Inflammatory cytokines and dendritic cells in acute graft-versus-host disease after allogeneic stem cell transplantation

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Cytokine & Growth Factor Reviews 19 (2008) 53–63 www.elsevier.com/locate/cytogfr

Inflammatory cytokines and dendritic cells in acute graft-versus-host disease after allogeneic stem cell transplantation Mohamad Mohty a,*, Be´atrice Gaugler b,* a

He´matologie Clinique, Centre Hospitalier et Universitaire de Nantes, INSERM UMR 601 and Universite´ de Nantes, Place Alexis Ricordeau, F-44093 Nantes Cedex 01, France b INSERM UMR 645, Bd. Fleming, F-25000 Besanc¸on, France Available online 20 December 2007

Abstract The wider use of allogeneic stem cell transplantation (allo-SCT) is still limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). The role of inflammatory cytokines such as TNF-alpha and IL-1, and their impact on immune effectors (mainly CD4+ and CD8+ T) cells has been extensively studied in the context of GVHD occurring after standard myeloablative allo-SCT. However, recent data suggested that GVHD pathophysiology is likely to involve more complex interactions where antigen-presenting cells, especially dendritic cells (DCs), may play a major role at time of initiation of acute GVHD. In addition, the wider use of reduced intensity and less toxic conditioning (RIC) regimens prior to allo-SCT would allow better visualization of the fine functions of immune effectors, thereby offering a window of opportunities to better decipher the intimate pathophysiological mechanisms underlying GVHD. The aim of this work is to review the available research evidence on the role of DCs as in vivo regulators of alloimmune reactivity, and their interactions with other immune effectors. # 2007 Elsevier Ltd. All rights reserved. Keywords: Cytokines; DCs; GVHD

1. Introduction Allogeneic hematopoietic stem cell transplantation (alloSCT) has proven to be an effective therapy for a variety of life-threatening malignancies. The beneficial effect of alloSCT is due to the graft-versus-tumor (GVT) reaction. However, allo-SCT is limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). GVHD continues to be a major source of morbidity and mortality following allo-SCT, which limits treatment of a broader spectrum of diseases and patients. The basic principles necessary for the development of GVHD were initially described by Billingham [1]. GVHD requires that (i) the host must be incapable of rejecting the graft, (ii) the graft must contain immunocompetent cells, and (iii) there must be incompatibilities in transplantation * Corresponding authors. E-mail address: [email protected] (M. Mohty). 1359-6101/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2007.10.010

antigens between the host and donor [1]. More recently, trafficking of alloreactive T cells to lymphoid tissues for activation by antigen-presenting-cells (APCs), followed by homing to specific organ sites, was recognized as another essential prerequisite to the induction and pathogenesis of acute GVHD [2], suggesting that GVHD is a T cell-mediated inflammatory disease (Table 1). Unfortunately, GVHD is often closely associated with the beneficial GVT activity, and much effort has been put forth to reduce GVHD while maintaining GVT. The role of adaptive immune effectors (mainly CD4+ and CD8+ T) cells has been extensively studied in the context of GVHD occurring after standard myeloablative allo-SCT. In the standard setting, GVHD is characterized by donor lymphocyte infiltrates in the gut, skin, and liver. Recruitment, activation and expansion of mature donor T cells at the site of inflammation represent the key processes during the initiation phase of GVHD that leads to tissue damage, and in worst case, death of the patient.

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Table 1 The revised Billingham criteria for the development of GVHD [1,2] (1) The host must be incapable of rejecting the graft (2) The graft must contain immunocompetent cells (3) There must be incompatibilities in transplantation antigens between donor and host (4) The effector cells must migrate to the target tissues

