Helper T-cell differentiation in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Arch. Immunol. Ther. Exp. DOI 10.1007/s00005-014-0284-z

REVIEW

Helper T-Cell Differentiation in Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation Jianing Fu • Jessica Heinrichs • Xue-Zhong Yu

Received: 23 September 2013 / Accepted: 27 January 2014 Ó L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2014

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic option for many malignant diseases. However, the efficacy of alloHSCT is limited by the occurrence of destructive graftversus-host disease (GVHD). Since allogeneic T cells are the driving force in the development of GVHD, their activation, proliferation, and differentiation are key factors to understanding GVHD pathogenesis. This review focuses on one critical aspect: the differentiation and function of helper T (Th) cells in acute GVHD. We first summarize well-established subsets including Th1, Th2, Th17, and T-regulatory cells; their flexibility, plasticity, and epigenetic modification; and newly identified subsets including Th9, Th22, and T follicular helper cells. Next, we extensively discuss preclinical findings of Th-cell lineages in GVHD: the networks of transcription factors involved in differentiation, the cytokine and signaling requirements for development, the reciprocal differentiation features, and the regulation of microRNAs on T-cell differentiation. Finally, we briefly summarize the recent findings on the J. Fu Cancer Biology PhD Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA J. Heinrichs Department of Pathology and Cell Biology, University of South Florida, Tampa, FL 33612, USA J. Fu J. Heinrichs X.-Z. Yu (&) Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA e-mail: [email protected] X.-Z. Yu Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA

roles of T-cell subsets in clinical GVHD and ongoing strategies to modify T-cell differentiation for controlling GVHD in patients. We believe further exploration and understanding of the immunobiology of T-cell differentiation in GVHD will expand therapeutic options for the continuing success of allo-HSCT. Keywords Allo-HSCT GVL GVHD T-cell differentiation

Introduction Allogeneic hematopoietic stem cell transplantation (alloHSCT), previously known as allogeneic bone marrow transplantation (BMT), is a transplant between two genetically non-identical individuals (Ferrara et al. 2009), which serves as an important therapeutic option to treat various malignant and non-malignant diseases, including acute and chronic leukemia, lymphomas, multiple myelomas, aplastic anemia, solid tumors, and severe immunodeficiency disorders (Goker et al. 2001). More than 20,000 allo-HSCTs have been estimated to be performed annually around the world. Some patients present with remission of primary diseases after allo-HSCT; however, in many cases patients develop a common secondary complication named graft-versus-host disease (GVHD), in which transplanted cells injure the host tissues. GVHD accounts for 15–30 % of deaths following allo-HSCT and is a major cause of morbidity in up to 50 % of transplant recipients (Ferrara et al. 2009). The effectiveness of allo-HSCT for many malignant diseases is due to T cell-mediated graft-versus-tumor (GVT) or graft-versusleukemia (GVL) effects. However, allo-HSCT benefits are frequently offset by the destructive GVHD which is also induced by donor T cells (Appelbaum 2001).

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GVHD has a complex etiology, but ultimately is the result of donor T cells recognizing disparate histocompatibility antigens (HA) of the recipient and causing injuries to normal tissues (Blazar and Murphy 2005). Murine models of GVHD are generally grouped into MHC-mismatched or minor HAmismatched situations. GVHD develops in response to a full (class I and II) MHC disparity, is dependent on CD4 T cells, and with additive pathology caused by CD8 T cells. Different from CD4-dependent GVHD, CD8-induced GVHD primarily depends on the cytolytic machinery. Either CD8 T cells or CD4 T cells, or both, may play a role in minor HAinduced GVHD depending on the strain combination. A majority of clinical allo-BMT recipients are MHC matched, but minor HA is disparate from the donor; however, there is no one single mouse model to fully represent the clinical setting of allo-HSCT (Reddy et al. 2008). There are two forms of clinical GVHD: acute GVHD (aGVHD) and chronic GVHD (cGVHD). Acute GVHD is characterized by damage to skin, liver, lung, and gastrointestinal tract, and is normally observed within the first 100 days post-transplantation. Patients with aGVHD have increased risk of morbidity and mortality following allo-HSCT. Chronic GVHD normally occurs after 100 days and has more diverse manifestations and many autoimmune-like characteristics. Moderate to severe cGVHD substantially diminishes longterm survival as well as quality of life of patients (Shlomchik 2007). GVHD has been postulated to occur in several stages (Blazar and Murphy 2005; Socie and Blazar 2009). The first stage involves the cytoreductive conditioning and/or immunosuppression, which allows for donor cell engraftment. Release of proinflammatory cytokines affects host antigen-presenting cells (APCs) and helps fuel the alloreactive donor T-cell response through alloantigen recognition. This is followed by the ‘‘induction phase’’ in which T-cell receptor (TCR) ligation and costimulation combine to achieve full T-cell activation and subsequent expansion. As the alloreactive T cells expand, they differentiate into different subsets; this is driven by the existence of certain transcriptional factors and cytokines. Activated T cells further home to various GVHD target organs, a process controlled by adhesion molecules, integrins, and chemokines. The production of chemokines in inflamed and injured tissue also results in the recruitment of other cell types (neutrophils, NK cells, monocytes/macrophages) to the GVHD target organs, further contributing to tissue injury. The ‘‘effector phase’’ is noted for the destruction of host tissue by immune effector molecules (e.g., Fas ligand, perforin, granzymes, interferon (IFN)-c, tumor necrosis factor (TNF)-a) and results in the continued production of proinflammatory cytokines that continually fuel the entire GVHD process (Fig. 1). The challenge of allo-HSCT for treatment of leukemia and other malignancies of the hematopoietic system is the

