4C3H.SW B6 mHAs CD8B6 BALB/b mHAs CD4B10.D2 DBA/2 mHAs CD4DBA/2 B10.D2 mHAs CD8B10.BR CBA mHAs CD8Chronic GVHD ModelsB10.D2 BALB/c mHAs CD4LP/J B6 mHAs CD4DBA/2 B6D2F1 I, II, mHAs CD4B6 (B6 × DBA/c)F1 I, II, mHAs CD4BALB/c BALB/c × A)F1 I, II, mHAs 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 minorHA (mHA), T cell dependence of subsequent GVHD (CD4 and/or CD8). Source: Biol Blood MarrowTransplantation 14:129–135(2008) PMID S1083–8791(07)00551–4.

3. Immunobiology

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 theyare undesirable. The donor lymphocytes that have been infused into the recipient function appropriately, given theforeign environment they encounter. Second, donor lymphocytes encounter tissues in the recipient that have oftenbeen profoundly damaged. The effects of the underlying disease, prior infections, and the intensity of conditioningregimen 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 topromote the activation and proliferation of inflammatory cells. Thus, the pathophysiology of acute GVHD may beconsidered a distortion of the normal inflammatory cellular responses (Reddy and Ferrara 2003). The developmentand evolution of acute GVHD can be conceptualized in three sequential phases (see Figure 1) to provide a unifiedperspective on the complex cellular interactions and inflammatory cascades that lead to acute GVHD: (1) activationof 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 diseaseand its treatment or infections that might be further exacerbated by the BMT conditioning regimens of variable intensitywhich include total body irradiation (TBI and/or chemotherapy) that are administered even before the infusion of donorcells (Clift et al., 1990; Gale et al., 1987; Hill and Ferrara, 2000; Paris et al., 2001; Xun et al., 1994). This first step resultsin activating the APCs. Specifically, damaged host tissues respond with multiple changes, including the secretion ofproinflammatory cytokines, such as TNF-α and IL-1, described as the “cytokine storm” (Hill and Ferrara, 2000; Xunet 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 andFerrara, 2000). These “danger signals” activate host APCs (Matzinger, 2002; Shlomchik et al., 1999). Damage to thegastrointestinal (GI) tract from the conditioning is particularly important in this process because it allows for systemictranslocation of immuno-stimulatory microbial products such as lipopolysaccaride (LPS) that further enhance theactivation of host APCs and the secondary lymphoid tissue in the GI tract is likely the initial site of interaction

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Figure 1. Three phases of GVHD immuno-biology.

between activated APCs and donor T cells (Hill and Ferrara, 2000; Paris et al., 2001; Cooke et al., 1998; Muraiet al., 2003). This scenario accords with the observation that an increased risk of GVHD is associated with intensiveconditioning regimens that cause extensive injury to epithelial and endothelial surfaces with a subsequent release ofinflammatory cytokines, and increases the expression of cell surface adhesion molecules (Hill and Ferrara, 2000; Pariset al., 2001). The relationship among conditioning intensity, inflammatory cytokine, and GVHD severity has beensupported by elegant murine studies (Paris et al., 2001; Hill et al., 1997). Furthermore, the observations from theseexperimental studies have led to two recent clinical innovations to reduce clinical acute GVHD: (a) reduced-intensityconditioning to decrease the damage to host tissues and, thus, limit activation of host APC and (b) KIR mismatchesbetween donor and recipients 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 speciesthat are exposed to infectious agents and in some parent into F1 mouse models, tissue stress and inflammation notcaused 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 thisphase; recent evidence suggests that donor type APCs exacerbate GVHD, but in certain experimental models donortype 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-Gretzingeret al., 2002; MacDonald et al., 2005). Amongst the cells with antigen-presenting capability, DCs are the most potentand play an important role in the induction of GVHD (Banchereau and Steinman, 1998). Experimental data suggestthat 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 persistenceof host DC after day 100 correlated with the severity of acute GVHD while elimination of host DCs was associatedwith reduced severity of acute GVHD (Auffermann-Gretzinger et al., 2002). The allo-stimulatory capacity of maturemonocyte derived DCs (mDCs) after reduced-intensity transplants was lower for up to six months compared to themDCs from myeloablative transplant recipients, thus suggesting a role for host DCs and the reduction in “dangersignals” secondary to less intense conditioning in acute GVHD (Nachbaur et al., 2003). Nonetheless this concept of