Based on the pioneering work by Ferrara and Reddy [4] and Holler et al. [3,5], an ever-growing body of data suggests that GVHD pathophysiology can be summarized into a three-step process. In step I, the conditioning regimen (high dose irradiation, chemotherapy, or both) leads to the damage of host tissues, especially the intestinal mucosa. This allows the translocation of LPS from the intestinal lumen to the circulation, stimulating the secretion of the inflammatory cytokines (such as TNF-alpha, IL-1, IL-6) from host tissues. These cytokines will increase the expression of MHC antigens and adhesion molecules on host tissues, enhancing the recognition of MHC and minor histocompatibility antigens (mHAgs) by mature donor T cells. Donor T-cell activation in step II is characterized by the predominance of T-helper 1 (Th1) cells and the secretion of IFN-gamma. In step III, effector functions of immune cells are triggered by the secondary signal provided by LPS and other stimulatory molecules that leak through the intestinal mucosa damaged during steps I and II. Activated macrophages, along with cytotoxic lymphocytes (CTL), secrete inflammatory cytokines that cause target cell apoptosis. CD8+ CTL also lyse target cells directly. Damage to the gastro-intestinal tract in this phase, principally by inflammatory cytokines, amplifies LPS release and leads to the so-called ‘‘cytokine storm’’ characteristic of severe acute GVHD. This damage results in the amplification of local tissue injury, and it further promotes an inflammatory response [4] (Fig. 1). Based on the above well-established data, studies on immune effectors during GVHD have concentrated on the adaptive T cell response and its hallmarks. However, the role of APCs, especially dendritic cells (DCs) is now well demonstrated as a factor of major importance in the hierarchy of the induction of immune reactions. The field has exploded in recent years, with insights into the role of different cell types and molecular mechanisms from recognition of pathogens to signal transduction and altered gene expression and functions. Also, interest in DCs has grown enormously, based on new knowledge of their integration with specific immune effectors and their key role in stimulating the subsequent clonal response. Thus, GVHD pathophysiology is likely to involve complex interactions where DCs and effectors from the innate immune system might play a major role prior to the establishment of the allogeneic adaptive immune response. Indeed, there is no doubt that activation of donor T lymphocytes is the central event in alloreactivity. However, the concomitant involvement of the innate immune system may play an important role. The latter is supported by findings on prevention of

GVHD by breeding mice under germ-free conditions that initiated a longstanding discussion on the role of antibiotic prophylaxis and the interactions between microbial and specific T cell activation. In addition, with description of cytokines as major effectors of at least acute GVHD, the endotoxin hypothesis postulated that the translocation of bacterial toxins across the gastrointestinal mucosa gave rise to co-stimulation of cytokine release and an alloimmune response [4]. The findings of Beelen et al. [6] demonstrating a strong effect of elimination of anaerobic bacteria on GVHD, and the observations that mice lacking Peyer’s patches or receiving prophylaxis with lactobacilli have a strongly reduced incidence of GVHD, suggested again a more direct interaction of bacterial ligands and activation of specific immune responses. More recently, a seminal work by Holler et al. showed that there is a significant association between the occurrence of single nucleotide polymorphisms (SNPs) within the NOD2/CARD15 gene and GVHD as well as transplant related mortality (TRM) [7,8]. The NOD2/ CARD15 gene had been identified as the first ‘‘susceptibility’’ or ‘‘disease’’ gene in Crohn’s disease, a form of inflammatory bowel disease [9,10]. It codes for an intracytoplasmatic sensor of the bacterial cell wall compound muramyldipeptide (MDP). Ligation of MDP to NOD2/CARD15 induces a signal transduction cascade finally leading to activation of the transcription factor NFKappa-B and subsequent inflammatory response in epithelial cells of the ileum as well as in monocyte/macrophage derived cells [11]. However, one should bear in mind that pathogens’ sensoring in the gut, and the subsequent immune events, are at the heart of APC and DC functions. Thus, these findings move forward our understanding of the clinical pathophysiology of acute GVHD after allo-SCT as they demonstrate a link between DCs and specific post-transplant complications, warranting comprehensive human studies on the major role of DCs in acute GVHD initiation and maintenance. While the above findings highlight the potential role of APCs in GVHD pathophysiology, the recent introduction of the so-called ‘‘non-myeloablative’’, ‘‘mini’’ or ‘‘reduced intensity conditioning’’ (RIC) regimens prior to allo-SCT added a supplementary level of complexity. Indeed, the use of RIC regimens has emerged as an attractive modality for allo-SCT, especially in elderly patients or those with comorbidities precluding the use of standard fully ablative regimens. One common link among the three cardinal organs (skin, gut and liver) affected by GVHD is exposure to the environment. Skin and gut have obvious barrier functions. The liver is the first line of defense downstream of the gut. All of these organs are rich in DCs, a probable prerequisite for antigen presentation. They are all subject to injury from conditioning, and breaches of the barrier would allow microorganisms into the circulation. Therefore, one prediction of the model is that less intense conditioning like in RIC regimens would be associated with less or modified features of GVHD. For instance, when donor lymphocyte

