Role of TH17 Cells and Interleukin 17 in Graft Versus Host Disease and Graft Versus Leukemia Reactivity

Chapter 14

Role of TH17 Cells and Interleukin 17 in Graft Versus Host Disease and Graft Versus Leukemia Reactivity Clint Piper1 and William R. Drobyski1, 2, 3, 4 1

Microbiology, Medical College of Wisconsin, Milwaukee, WI, United States; 2Blood and Marrow Stem Cell Transplant Program, Medical College of

Wisconsin, Milwaukee, WI, United States; 3Departments of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United States; 4Medicine, Medical College of Wisconsin, Milwaukee, WI, United States

Chapter Outline Discovery of TH17 Cells Differentiation and Actions of TH17 Cells Interaction of TH17 Cells with TH1, TH2, and Tregs Role of IL-17 and TH17 Cells in Preclinical Models of Acute GVHD Role of IL-17 and TH17 Cells in Chronic GVHD Role of Cytokines That Induce TH17 Differentiation in GVHD Interleukin 23 Interleukin 6 TGF-b Role of Transcription Factors Involved in TH17 Cell Differentiation in GVHD

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Role of IL-17 and TH17 Cells in Clinical GVHD Role of IL-17 and IL-23R Polymorphisms in GVHD Role of Other TH17 Cytokines in GVHD Biology Interleukin 21 Interleukin 17F Interleukin 22 Role of IL-17 and TH17 Cells in GVL Reactivity Summary Unanswered Questions Acknowledgments References

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DISCOVERY OF TH17 CELLS CD4þ helper T cells play an important role in the initiation and regulation of immune responses in vivo. Naïve CD4þ T cells proliferate and differentiate into different effector subsets when activated and this is largely influenced by the local cytokine milieu at the time of activation. Historically, T helper cells have been divided into two distinct subsets, termed T helper 1 (TH1) and T helper 2 (TH2) based on their distinct cytokine profiles and effector functions [1]. TH1 cells secrete large amounts of interferon-g (IFN-g) upon activation. These cells are important in mediating cell-mediated immune responses and are highly effective in eradicating intracellular pathogens. On the other hand, TH2 cells secrete IL-4, IL-5, and IL-13 and are mainly involved in humoral immunity such as allergic immune responses and antiparasitic immunity [2]. For some time, this TH1 and TH2 classification has formed the basis for our understanding of CD4þ T-cell biology. This paradigm, however, began to be altered approximately 25 years ago when Rouvier [3] and then Yao and colleagues [4] described a new cytokine in mice which they termed IL-17 and which had homology to a gene in herpesvirus saimiri. The human counterpart of murine IL-17 was soon cloned by Fossiez and colleagues [5]. A number of studies quickly established that IL-17 had proinflammatory properties and could induce the production of inflammatory cytokines such as IL-1b, TNF-a, IL-6, IL-8 along with hematopoietic cytokines such as G-CSF [5,6]. The elucidation of TH17 cells as another CD4þ T cell subset, however, did not come for several more years and was facilitated by the discovery of interleukin 23 (IL-23), which was identified as a member of the IL-12 family. IL-12 is important in driving naïve CD4þ

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T-cell differentiation into the TH1 pathway [7] and is composed of two subunits, termed p35 and p40. Oppmann and colleagues in 2000 reported a new cytokine, IL-23, in which the p40 subunit previously assumed to be exclusive to IL-12 paired with a new p19 subunit to form a heterodimeric cytokine [8]. Thus, IL-12 and IL-23 share the common p40 subunit, but they also have unique subunits, p35 for IL-12 and p19 for IL-23. The subsequent investigation into the role of IL-23 led to the seminal studies performed by Cua and colleagues [9] that showed that mice deficient in the IL-23 p19 subunit were highly resistant to the development of autoimmunity, whereas those mice deficient only in the IL-12 p35 subunit were highly susceptible to experimental autoimmune encephalomyelitis (EAE) induction. Moreover, IL-23 gene transfer vectors delivered into the central nervous system (CNS) reconstituted EAE in both p19- and p40-deficient mice while IL-12 gene transfer into the CNS did not facilitate disease in p40-deficient animals. Thus, IL-12 and TH1 cells were not required for induction of EAE, and IL-23 was shown to be a crucial cytokine for the development of CNS autoimmune inflammation [9]. These observations revealed IL-23 as a critical driver of autoimmune inflammation and suggested that T cells that differentiate under the influence of IL-23 are key players in the induction of autoimmunity. A link between IL-23 and IL-17 was then established by studies showing that IL-23 could promote the secretion of IL-17 by activated T cells [10], and that IL-23 enhanced the expansion of CD4þ IL-17 secreting T cells in various murine models of autoimmunity [11e13]. These investigations also demonstrated a unique cytokine and gene expression profile for these cells that distinguished them both TH1 and TH2 cells [12]. Finally, elegant work identified that TH17 cells expressed a transcription factor termed retinoic aciderelated orphan receptor gt (RORgt) [14] which distinguished them from TH1 and TH2 cells and clearly demarcated these cells as a unique T helper population.

DIFFERENTIATION AND ACTIONS OF TH17 CELLS The differentiation of helper T cells requires coordinated cytokine signaling that induces the activation of specific transcription factors to promote lineage-specific CD4þ T-cell differentiation. While T-box-containing protein expressed in T cells (T-bet) is activated by IL-12 and IFN-g and is exclusively expressed during TH1 cell differentiation [15], GATAbinding protein 3 (GATA-3) is required for TH2 cell polarization [16]. TH17 cells have a specialized developmental pathway that is distinct from TH1 or TH2. It has been shown that TH17 cells differentiation from naïve T-cell precursors does not depend on the transcription factors, signal transducer and activator of transcription 1 (STAT1), STAT4, and/or STAT6, and they do not express any conventional transcriptional factors involved in TH1 and TH2 differentiation [17,18]. Initially, it was reported that IL-23 was an important factor for the development of TH17 cells [10,19]. However, naïve murine T cells do not express the IL-23 receptor and do not differentiate into TH17 cells in the presence of IL-23 in vitro [20]. Therefore, IL-23 is not required for the initial differentiation of TH17 cells from naïve CD4 T cell precursors, although it appears to have a role in the maintenance of effector function once these cells are induced [21]. Three independent groups made the observation that a combination of transforming growth factor b (TGF-b) plus IL-6 induced the differentiation of naïve T cells into TH17 cells [20,22,23]. This cytokine combination activates STAT3 and induces the expression of the transcription factor RORgt, which in turn induces transcription of the IL-17 gene in naïve T cells [14,24]. A deficiency of suppressor of cytokine signaling 3 (SOCS3), which is a negative regulator of STAT3 phosphorylation, significantly enhances TH17 generation [25e28]. IL-21 has also been reported to play an important role in the generation of TH17 cells. IL-21 is potently induced by IL-6 [29] and in vitro differentiated TH17 cells express much higher levels of IL-21 mRNA and protein [30,31]. In fact, in the absence of IL-6, IL-21 in combination with TGF-b can function as an alternative signal for the induction of TH17 cells [31]. Other proinflammatory cytokines such as TNF-a and IL-1b have also been shown to facilitate TH17 cell differentiation [32,33]. TH17 cells are characterized by the production of several distinct cytokines that are not typically secreted by TH1 and TH2 cells. In addition to the signature cytokine IL-17A, TH17 cells also preferentially secrete IL-17F, IL-21, IL-22, and, in humans, IL-26 [34]. These cytokines play an important and nonredundant role in enhancing the host defense against extracellular pathogens, such as Klebsiella pneumoniae, Candida albicans, and Citrobacter rodentium [34]. It is thus believed that TH17 cells have evolved primarily to provide inflammatory protection against extracellular pathogens. Signaling by IL-17, in general terms, occurs through a receptor complex composed of IL-17RA and IL-17RC. IL-17RA is ubiquitously expressed but preferentially on hematopoietic cells, while IL-17RC is expressed only on nonhematopoietic tissues [35]. There are differences, however, between IL-17 signaling pathways between the mouse and humans. In man, IL-17A and IL-17F can bind to both IL-17RA and IL-17RC. In contrast, in mice, IL-17A signals through IL-17RA, whereas IL-17F binds to both IL-17RA and IL-17RC [36]. These differences may be responsible for some of the unique actions of each cytokine.