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prevention of GVHD without losing the GVL effect. GVL also depends on donor T cells that recognize alloantigens and tumor-associated antigens. Various strategies have been used in different murine models to successfully separate GVHD from GVL, such as: depletion of alloreactive T cells, inhibition of inflammatory cytokines, interfering with T-cell cytolytic pathways, co-stimulatory pathways and trafficking, infusing certain subsets of T cells, and using immunosuppressive cell populations, including T-regulatory cells (Tregs) and NKT cells (Jenq and van den Brink 2010; Kolb 2008; Vincent et al. 2011). Despite its paradigmatic and clinical relevance, the mechanisms involved in the GVL effect are still not completely understood and many currently used strategies to prevent GVHD impair T-cell function with deleterious GVL effects. However, advances are being made gradually by documenting the potential of GVHD prevention and even with GVL augmentation, such as the strategy of adoptively transferring murine or human TRAIL? T cells (Ghosh et al. 2013). T-cell activation, proliferation, and differentiation are considered determining factors for GVHD development. It is well established that following activation, CD4 T cells will differentiate into either Th1 (secreting interleukin (IL)-2 and IFN-c), Th2 (secreting IL-4, IL-5, and IL-13), Th17 (secreting IL-17, IL-21, and IL-22), or Tregs (secreting IL-10 and transforming growth factor (TGF)-b) (Tato and O’Shea 2006). Several newly reported subsets of Th cells, such as Th9, Th22, and T follicular helper cells (Tfh), also enriched our understanding about T-cell differentiation. (Duhen et al. 2009; Nurieva et al. 2008; Veldhoen et al. 2008b). Acute GVHD has been considered a Th1-type disease based on the predominance of cytotoxic T lymphocyte (CTL)-mediated pathology and the increased production of Th1-type cytokines, including IFN-c (Antin and Ferrara 1992; Blazar et al. 1997). However, Th17 cells have also been implicated in the induction of experimental murine aGVHD (Carlson et al. 2009; Iclozan et al. 2010). In this review, we summarize the discovery, functions, characteristics, and relationships among Th cells. We focus on the experimental and translational studies of Th subsets in aGVHD, including the roles of important transcription factors and cytokines and the effect of microRNAs (miRNAs) on T-cell differentiation. We also discuss the current findings of the role of T-cell subsets in clinical GVHD and the new approaches regarding T-cell differentiation being used in clinical trials.

Helper T-Cell Differentiation: General Pathways and Characteristics Naı¨ve CD4? T cells can commit to particular lineages on the basis of different modes of stimulation, antigen


Fig. 1 Three phases of GVHD pathophysiology. Acute GVHD has been postulated to occur in three sequential phases. (1) The initiation phase starts from conditioning regimen (e.g., irradiation) that makes ‘‘space’’ to allow for donor cell engraftment, but also causes host tissue damage. Release of proinflammatory cytokines leads to increased expression of MHC and adhesion molecules that enhance alloantigen presentation by host APCs to donor CD4 and CD8 T cells. (2) The induction phase involves TCR ligation and costimulation to achieve full T-cell activation and subsequent proliferation. As the alloreactive T cells expand, they differentiate into different subsets.

This process is driven by the cytokine milieu through lineage-specific transcription factors. Activated T cells release additional inflammatory cytokines, express chemokine receptors, and migrate to various GVHD target organs. Chemokine production in inflamed tissues recruits other cell types (natural killer cells, neutrophils, monocytes) to the target organ sites, further contributing to GVHD pathology. (3) The effector phase is noted for the destruction of host tissue by immune effector molecules, such as perforin, granzymes, IFN-c, and TNF-a, culminating in clinically evident GVHD

concentration, costimulation, and cytokine milieu. In this section, we summarize the general paradigms and characteristics among Th cells: the standard model that includes Th1, Th2, Th17, and Tregs; their flexibility, plasticity, and epigenetic regulation; and newly identified subsets, such as Th9, Th22, and Tfh (Fig. 2).

antigen specificity, but also by their functional skill sets, defined initially by differential cytokine secretion profiles (Mosmann et al. 1986). This paradigm was further strengthened by equivalent findings from other groups, proving that Th1 cells distinctively produce IFN-c, lymphotoxin, IL-2, and TNF-a. In contrast, Th2 cells produce their signature cytokines: IL-4, IL-5, and IL-13. However, Th2 cells do produce lower levels of TNF-a, granulocyte macrophage-colony stimulating factor, and IL-2 than Th1 cells (Cherwinski et al. 1987; Mosmann and Coffman 1989). Beyond the studies on lineage-specific cytokines, critically important work from the groups of Zheng and Flavell (1997) and Szabo et al. (2000) further identified lineage-specific transcription factors for Th1 and Th2 cells in mice. T-bet (Tbx21) was found to be the critical transcription factor for the generation of Th1 cells, whereas GATA3 served that role for Th2 cells. Evidence provided

The Standard Model: Th1, Th2, Th17, Tregs The Th1/Th2 Cell Paradigm Th1 and Th2 cells have two distinct cytokine secretion patterns that have been defined for both murine and human Th cells (Romagnani 1992). Mosmann and Coffman initiated Th1/Th2 paradigm in 1986, when their seminal work was published, which showed that murine CD4? T-cell clones could be characterized not only on the basis of their

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Fig. 2 Pathways of helper T-cell differentiation: cytokines, transcription factors, epigenetic modifications, and pathogenicity in GVHD. Upon encountering cognate antigens presented by APCs, naı¨ve CD4 T cells differentiate into different T helper subsets, including the classic Th1, Th2, Th17, Tregs, or more recently defined Th9, Th22, or Tfh effector cells. Cytokines secreted in the microenvironment during differentiation play a major role in dictating the phenotype of activated CD4 T cells. T-cell lineages are further defined depending on the expression of transcription factors, chemokine receptors, and effector cytokines, which are influenced by epigenetic modifications. In the standard model, CD4 T cells can differentiate into Th1, Th2, Th17, or Treg cells when activated in the presence of IL-12/IFN-c, IL-4, IL-6/TGF-b, or TGF-b, respectively. T-bet, GATA3, RORct, and Foxp3 have been well characterized and considered as the master lineage determinants of Th1, Th2, Th17 and

Treg cells, accordingly. T-bet leads to gene silencing of Th2 and Th17 effector cytokines, IL-4 and IL-17, thus opening chromatin at Th1 cytokine or chemokine receptor gene loci, such as IFN-c and CXCR3 activating transcription. Likewise, GATA3 causes gene silencing of Th1 and Th17 effector cytokines: IFN-c and IL-17, thus activating Th2 effector cytokines IL-4 and IL-13. CD4 T cells may also differentiate into Th9, Th22, or Tfh subsets when activated in the presence of IL-4/TGF-b, IL-6/TNF-a, and IL-21, respectively. They express lineage-specific transcription factors, chemokine receptors, and produce effector cytokines. Based on the respective cytokine profiles, responses to chemokines, and interactions with other cells, these fully functional Th cells exert specific functions and contribute to immune response. Different symbols represent the pathogenicity of different Th subsets in GVHD. ‘‘?’’ pathogenic effect; ‘‘-’’ protective effect; ‘‘?’’ non-conclusive effect

by different studies strongly suggests that the cytokines play a major role in inducing the transcription factors that determine T-cell differentiation (Grogan et al. 2001; Mullen et al. 2001). Th1 cells are triggered by IL-12 and IFN-c, and Th2 cells are triggered by IL-4. With growing experience it has become clear that for Th1 and Th2 cells, specialization in patterns of cytokine production, transcription factors, and other phenotypic characteristics do occur in mice and in humans. The Th1/Th2 paradigm has been accepted for more than 20 years (Mosmann and Coffman 1989). The differential development of these subsets allows for protective immunity against specific pathogens. Th1 cells are important in mediating cellular

immune responses and play a critical role in the clearance of intracellular pathogens, whereas Th2 cells are mainly involved in humoral immunity such as allergic immune responses and anti-parasitic immunity (Abbas et al. 1996).