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Fig. 1. Schematic representation of the pathophysiology of GVHD. A body of data suggests that GVHD induction relies on a three sequential phases process: (1) injury to the host tissues mediated by the conditioning regimen, resulting in release of inflammatory cytokines (the so-called ‘‘cytokine storm); (2) DCs/ APCs encounter with donor T-cells leading to their activation, proliferation, and differentiation; (3) activation of a myriad of immune effectors leading to GVHD target tissues destruction through cytotoxic and inflammatory attacks.

infusions (DLI) were administered without pharmacologic GVHD prophylaxis, the resultant GVHD has been less frequent and severe than would be expected in the absence of immunosuppressive drugs [12]. Similarly, the natural history and pathophysiology of GVHD after RIC regimens should be modified, as a result of diminished cellular injury from reduced endotoxin exposure due to less mucosal injury. Overall, the combination of an ever growing body of fundamental knowledge on DCs and their interactions with adaptive and innate immunity effectors, associated to a wider use of reduced intensity and less toxic conditioning regimens prior to allo-SCT allowed better visualization of the fine functions of immune effectors, offering a window of opportunities to better decipher the intimate pathophysiological mechanisms underlying GVHD. The aim of this work is to review the available research evidence (mainly from human studies) on the role of DCs as in vivo regulators of alloimmune reactivity, and their interactions with other immune effectors.

2. Clinical aspects of acute GVHD Historically, GVHD is clinically divided as acute and chronic GVHD based on the time of onset, and usually different clinical presentations and target organs. GVHD occurring within the first 100 days following allo-SCT is called acute GVHD. Chronic GVHD is defined as GVHD

that occurs after 100 days after allo-SCT (with or without preceding acute GVHD), though this time distinction is not always so clear-cut, especially at the era of RIC regimens. Acute GVHD is a clinico-pathological syndrome involving mostly three organs: the skin (Fig. 2), the gastrointestinal tract, and the liver. Any one organ or combination of these organs may be affected. Acute GVHD reaction is directed against many cells of the host including epithelial cells of skin and mucosa, bile ducts of liver, crypt cells of intestinal tract, airways, mucous membranes, bone marrow, and immune system [13]. Clinically significant acute GVHD defined as grades 2–4, occurs in 9–50% of patients who receive an HLA-matched allo-SCT, even when intensive prophylaxis with immunosuppressive drugs such as methotrexate, cyclosporine A, and corticosteroids is used [13]. This incidence may be even higher in unrelated HLAmatched allo-SCT, and even as high as 80–90% in HLAhaploidentical (MHC-mismatched) transplantation [14,15]. Risk of acute GVHD can reach 100% if no GVHD prophylaxis has been used [16]. The incidence of acute GVHD varies also with recipient age, source and number of infused donor T lymphocytes, and GVHD prophylaxis strategy [17]. Other factors play a role in the induction of GVHD. Such other risk factors include the intensity of conditioning regimen, dose of total body irradiation (TBI), underlying primary disease, state of primary alloimmunization (multiple transfusions), gender (such as female multiparous donors), prior splenectomy, and viral infections [17].

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Fig. 2. Example of severe cutaneous GVHD.

3. Dendritic cells as in vivo regulators of alloimmune reactivity The central role of APCs in GVHD induction has been established by elegant murine studies which demonstrated that host APCs alone are sufficient to activate donor T cells [18]. Although alloantigens can be presented directly by host-derived and indirectly by donor-derived APCs, hostderived APCs appear to be critical in inducing GVHD across both minor and MHC mismatches [18,19]. Indeed, using a minor-histocompatibility-antigen disparate models, Shlomchik et al. have demonstrated that the initiation of CD8+ T cell-mediated GVHD and GVT requires donor T cell recognition of host antigen in the context of host APCs [18]. Donor-derived APCs are then able to augment CD8+ T cellmediated GVHD, presumably by acquiring and presenting host antigens (cross-priming) [20]. In full MHC or single MHC-disparate murine bone marrow transplants, antigen presentation by host APCs is critical for the induction of GVT [20–22]. In contrast, there are conflicting data regarding the ability of donor APCs to participate in antitumor responses, which may be related to the differences in the experimental models tested [23]. Furthermore, another murine study identified the enhanced allostimula-