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enhanced host APC activation explains a number of clinical observations, such as increased risks of acute GVHDassociated with advanced stage malignancy, conditioning intensity and histories of viral infections. This has beenfurther 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 rolein this phase. For example, recent data suggests that host type B cells might play a regulatory role under certaincontexts (Rowe, 2006). Also host or donor type nonhematopoietic stem cells, such as mesenchymal stem cells orstromal cells when acting as APCs have been shown to reduce T cell allogeneic responses, although the mechanismfor such inhibition remains unclear. The relative contributions of various APCs, professional or otherwise, remain tobe elucidated.

The other aspects of the innate immune system such as complement activation, PMNs, and defensins remainpoorly 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 ofacute 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. Thisphase includes antigen presentation by primed APCs, the subsequent activation, proliferation, differentiation andmigration 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 recognizealloantigens 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 MHCmolecules or allogeneic MHC molecules without peptide (Lechler et al., 2001; Sayegh and Carpenter, 1996). Duringindirect presentation, T cells respond to the peptide generated by degradation of the allogeneic MHC moleculespresented on self-MHC (Sayegh and Carpenter, 1996). An experimental study demonstrated that APCs derived fromthe host, rather than from the donor, are critical in inducing GVHD across MiHA mismatch (Shlomchik, 2003). Recentdata suggest that presenting distinct target antigens by the host and donor type APCs might play a differential role inmediating target organ damage (Shlomchik, 2003; Anderson et al., 2005; Kaplan et al., 2004). In humans, most casesof acute GVHD developed when both host DCs and donor dendritic cells (DCs) were present in peripheral blood afterBMT (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 insufficientto 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 achieveT proliferation, differentiation and survival (Sharpe and Freeman, 2002). The danger signals generated in phase 1augment 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 negativesignals and molecules from two major families; the B7 family and the TNF receptor (TNFR) family play pivotalroles in GVHD (Greenwald et al., 2005). Interrupting the second signal by blockade of various positive costimulatorymolecules (CD28, ICOS, CD40, CD30, 4–1BB and OX40) reduces acute GVHD in several murine models whileantagonism 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 etal., 2003). The various T cell and APC costimulatory molecules and the impact on acute GVHD are summarized inTable 2. The specific context and the hierarchy in which each of these signals play a dominant role in the modulationof 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 effectorfunctions. The alloantigen composition of the host determines which donor T cell subsets proliferate and differentiate.

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T cell costimulation T cell APC

Adhesion ICAMs LEA-1LEA-1 ICAM∼CD2 (LEA-2) LFA-3

Recognition TCR/CD4 NIIIC hiTCR/CD8 Mi-lcc l

Costimulation CD28 CD80/86CD152 (CTLA-4) CD80/86ICOS B7H/B7RP-1PD-1 PD-L1, PD-L2Unknown B7-H3CD 154 (CD4OL) CD40CD134 (0X40) CD13L(OX40L)CD137 (4-IBB CD137L (4-1IBBLHVEM 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 MHCclass II (HLA-DR -DP, -DQ) differences stimulate CD4+T cells (Csencsits and Bishop, 2003; Ferrara et al., 1993;Korngold and Sprent, 1982; Korngold and Sprent, 1985; Korngold and Sprent, 1987). But clinical trials of CD4+ orCD8+ depletion have been inconclusive (Wu and Ritz, 2006). This may not be surprising because GVHD is inducedby MiHAs in the majority of HLA-identical BMT, which are peptides derived from polymorphic cellular proteins thatare presented by MHC molecules (Goulmy, 2006). Because the manner of protein processing depends on genes of theMHC, two siblings will have many different peptides in the MHC groove (Goulmy, 2006). Thus, in the majority ofHLA-identical BMT, acute GVHD may be induced by either or both CD4+ and CD8+ subsets in response to minorhistocompatibility antigens (Wu and Ritz, 2006). The peptide repertoire for class I or class II MHC remains unknownand likely to be distinct between one individual to the next (Spierings et al., 2006). But it is plausible that only a fewof 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 eliminatecontinued thymic production of both CD4+ and CD8+ 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 CD8+T cells appears to be distinct (Wells et al., 1999; Wells et al., 2001). The T cell apoptosis pathways by which peripheraldeletion 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 (CD8+) pathways in Th1 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 torapid downregulation of Bcl-2 and appears to be critical in non-irradiated, but not after irradiated BMT (Drobyski etal., 2002). Thus, distinct mechanisms of tolerance induced by apoptosis have a dominant role depending on the T cellsubsets, the conditioning regimens and the histocompatibility differences. Nonetheless strategies aimed at selectiveelimination of donor T cells in vivo after HCT, either by targeting a suicide gene to the allo-T cells or by photodynamiccell 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).