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INTERACTION OF TH17 CELLS WITH TH1, TH2, AND TREGS A balanced interaction between different T-cell subsets is very important in maintaining immune homeostasis in vivo. Earlier studies on naïve CD4þ T-cell differentiation into TH1 and TH2 cells revealed that these subsets were capable of considerable cross-regulation. Thus, TH1 and TH2 cells inhibit the development of one another through the action of their lineage-specific cytokines IFN-g and IL-4, respectively [37,38]. This principle also holds true for TH17 cells as it has been shown that the differentiation of TH17 cells can be inhibited by IFN-g and IL-4 [17,18]. However, once fully matured, TH17 cells are resistant to the suppressive effects of IFN-g and IL-4 in vitro [17,18]. TH17 and TH1 cells have been found to coexist in inflammatory lesions that develop in a number of autoimmune diseases [39], suggesting the possibility of redundant functional relationships between these lineages in disease pathology [40]. Emerging evidence has suggested that mature TH17 exhibit significant lineage plasticity. Three independent groups simultaneously observed that in vitro polarized TH17 cells which initially express IL-17 can become IL-17þ/IFN-gþ double producing cells or lose IL-17 production capacity entirely and become IFN-gþ “nonclassical TH1” or “ex-TH17” cells [41e43]. Evidence for this population was further bolstered with the development of an IL-17 fate reporter mouse, in which cells that express IL-17 at any point in their lineage are permanently labeled with YFP [44]. Since their discovery, ex-TH17 cells have been noted in mouse models and human patients with autoimmune disease, including rheumatoid arthritis [45,46], EAE [44], and colitis [47] and have been observed to exhibit pathogenic features, including reduced suppression by regulatory T cells. The switch from an IL-17 producing TH17 phenotype to an IFN-g producing phenotype appears to be at least partially mediated by signaling through the IL-23 receptor. Though this population has not been studied in the context of GVHD, it may be one reason that therapies targeting the IL-23 signaling pathway have been met with moderate success in autoimmune disease. Recent studies have also demonstrated that TH17 cells can undergo phenotype switching to become either IFN-g expressing or IFN-g/IL-17 coexpressing cells when exposed to certain cytokine milieus at the inflammation site or during in vitro culture under TH1 polarization condition [42,48]. These studies thus suggest that changes in the cytokine milieus during T helper cell differentiation can mediate significant phenotypic shifts related to cytokines expressed by TH17 cells. An important aspect of TH17 cell differentiation is the relationship that the differentiation of these cells has with regulatory T cells (Tregs). Tregs are critical for the maintenance of immunological homeostasis and self-tolerance in vivo [49]. These cells have been classically defined as being CD4þ and expressing the transcription factor Foxp3 [50]. This population of cells is comprised of two major subsets which have been termed natural (nTregs) and induced (iTregs) based on the unique ontological and developmental characteristics that are specific for each cell population [51]. nTregs are generated in the thymus and then migrate to the periphery to regulate immune responses, whereas iTregs derive from the conventional T-cell pool and undergo conversion in the periphery after encounter with antigen in the presence of TGF-b [52,53]. Work by Kuchroo and colleagues established that IL-6 is a pivotal cytokine that controls the differentiation decision of naïve T cells to become either iTregs or TH17 cells. In the presence of IL-6, T cells differentiate into TH17 cells whereas in the absence of this cytokine the development of iTregs is fostered [23] (Fig. 14.1). Moreover, Foxp3 itself is able to associate with RORgt and inhibit RORgt transcriptional activation [24]. Thus, IL-6 acts as a potent

FIGURE 14.1 Differentiation of CD4D T-cell lineages. The cytokines associated with arrows indicate dominant cytokines involved in the specification of each of the indicated lineages. The cytokines listed below each cell type indicate key effector or regulatory cytokines produced by differentiated cells of that lineage or, in the case of nTregs, a contact-dependent mechanism of suppression. Tn, naïve post-thymic CD4 T cell precursors; Tp, thymic precursors. Dotted lines represent less well-defined lineage relationships. Reprinted with permission.

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proinflammatory cytokine through promotion of TH17 differentiation and inhibition of Treg differentiation, altering the balance between effector and regulatory T cells.