tory activity of host APCs in aged mice as one of the important reasons for greater severity of GVHD in aged recipients [24]. DCs are professional APCs that can be activated by (i) inflammatory cytokines such as TNF-alpha and IL-1, (ii) microbial products such as LPS and toll-like receptors (TLR) ligands entering systemic circulation from intestinal mucosa damaged by conditioning, and (iii) necrotic cells that are damaged by recipient conditioning [25]. These effects are extremely important in producing the ‘‘danger signals’’ that mature DCs and induce T-cell activation, whereas immature DCs induce T-cell tolerance. However, the relative contribution of the different DC subsets, and other ‘‘less’’ professional APCs such as monocytes/ macrophages and B cells in inducing acute GVHD, remains to be elucidated. A working paradigm for DC function holds that immature DCs are distributed in peripheral tissues, and that they are specialized for uptake of pathogen-derived antigens via endocytosis, and an array of surface receptors such as C-type lectins [26,27]. Pathogen-derived products can activate DCs via TLRs to migrate to secondary lymphoid organs and mature into a form specialized for T cell activation rather than antigen uptake [28]. In concert with this, DCs increase expression of MHC and costimulatory molecules, and cytokines that promote adaptive immunity. On the other hand, one should bear in mind that these DCs are also the target of the immune response, and will be eliminated as soon as the infection is controlled. In the context of alloimmune responses, there is not necessarily a defined population of DCs presenting immunogenic antigens. Instead, every APC can present self-peptides, although it is possible that DCs from specific tissues might acquire exogenous antigen from that tissue for presentation to donor T cells. In addition, DC maturation and migration to secondary lymphoid organs is no longer linked to a specific pathogen. In alloimmune responses, DCs stimulated to mature for any reason might become effective APCs for promoting GVHD. And, unlike responses to infection where antigen is largely eliminated, in GVHD, the non-hematopoietic self-antigens are limitless. While GVHD may promote elimination of host hematopoiesis, donorderived DCs will retain their ability to acquire and present host antigens. In this sense, GVHD resembles autoimmune diseases. The maturation status of DCs has also been proposed to play a role in the induction and maintenance of peripheral tolerance, with immature DCs being considered as ‘‘tolerogenic’’ [29]. Thus, one can imagine donor immune effectors entering secondary lymphoid tissues in which they will encounter a mix of mature and immature DCs, but also different DC subsets. In humans, at least two subsets of peripheral blood circulating DCs have been characterized thus far: the myeloid (MDC) and plasmacytoid (PDC) subsets. MDC demonstrate remarkable plasticity and, depending on cytokine environment, can differentiate into either macrophages (with

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M-CSF), or distinct subsets of tissue-localized DC, i.e., epithelial Langerhans cells (with IL-15 or TGF-b) or interstitial DCs (with IL-4). Upon activation, MDCs secrete IL-12 and mature into APCs able to prime T cells. On the other hand, PDC, also known as the type I IFN-producing cells, are a major member of the innate immunity effectors, and represent 0.1–1% of peripheral blood mononuclear cells in both humans and mice. PDC display plasma cell morphology and selectively express TLR7 and TLR9, and are specialized in rapidly secreting massive amounts of type I IFN following viral stimulation. PDC can promote the function of NK cells, B cells, T cells, and MDC through type 1 IFN during an antiviral immune response (for a detailed review, see Ref. [30]. From a functional point of view, Kuwana et al. have reported that circulating PDC can induce Ag-specific anergy in CD4+ T-cell lines [31]. In addition, human PDC activated by phosphorothioated CpG-oligodeoxynucleotides (ODN) prime CD4+ T cells to produce a CD4+CD25+ T cells with regulatory properties [32]. Moreover, human naı¨ve CD8+ T cells primed in vitro with CD40L-activated allogeneic PDC, differentiate into IL-10high-IFN-gammalow-producing regulatory T cells [33]. The mouse homologue of human PDC has been identified in various organs. Freshly isolated mouse PDC precursors (CD11clow-B220+CD11b CD19 ) have poor stimulatory capacity for naı¨ve allogeneic and Ag-specific T cells, consistent with their low surface levels of MHC class II and costimulatory molecules (CD40, CD80, and CD86) [30,34]. Several groups have demonstrated that immature mouse PDC induce T regulatory cells in vitro [35,36] and ex vivo [37]. Martin et al [35] reported that mouse PDC are endowed with the capacity to induce a state of T cell unresponsiveness that involves differentiation of T regulatory cells capable of suppressing model Ag (ovalbumin)-specific T-cell proliferation. In an asthma model, PDC induced differentiation of T regulatory cells capable of suppressing Ag-specific T-cell proliferation [37]. In addition, expression of indoleamine2,3-dioxygenase (IDO), which initiates the tolerogenic pathway of tryptophan catabolism, can be induced in mouse spleen PDC and inhibits Ag-specific T-cell proliferation in vitro and in vivo [38]. Recently, Fallarino et al. [39] have demonstrated that expression and function of IDO are induced in PDC in vivo and that these cells suppress Agspecific responses in response to CD200 receptor engagement. Thus far, only few studies have examined PDC function in vivo. Surprisingly, some groups have demonstrated that PDC do not induce either regulatory activity or tolerance in vivo [40,41]. By contrast, adoptive transfer of PDC has been reported to prevent disease in a mouse asthma model [37]. Most convincingly, Ochando et al. identified PDC as phagocytic APCs essential for tolerance to vascularized cardiac allografts [42]. Tolerizing PDC acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting PDC induced the generation of CCR4+CD4+CD25+Foxp3+ Treg cells. Deple-