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Discovery of Th17 Cells Classification and lineage-specific function of Th1 and Th2 cells formed the basis of our understanding of CD4? T-cell biology, until the paradigm began to be altered and expanded in the mid-1990s when Rouvier et al. (1993) and Yao et al. (1995) described a new cytokine, IL-17, in mice. Soon after, Fossiez et al. (1996) cloned the human


counterpart of murine IL-17 increasing its clinical relevance. Subsequent studies established that human IL-17 displayed proinflammatory properties and could induce the production of inflammatory cytokines such as IL-1b, TNFa, IL-6, and IL-8 along with hematopoietic cytokines such as granulocyte-colony stimulating factor (G-CSF) (Fossiez et al. 1996; Jovanovic et al. 1998). The divergence of a new cell subset that displayed similar proinflammatory phenotype as Th1, but produced different cytokines was initially introduced by the IL-12/IL-23 axis. The axis was seminally described by findings (Becher et al. 2002) that mice lacking the IL-12 p40 chain act differently in a model of experimental autoimmune encephalomyelitis (EAE) than mice lacking the IL-12 p35 chain; this led to the initial description of a subset of CD4? T cells separate from Th1 cells. This axis was further explained by the finding that p40 pairs with a second chain p19 to generate the cytokine IL-23 (Oppmann et al. 2000). IL-23 was shown to be important for the maintenance of T cells producing IL-17 (Aggarwal et al. 2003), and IL-23 enhanced the expansion of CD4? IL-17-secreting T cells in various murine models of autoimmunity (Langrish et al. 2005). However, whether T cells producing IL-17 differed from Th1 cells was still not clear, until two seminal studies established the murine Th17 lineage as independent and distinct (Harrington et al. 2005; Park et al. 2005). By using murine inflammatory disease models, another group quickly published their elegant work identifying Th17 cells uniquely expressing a transcription factor termed retinoic acid-related orphan receptor ct (RORct) (Ivanov et al. 2006), which further distinguished Th17 from Th1 and Th2 cells and clearly demarcated these cells as a unique T-helper population.

cytokines such as IL-6 and IL-21 are implicated in the regulation and induction of IL-17 production by using gene-deficient mice (Korn et al. 2007; Zhou et al. 2007). In vitro, IL-6 is a potent inducer of Th17 cells, but only when combined with other cytokines including TGF-b (Veldhoen et al. 2006). Thus, it appears the conjunction of TGF-b with IL-6 is the initial drivers of Th17 specification, and they can also activate the transcription factor STAT-3, which is required for murine Th17 differentiation (Mathur et al. 2007). However, full expression of the Th17 phenotype depends on RORct, which induces the expression of IL-17 and IL-17F, contributes to the generation of IL-23R, and mediates IL-22 production in mice (Ivanov et al. 2006). 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 (Korn et al. 2007). In disease models such as EAE that depend on Th17 induction, IL-6 seems to be more relevant than IL-21 (Samoilova et al. 1998). Other proinflammatory cytokines such as TNF-a and IL-1b have also been shown to facilitate Th17 differentiation (Kidoya et al. 2005; Sutton et al. 2006). 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-17, Th17 cells also preferentially secrete IL-17F, IL-21, IL-22, and, in humans, IL-26 (Ouyang et al. 2008). These cytokines play important and non-redundant roles in enhancing the host defense against extracellular pathogens. The receptors for IL-17 and IL-22 are broadly expressed on various epithelial tissues. Thus, Th17 cells play critical roles in tissue immunity by mediating the crosstalk between immune system and parenchymal tissues.

Differentiation and Characteristics of Th17 Cells

Interaction of Th17 Cells with Th1, Th2, and Tregs

Th17 cells are highly proinflammatory cells that are part of the adaptive immune response mounted against extracellular pathogens and also have been implicated in the induction of several autoimmune diseases in mice (Bettelli et al. 2007). In the past 8 years, an accumulation of information on this popular T-cell subset (Th17) has been witnessed in both humans and mice: The cytokines for its differentiation and expansion have been identified and the key transcription factors that are involved in its generation have been elucidated (Laurence and O’Shea 2007). Murine IL-23 has long been recognized to be an inducer of IL-17 (Aggarwal et al. 2003). However, naı¨ve murine T cells do not express the IL-23 receptor (IL-23R) and do not differentiate into Th17 cells in the presence of IL-23 in vitro (Mangan et al. 2006). Therefore, IL-23 cannot be the sole inducer of Th17 differentiation, although it appears to have a role in the maintenance of effector function once these cells are induced (McGeachy et al. 2009). Other

A balanced interaction among various T-cell subsets is very important in maintaining immune homeostasis. Th1 and Th17 cells have a proinflammatory role and have been implicated in many inflammatory conditions in humans and mice (Dardalhon et al. 2008b), while an antiinflammatory role is attributed to Th2 and Treg cells. Th1 and Th2 cells inhibit the development of each other through the actions of their lineage-specific cytokines IFN-c and IL-4, respectively (O’Garra 1998; Murphy and Reiner 2002). This principle fits for Th17 cells as well, since the differentiation of Th17 cells can be inhibited by IFN-c or IL-4. However, once fully differentiated, Th17 cells are resistant to the suppressive effects of IFN-c and IL-4 in vitro (Harrington et al. 2005; Park et al. 2005). Coexistence of Th17 and Th1 cells in inflammatory lesions suggest their possible redundant function in pathology of murine experimental autoimmune uveitis (Luger et al. 2008).