ROLE OF IL-17 AND TH17 CELLS IN PRECLINICAL MODELS OF ACUTE GVHD The critical role that TH17 cells and IL-17, more specifically, play in inflammation was a natural impetus for the subsequent inquiry into the role that these cells play in GVHD biology. When considering the effect of IL-17 in GVHD biology, one needs to distinguish between the cytokine itself and the helper T-cell population known as TH17 cells. This distinction is important due to the fact that TH17 cells secrete a number of cytokines (e.g., IL-22, IL-21, IL-17F) in addition to IL-17A. Thus, simply equating TH17 cells with the secretion of IL-17 is an oversimplification of the role that these cells may indeed play in the pathophysiology of GVHD. For the purposes of this review, unless otherwise specified, IL-17 refers to IL-17A and should be considered to be distinct from other IL-17 cytokine family members. A requirement for IL-17 in the pathogenesis of acute GVHD has been formally examined in several studies in which mice deficient in the secretion of this cytokine were employed as donor animals in transplant experiments. The first report by Yi and colleagues [54] employed a B6/Balb/c transplantation model and somewhat surprisingly showed that animals transplanted with IL-17/ grafts had more severe diarrhea, weight loss, and significantly reduced survival compared to recipients transplanted with grafts from wild-type donors. Furthermore, serum IFN-g and TNF-a levels were significantly elevated in the former animals and this was accompanied by an increase in donor IFN-g-secreting CD4þ and CD8þ T cells in both mesenteric lymph nodes (MLNs) and spleen, indicative of enhanced TH1 differentiation by IL-17/ donor T cells. Neutralization of IFN-g by in vivo administration of a blocking antibody or supplementation of exogenous IL-17 protected mice from an exacerbation of GVHD lethality by inhibiting TH1 differentiation. Thus, whereas the selective absence of donor IFN-g production has been shown to exacerbate GVHD in certain tissue sites such as the GI tract [55], blockade of IFN-g signaling under conditions where there was concurrent IL-17 deficiency was actually able to mitigate the severity of GVHD. Similarly, the fact that the exogenous administration of IL-17 was able to blunt the severity of GVHD was evidence that there was to some extent reciprocal regulation between IFN-g and IL-17, and by extension, TH1 and TH17 cells. The concept that these two cytokines may serve to regulate each other was bolstered by data showing that pulmonary inflammation induced in the absence of IFN-g results in the emergence of pathogenic IL-17-producing CD4þ T cells [56]. In a subsequent paper, Kappel and colleagues [57] confirmed that donor-derived IL-17 secreting CD4þ T cells were generated early post transplantation, and that a significant percentage of them secreted both IL-17 and IFN-g. A new finding of this paper was that other TH17 cellederived cytokines such as IL-22 and IL-17F were also increased in secondary lymphoid tissue of GVHD recipients. Notably, this was only observed early post transplantation within the first 7e14 days and was not seen in all tissue sites. Unlike the study by Yi et al., however, there was no difference in GVHD mortality between recipients that received whole T cells from wild-type versus IL-17/ animals. When purified CD4þ T cells were examined as a donor T-cell source, there was a delay noted in the onset of mortality in recipients of IL-17deficient grafts, but overall GVHD mortality was unaffected. Thus, one would conclude from this work that the absence of donor-derived IL-17 had no appreciable effect on GVHD severity and, in particular, did not lead to an exacerbation of disease. The reason for these disparate results is not entirely clear since a similar murine model was employed for at least some of the studies in both of the reports. An alternative approach to investigate the role of IL-17 was employed by Carlson and colleagues who used an in vitro culture system to generate highly purified TH17 cells for subsequent transplantation into recipient animals [58]. Notably, the majority of these cells expressed IL-17A and IL-17F by intracellular cytokine staining indicating that at least two IL-17 family members were induced under these conditions. When these highly purified cells from B6 mice were adoptively transferred into lethally irradiated MHC-incompatible recipients, they were sufficient to induce lethal GVHD and also acted synergistically with naïve T cells to mediate GVHD. The most significant finding was that GVHD induced by these in vitroegenerated cells was characterized by preferential pathological damage in the skin and lung of recipient mice when compared to either whole T cells or purified CD4þ T cells. It is noteworthy that in vitroedifferentiated TH17 cells also expressed both IL-21 and IL-22 so it remains uncertain whether these cytokines might contribute to some of the pathological manifestations observed under these conditions. The ability of in vitroepolarized TH17 cells to induce lethal GVHD and, more precisely, augment pathology in the lung relative to TH1 cells was confirmed by Iclozan and colleagues [59]. They also noted instability of the cytokine profile in vivo after transfer, with increased production of both IFN-g and TNF-a but, as Carlson et al. observed, IFN-g was not required for GVHD mediated by TH17 cells. In this report, the authors parenthetically noted that skin pathology was not increased, but this may have been attributable to different murine models being employed. More recently, Uryu and colleagues have shown that a-mannan, a cell wall component of

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Candida albicans, is capable of inducing TH17 cells which can cause GVHD in the lung, providing an alternative pathway by which these cells may contribute to GVHD in this tissue site [60]. While a majority of early studies focused on CD4þ IL-17þ T cells, Gartlan and colleagues [61] recently have identified a population of IL-17-producing CD8þ T cells that emerges early during GVHD. Interestingly, these cells fail to maintain lineage stability, but produce an array of inflammatory cytokines and exacerbate lethal GVHD. Notably these cells are noncytolytic and do not mediate graft versus leukemia (GVL) effects, raising the possibility that they might be a viable target population to separate GVH and GVL reactivity. Finally, a recent publication [62] has indicated a protective role for recipient IL-17 production within the gastrointestinal tract. Animals deficient in either IL-17 or IL-17 receptor subunits had increased GVHD lethality. The inability to signal through IL-17 resulted in an alteration in the microbiome which could be transferred in cohoused wild-type animals resulting in enhanced acute GVHD severity. Thus, the effects of IL-17 in GVHD biology appears to extend beyond direct actions that influence immune cell populations and inflammatory cytokine production to those that modulate and alter the microbiome. Moreover, IL-17 may have discordant effects depending upon whether production is from the donor or recipient. The prior studies raised the interesting question as to whether various T helper populations (i.e., TH1, TH2, TH17) might have unique and nonredundant roles in mediating GVHD pathology in specific target organs. To address this question, Yi and colleagues [63] used cytokine double knockout donor T cells (IFN-g/IL-4/ and IFN-g/IL-17/) to delineate the relative contribution of TH1, TH2, and TH17 cells to the pathogenesis of acute GVHD and to study how different subsets of CD4þ T cells induced GVHD in different target organs. Using a standard B6/Balb/c GVHD model, they reaffirmed that acute GVHD in this setting is primarily a TH1-mediated event. In the absence of the TH17-associated cytokine IL-17, or TH2-associated cytokine IL-4, TH1 differentiation was further augmented, which led to an exacerbation of tissue damage in the gut and liver. In contrast, absence of IFN-g in CD4þ T cells resulted in enhanced TH2 and TH17 differentiation and exacerbated tissue damage in both the lung and skin. The absence of both IL-4 and IFN-g resulted in increased TH17 differentiation and preferential tissue damage in skin, whereas absence of both IFN-g and IL-17 led to further augmentation of TH2 differentiation and lung damage. The tissue specificity mediated by TH1, TH2, and TH17 cells was attributed, in part, to the differential expression of chemokine receptors. TH1 cells, which preferentially cause GVHD in gut and liver, expressed high levels of the gut-homing receptor a4b7 and CCR9 as well as the liver-homing receptors CCR5 and CXCR6. In contrast, TH2 and TH17 cells, which preferentially caused GVHD in skin and lung, expressed high levels of lung- and skin-homing molecules CCR3, CCR4, and CCR6. In addition, the expression of ligands for different homing molecules was increased in the respective target organs. The major caveat with these studies is that the forced expression of T cells along select differentiation pathways may not necessarily be an accurate reflection of what occurs when CD4þ T cells have unrestricted options available to them.

ROLE OF IL-17 AND TH17 CELLS IN CHRONIC GVHD Clinically, GVHD has been divided into acute and chronic phases that have historically been distinguished primarily by their temporal onset. In contrast to acute GVHD, where pathology is generally restricted to the skin, liver, and intestinal tract, chronic GVHD has unique clinical features that include more extensive organ involvement. Frequently, this disease involves the lung, eyes, and mucous membranes, which are tissue sites that are generally unaffected during acute GVHD [64]. Moreover, chronic GVHD often presents with clinical manifestations that resemble those seen in autoimmune diseases such as systemic lupus erythematosus, Sjogren’s syndrome, scleroderma, and rheumatoid arthritis [65e67]. These distinguishing clinical features of chronic GVHD have been the impetus for the reclassification of this disease with diagnostic criteria that, in part, highlight the similarities that this syndrome has with other autoimmune disorders [68]. These new criteria are meant to emphasize that chronic GVHD is not merely an extension of antecedent acute GVHD, but is a distinct clinical and pathophysiological entity. Whereas the pathophysiology of acute GVHD has been extensively examined using a variety of murine BMT models, the pathogenesis of chronic GVHD is much less well understood. That being said, a number of studies have recently been conducted in an effort to not only uncover the pathophysiology of chronic GVHD but also to delineate the role that IL-17 and TH17 cells may play in this disorder. It should be emphasized, however, that since there is no animal model that recapitulates all of the varied manifestations of chronic GVHD, the studies which have been conducted have generally examined select, yet important, features of this protean disorder. A potential pathogenic role for IL-17-secreting CD4þ T cells was initially supported by studies which were conducted in an effort to understand how acute GVHD which is a proinflammatory syndrome transitions into chronic GVHD which is characterized by features of autoimmunity [69,70]. Using an adoptive transfer model, we observed that spleen cells from