• Naı̈ve and Memory Subsets

Several independent groups have intriguingly found that, unlike memory (CD62L−) T cells, the naı̈ve (CD62L+)T cells were alloreactive and caused acute GVHD across different donor/recipient strain combinations (Anderson etal., 2003; Chen et al., 2004; Maeda et al., 2007; Zhang et al., 2004). Furthermore, expression of the naı̈ve 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 theirprecursor 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+CD25+, CD4+CD25−IL10+ Tr cells, γ δTcells, DN− T cells, NK T cells and regulatory DCs control immune responses by inducing anergy or active suppressionof alloreactive T cells (Sato et al., 2003; Blazar and Taylor, 2005; Cohen et al., 2002; Cohen and Boyer, 2006; Hoffmannet al., 2002; Johnson et al., 2002; Lowsky et al., 2005; Maeda et al., 2005; Roncarolo, 1997; Young et al., 2003; Zenget al., 1999; Zhang et al., 2004). Several studies have demonstrated a critical role for the natural donor CD4+CD25+Foxp3+ regulatory T (Treg) cells, obtained from naı̈ve animals or generated ex-vivo, in the outcome of acute GVHD.Donor CD4+CD25+ T cells suppressed the early expansion of alloreactive donor T cells and their capacity to induceacute GVHD without abrogating GVL effector function against these tumors (Edinger et al., 2003; Nguyen et al.,2007). CD4+CD25+ T cells induced/generated by immature or regulatory host type DCs and by regulatory donortype myeloid APCs were also able to suppress acute GVHD (Sato et al., 2003). One of the clinical studies thatevaluated the relationship between donor CD4+CD25+ cells and acute GVHD in humans after matched sibling donorgrafts and found that, in contrast to the murine studies, donor grafts containing larger numbers of CD4+ CD25+Tcells developed more severe acute GVHD (Stanzani et al., 2003). These data suggest that coexpression of CD4+ andCD25+ is insufficient because an increase in CD25+ T cells in donor grafts is associated with greater risks of acuteGVHD after clinical HCT. Another recent study found that Foxp3 mRNA expression (considered a specific markerfor naturally occurring CD4+CD25+Tregs) was significantly decreased in peripheral blood mononuclear cells frompatients with acute GVHD (Miura et al., 2004; Zorn et al., 2005). But Foxp3 expression in humans, unlike mice, maynot be specific for T cells with a regulatory phenotype (Ziegler, 2006). It is likely that the precise role of regulatory Tcells in clinical acute GVHD will, therefore, not only depend upon identifying specific molecular markers in additionto Foxp3, but also on the ability for ex vivo expansion of these cells in sufficient numbers. Several clinical trials areunderway in the United States and Europe to substantially expand these cells ex vivo and use for prevention of GVHD.