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tion of PDC or prevention of PDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized PDC induced Treg cell development and prolonged graft survival, highlighting the role of PDC in allograft tolerance and the existence of a unique partnership between Treg cells and PDCs [42]. The latter is further emphasized if considering data showing that human PDC exploit FcgammaRII (CD32) to internalize antigen–antibody complexes, resulting in the presentation of exogenous antigen to T cells. Such antigen presentation by PDCs was shown to be of particular relevance when circulating antibodies were present, suggesting that antigen presentation by PDC may modulate the strength and quality of the secondary phase of an immune response [43]. However, little is known regarding the influence of PDC on alloimmune responses in humans, especially after alloSCT. Indeed, given their broad spectrum of functional properties, the role of PDC in the outcome of allo-SCT is increasingly becoming an area of considerable interest. Although the evidence is still not conclusive, recent reports suggest that recipient as well as donor DCs may play important roles in the outcome of allo-SCT. Waller et al. [44] reported that larger numbers of PDC in a bone marrow graft resulted in reduced event-free survival and a lower incidence of chronic GVHD. In addition, use of G-CSF (which induces in vivo mobilization of PDC [45]) to expand donor peripheral blood stem cells, did not increase the incidence of acute GVHD despite a higher number of infused T cells [45]. These data suggest that donor PDC have a role in regulating GVHD responses. Also, it is likely that the reconstitution of PDC after allo-SCT is important for immune responses against allo-antigens and pathogens. In healthy donors [45], G-CSF administration selectively increases PDC numbers. In contrast, G-CSF administration after allo-SCT did not influence the reconstitution pattern of PDC and MDCs [46]. Other investigators have shown that reconstitution of PDC after allo-SCT correlates with plasma levels of Flt3L, not G-CSF [47]. Several other studies suggested that the impairment of recovery of DCs significantly increased the risk of relapse and acute GVHD and predicted death after allo-SCT [48]. We previously demonstrated that the reconstitution of the PDC compartment might influence outcome after allo-SCT. The impact of blood PDC recovery was measured at the third month after a RIC regimen in 54 patients who received an HLA-identical sibling allo-SCT. The absence of clinically severe acute GVHD was associated with an improved PDC count. In a multivariate analysis, only a ‘‘high’’ PDC count was significantly predictive of a decreased risk of death [48]. In addition, though one group observed that numbers of PDC were higher at the onset of acute GVHD [46], low PDC counts at engraftment were associated with acute GVHD [49] and acute GVHD impaired PDC recovery 1–3 months after allo-SCT [48,50–52]. Corticosteroid administration also impaired PDC recovery [46,51,52], and patients with