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mice undergoing acute GVHD could induce pathological damage in secondary immunodeficient animals of the same donor major histocompatibility complex (MHC) type. This was determined to be mediated by CD4þ T cells which were responsible for inducing pathological damage in the colon and liver. The fact that T cells in both primary GVHD animals and secondary immunodeficient mice were of the same MHC type was evidence that these T cells were reactive against self-antigens [69]. Subsequent studies demonstrated that GVHD-associated autoimmunity and, by extension, chronic GVHD was attributable to the progressive loss of CD4þCD25þFoxp3þ regulatory T cells during the course of acute GVHD [63]. This led to the expansion of donor-derived CD4þ T cells with both TH1 and TH17 cytokine phenotypes along with the overproduction of proinflammatory cytokines, including IFN-g and IL-17. Intracellular cytokine staining confirmed the presence of TH1 and TH17 cells in spleen and target organs of autoimmune mice after adoptive transfer. The cotransfer of purified donor-type Tregs completely protected mice and led to a substantial reduction in the absolute number of IFN-g and IL-17-producing CD4þ T cells, confirming that loss of effective regulation was responsible for autoimmunemediated pathology. Notably, the majority of TH17 cells identified in this chronic GVHD model produced both IFN-g and IL-17. This finding has been observed in acute GVHD [49] as well as other autoimmune disease models where CD4þIL17þIFN-gþ T cells have been postulated to have an etiological role in the pathogenesis of the disease [71]. Whether this population has a unique inflammatory role in GVHD biology will require further study. In subsequent studies, we examined whether there was an obligate requirement for donor-derived IL-17 production in GVHD-associated autoimmunity which manifests a proinflammatory syndrome in this model. Using a combination of antibody-based and genetic approaches, we observed that IL-17 was not required for the loss of self-tolerance and resulting CD4þ T cell-dependent pathologic damage that occurs during the evolution from acute to chronic GVHD [72]. Specifically, anti-IL-17 antibody treatment had no effect on the development of autoimmunity in recipient mice. Moreover, no difference was observed in the percentage of CD4þIL-17þ or CD4þIFN-gþ T cells in either the spleen or the colon of anti-IL-17 versus isotype antibody-treated mice, indicating that anti-IL-17 antibody administration did not appear to alter the differentiation of naïve T cells into IL-17 or IFN-g-secreting CD4þ T cells. These studies, did not, however, define the precise role of IL-17 in this process or the extent to which IL-17 and IFN-g may cooperatively function to induce disease. G-CSF is a cytokine widely used in allogeneic hematopoietic stem cell transplantation. G-CSF mobilized peripheral stem cell transplantation is associated with several favorable outcomes such as faster hematopoietic reconstitution, improved GVL effect, and similar severity of acute GVHD despite higher T-cell number infused when compared with traditional bone marrow transplantation. However, this treatment is also associated with increased chronic GVHD [73]. A study by Hill and colleagues has provided a potential explanation for some of these observations [74] and thereby delineated a new role for IL-17 in chronic GVHD. In this study, the authors observed that G-CSF treatment of donor mice promoted the generation of TH17 cells as demonstrated by enhanced production of the signature cytokines IL-17A and IL-17F. G-CSF triggered IL-17A production in both CD4þ and CD8þ T-cell populations with enhanced production of IL-17A in the absence of CD8þ T cells. Interestingly, G-CSF promoted TH17 differentiation independent of cytokines TGF-b and IL6, two cytokines widely reported to induce TH17 cell generation. Instead, IL-21 was critically important because the absence of IL-21 signaling completely abolished the enhancement of IL-17 production. When G-CSF-mobilized cells were used for transplantation, there was IL-17A-dependent pathologic damage observed in the skin of recipient animals, whereas GVHD of the gastrointestinal tract was IL-17A independent. Notably, donor CD8þ, and not CD4þ, T cells were the primary source of IL-17 and IL-21 production after transplantation. Donor CD8þ T cellederived IL-17A production after G-CSF treatment was shown to be important for promoting macrophage infiltration and the subsequent development of cutaneous fibrosis, which is one the hallmarks of chronic GVHD in man. A follow-up study by this group [75] demonstrated that donor-derived CSF-1-dependent macrophages are the downstream mediators of IL-17-dependent chronic GVHD manifestations in both the skin and lung. Thus, these studies provide evidence of a pathophysiological role for IL-17 in the induction of fibrosis, which is a hallmark of chronic GVHD. A putative role for IL-17 in the pathogenesis of chronic GVHD of the skin is also supported by studies which were conducted using a B10.D2/Balb/c murine model in which recipients develop skin pathology that is also observed in human chronic GVHD. The authors demonstrated that systemic manifestations, and more specifically, skin changes were significantly attenuated in recipients that received IL-17/ donor T cells [76]. Thus, unlike in acute GVHD, transplantation with IL-17-deficient grafts reduced the severity of GVHD. However, this was only observed in the skin and salivary glands, whereas there was no difference in pathological damage in the lung, liver, or colon. Studies by both groups have therefore implicated a role for IL-17 in the induction of fibrosis in the skin, but whether pathology in other tissues sites is attributable to IL-17 is still unclear.

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ROLE OF CYTOKINES THAT INDUCE TH17 DIFFERENTIATION IN GVHD The cytokines TGF-b, IL-23, and IL-6 have all been shown to be involved in the differentiation of TH17 cells from naïve T cells, and/or the subsequent expansion and maintenance of this helper T cell population. Within recent years, a number of studies have been performed in an attempt to determine the role of these cytokines in the pathophysiology of GVHD biology.