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Another important aspect of Th17 cell differentiation is their relationship with Tregs. Tregs can follow a developmental path similar to other T cells and emerge from the thymus (referred to as tTreg or frequently as nTreg, for natural) or differentiate extrathymically from naı¨ve CD4? T cells; Tregs in the latter subset are known as peripherally induced Treg cells (referred to as pTreg, or frequently as iTreg, for induced) (Abbas et al. 2013; Sakaguchi et al. 2001). Tregs are critical for the maintenance of immunological homeostasis and self-tolerance in humans and mice (Sakaguchi 2005). These cells have been classically defined as being CD4?CD25? and expressing the transcription factor Foxp3 (Fontenot et al. 2005). Development of Th17 or Treg cells from induced murine naı¨ve T cells has been suggested in a mutually exclusive manner (Bettelli et al. 2006). IL-6 plays an important role in regulating the balance between Th17 cells and Tregs in humans and mice. IL-6 induces the development of Th17 cells from naı¨ve T cells together with TGF-b; in contrast, IL-6 inhibits TGF-b-induced Treg differentiation (Kimura and Kishimoto 2010). Thus, IL-6 acts as a potent proinflammatory cytokine through promotion of Th17 differentiation and inhibition of Treg differentiation, indicating that this cytokine alters the balance between effector and regulatory T cells. Moreover, Foxp3 itself is able to associate with RORct and inhibit RORct transcriptional activation in mice (Zhou et al. 2008). Plasticity among Th Subsets: Th1, Th2, Th17, and Tregs CD4? T-cell differentiation was originally thought to be a rigid process, centered on the idea that naı¨ve cells differentiate into terminal phenotypes. However, the emerging concept of plasticity of the different Th subsets states that depending on the cytokine milieu and the triggering antigen, a specific Th subset can adopt the phenotype of another subset. Plasticity among Th cells is achieved by signal pathway reprogramming, driven by cytokines and master transcription regulator, that causes gene expression changes (Yang et al. 2008b). This process involves epigenetic modification and chromatin remodeling as well (Wilson et al. 2009). Flexibility of Th2 Cells The established Th1/Th2 paradigm seems to imply that these T-cell subsets conform to a lineage commitment model. This is correct to some extent, since generally Th1polarized cells produce IFN-c, but not IL-4 or IL-17. Similarly, CD4? nTreg cells behave as a reasonably stable lineage in vivo (Rubtsov et al. 2010). However, other findings indicate more flexibility and provide mechanisms

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involved in expression of key transcription factors. Hegazy et al. (2010) showed that committed murine GATA3? Th2 cells could be reprogrammed to adopt GATA3? T-bet? and IL-4? IFN-c? phenotype by lymphocytic choriomeningitis virus (LCMV) infection; these GATA3 T-bet double-positive cells were critical to LCMV exclusion. A population of murine IL-9-producing cells, derived from Th2 cells by treatment with TGF-b, has also been described and later was defined as a new cell subset, Th9. However, Th2 cells can still produce IL-9 lending to their flexibility (Veldhoen et al. 2008b). Intrinsic Instability of Th17 Cells Double-positive Th cells were identified among both human (Annunziato et al. 2007) and murine Th cells (Harrington et al. 2005). Studies demonstrated that Th17 cells can undergo phenotype switching to become either IFN-c-expressing or IFN-c/IL-17-coexpressing cells when exposed to certain cytokine milieus at inflammation sites or during in vitro culture under Th1 polarization conditions (Lee et al. 2009; Shi et al. 2008). Especially relevant for GVHD pathophysiology, this process occurs more readily in a lymphopenic environment (Martin-Orozco et al. 2009). Recently, IFN-c/IL-17 double-positive secreting Th cells were also described exclusively after the allogeneic murine model of cGVHD (Nishimori et al. 2012). These studies suggest that changes in the cytokine milieu during T helper cell differentiation can mediate significant phenotypic shifts related to Th17-expressed cytokines. Similarly, Th17-mediated tumor immunity required IFN-c expression (Muranski et al. 2008). By using a fate mapping reporter strategy, Stockinger and colleagues demonstrated that this conversion occurs physiologically in mice (Hirota et al. 2011). Recent work indicates that murine T-bet, the key Th1 transcription factor, suppresses Th17 differentiation by preventing Runx1-dependent transactivation of Rorc (Lazarevic et al. 2011). The rapid appearance of RORct T-bet double-positive cells has been identified by different groups (Ghoreschi et al. 2010; Nistala et al. 2010). T-bet? RORct? cells are more potent at inducing EAE pathogenicity compared with RORct? cells (Ghoreschi et al. 2010). Although Th17 and Th2 cells are both dependent on the transcription factor interferon regulatory factor 4 (IRF4), there was relatively little evidence of conversion between these two subsets. IRF4 has been studied as a therapeutic target for IL-17-dependent murine autoimmune diseases with promising results, leading to its potential as a target for GVHD (Brustle et al. 2007; Chen et al. 2008). A subset of GATA3? RORct? cells was recently identified and reported to play a role in the development of experimental asthma (Wang et al. 2010). Previous studies showed that induced Tregs (iTregs) can convert under certain


conditions into Th17 cells and vice versa; this led investigators to evaluate the mechanism underlying this conversion (Mangan et al. 2006). Both Th17 and Tregs can develop from naı¨ve CD4? T-cell precursors under the influence of the same cytokine TGF-b. However, IL-6 plays a very important role in regulating Th17/Tregs balance. IL-6 induces the development of Th17 cells, but inhibits TGF-b-induced Treg differentiation (Kimura and Kishimoto 2010). IL-2 also shows the reciprocal effects on Treg and Th17 differentiation. IL-2 promotes Tregs differentiation, while negatively regulating Th17 differentiation in mice. Both of these effects are found to be dependent on STAT5 (Laurence et al. 2007; Yang et al. 2011). Stability of Treg Cells: the Ongoing Debate As a unique T-cell lineage, Treg cells are committed to suppressive functions and play a critical role in maintaining self-tolerance and immune homeostasis. Foxp3? Treg cells are produced in the thymus and are also generated from conventional CD4? T cells in the periphery. Adoptive transfer of Tregs is a promising cellular therapy for patients receiving transplantation or suffering from various autoimmune diseases (Welniak et al. 2007). Different murine models used for preclinical evaluation have elucidated many interesting findings, for example, ex vivo activated and expanded donor Tregs can reliably suppress GVHD in mice (Hoffmann et al. 2002; Taylor et al. 2002) and possibly spare GVL (Edinger et al. 2003). The first study to test the safety of human umbilical cord blood-derived Tregs was reported by Brunstein et al. (2011). The most promising outcome was a decreased incidence of GVHD, at least in individuals achieving the highest target Treg dose (Brunstein et al. 2011). Although it is still controversial, many studies have indicated that Foxp3? Treg cells might become unstable and lose their identity or be reprogrammed to various effector Th cells under certain conditions (Sakaguchi et al. 2013). For example, in the absence of exogenous TGF-b, Treg cells can lose Foxp3 expression and have the capacity to reprogram into IL-17secreting cells that could induce tissue destruction in mice (Xu et al. 2007). Furthermore, Treg cells can produce IFNc in the setting of lethal infection or colitis (Feng et al. 2011; Oldenhove et al. 2009). Many studies have found that Tregs will acquire the similar transcription factor as effector Th subset that Tregs intend to suppress. For example, a unique murine Treg subset that expresses both Foxp3 and T-bet has been reported to exist at inflammation sites, which can inhibit Th1 inflammation (Koch et al. 2009). Similarly, mice that lack the Th2-associated transcription factor IRF4 in Treg cells have selective dysregulation of Th2 responses and develop Th2