Host NK1.1+ T cells are another T cell subset with suppressive functions that have also been shown to suppressacute 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 mediatedGVL in an IFN-γ dependent manner. Recent clinical data suggests that enhancing recipient NKT cells by repeated TLIconditioning promoted Th2 polarization and dramatically reduced GVHD (Lowsky et al., 2005). Experimental dataalso show that activated donor NK cells can reduce GVHD through the elimination of host APCs or by secretion oftransforming growth factor-β (TGF-β; Morris et al., 2006). A murine BMT study using mice lacking SH2-containinginositol phosphatase (SHIP), in which the NK compartment is dominated by cells that express two inhibitory receptorscapable of binding either self or allogeneic MHC ligands, suggests that host NK cells may play a role in the initiationof 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 genesincluding cytokines and their receptors. The Th1 cytokines (IFN-γ , IL-2 and TNF-α) have been implicated in the patho-physiology of acute GVHD (Antin and Ferrara, 1992; Ferrara and Krenger, 1998; Liu et al., 2007; Ratanatharathorn etal., 1998; Reddy, 2003). IL-2 production by donor T cells remains the main target of many current clinical therapeuticand prophylactic approaches, such as cyclosporine, tacrolimus and monoclonal antibodies (mAbs) against the IL-2 andits receptor to control acute GVHD (Ferrara and Krenger, 1998; Liu et al., 2007; Ratanatharathorn et al., 1998; Reddy,2003; Ferrara, 1994). But emerging data indicate an important role for IL-2 in the generation and maintenance ofCD4+CD25+ Foxp3+ Tregs, suggesting that prolonged interference with IL-2 may have an unintended consequenceof 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).

Similarly the role of other Th1 cytokines IFN-γ or their inducers as regulators or inducers of GVHD severitydepends on the degree of allo-mismatch, the intensity of conditioning and the T cell subsets that are involved afterBMT (Reddy, 2004; Sykes et al., 1999; Yang et al., 1997). Thus, although the “cytokine storm” initiated in phase 1and amplified by the Th1 cytokines correlates with the development of acute GVHD, early Th1 polarization of donorT cells to HCT recipients can attenuate acute GVHD suggesting that physiological and adequate amounts of Th1

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cytokines are critical for GVHD induction, while inadequate production (extremely low or high) could modulate acuteGVHD 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 NK1.1+ 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 Th1 and Th2 subsets cause injury of distinct acute GVHD target tissues, and some studies failed to showa beneficial effect of Th2 polarization on acute GVHD (Nikolic et al., 2000). Thus the Th1/Th2 paradigm of donorT cells in the immuno-pathogenesis of acute GVHD has evolved over the last few years and its causal role in acuteGVHD is complex and incompletely understood.

IL-10 plays a key role in suppressing immune responses and its role in regulating experimental acute GVHDis unclear (Blazar et al., 1998). Recent clinical data demonstrate an unequivocal association between IL-10 polymor-phisms and the severity of acute GVHD (Lin et al., 2003). TGF-β, another suppressive cytokine, was shown to suppressacute GVHD, but to exacerbate chronic GVHD (Banovic et al., 2005). The roles of some other cytokines, such asIL-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 Th17 cells, a recently described novel T cell differentiationin many immunological processes, is not yet known (Weaver and Murphy, 2007). In any case, all of the experimentaldata so far collectively suggest that the timing of administration, the production of any given cytokine, the intensityof the conditioning regimen and the donor-recipient combination may all be critical to the eventual outcome of acuteGVHD.