Interleukin 23 IL-23 was initially thought to be required for the differentiation of naïve T cells into TH17 cells when it was first described [10], but subsequent studies determined that this was incorrect [20,22,23]. Rather, IL-23 appears to be necessary for the maintenance of effector function in vivo [21]. This cytokine has also been shown to play a critical role in mediating pathological damage in several mouse models of autoimmunity [11e13] which is a characteristic of GVHD. Furthermore, a linkage between IL-23 and autoimmunity has been strengthened by a considerable body of genetic evidence in man that links IL-23R polymorphisms with susceptibility to a range of autoimmune diseases such as Crohn’s disease, multiple sclerosis, and psoriasis [77e79]. For these reasons, we conducted studies to examine the role of IL-23 in the biology of GVHD. We observed that IL-23 has a unique and selective role in the induction of colonic inflammation during acute GVHD and serves as a critical mediator linking conditioning regimeneinduced mucosal injury and LPS translocation to subsequent proinflammatory cytokine production and GVHD-associated pathological damage [80]. Specifically, transplantation of IL-23-deficient marrow grafts significantly reduced the severity of acute GVHD, which was attributable to the preferential reduction in colonic GVHD and a decrease in the production of proinflammatory cytokines within this tissue site. Secretion of IL-23 by donor, and not host, antigen-presenting cells (APCs) was shown to be a critical event in the induction of GVHD of the colon. These findings indicated a novel organ-specific role for IL-23 in the pathophysiology of GVHD and demonstrated that IL-23 can direct tissue-specific pathology within the context of a systemic inflammatory disorder. In a follow-up study, we identified a CD4þ IL-23Rþ T-cell population that expresses the beta 2 integrin, CD11c, and is a primary driver of colonic inflammation during GVHD [81]. Interestingly, while the majority of experimental colitis models have implicated TH17 cells as the downstream effectors of IL-23-induced mucosal pathology [11,82,83], these studies did not reveal a role for IL-17 in the pathophysiology of GVHD in the colon. Elevated levels of IL-23 were not accompanied by corresponding increases in IL-17 in the colon microenvironment and IL-17 mRNA levels were virtually undetectable in the colons of these same animals. CD4þIL-17þ cells were also present only in negligible numbers in the spleen, liver, and colon of GVHD animals. Furthermore, transplantation with marrow grafts from IL-17/ donors had no protective effect on either overall or colon-specific GVHDassociated pathology, providing evidence that the proinflammatory effects of IL-23 were independent of IL-17. Rather, these studies indicated that the downstream proinflammatory effects of IL-23 were dependent upon donor-derived secretion of IFN-g as the absolute number of CD4þ IFN-gþ cells in the colon was significantly increased relative to CD4þIL-17þ cells. These results confirmed that mucosal pathology mediated by IL-23 could occur in a TH17independent manner which has been reported in nontransplant studies, supporting the existence of an alternative pathway whereby the proinflammatory effects of IL-23 in the colon are mediated through secretion of IFN-g [84,85]. Notably, blockade of IL-23 signaling as a strategy to prevent GVHD may soon be able to be examined in patients, given that several p19-specific antibodies are in clinical development for the treatment of a variety of autoimmune diseases [86,87].

Interleukin 6 IL-6 is a proinflammatory cytokine that has been shown to be crucial in initiating a TH17 immune response. In the presence of IL-6 and TGF-b, naïve T cells differentiate into Th17 cells, whereas in its absence these same cells are induced to become Tregs [22,23]. Furthermore, IL-6 produced by dendritic cells after activation through Toll-like receptors is able to inhibit the suppressive function of natural Tregs [88,89]. Thus, IL-6 appears to have a pivotal role in directing the immune system toward an inflammatory response. This is of particular interest with respect to GVHD biology since the pathogenesis of GVHD is characterized by an imbalance between the effector and regulatory arms of the immune system. The role of IL-6 in GVHD biology in mice has been examined by two groups. Chen and colleagues [90] performed experiments to examine how IL-6 contributed to the pathophysiology of GVHD using antibody-mediated blockade of the IL-6 receptor (IL-6R). These studies determined that blockade of IL-6 signaling markedly reduced pathologic damage attributable to GVHD. Notably, the colon was the site of most pronounced protection after blockade of IL-6 signaling, suggesting that

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IL-6 has a pivotal role in mediating GI tract inflammation. This was accompanied by a significant increase in the absolute number of regulatory T cells (Tregs) that was due to augmentation of both thymic-dependent and thymic-independent Treg production. Blockade of IL-6 signaling was also associated with a significant reduction in the number of TH17 cells in all tissue sites. These data indicate that blockade of IL-6 signaling was able to divert the differentiation of naïve T cells away from the TH17 cell lineage into the Treg lineage, and thereby serve to recalibrate the effector and regulatory arms of the immune system. There was also a significant reduction in the absolute number of CD4þIFN-gþ TH1 cells as well in GVHD target organs, implying that blockade of IL-6 inhibited the expansion and/or differentiation of TH1 cells. Similar results have been reported before in a model of experimental allergic encephalomyelitis [91]. Protection from GVHD by way of IL-6 blockade was confirmed by Tawara and colleagues [92]. In addition, they demonstrated that the GVL effect could also be preserved in animals that were protected from lethal GVHD. However, a notable difference between the two studies was that the latter did not observe an increase in Treg reconstitution after BMT. This discrepancy could be due to a different antibody dosing and administration schedule which could have affected Treg regeneration. It should be noted that blockade of IL-6/IL-6R interactions is currently a clinically feasible option given that Tocilizumab is an FDA-approved, humanized anti-IL-6R antibody that has been administered to patients with rheumatoid and juvenile arthritis. In fact, Tocilizumab has been shown to have activity in patients with steroid refractory GVHD [93] and may also be effective for the prevention of acute GVHD in allogeneic stem cell transplant recipients [94].

TGF-b The role of TGF-b in experimental GVHD has been somewhat controversial and complicated by the fact that this cytokine may have different roles depending upon when it is secreted post transplantation. Early studies in a murine transplantation model of skin fibrosis (B10.D2/Balb/c) demonstrated that antibody blockade of TGF-b could prevent skin and lung fibrosis in recipient animals [95]. While this is viewed as a model of chronic GVHD, it should be noted that fibrotic changes were apparent within 21 days of transplantation, which is indicative of a much more rapid disease course than seen in patients. Fibrosis in this model was prominently associated with infiltration of mononuclear cells. Subsequent work by Banovic et al. [96] indicates that the role of TGF-b may be more complex than initially appreciated. They observed that TGF-b had a protective effect early post transplantation that was mediated by donor T cells, whereas in the later stages of GVHD, TGF-b production, primarily by mononuclear cells, was responsible for manifestations of chronic GVHD. Finally, a protective effect of TGF-b during acute GVHD is supported by more recent studies which have investigated the role of Smad 3 in GVHD pathogenesis. Smad 3 is a critical component of the TGF-b signaling pathway. These studies [97] demonstrated that transplantation with Smad 3/ marrow grafts resulted in an acceleration in GVHD lethality, and this was accompanied by an increase in TH1 cells and granulocytes which together released proinflammatory cytokines and promoted tissue damage.

ROLE OF TRANSCRIPTION FACTORS INVOLVED IN TH17 CELL DIFFERENTIATION IN GVHD TH17 cell differentiation is regulated by a series of transcription factors, specifically RORgt, RORa, IRF-4, and Stat3 [98,99]. The primary transcription factor is RORgt which has been shown to be sufficient for TH17 cell differentiation and whose function is modified by interactions with Runx1 which can act in concert with RORgt to activate IL-17 gene expression [100]. The role of RORgt in GVHD pathogenesis has been examined using genetically modified animals that lack either T-bet and RORgt or both transcription factors [101]. In these studies, RORgt/ T cells had comparable ability to cause GVHD as wild-type T cells, whereas T-bet/ T cells were less pathogenic, and mice transplanted with these cells had attenuated GVHD and improved survival. The results in this model system suggested a predominant role of TH1 versus TH17 cells in GVHD induction. Importantly, T cells deficient for both RORgt and T-bet induced minimal GVHD with all recipient mice surviving long term with only moderate weight loss. Consistent with these observations, pathologic examination of GVHD target organs revealed significantly reduced pathology scores in recipients transplanted with RORgt//T-bet/ T cells compared to recipients of wild-type and RORgt/ T cells, suggesting that T-bet and RORgt are required for GVHD induction in vivo. Furthermore, the absence of T-bet and RORgt was associated with a significant reduction in absolute numbers of TH1 and TH17 cells in GVHD target tissues, whereas there was a corresponding increase in Treg reconstitution. However, even with combined deficiency of T-bet and RORgt in donor T cells, recipient mice were not completely free of GVHD. This is similar to prior studies using IFN-g/ IL-17/ animals where GVHD could still be mediated through TH2 pathways [63]. A major caveat from these studies, however, is that there are other immune populations that are regulated by expression of RORgt. In particular, innate lymphoid cells and NK-like cells have been shown

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to be RORgtþ [102]. Thus, one cannot strictly equate RORgtþ cells with TH17 cells. The interpretation of these studies therefore needs to be qualified with the understanding that other RORgtþ cells, in addition to TH17 cells, may play a role in mediating GVHD pathophysiology.