inflammation (Cretney et al. 2011; Zheng et al. 2009) The ongoing controversy between phenotypic stability and considerable plasticity raises the question: what factors determine the phenotypic preservation of Treg cells? Studies have shown many key factors and signals that regulate the development and survival of Tregs, including IL-2, TGF-b, and co-stimulatory molecules, such as CD28 (Horwitz et al. 2008; Soligo et al. 2011). Further studies are needed to explore the key factors that can be manipulated to achieve functionally stable Tregs. Epigenetic Regulation of Helper T-Cell Differentiation Beyond the cytokine milieu, signal network, and transcription regulations, epigenetic modification also plays an important role in controlling T-cell differentiation (Wilson et al. 2009). Epigenetic modification and chromatin remodeling play critical roles in determining specific gene expression induced by common transcription factors. Epigenetic modifications include DNA CpG methylation, histone methylation, acetylation, and DNase I hypersensitive sites induction. Trimethylation of histone H3 lysine 4 (H3K4me3) is a permissive mark, which appears at the promoters and enhancers of active genes, whereas H3K27me3 is present in broad domains that surround inactive genes. H3K4me3 and H3K27me3 modifications are both present at some genomic regions and control the gene status for either activation or repression during differentiation. The bivalent H3K4me3 and H3K27me3 modifications reveal the flexibility of master regulator expression in different lineages, particularly for the murine Tbx21 locus (Wei et al. 2009). T-bet associates with RbBp5, a core component of H3K4 methyltransferase complexes, and with JMJD3, a H3K27 demethylase (Miller et al. 2008). Epigenetic studies showed that murine T-bet is able to mediate the removal of the repressive H3K27me3 mark at the Ifng and Cxcr3 promoters and establish the permissive H3K4-methylation state (Miller et al. 2008). Through transcription factor binding and epigenetic modification studies, many critical regulatory elements have been identified at each signature cytokine gene locus. Schoenborn et al. (2007) identified several important conserved noncoding sequence (CNS) sites within 100 KB of the murine Ifng gene representing enhancers and insulators. Takemoto et al. (2000) revealed that murine GATA3 induces the chromatin remodeling at the IL-4/IL-13 intergenic regulatory region, which indicates the coordinate expression of the clustered Th2-specific cytokine genes. The role of STAT3 in the regulation of epigenetic modifications has been investigated under Th17 condition in mice (Durant et al. 2010). In the absence of STAT3, permissive histone active marks H3K4m3 are either absent or reduced at the gene loci of Il17a, Il17f, Il21, and Il6ra,

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suggesting that STAT3 regulates the chromatin accessibility of these genes during the Th17 differentiation process. STAT3 is also required for the acquisition of permissive histone active marks at the gene loci of Rorc, Rora, and Batf, which encode key transcriptional factors for Th17 linage specification. Akimzhanov et al. (2007) studied chromatin remodeling of Il17a/Il17f in murine Th17 cells and found eight CNS sites, all associated with histone 3 acetylation. The epigenetic marks found at the Ifng, Il4, and Il17 gene loci correlated precisely with Th1 and Th2 cell lineages, with H3K4me3 at the Ifng locus and H3K27me3 at the Il4 and Il17 loci in Th1 cells, and H3K4me3 at the Il4 locus and H3K27me3 at the Ifng and Il17 loci in Th2 cells. Studies on epigenetic modification and chromatin remodeling emphasize the plasticity of Thcell differentiation programs. Specific T-cell lineage is determined not only by the epigenetic status of signature cytokines, but also relies on that of master transcription factors. Hopefully, with innovation in scientific technology, researchers will be able to provide a comprehensive epigenetic profile of Th-cell lineages in the near future. Novel Subsets: Th9, Th22, Tfh Th9 IL-9, a cytokine previously associated with the Th2 lineage, has been implicated to have an important role in the pathogenesis of asthma, IgE class switch recombination, and resistance to parasites in either human or mice studies (Goswami and Kaplan 2011; Soroosh and Doherty 2009). Interestingly, IL-4, a critical Th2 inducer, has a minimal effect on IL-9 expression during naı¨ve T-cell differentiation; however, IL-4 plus TGF-b can greatly enhance IL-9 production and inhibit the production of Th2 cytokines in mice (Dardalhon et al. 2008a; Veldhoen et al. 2008b). Therefore an additional Th subset termed Th9 was established, which is characterized as the most consistent IL-9-producing T cells distinguished from classical Th2 cells (Noelle and Nowak 2010). Murine Th9 cells can generate abundant IL-9, IL-10, CCL17, and CCL22, with minimal expression of IL-4, IL-5, IL-13, IFN-c, or TNF-a (Dardalhon et al. 2008a; Veldhoen et al. 2008b). Additionally, Th9 cells were not found to express any of the known T-cell transcription factors that define the Th1, Th2, iTreg, or Th17 lineages (T-bet, GATA3, Foxp3, or RoRct, respectively). Following activation, naı¨ve CD4? T cells differentiate into Th9 cells in the presence of TGF-b and IL-4. These Th9-produced cytokines can induce the expression of transcription factors PU.1/Spi-1 and IRF4, which subsequently regulate expression of the IL-9 gene in humans and mice (Chang et al. 2010; Staudt et al. 2010). Additional cytokines, such as IL1b, IL-6, IL-21, and type I interferons, have been shown to