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lower numbers of PDC were more susceptible to viral infection [48,52]. In contrast to findings in acute GVHD, Clark et al. [53] reported that chronic GVHD might be associated with increased numbers of PDC. However, other studies did not find a correlation between PDC number and chronic GVHD [48,50,51]. The worsened outcome in patients with altered DC recovery, further establishes the crucial role of DCs for efficient immune defenses. Unfortunately, the numerous conflicting data in the field of GVHD highlight the need for a comprehensive analysis on the role of DCs in GVHD induction and maintenance of tolerance that would temper the severity of GVHD. Another level of complexity resides in the fact that blood DCs might not completely reflect the status of DCs in GVHD target organs. In this context, another determinant for GVHD development and severity might be the migration and functional status of DCs in target tissues of GVHD. The importance of the latter postulate was recently shown at the level of Langerhans cells (LCs), the only DC subset residing in the epidermis. Confocal microscopy of epidermal sheets showed that full intensity standard myeloablative allo-SCT depletes LCs more rapidly than RIC allo-SCT at day of graft infusion (day 0), although the nadir is similar in both at 14–21 days. Recovery of LCs occurs rapidly within 40 days in the absence of acute GVHD, but is delayed beyond day 100 after allo-SCT when GVHD is active. Acquisition of donor chimerism at day 40 was found to be more rapid after standard myeloablative allo-SCT (97%) than RIC allo-SCT (36.5%), irrespective of blood myeloid engraftment. In this study, at day 100, all patients achieved at least 90% LC donor chimerism and over half achieved 100%. Complete donor chimerism was associated with prior acute cutaneous GVHD, suggesting a role for allogeneic T cells in promoting LC engraftment [54]. On the other hand, and during homeostasis, PDCs are encountered exclusively in the blood and lymphoid organs. However, viral infections lead to an active recruitment of PDCs from the blood into peripheral sites of infection [55]. Recent studies have shown that PDCs may also accumulate in peripheral tissues during certain non-infectious inflammatory disorders, including psoriasis, lupus (SLE), as well as in tumor cells [56–59]. In SLE patients, the numbers of circulating PDCs are decreased, but large numbers of activated PDCs infiltrate the skin lesions and actively produce type I IFN in these patients [56,60,61]. PDCs appear to be activated by immune complexes consisting of antidouble-strand DNA antibodies and DNA derived from apoptotic cells [62]. The high levels of IFN-alpha in the sera of SLE patients were found to activate MDCs to trigger Tcell mediated autoimmunity [62,63], as well to promote differentiation of B cells into antibody-secreting plasma cells [64]. Moreover, PDC-derived IFN-alpha seems to be essential to drive the development of psoriasis in vivo [65]. More recently, activation of IFN pathways and PDC recruitment in target organs of primary Sjo¨gren’s syndrome has been demonstrated, providing new insights into the

pathogenesis of this autoimmune disease [66]. The importance of PDCs in linking innate and adaptive immune responses, and their implication in a number of pathological conditions such as autoimmune diseases, provide a rationale to suspect a role for PDCs in the pathophysiology of acute GVHD that curiously can mimic an autoimmune disorder from a clinical point of view.

4. Inflammatory cytokines, dendritic cells, and effector lymphocytes In standard myeloablative allo-SCT, several lines of evidence have suggested that inflammatory cytokines, particularly TNF-alpha and IL-1, act as mediators of acute GVHD. Perturbation of the cytokine network may function as a final common pathway of target organ damage, and the rapid onset of severe GVHD can be considered a ‘‘cytokine storm’’. The inflammatory cytokines TNF-alpha and IL-1 are usually produced by monocytes, usually considered as the circulating pool of MDCs [67], and macrophages. The stimulus for this secretion may be provided through TLRs by microbial products such as LPS and other microbial components, which can leak through the intestinal mucosa or skin damaged by the conditioning regimen. The role of LPS in acute GVHD has been elucidated in several experimental models [12]. Other murine studies demonstrated that TNF-alpha production by donor cells in response to LPS predicts the severity of GVHD and that direct antagonism of LPS reduces GVHD [68]. Therefore, the gastrointestinal tract plays a major role in the amplification of systemic acute GVHD. In addition to LPS, TNF-alpha plays a critical role in the pathophysiology of acute GVHD both in murine and human studies [69]. Target organ damage could be inhibited by infusion of anti-TNFalpha mAbs [70]. Also, a role for TNF-alpha in clinical acute GVHD has been suggested by studies demonstrating elevated levels of TNF-alpha in the serum of patients with acute GVHD [3]. Given its biology, TNF-alpha may be involved in a multistep process of GVHD pathophysiology: (i) TNF-alpha is known to activate DCs and enhances alloantigen presentation [28]; (ii) TNF-alpha can induce inflammatory chemokines allowing migration of immune effectors [71]; (iii) TNF-alpha can cause direct tissue damage by inducing apoptosis and necrosis [72]. The other well-studied proinflammatory cytokine that plays a role in acute GVHD is IL-1. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD of the spleen and skin, two major GVHD target organs [73]. Mice receiving IL-1 after allo-SCT displayed an accelerated form of GVHD [74]. Although administration of an IL-1 receptor antagonist to recipients reduces GVHD mortality in animal models [75], human studies failed to demonstrate any significant benefit against acute GVHD [76], suggesting that IL-1 might have rather a pleiotropic role in acute GVHD that may be synergistic with TNF-alpha. Such synergy was suggested in a murine study wherein