ROLE OF IL-17 AND TH17 CELLS IN CLINICAL GVHD The role of TH17 cells in patients with GVHD still remains unclear. Ratajczak and colleagues have performed the most extensive analysis to date with approximately 100 patients in 3 separate cohorts [103]. In the first cohort, they found an increased percentage of CD4þFoxp3þ Tregs, but a decreased percentage of CD4þIL-17þ TH17 cells in duodenal biopsies of patients with more severe GVHD (grade 2 or above). This resulted in a Th17/Treg ratio less than 1 (medium ratio 0.5) which correlated with more severe GVHD both clinically and pathologically. This ratio was also associated with increased apoptosis of epithelial cells as demonstrated by quantitative TUNEL assay and with the increased expression of TNF, TNF receptor 1, and TNFR2. The authors performed a similar analysis in a second cohort of patients receiving either a myeloablative or reduced intensity conditioning regimen. They were able to confirm the results obtained from the first group of patients regarding the association of the TH17/Treg ratio with severity of GVHD. Furthermore, they demonstrated that neither TH17 and Treg percentages nor the TH17/Treg ratio was affected by the type of conditioning regimen. In a third cohort, the authors analyzed data from additional patients with clinical manifestations of skin GVHD, since animal studies have suggested that TH17 cells play a pathogenic role [74]. This was a mixed population that included patients with acute and chronic lichenoid GVHD. The authors found no evidence that TH17 cells were increased in patients with cutaneous GVHD. Finally, they analyzed TH17 and Treg CD4 T cell subsets from peripheral blood mononuclear cells of patients with GVHD. In patients with acute GVHD, the percentage of Tregs was significantly reduced in comparison to patients without GVHD. However, there was no correlation between the percentage of TH17 cells and the occurrence of GVHD, and no difference in the peripheral blood TH17/Treg ratio between patients with or without GVHD. The finding that patients with more severe GVHD (grade 2 or above) had an increased percentage of CD4þFoxp3þ cells in tissue biopsies is somewhat surprising since Tregs have been shown to play an important role in controlling GVHD target organ damage. These data therefore differ from a previous report by Rieger and colleagues [104] who found increased numbers of Tregs in intestinal biopsies from patients without GVHD. However, the observation that the peripheral blood Treg percentage was significantly lower in patients with GVHD than in patients without GVHD is consistent with the prior study. Notably, although these studies failed to establish an association between TH17 expansion and skin GVHD in humans as previously suggested by preclinical GVHD models, only five patients had chronic GVHD and none of them were reported to have sclerodermatous GVHD, which is what has been most supported as being TH17-mediated in murine studies. Thus, it remains an open question as to whether TH17 cells may be proximate mediators of skin fibrosis in man. Similar results were observed in another recent study examining the role of TH17 cells in human cutaneous GVHD. In this report, Broady and colleagues [105] collected peripheral blood samples from patients receiving allogeneic stem cell transplantation weekly and at onset of acute GVHD. They then analyzed IL-17 production in samples from patients at onset of acute GVHD and time-matched samples from patients without GVHD. The frequency of IL-17-producing T cells (including both CD4þ and CD8þ T cells) from patients with GVHD did not differ from that in patients without GVHD, although the percentage of IL-17þCD3þ T cells in these patients was significantly higher than in healthy donors. Of note, the frequency of IL-17-producing peripheral circulating T cells was very low with mean value of about 0.5%. Analysis of another TH17-associated cytokine, IL-22, revealed no difference in the proportion of IL-22-producing CD3þ T cells between patients with GVHD, without GVHD, or healthy donors. However, in contrast to intracellular cytokine data, when amounts of IL-17 and IL-22 in the supernatants from peripheral blood mononuclear cells activated via aCD3/CD28 antibody were measured, the levels of IL-17 were significantly decreased in patients with GVHD compared with patients without GVHD. In keeping with decreased IL-17 production, lower levels of IL-22 were detected in patients with GVHD compared with patients without GVHD. The authors reasoned that the lack of circulating IL-17-producing T cells might be attributable to preferential homing of these cells to GVHD target tissues such as the skin. However, when skin-resident T cells isolated from cultured explants from skin biopsies of patients with acute GVHD were analyzed, few IL-17þ or IL-22þ CD4þ T cells were present in the skin of patients with acute GVHD. The proportion of CD4þ T cells expressing CCR4 or CCR6, characteristic homing markers of TH17 cells, was also significantly lower in skin from patients with GVHD compared with that from healthy controls. The notion that TH17 cells are not responsible for the tissue damage in this setting was reinforced by immunohistochemical analysis of skin biopsies showing very few T cells from GVHD lesions produced IL-17 or IL-22. Finally, the authors demonstrated that in patients with acute GVHD, there were actually a higher percentage of IFN-gþCD4þ TH1 cells in the skin

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compared with T cells from healthy donor skin and infiltrating T cells expressed CCR5, a TH1-associated chemokine receptor. Thus, in both studies, there was no evidence of an increased percentage or number of TH17 cells in acute GVHD of the skin. In contrast to these reports, there has been one study that has reported an augmented TH17 response in patients with GVHD [106]. In this publication, the authors found a significant increase in IL-17-producing CD4þ T cells in patients with acute GVHD compared with healthy donors or patients without GVHD. Furthermore, in patients with active chronic GVHD, but not in patients with inactive chronic GVHD, a significant increase in TH17 cells were observed. Of note, there was an increased population of IFN-g and IL-17 coexpressing TH17 cells, which is an observation made in several animal studies [70]. Plasma IL-17 levels were also significantly increased in patients with either acute or active chronic GVHD compared with those from healthy donors. Interestingly, there was a reciprocal relationship between TH17 cells and Tregs. In samples from patients with active GVHD, high levels of TH17 cells but low levels of Treg cells were observed. Conversely, the inactive phase of the disease was associated with an enhanced frequency of Treg cells but a decreased frequency of TH17 cells. The most interesting aspect of the study was the observation that T cells that coexpressed IFN-g and IL-17 were detected in skin lesions of patients with active chronic GVHD, whereas these cells were absent in normal skin or skin from patients with inactive chronic GVHD. Thus, these results are supportive of the premise that TH17 cells may play a role in some of the cutaneous manifestations of chronic GVHD. In recent years, more sophisticated approaches have been employed to examine the role of TH17 cells and IL-17 in GVHD biology. Furlan and colleagues [107] used a systems analysis approach to delineate the transcriptional signature of T cells during acute GVHD that developed on immune suppressive medications. They noted that patients who developed GVHD had enrichment of Th/Tc17 modules by gene set enrichment analysis. These results were substantiated more fully in a corresponding nonhuman primate model where a robust IL-17-based transcriptional signature was also evident. Using proteomic profiling, Li et al. [108] identified a CD4þ CD146þCCR6þ T-cell population that had a biased TH17 transcriptional profile and was detectable early before the onset of acute GVHD in patients destined to develop GVHD of the GI tract. Collectively, these studies provide support for the premise that IL-17 plays a role in the evolution of GVHD which arises while on concurrent immune suppressive therapy. Finally, IL-17-producing CD8þ T cells, which were reported to contribute to acute GVHD early post transplantation in murine recipients [61] have been identified in the skin of patients with chronic GVHD-associated lichenoid skin changes [109].