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enhance Th9 differentiation under polarizing conditions, and others , such as IL-25 and IL-2, can promote IL-9 secretion (Kaplan 2013). IL-9 production has also been linked to other T-cell subtypes, including Th17 cells and iTregs, and has been shown to mediate both proinflammatory and immunomodulatory effects in vivo (Lu et al. 2006; Nowak et al. 2009). Despite various roles of IL-9, Th9 cells are primarily proinflammatory and appear to function in a broad spectrum of autoimmune diseases and allergic inflammation in both mice and humans (Nowak et al. 2009; Soroosh and Doherty 2009). Apart from this, IL-9 has been reported to promote the maintenance of tolerant environment by either enhancing immunosuppressive function of Tregs (Elyaman et al. 2009) or limiting the pathogenic activity of Th17 cells (Stephens et al. 2011). In addition, IL-9 is associated with an impaired Th1 immune response in patients with tuberculosis due to the decreased production of IL-12 and limited APC functions (Wu et al. 2008). Therefore, these studies suggest that IL-9 is a pleiotropic cytokine that can function as both a positive and negative regulator of immune responses. However, mechanisms involved in Th9 subset regulation and Th9-mediated diseases remained poorly defined, until Xiao et al. (2012) found that the costimulatory receptor OX40 was a powerful inducer of Th9 cells in vitro and Th9 cell-dependent airway inflammation in vivo. Engagement of the receptor OX40 on T cells increases expression of the transcription factor TRAF6, activates the alternative transcription factor NF-jB pathway, and induces the production of IL-9 in mice (Xiao et al. 2012). Most recently, the role of Th9 cells in cancer immunity has been reported. Lu et al. (2012) found that Th9 cells have the capacity to induce protective antitumor immunity by eliciting a tumor-specific CTL response. The role of Th9 cells in the pathogenesis of GVHD remains to be clarified. In solid organ transplantation models, IL-9 expression by iTregs has been shown to promote graft tolerance through the recruitment of immunosuppressive mast cells (Lu et al. 2006), which suggests a potential protective role for IL-9 in the allo-HSCT setting. Conversely, Th9 cells might make aGVHD worse by directly exacerbating intestinal injury (Dardalhon et al. 2008a), or it could potentiate cGVHD responses by promoting pathogenic B-cell expansion or autoantibody production (Vink et al. 1999). Evaluation for the presence of Th9 cells, especially in the gastrointestinal tract in both animal models and clinical samples, are needed in future work. Th22 A specific T-cell lineage that produces IL-22 was first described by Eyerich et al. (2009). These studies evaluated human T cells from patients with psoriasis, atopic eczema,


and allergic contact dermatitis and found that Th22 clones generate IL-22, IL-10, and TNF-a. Sorting for the skin homing chemokine receptor CCR10 can enrich IL-22expressing memory T cells. Transcriptome analysis further indicated that Th22 cells produce CCL7 and CCL15. Th22 cell clones express higher levels of the transcription factors Bnc2 and Foxo4, but lower levels of Rorc, Gata3, and Tbox21 compared with Th1, Th2, or Th17 cells. IL-22 is a cytokine that is a member of the anti-inflammatory IL-10 cytokine family and is expressed by innate and adaptive lymphocytes (Ouyang et al. 2011). IL-22 functions as a signaling mediator that can connect lymphocytes and epithelial cells. IL-22 was initially speculated to be produced by Th17 cells due to the effect of IL-23 on the expansion of murine IL-22 producing cells (Liang et al. 2006). IL-22 in conjunction with IL-17, IL-17F, or both is important in the generation of small antimicrobial peptides, which play a vital role in skin integrity. IL-22 also induces inflammatory molecules such as chemokines and cytokines including IL6. The function of IL-22 is complex because it has been shown to mediate both proinflammatory and anti-inflammatory activity (Tian et al. 2013; Zhang et al. 2011). Recent studies of IL-22 activity on either donor or recipient compartments during GVHD make it an interesting topic for further investigation. Early this year, Zhao et al. (2013) reported that the frequency of IL-22-producing CD4? T cells showed dynamic changes with the development of GVHD in mice. Van den Brink’s group (Couturier et al. 2013; Hanash et al. 2012) showed that IL-22 deficiency in donor T cells could decrease the severity of murine aGVHD by limiting systemic and local inflammation in aGVHD target organs. Likewise, Tregs were increased in recipient mice that received IL-22-deficient T cells. Furthermore, GVL effect mediated by donor T cells was preserved in the absence of IL-22. These data suggest that targeting IL-22 may represent a valid approach toward decreasing aGVHD severity after allo-HSCT while preserving the GVL effect. In contrast, Hanash et al. (2012) demonstrated that deficiency of recipient-derived IL-22 increased aGVHD tissue damage and mortality. Intestinal IL-22 was produced after BMT by IL-23-responsive innate lymphoid cells (ILCs) from the recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of intestinal stem cells (ISCs), and loss of epithelial integrity. Their findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage. Although the predominant effect seems to be epithelial protection by IL-22, given the worse outcome of GVHD in recipient mice treated with IL-22 neutralizing antibody, these findings indicate that the same