3.2.4. Leukocyte migration

Donor T cells migrate to lymphoid tissues; recognize alloantigens on either host or donor APCs and become activated.They then exit the lymphoid tissues and traffic to the target organs causing tissue damage (Wysocki et al., 2005). Themolecular interactions necessary for T cell migration and the role of lymphoid organs during acute GVHD have recentlybecome the focus of a growing body of research. Chemokines play a critical role in the migration of immune cells tosecondary lymphoid organs and target tissues (Cyster, 2005). T-lymphocyte production of macrophage inflammatoryprotein-1alpha (MIP-1α) is critical to the recruitment of CD8+ but not CD4+ T cells to the liver, lung and spleenduring acute GVHD (Serody et al., 2000). Several chemokines such as CCL2–5, CXCL2, CXCL9–11, CCL17 andCCL27 are overexpressed and might play a critical role in the migration of leukocyte subsets to target organs liver,spleen, skin and lungs during acute GVHD (Wysocki et al., 2005; Cyster, 2005; Serody et al., 2000; Mapara et al.,2006). CXCR3+ T and CCR5+ T cells cause acute GVHD in the liver and intestine (Wysocki et al., 2005; Duffneret al., 2003; Murai et al., 1999; Wysocki et al., 2004). CCR5 expression has also been found to be critical for Tregmigration in GVHD (Wysocki et al., 2005). In addition to chemokines and their receptors, expression of selectinsand integrins and their ligands also regulate the migration of inflammatory cells to target organs (Murai et al., 2003;Cyster, 2005; Waldman et al., 2006). For example, interaction between α4β7 integrin and its ligand MadCAM-1 areimportant for homing of donor T cells to Peyer’s patches and in the initiation of intestinal GVHD (Murai et al., 2003;Waldman et al., 2006). αLβ2/ICAM1, 2, 3 and α4β1/VCAM-2 interactions are important for homing to the lung andliver after experimental HCT (Wysocki et al., 2005). Expressing CD62L on donor Tregs is critical for their regulationof acute GVHD, suggesting that their migration in secondary tissues is critical for their regulatory effects (Beilhack etal., 2005). The migratory requirement of donor T cells to specific lymph nodes (e.g., Peyer’s patches) for the inductionof GVHD might be dependent on other factors such as the conditioning regimen, inflammatory milieu etc. (Murai etal., 2003; Welniak et al., 2006). Furthermore, FTY720, a pharmacologic sphingosine-1-phosphate receptor agonist,inhibited GVHD in murine, but not in canine models of HCT (Kim et al., 2003; Lee et al., 2003). Thus, there mightalso be significant species differences in the ability of these molecules to regulate GVHD.

3.3. Phase 3: Effector phase

The effector phase that leads to the GVHD target organ damage is a complex cascade of multiple cellular andinflammatory effectors that further modulate each others’ responses either simultaneously or successively. Effectormechanisms of acute GVHD can be grouped into cellular effectors (e.g., CTLs) and inflammatory effectors such ascytokines. Inflammatory chemokines expressed in inflamed tissues upon stimulation by proinflammatory effectorssuch as cytokines are specialized for the recruitment of effector cells, such as CTLs (Sallusto et al., 2000). Furthermorethe spatio-temporal expression of the cyto-chemokine gradients might determine not only the severity, but also theunusual cluster of GVHD target organs (skin, gut, and liver; Wysocki et al., 2005; Cyster, 2005; Serody et al., 2000;Mapara et al., 2006; Duffner et al., 2003; Murai et al., 1999; Wysocki et al., 2004; Waldman et al., 2006; Welniak etal., 2006; Kim et al., 2003; Lee et al., 2003; Sallusto et al., 2000; Sackstein, 2006; Wysocki et al., 2003).

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3.3.1. Cellular effectors

Cytotoxic T cells (CTLs) are the major cellular effectors of GVHD (Kagi et al., 1994; Van Den Brink and Burakoff,2002). The Fas-Fas ligand (FasL), the perforin-grazyme (or granule exocytosis) and TNFR-like death receptors (DR),such as TNF-related apoptosis-inducing ligand (TRAIL: DR4, 5 ligand) and TNF-like weak inducers of apoptosis(TWEAK: DR3 ligand), are the principle CTL effector pathways that have been evaluated after allogeneic BMT (VanDen Brink and Burakoff, 2002; Chicheportiche et al., 1997; Jiang et al., 1998; Jiang et al., 2004; Maeda et al., 2005;Pan et al., 1997). The involvement of each of these molecules in GVHD has been testing by utilizing donor cells thatare unable to mediate each pathway. Perforin is stored in cytotoxic granules of CTLs and NK cells, together withgranzymes and other proteins. Although the exact mechanisms remain unclear, following the recognition of a target cellthrough the TCR-MHC interaction, perforin is secreted and inserted into the cell membrane, forming “perforin pores”that allow granzymes to enter the target cells and induce apoptosis through various downstream effector pathwayssuch as caspases (Voskoboinik et al., 2006). Ligation of Fas results in a death-inducing signaling complex (DISC) andalso activates caspases (Chinnaiyan et al., 1996; Krammer, 2000).