ROLE OF IL-17 AND IL-23R POLYMORPHISMS IN GVHD HLA matching between donor and recipient is the major genetic determinant of clinical outcome after allogeneic HSCT, in particular the severity of GVHD. However, studies have also demonstrated that non-HLA genes associated with immune function are also involved in determining clinical outcomes after transplantation [110]. In this regard, single nucleotide polymorphisms (SNPs) in genes involved in the allogeneic immune response and the amplification of inflammatory reactions have been identified as additional predictive markers for the severity of GVHD. For example, polymorphisms of cytokine genes including TNF-a, IL-10, IFN-g, and IL-6 are associated with more severe acute GVHD [111e113]. The potential role of TH17 cells in the pathophysiology of GVHD has been studied by examining the SNPs of cytokines that are crucial for TH17 differentiation and expansion. A polymorphism in the promoter region of the IL-17 gene which is the result of an SNP rs2275913 (G197A) has been reported to be associated with the susceptibility to rheumatoid arthritis [114] and ulcerative colitis [115]. Espinoza and colleagues [116] examined the impact of IL-17 gene polymorphism in a total of 510 recipients with hematologic malignancies and their unrelated donors on GVHD in HLA-matched myeloablative and nonmyeloablative BMT (Table 14.1). They found that the 197A allele in the recipient was associated with an increased incidence of acute GVHD. A subsequent report also demonstrated that the donor IL-17 197A allele was associated with a higher risk of GVHD after unrelated fully HLA-matched BMT [117]. 197A allele positive PBMCs produced significantly more IL-17 than 197A allele negative PBMCs upon in vitro stimulation, implying that the high production of IL-17 might be associated with the development of acute GVHD. Furthermore, the 197A allele displayed a higher affinity for the nuclear factor of activated T cells (NFAT), a critical transcription factor involved in IL-17 regulation. IL-23 is a key regulator of inflammation and influences the survival and/or expansion of TH17 cells in vivo, and previous studies have demonstrated that variants in the gene coding for IL-23R are strongly associated with inflammation of the gut such as in Crohn’s disease [77]. Since the colon is one of the target organs in GVHD pathogenesis, several studies have examined whether IL-23 receptor (IL-23R) gene polymorphisms affect the severity of GVHD. The polymorphism in the IL-23R that has been studied is at position 1142 where guanine replaces adenine. This results in a protein substitution at position 381 in which glutamine supplants arginine. In the general population, it appears that

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TABLE 14.1 IL-17 and IL-23 Single Nucleotide Polymorphisms and Risk of GVHD Single Nucleotide Polymorphism

Number of Patients

Donor/Recipient Histocompatibility

Espinoza et al. [110]

IL-17 G197A (Donor)

510

URD

Increased incidence of acute GVHD

Espinoza et al. [111]

IL-17 G197A (Donor)

438

URD

Increased incidence of acute GVHD

Elmaagacli et al. [113]

IL-23 1142G > A (Donor)

407

MSD, URD

Decreased incidence of acute GVHD, Decreased TRM, No difference in survival

Gruhn et al. [114]

IL-23 1142G > A (Donor)

231

MSD, URD, MMD

Decreased incidence of acute GVHD, No difference in survival

Wermke et al. [115]

IL-23 1142G > A (Donor)

304

MSD, URD

Decreased incidence of acute GVHD, No difference in survival

Nguyen et al. [116]

IL-23 1142G > A (Donor)

390

URD

No difference in acute GVHD or survival

Broen et al.

IL-23 1142G > A (Donor)

161

MSD (Partial T Cell Depletion)

No difference in acute GVHD, chronic GVHD, or survival

Study (References)

Comment

MMD, mismatched family donor; MSD, HLA-identical sibling donor; TRM, transplant-related mortality; URD, unrelated donor.

w10% of patients are heterozygous for this polymorphism, whereas very few are homozygous (<1%). To date, there have been a total of four studies in which the association between this specific IL-23R polymorphism and GVHD has been examined. Three of these studies were performed in Germany with two focused primarily on adults and one on children [118e120]. The results from these studies were remarkably consistent. Specifically, all demonstrated that there was no effect on the severity of GVHD when the recipient possessed the IL-23R polymorphism. In contrast, when the gene variant was present in the donor, there was a significant decrease in the incidence and severity of acute GVHD. One study also reported a significant reduction in transplant-related mortality, although this could not be confirmed in the other two reports. Despite the observed reduction in acute GVHD, none of these studies were able to document any improvement in overall survival. Moreover, there was no difference in the incidence of chronic GVHD or relapse. Blockade of IL-23 signaling in experimental murine models has been associated with selective protection in the colon microenvironment [80]. None of these studies, however, provided detailed information as to organ-specific responses in this cohort, although it was noted parenthetically in one study that there was no difference in organspecific pathology. It should be noted that there have been two studies that differ from the above reports [121]. The first was facilitated by the National Marrow Donor Program and analyzed 390 primarily adult patients. All patients in this study received stem cell grafts from unrelated donors, which distinguished this study from the European reports where the majority of transplantations were performed with family donors. The gene frequency for the IL-23R polymorphism was 13% in donors and 16% in recipients, which is similar to that reported by the European groups. The authors noted that the IL-23R donor genotype had no effect on the incidence or severity of GVHD after transplantation. The reason for the difference between these results and those reported in the other studies were speculated to be attributable to different patient populations, racial diversity, and less common use of gut decontamination. Similar results were reported by Broen and colleagues [122] where no difference in acute GVHD was observed in patients who had the IL-23R polymorphism. In this latter study, however, it is important to note that patients received partially T-cell-depleted grafts, which itself can affect GVHD incidence. When viewed in the aggregate, there is a foundation of preclinical and epidemiologic data that support the continued investigation of the role of IL-23 in GVHD pathogenesis in humans.

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ROLE OF OTHER TH17 CYTOKINES IN GVHD BIOLOGY Interleukin 21 In addition to the signature cytokine, IL-17A, TH17 cells also secrete a number of other cytokines which include IL-21, IL22, and IL-17F. IL-21 which is produced by CD4þ T cells, CD8þ T cells, and NKT cells is the one which has been most extensively examined with respect to GVHD biology. The receptor for IL-21 is expressed in a variety of cell types including T cell, B cells, NK cells, dendritic cells, macrophages, and epithelial cells [123]. Relevant to this review is that fact that IL-21 is secreted by TH17 cells and also aids in the differentiation of these cells via an alternative pathway that is IL-6 independent [30]. In the context of GVHD, several groups have examined the role of IL-21 in GVHD biology in murine transplantation models [124e127]. For the most part, all studies have demonstrated that interruption of IL-21 signaling by either antibody-based strategies or genetic approaches is able to attenuate the severity of GVHD. The mechanisms by which this occurs have varied and included augmentation of Treg reconstitution and decreased expansion of donor effector T cells, indicating that blockade of IL-21 signaling appears to result in a recalibration of the effector and regulatory arms of the immune system. The augmentation of Tregs in the setting of IL-21 blockade has been reported for both nTregs and iTregs. Notably, GVHD protection has been observed selectively in the gastrointestinal tract in at least one study [126] which is similar to what has been reported for IL-23. Of interest, both cytokines signal though Stat 3, raising the question as to whether this pathway is particularly important in mediating pathological damage in the colon microenvironment. More recently, IL-21 blockade has also been shown to be effective in preventing GVHD mortality in a xenogeneic model of GVHD [128]. Notably, in contrast to prior mouse models, blocking of IL-21 resulted in a decreased percentage of IL-17þ cells, indicating that IL-21 inhibition may reduce GVHD in humans, in part, by preventing differentiation of TH17 cells. Furthermore, colon and skin samples obtained from patients with active GVHD revealed increased IL-21 expression in mononuclear cells relative to samples acquired from patients with no GVHD. Collectively, these results provide support for the premise that IL-21 has a role in mediating GVHD pathology in humans as well.