cytokine produced by donor versus recipient-derived sources may have opposing effects on GVHD-related tissue damage. Further investigation will be required to determine which effect is dominant and whether exogenous IL-22 administration would yield a protective outcome in clinical settings. T Follicular Helper Cells Tfh cells in humans were initially described 14 years ago when several groups reported that a large proportion of CD4 T cells in tonsils have a unique phenotype, expressing high levels of chemokine receptor CXCR5, which is critical for their migration into the germinal center (GC) where antibody generation and class switching occur (Kim et al. 2001; Schaerli et al. 2000). IgG and IgA generation by B cells was significantly increased in the presence of Tfh cells. Transcriptional repressor Bcl-6 is critical for Tfh activity. Forced expression of Bcl-6 in T cells generates Tfh features and diminishes the expression of Tbox21 and Gata3, suggesting that T cells undergoing Tfh generation suppress transcription factors critical for Th1 or Th2 polarization. Absence of murine Bcl-6 leads to complete loss of GC B cells and GC reaction, which indicates the inability of Tfh generation (Yu et al. 2009). Tfh cells, compared with non-Tfh cells, express increased levels of CD40L, inducible T-cell costimulator (ICOS), and IL-10 (Kim et al. 2001; Schaerli et al. 2000), which are the molecules that positively regulate B-cell differentiation. Studies indicated that humans or mice with CD40L or ICOS gene mutations present compromised humoral immune responses (Grimbacher et al. 2003; Mak et al. 2003). In addition, IL-21 serves as a helper cytokine or even as an autocrine factor produced by Tfh cells that stimulates B cells through IL-21R. Thus, abnormal expression of Tfh-associated molecules, such as ICOS or IL-21, leads to dysregulation of Tfh cell function and likely accounts for the pathogenesis of immunodeficiencies or certain autoimmune diseases in mice or humans (King et al. 2008). However, as the newest ‘‘lineage’’, it is not surprising that there is considerable controversy regarding the origin and fate of Tfh. Whether Tfh cells represent a distinct lineage parallel to other subsets of Th cells or, as a temporary ‘‘state’’ of differentiation, their phenotype can be superimposed upon Th1, Th2, Th17, or Treg cells remains an open question. Activated CD4? T cells are not devoid of Bcl-6. Tfh can differentiate from Th2 cells in response to helminth infection, which results in GATA3 Bcl-6 double-positive Tfh cells (Glatman Zaretsky et al. 2009). Moreover, Foxp3? T cells to Peyer’s patches can convert to Tfh cells (Tsuji et al. 2009). The signature Tfh cytokine IL-21 is not exclusively produced by Tfh cells (Wei et al. 2007; Wurster et al. 2002), and more

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complicated Tfh cells have the capacity to produce cytokines characteristic of Th1, Th2, and Th17 (Nurieva et al. 2008). Although blocking IL-21 by neutralizing monoclonal antibody (mAb) has been shown to attenuate GVHDrelated weight loss and prolong survival in both allogeneic and xenogeneic murine models by Blazar’s group (Bucher et al. 2009; Hippen et al. 2012), further investigation is required to clearly define the roles of Tfh cells in GVHD pathogenesis.

Helper T-Cell Subsets in GVHD: Experimental and Translational Evaluations GVHD is a major complication of allo-HSCT. Most of the knowledge about the pathophysiology of GVHD has been derived from studies in mouse models. The differentiation of Th cells requires coordinated cytokine signaling that induces the activation of specific transcription factors and the production of linage-specific cytokines to promote lineage-specific CD4? T-cell differentiation. Here, we summarize the experimental and translational evaluations of Th-cell subsets in GVHD, including the roles of important transcription factors and effector cytokines, their reciprocal differentiation features, and the effect of miRNAs on T-cell differentiation. Transcription Factors Involved in T-Cell Differentiation in GVHD The molecular mechanism by which a Th precursor adopts a Th1, Th2, Th17, or Treg phenotype involves a complex interplay between the induction of lineage-specific transcription factors, such as T-bet, GATA3, RoRct, and Foxp3, and the stimulatory effects of the cytokine milieu which are largely provided by host APCs (Ho and Glimcher 2002). Th1: T-bet, STAT1, STAT4, and c-Rel Several transcription factors in coordination induce full Th1 differentiation, including T-box transcription factor (T-bet), STAT1, STAT4, and c-Rel. As one of the master regulators that shape immune responses, T-bet has a unique role in the differentiation of all three subsets (Th1, Th2, Th17) of CD4? Th cells by promoting Th1 differentiation, while simultaneously inhibiting the opposing Th2 and Th17 lineage commitment (Lazarevic and Glimcher 2011). T-bet also controls the expression of a subset of genes encoding molecules that influence the migration and homeostasis of Tregs during Th1 cell-mediated immune responses (Lazarevic and Glimcher 2011). T-bet is a transcriptional activator of IFN-c (Szabo et al. 2000), and

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the key regulator for the Th1 differentiation program. T-bet orchestrates Th1 migration by directly controlling expression of chemokine receptor CXCR3 and chemokines CCL3 and CCL4 (Jenner et al. 2009; Lord et al. 2005). T-bet can also function as a repressor of certain genes such as Socs1, Socs3, and Tcf7, which promotes the development of Th1 fate. T-bet physically associates and functionally recruits the transcriptional repressor Bcl-6 to the Socs1, Socs3, and Tcf7 promoter regions and represses their transcription (Oestreich et al. 2011). The repression of Socs family members Socs1 and Socs3 is necessary to create functional Th1 cells. The repression of Tcf7 will inhibit Th2 fate by sequestering the Th2-specific transcription factor GATA3 away from the Il5 and Il13 promoters. Runt-related transcription factors also participate in the differentiation process. Runx1 and Runx3 were found to promote Th1 cell differentiation in coordination with T-bet (Djuretic et al. 2007; Komine et al. 2003). T-bet and Runx3 bind to Il4 silencer and prevent Il4 expression (Lazarevic and Glimcher 2011). In Th17 cells, T-bet interacts with Runx1 and blocks Runx1-mediated transactivation of Rorc (which encodes RORct) and then suppresses the Th17 lineage development (Lazarevic and Glimcher 2011). Our group previously showed targeting T-bet and RORct can prevent GVHD without offsetting GVL activity in allo-BMT murine models (Yu et al. 2011). Our unpublished study (100th AAI Meeting Abstract P2142) also found that T-bet is critical for CD4 T cell-mediated GVHD by controlling T-cell differentiation and function; also the involvement of T-bet-dependent but IFN-c independent genes can partially explain the compromised ability of T-bet deficient, but IFN-c sufficient CD4 T cells in murine GVHD induction. STAT1 is another main signal transducer for IFN-c (Schindler and Plumlee 2008). In response to IFN-c stimulation, STAT1 is activated and then initiates Th1 development. T-bet is induced by IFN-c and STAT1 signal pathway upon T-cell activation. Once triggered, T-bet in turn activates IFN-c expression, leading to autocrine and paracrine positive feedback effects on IFN-c/STAT1 signaling and further controls Th1 differentiation (Afkarian et al. 2002). STAT1 activation in GVHD target tissue and secondary lymphoid organs is one of the earliest events in GVHD induction (Leng et al. 2006). Ma et al. (2011b)showed that absence of STAT1 in donor CD4? T cells promotes the expansion of Tregs and reduces GVHD in mice. STAT4 is another important transcription factor involved in the Th1 cell differentiation (Thierfelder et al. 1996). By inducing IFN-c production, STAT4 creates a positive feedback loop for further T-bet and IL-12Rb2 expression. At later stages of differentiation, IL-12/STAT4 pathway upregulates the expression of IL-18Ra. IL-12 along with IL-18 induces IFN-c production, independent of