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mortality and target organ injury was prevented by the neutralization of TNF-alpha and IL-1 [19]. This neutralization was particularly effective for CD4-mediated acute GVHD but also partially for CD8-mediated disease [19]. In addition to TNF-alpha and IL-1, development of acute GVHD is preceded by an increase in serum levels of oxidation products, such as NO [77], that also contributes to the deleterious effects on GVHD target tissues [78]. In order to investigate the ‘‘cytokine storm’’ dogma already established in the standard myeloablative allo-SCT setting, we have studied the role of inflammatory cytokines in acute GVHD incidence and severity in patients who underwent a RIC regimen prior to allo-SCT [79]. Among all tested cytokines in the first 3 months after allo-SCT, only IL12p70 tested within the first month after allo-SCT, was significantly associated with clinically severe GVHD development, and probably through activation of Th1 polarized T lymphocytes and NK cells. Prior to acute GVHD onset, blood monocytes, the main precursor pool of IL-12p70-secreting DCs, recovered more rapidly, in patients with clinically severe acute GVHD. Similarly, at the effectors level, there was a more robust reconstitution of naı¨ve CD3 + CD4 + CD45RA + CD27 + T-cells in patients developing clinically severe acute GVHD [79]. So far, such precise dissection of the pathophysiology of GVHD was rather very difficult in the standard myeloablative allo-SCT setting, and our findings emphasize the fact that the use of minimally toxic conditioning regimens would allow a better definition of the fine mechanisms implicated in alloimmune responses. However, one should bear in mind that IL-12p70 is mainly produced by MDCs following stimulation through several pattern recognition receptors such as TLR or via CD40-CD40L interactions, further supporting a major role of the DC network in the pathophysiology of GVHD. In fact, DCs are able to integrate multiple signals from pathogens, injured tissues and innate leukocytes to direct and ‘‘finetune’’ immune responses [80,81]. DCs are able to recognize

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pathogen-derived molecules including microbial RNA and DNA, peptidoglycans and LPS derived from bacteria in the intestine through several pattern recognition receptors consisting in TLR, c-type lectins [27] or NOD proteins. The NOD2/CARD15 protein belongs to a large family of proteins involved in intracellular pathogen recognition [82]. As seen above, mutations in NOD2/CARD15 and TLR-4 genes are associated with an increased incidence and severity of GVHD following allo-SCT [83], due to a diminished monocyte/macrophage response to bacterial cell wall products. DCs in the intestine are in intimate contact with stromal and epithelial cells. Tissue injury intrinsic to the administration of the conditioning regimen may initiate the breakdown of mucosal barriers, allowing endotoxin into tissues. Therefore, an orchestrated ‘‘dialogue’’ between DCs, T cells and host cells represents a requisite for GVHD induction, and would profoundly affect GVHD severity and overall mortality. Dose and timing of cytokine production are critical factors with regard to their role in the induction of acute GVHD. This is illustrated by the case of IL-10 which is produced by DCs and CD4+ Th2 cells, and is critical for the induction of regulatory T cells (Treg) [84]. Higher production of IL-10 [85], or the presence in recipients of a polymorphism linked with increased IL-10 production [86] is associated with reduced incidence and severity of acute GVHD. Paradoxically, high-serum IL-10 levels in patients after allo-SCT are associated with a fatal outcome [87], whereas administration of low doses of IL-10 is protective in murine acute GVHD [88], highlighting the pleiotropic, but also opposing, nature of cytokines during the different phases of GVHD pathogenesis.

5. Conclusions and perspectives Acute GVHD is a dysregulated immune reaction of the donor immune system to tissue damage that is intrinsic to

Table 2 Potential novel ‘‘pathways’’ for the prevention and treatment of GVHD ‘‘Pathway’’

Treatment

Reduction of the inflammatory component of the conditioning

Nonmyeloablative or reduced intensity conditioning regimens TNF-a inhibitors IL-2 inhibitors

Targeting DCs and APCs

Extracorporeal photophoresis (ECP) Alemtuzumab (anti-CD52 mAb) Antithymocyte globulins (ATG)

Interference with immune effectors traffic and migration

FTY720 Chemokine receptors blockade

Interference with costimulatory molecules (CD28:B7 and TNF:TNF-R super families)