Transplantation of perforin deficient T cells results in a marked delay in the onset of GVHD in transplants acrossMiHA disparities only, both MHC and MiHA disparities (Zeng et al., 1999), and across isolated MHC Class I or ClassII disparities (Zeng et al., 1999; Van Den Brink and Burakoff, 2002; Baker et al., 1997; Braun et al., 1996; Graubertet al., 1996; Graubert et al., 1997). However, mortality and clinical and histological signs of GVHD were still inducedeven in the absence of perforin-dependent killing in these studies, demonstrating that the perforin-granzyme pathwaysplay little role in target organ damage. A role for the perforin-granzyme pathway for GVHD induction is also evidentin studies employing donor T cell subsets. Perforin- or granzyme B-deficient CD8+ T cells caused less mortality thanwild type T cells in experimental transplants across a single MHC Class I mismatch. This pathway, however, seemsto be less important compared to the Fas/FasL pathway in CD4-mediated GVHD (Graubert et al., 1996; Graubertet al., 1997; Ueno et al., 2000). Thus, it seems that CD4+ CTLs preferentially use the Fas-FasL pathway, whereasCD8+CTLs primarily use the perforin-granzyme pathway.

Fas, a TNF-receptor family member, is expressed by many tissues, including GVHD target organs (Aggarwal,2003). Its expression can be upregulated by inflammatory cytokines such as IFN-γ and TNF-α during GVHD, and theexpression of FasL is also increased on donor T cells, indicating that FasL-mediated cytotoxicity may be a particularlyimportant effector pathway in GVHD (Van Den Brink et al., 2000; Via et al., 1996). FasL-defective T cells cause lessGVHD in the liver, skin and lymphoid organs (Baker et al., 1997; Ueno et al., 2000; Van Den Brink et al., 2000; Viaet al., 1996). The Fas-FasL pathway is particularly important in hepatic GVHD, consistent with the keen sensitivityof hepatocytes to Fas-mediated cytotoxicity in experimental models of murine hepatitis (Van Den Brink et al., 2000).Fas-deficient recipients are protected from hepatic GVHD, but not from other organ GVHD, and administration ofanti-FasL (but not anti-TNF) MAbs significantly blocked hepatic GVHD damage occurring in murine models (VanDen Brink and Burakoff, 2002; Van Den Brink et al., 2000; Hattori et al., 2005). Although the use of FasL-deficientdonor T cells or the administration of neutralizing FasL MAbs had no effect on the development of intestinal GVHDin several studies, the Fas-FasL pathway may play a role in this target organ, because intestinal epithelial lymphocytesexhibit increased FasL-mediated killing potential (Lin et al., 1998). Elevated serum levels of soluble FasL and Fashave also been observed in at least some patients with acute GVHD (Das et al., 1999; Liem et al., 1998).

Using a perforin-granzyme and FasL cytotoxic double-deficient (cdd) mouse provides an opportunity to addresswhether other effector pathways are capable of inducing GVHD target organ pathology. An initial study demonstratedthat cdd T cells were unable to induce lethal GVHD across MHC Class I and Class II disparities after sublethalirradiation (Braun et al., 1996). However, subsequent studies demonstrated that cytotoxic effector mechanisms ofdonor T ells are critical in preventing host resistance to GVHD (Maeda et al., 2005; Martin et al., 1998). Thus, whenrecipients were conditioned with a lethal dose of irradiation, cdd (CD4+ T cells produced similar mortality to wild typeCD4+ T cells; Maeda et al., 2005). These results were confirmed by a recent study demonstrating that GVHD targetdamage can occur in mice that lack alloantigen expression on the epithelium, preventing direct interaction betweenCTLs and target cells (Maeda et al., 2005).