Interleukin 17F There are a number of other IL-17 family members which have been termed IL-17B, C, D, E, and F. The most homologous to IL-17A is IL-17F, which is also produced by TH17 cells as well as innate immune cells and epithelial cells. IL-17A and IL-17F can form heterotrimers, and the pairing of IL-17A and IL-17F as homodimers or heterotrimers can affect their immune function [129]. The roles of these cytokines have been shown to be overlapping yet distinct with respect to the induction of autoimmunity and production of cytokines [34]. IL-17F has not been specifically examined within the context of GVHD, although as noted previously, in vitroegenerated TH17 cells that mediated lethal GVHD were comprised of a subpopulation that secreted IL-17F [58]. However, transferred TH17 cells recovered from GVHD target organs in this study had lost expression of IL-17F. Thus, whether IL-17F promotes pathological damage or synergizes with IL-17A is not known.

Interleukin 22 IL-22 is a novel cytokine in that it is secreted by immune cells such as TH17 cells, NK cells, LTi cells, and NKT cells, but acts specifically on epithelial cells [130]. Prior studies have defined both proinflammatory and anti-inflammatory roles for this cytokine in various tissue sites such as the liver and GI tract [131,132]. Notably, these tissues are also target organs during GVHD, thus making the examination of this cytokine a reasonable area of inquiry. IL-22 has been shown to play a critical role in thymic regeneration occurring after irradiation or other depletion approaches [133]. In addition, host IL-22 production is critical for the preservation of intestinal stem cells from alloreactive donor T cells and protecting animals from lethal GVHD [134,135]. The mechanism by which this is postulated to occur is via the secretion of IL-23 which induces innate lymphoid cells to secrete IL-22 which has protective effects on the gastrointestinal tract epithelium. The role of donor-derived IL-22 has been more controversial with studies demonstrating both proinflammatory [134] and antiinflammatory effects [136,137] in murine GVHD models.

ROLE OF IL-17 AND TH17 CELLS IN GVL REACTIVITY Because of the proinflammatory effects of IL-17, a number of investigators have examined the role of both IL-17 and TH17 cells to determine whether they have a role in promoting antitumor immunity in nontransplant tumor models. IL-17

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has been shown to be capable of promoting tumor growth and metastasis in several different tumor models [138,139]. This has been associated with increased angiogenesis and upregulation of prosurvival genes. Interestingly, in one report, commensal organisms in the gut were able to activate TH17 cells which then led to the development of colon tumors in predisposed animals [140]. Conversely, tumor-specific polarized TH17 cells can mediate potent antitumor effects indicating that augmented production of IL-17 can in some instances eliminate malignant cells [141]. Thus, it appears that IL17 and TH17 cells can both promote tumorigenesis and mediate antitumor immunity depending upon the experimental system. In allogeneic stem cell transplantation, the role of IL-17 and TH17 cells in mediating a GVL effect has been much less well characterized than corresponding studies that have been performed to elucidate the effect of this cytokine in the pathogenesis of GVHD. There have been reports [64,108] that have examined GVL responses in the larger context of the role of TH17 cells and IL-17 in GVHD biology. These studies demonstrated that neither IL-17 production nor the presence of RORgt was required for donor T cells to mediate a GVL response. In addition, a more recent study described a population of CD8þ Tc17 cells which contribute to GVHD lethality but fail to mediate a GVL effect [61]. With respect to other TH17-related cytokines, several studies have shown similar preservation of GVL effects when signaling through either IL-21 or IL-23 is impaired by either antibody-based or genetic approaches [124,142]. Thus, based on limited data in animal models, there appears to be no obligate requirement for TH17-related cytokines to maintain an effective GVL response.

SUMMARY Results from a number of studies in experimental GVHD models have now established that IL-17 and TH17 cells contribute to the pathophysiology of both acute and chronic GVHD, although the extent and magnitude of this contribution is still not resolved. Moreover, how these cells interact with other resident T-cell populations in promoting the inflammatory cascade that is the hallmark of GVHD remains to be elucidated. Perhaps the most intriguing aspect of these initial studies has been the emerging awareness that TH17 cells may have a unique role in mediating tissue-specific pathology, such as cutaneous fibrosis and lung injury, which are particularly severe manifestations of chronic GVHD in patients. These observations could have significant clinical implications and could open the door for IL-17-directed therapies designed to address these complications. While the role of TH17 cells has yet to be validated in human GVHD, there are some data that suggest that these cells may also contribute to the skin pathology observed in chronic GVHD. Moreover, genetic polymorphism studies in humans support a role for IL-17 and IL-23 in the pathophysiology of GVHD. Continued examination of this T helper population is likely to yield new insights into GVHD biology and hopefully provide opportunities for translational strategies designed to reduce complications from GVHD in man.

UNANSWERED QUESTIONS The precise role of TH17 cells and IL-17 in the pathophysiology of GVHD is still an evolving issue. There remain many important questions that need to be addressed before the contribution of these cells can be resolved. Specifically, these would include, but not be limited to, the following: 1. Does blockade of IL-17 augment or ameliorate acute GVHD severity? While multiple studies clearly demonstrate the presence of these cells during GVHD, the precise role of IL-17 in animal models of GVHD have been inconclusive. Is this a rational approach to be considered in humans? 2. Is IL-17 responsible for some of the autoimmune-like manifestations observed in chronic GVHD, particularly skin and lung fibrosis? More specifically, does IL-17 direct tissue-specific pathology within the context of GVHD? 3. What is the role of IFN-yþ/IL-17 double producing or IFN-y producing “ex-Th17” cells in GHVD? Do they represent a unique cell population or is this an intermediary stage of differentiation? Given the pathogenicity of “ex-Th17” cells observed in many autoimmune diseases, are these cells pathogenic in the context of allogenic HSCT? 4. What are the contributions of other IL-17-secreting cells (e.g., CD8þ T cells, gd T cells, and granulocytes) in GVHD biology? 5. What is the role of other IL-17 cytokine family members (e.g., IL-17F) in GVHD biology? 6. Given the differences in IL-17 family cytokine signaling between humans and mice, to what extent do animal results replicate IL-17-mediated inflammatory processes in man? 7. To what extent does the IL-17-dependent regulation of the microbiome contribute to the pathophysiology of GVHD?

244 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

ACKNOWLEDGMENTS The work from the authors’ laboratory was supported by NIH R01 HL64603, HL126166, and DK083358 and by awards from the Midwest Athletes Against Childhood Cancer Fund.

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