Anti-CD154 mAb

Manipulation of ‘‘regulatory’’ or ‘‘suppressive’’ cells

Total lymphoid irradiation associated with ATG In vitro/in vivo generation of regulatory T cells

Novel relatively ‘‘more selective’’ immunosuppressive agents

Mycophenolate mofetil Pentostatin Sirolimus

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allo-SCT. The donor’s immune system (both innate and adaptive components) reacts as if there is a massive ‘‘attack’’ by ‘‘danger signals’’. Subsequent tissue injuries are correlated among other factors to the intensity of the conditioning regimen that can initiate the breakdown of mucosal barriers, allowing endotoxin into tissues. Undoubtfully, TLRs on DCs bind to endotoxin and activate signal transduction pathways that lead to DC maturation, upregulation of costimulatory molecules, MHC molecules, adhesion molecules, chemokines secretion, fueling the fire. Identification of specific GVHD effectors that can be targeted in vivo will pave the way for therapeutic interventions. Indeed, the success and feasibility of allo-SCT owes much to improvements in the immunosuppressive regimens that prevent or control GVHD. Nevertheless, the potent immunosuppressive drugs that are most widely used (corticosteroids, cyclosporine A and mycophenolate mofetil) increase susceptibility to infection and have adverse effects not directly related to immunosuppression. Corticosteroids for example, affect not only immune cells, but can cause a myriad of harmful side effects. In addition, the mainstay of immunosuppression has been the calcineurin antagonist cyclosporine A. Its immunosuppressive effects can be unpredictable, making monitoring of sera levels essential. Polyclonal antibodies such as antithymocyte globulin can be used, but the combination of these immunosuppressive antibodies with other immunosuppressive drugs may be associated with an increased risk of post-allo-SCT opportunistic infections. The immune system itself holds a clue to what may be a new era in immunosuppression, based on selective, and relatively nontoxic immunosuppressive agents (Table 2). As DCs can mediate both immunostimulation and immune tolerance, their manipulation in allo-SCT holds promise for modifying the alloimmune response to achieve a clinical benefit. Moreover, it makes sense to manipulate T-cell responses from the first steps, when T cells initially encounter DCs. Identification of such new immunosuppressive agents will likely come from systematic approaches, aiming to understand which molecules, immune effectors, and/or signaling networks cooperate to regulate immunity when donor effector cells encounter host cells.

Acknowledgements We would like to apologize for the colleagues whose work could not be cited because of space restrictions. We thank the ‘‘Association pour la Recherche sur le Cancer (ARC)’’, the ‘‘Ligue Nationale contre le Cancer’’, the ‘‘Fondation de France’’, the ‘‘Fondation contre la Leuce´mie’’, the ‘‘Agence de Biome´decine’’, the ‘‘Association Cent pour Sang la Vie’’, and the ‘‘Association Laurette Fuguain’’, for their generous and continuous support for our clinical and basic research work.

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M. Mohty, B. Gaugler / Cytokine & Growth Factor Reviews 19 (2008) 53–63 reviewer in different immunology, hematology and cancer journals. Dr Mohty is member of the American Society of Hematology (ASH), American Society for Clinical Oncology (ASCO), European Hematology Association (EHA), International Society for Experimental Hematology (ISEH), and EBMT. Be´atrice Gaugler, PhD is a senior researcher at the ‘‘Institut National de la Sante´ et de la Recherche Me´dicale (INSERM UMR 645) in Besanc¸on, France. Dr. Gaugler obtained her PhD degree from the University of Marseille, France, where she trained at the ‘‘Centre d’Immunologie de Marseille-Luminy’’ (CIML). She also trained for 4 years as a post-doc at the Brussels Branch of the Ludwig Institute for

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Cancer Research (Belgium) directed by Prof. T. Boon, where she actively contributed to the characterization of human tumor antigens. She later joined as a post-doc followed by a senior researcher position the Immunology of tumors Laboratory at the Institut Paoli-Calmettes, the regional cancer centre in Marseille, INSERM UMR 599, where she stayed for 10 years before moving to Besanc¸on. Dr. Gaugler’s basic and translational research activities are currently focused on different aspects of the immunbiology of stem cell transplantation (immune reconstitution, donor-host interactions, anti-viral immunity, etc.), especially the pathophysiology of graft-versus-host disease (GVHD) and the immunobiology of normal and pathological antigen-presenting cells in the context of haematological malignancies and stem cell transplantation.