The participation of other death ligand receptor signaling pathway, TNF/TNFRs, has also been evaluated.Experimental data suggests that this pathway is crucial for GI GVHD (discussed more below). Recently, severaladditional TNF family apoptosis-inducing receptors/ligands have been identified, including TWEAK, TRAIL andLTβ/LIGHT, and are all assumed to play a role in GVHD and GVL responses (Welniak et al., 2007; Brown and Thiele,2000; Brown et al., 2002; Brown et al., 2005; Sato et al., 2005; Schmaltz et al., 2002; Xu et al., 2006; Zimmermanet al., 2005). However, whether these distinct pathways play a more specific role for GVHD mediated by distinct

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T cell subsets in certain situations remains unknown. Intriguingly, recent data suggest that none of these pathwaysmight be critical for mediating the rejection of donor grafts (Zimmerman et al., 2005; Zimmerman et al., 2005). Thus,it is likely that their role in GVHD might be modulated by the intensity of conditioning and by the recipient T cellsubsets. Existing experimental data suggest that perforin and TRAIL cytotoxic pathways are associated with CD8+ Tcell–mediated GVL (Van Den Brink and Burakoff, 2002). The available experimental data are strongly skewed towardCD8+ T cell–mediated GVL based on the dominant role of this effector population in most murine GVT models;however, CD4+ T cells can mediate GVL and might be crucial in clinical BMT depending on the type of malignancyand the expression of immuno-dominant antigens.

Taken together, although experimental data suggest there might be some distinction between the use of differentlytic pathways for the specific GVHD target organs and GVL, the clinical applicability of these observations is as yetlargely unknown

3.3.2. Inflammatory effectors

Inflammatory cytokines synergize with CTLs resulting in the amplification of local tissue injury and further promotionof an inflammation, which ultimately leads to the observed target tissue destruction in the transplant recipient (Antinand Ferrara, 1992). Macrophages, which had been primed with IFN-γ during step 2, produce inflammatory cytokinesTNF-α and IL-1 when stimulated by a secondary triggering signal (Ferrara et al., 1999). This stimulus may be providedthrough Toll-like receptors (TLRs) by microbial products such as LPS and other microbial particles, which can leakthrough the intestinal mucosa damaged by the conditioning regimen and gut GVHD (Hill and Ferrara, 2000; Iwasakiand Medzhitov, 2004). It is now apparent that immune recognition through both TLR and non-TLRs (such as NOD)by the innate immune system also controls activation of adaptive immune responses (Iwasaki and Medzhitov, 2004;Fritz et al., 2006). Recent clinical studies of GVHD suggested the possible association with TLR/NOD polymorphismsand the severity of GVHD (Lorenz et al., 2001; Dickinson et al., 2004; Holler et al., 2006). LPS and other innatestimuli may stimulate gut-associated lymphocytes, keratinocytes, dermal fibroblasts, and macrophages to producepro-inflammatory effectors that play a direct role in causing target organ damage. Indeed experimental data withMHC-mismatched BMT suggest that, under certain circumstances, these inflammatory mediators are sufficient incausing GVHD damage even in the absence of direct CTL-induced damage (Teshima et al., 2002). The severityof GVHD appears to be directly related to the level of innate and adaptive immune cell priming and release ofproinflammatory cytokines such as TNF-α, IL-1 and nitric oxide (NO; Teshima et al., 2002; Hill and Ferrara, 2000;Hill et al., 1999; Krenger et al., 1996; Nestel et al., 1992).

The cytokines TNF-α and IL-1 are produced by an abundance of cell types during processes of both innate andadaptive immunity; they often have synergistic, pleiotrophic, and redundant effects on both activation and effectorphases of GVHD (Reddy, 2003). A critical role for TNF-α in the pathophysiology of acute GVHD was first suggestedover 20 years ago because mice transplanted with mixtures of allogeneic BM and T cells developed severe skin, gut,and lung lesions that were associated with high levels of TNF-α mRNA in these tissues (Piguet et al., 1987). Targetorgan damage could be inhibited by an infusion of anti-TNF-α MAbs, and mortality could be reduced from 100% to50% by the administration of soluble TNF-α receptor (sTNFR), an antagonist of TNF-α (Xun et al., 1994; Hill et al.,1997; Hill et al., 1999). Accumulating experimental data further suggest that TNF-α is involved in a multi-step processof GVHD pathophysiology. TNF-α can (1) cause cachexia, a characteristic feature of GVHD, (2) induce maturationof DCs, thus enhancing alloantigen presentation, (3) recruit effector T cells, neutrophilis and monocytes into targetorgans through the induction of inflammatory chemokines and (4) cause direct tissue damage by inducing apoptosisand necrosis. TNF-α also involves in donor T cell activation directly through its signaling via TNFR1 and TNFR2 onT cells. TNF-TNF1 interactions on donor T cells promote alloreactive T cell responses, and TNF-TNFR2 interactionsare critical for intestinal GVHD (Brown et al., 2002; Hill et al., 2000). TNF-α also seems to be an important effectormolecule in GVHD in skin and lymphoid tissue (Piguet et al., 1987; Ferrara and Burakoff, 1990). Additionally, TNF-αmight also be involved in hepatic GVHD, probably by enhancing effector cell migration to the liver via the inductionof inflammatory chemokines (Tanaka et al., 1993) An important role for TNF-α in clinical acute GVHD has beensuggested by studies demonstrating elevated serum levels or TNF-α or elevated TNF-α or elevated TNF-α mRNAexpression in peripheral blood mononuclear cells in patients with acute GVHD and other endothelial complications,such as hepatic veno-occlusive disease (VOD; Tanaka et al., 1993; Holler et al., 1993; Tanaka et al., 1993). Phase I-IItrials using TNF-α antagonists reduced the severity of GVHD suggesting that it is a relevant effector in causing targetorgan damage (Herve et al., 1992; Uberti et al., 2005).

The second major proinflammatory cytokine that appears to play an important role in the effector phase of acuteGVHD is IL-1 (Antin and Ferrara, 1992). Secretion of IL-1 appears to occur predominantly during the effector phase

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of GVHD of the spleen and skin, two major GVHD target organs (Abhyankar et al., 1993). A similar increase inmononuclear cell IL-1 mRNA has been shown during clinical acute GVHD. Indirect evidence of a role for IL-1 inGVHD was obtained with administering this cytokine to recipients in an allogeneic murine BMT model. Mice receivingIL-1 displayed a wasting syndrome and increased mortality that appeared to be an accelerated form of disease. Bycontrast, intraperitoneal administration of IL-1ra starting on day 10 post-transplant reversed the development of GVHDin the majority of animals, giving treated animals a significant survival advantage (McCarthy et al., 1991). However,the attempt to use IL-1ra to prevent acute GVHD in a randomized trial was not successful (Antin et al., 2002). As aresult of activation during GVHD, macrophages also produce Nitric Oxide (NO), which contributes to the deleteriouseffects on GVHD target tissues, particularly immunosuppression (Krenger et al., 1996; Falzarano et al., 1996). NOalso inhibits the repair mechanisms of target tissue destruction by inhibiting proliferation of epithelial stem cells in thegut and skin (Nestel et al., 2000). In humans and rats, the development of GVHD is preceded by an increase in serumlevels of NO oxidation products (Bogdan, 2001; Langrehr et al., 1992; Langrehr et al., 1992; Weiss et al., 1995)

Existing data derived from mouse models demonstrate important role for various inflammatory effectors inGVHD. Although some have shown to be of therapeutic relevance (TNF-α) and the role of some others (IL-1) has notbeen clinically validated. The relevance of currently studied, or as yet unknown specific effectors, might however, bedetermined by other factors, including the intensity of preparatory regimens, the type of allograft, the T cell subsetsand the duration of BMT.

4. Conclusion

Studies from murine models have contributed to the outstanding progress in understanding the biological basisof GVHD. The three phase model, developed largely on the basis of murine studies, allows for an easy perspectiveof the complex process of GVHD but is not meant to suggest that GVHD actually occurs to such discrete phases orsteps. The mouse models have allowed for a more refined understanding of the cellular interactions and networks thatimpact upon GVHD. Further improvements and greater sophistication in analysis of the existing mouse models overthe next few years will undoubtedly bring better refinement and greater understanding of this complex immunologicalprocess.

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Mouse models of graft-versus-host disease€¦ · In addition to these seminal postulates on GVH...

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