TCR activation, thus creating a pathway for enhancing Th1 response (Thieu et al. 2008). Nikolic et al. (2000) evaluated the role of STAT4 or STAT6 in the induction of aGVHD using gene-deficient mice as donors and found that STAT4-/- donor T cells mediated aGVHD with delayed kinetics compared with wild-type (WT) or STAT6-/- T cells. On the other hand, STAT6 was indispensable for liver and skin GHVD. c-Rel controls CD4 T-cell differentiation during diverse immune responses by using both T-cell autonomous and nonautonomous mechanisms (Gerondakis et al. 2006). Hlx, another transcription factor and Th1 cell-specific homeobox protein, has been shown to synergize with T-bet in promoting the transcription of IFN-c (Mullen et al. 2002). One explanation is that c-Rel might indirectly regulate the expression of IFN-c by controlling the levels of Hlx. The defect in IFN-c production by c-Rel-deficient Th1 cells could also be due to a remodeling failure at IFN-c locus (Agarwal and Rao 1998). Alternatively, c-Rel could be involved in a pathway parallel to T-bet or serve as a common effector molecule downstream of T-bet and STAT4 pathways. Thus, the precise position of c-Rel in the hierarchy of the Th1 transcriptional cascade leading to the production of IFN-c has not been defined yet. Our group recently found that (Yu et al. 2013) T cells deficient in c-Rel have a dramatically reduced ability to cause aGVHD after allo-BMT by using major and minor histocompatibility mismatched murine models. Our data showed that c-Rel-/T cells had a reduced ability to expand in lymphoid organs and to infiltrate in GVHD target organs in allogeneic recipients. c-Rel-/- T cells were defective in the differentiation into Th1 cells after encountering alloantigens, but were enhanced in the differentiation toward Foxp3? Treg cells. Furthermore, c-Rel-/- T cells had largely preserved activity to mediate GVL response. Thus, c-Rel plays an essential role in T cells in the induction of aGVHD. Th2: STAT6, GATA3, STAT5, STAT3 STAT6 is one of the major transcription factors involved in Th2 lineage differentiation (Shimoda et al. 1996) and it also upregulates the expression of Th2 master regulator GATA3 (Kaplan et al. 1996; Zhu et al. 2001). STAT6 is activated after IL-4 and IL-13 stimulation and is required for mediating Th2 development. Therefore, T cells deficient in STAT6 impair IL-4 and IL-13 responsiveness and cause almost complete loss of Th2 responses, but enhance Th1 responses (Kaplan et al. 1996; Takeda et al. 1996). Interestingly, STAT6-mediated Th2 response is also required in murine GVHD prophylaxis mediated by NKT cells (Hashimoto et al. 2005). Studies have postulated three distinct mechanisms of GATA3 involvement in Th2 differentiation: induction of

Th2 cytokine production, selective growth of Th2 cells through recruitment of Gfi-1, and inhibition of Th1 differentiation by controlling its lineage-specific factors (Usui et al. 2003; Zhu et al. 2006). In GATA3-deficient mice, differentiation of naı¨ve cells was diverted toward the Th1 lineage and Th2 differentiation was interrupted, which confirmed the indispensable role of GATA3 in Th2 response and also showed the importance of basal GATA3 expression in inhibiting Th1 differentiation (Pai et al. 2004; Zhu et al. 2004). STAT5 also plays a critical role in the Th2 lineage commitment. STAT5 activation is predominantly activated by IL-2, but not IL-4, and it does not induce GATA3 expression (Zhu et al. 2003). However, since GATA3 and STAT5 bind to different sites of the IL-4 locus and GATA3 alone cannot induce the production of IL-4, the coordinated activity of STAT5 and GATA3 are required for differentiation of Th2 cells (Zhu et al. 2003, 2006). Blazar’s group described a murine model of GVHD in which overexpression of a constitutively active form of STAT5 increases the overall Treg numbers. Interestingly, in the absence of Tregs, Th2 anti-inflammatory cytokines increased, thus proving the dual role of STAT5 in Tregs and Th2 cells (Vogtenhuber et al. 2010). More recently, a clinical trial in patients with cGVHD showed that low dose of IL-2 treatment increased phosphorylation of STAT5 resulting in an overall increase in Tregs and inhibited progression of cGVHD in all patients (Matsuoka et al. Matsuoka and al 2013). STAT3 is mainly considered to be an important transcription factor for Th17 and Tfh cells; however, recent studies identified the role of STAT3 in Th2 differentiation (Stritesky et al. 2011). STAT3 cooperates with STAT6 in promoting Th2 lineage development. STAT3 binds directly to Th2-relevant gene loci and is required by STAT6 for interaction with target genes in developing T cells. STAT6 is normally activated, but displays impaired ability to interact with loci in the absence of STAT3, indicating STAT3 serves as a mediator for STAT6 to access to the loci. Allergic inflammation was eliminated in STAT3-deficient mice suggesting the importance of the presence of STAT3 for the proper development of Th2 cells (Stritesky et al. 2011). Recently, Fowler’s group was also able to show that STAT3-deficient donor T cells attenuated murine aGVHD, mainly by increasing overall iTregs; this is in line with the known reciprocal differentiation of Tregs and Th17 cells, which is discussed in detail later in this review (Laurence et al. 2012). In general, Th2 cells are not pathogenic and are even protective in the development of GVHD, as suggested by many published works (Krenger et al. 1995; Pan et al. 1995; Tawara et al. 2008). Studies showed that donor Th2 cells do not initiate aGVHD and can regulate the GVHD mediated by unmanipulated donor T cells without

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impairing allo-engraftment (Fowler and Gress 2000). One study investigated the effects of atorvastatin (AT), the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme-A) reductase inhibitor, on murine aGVHD model. AT treatment of donor T cells induced a Th2 cytokine profile and reduced their in vivo expansion, which translated into significantly reduced aGVHD lethality while maintaining GVL (Zeiser et al. 2007). STAT6 signaling and the L-mevalonate pathway are associated with AT-mediated aGVHD protection, given that the AT effect is partially reversed in STAT6-/- donors and abrogated by L-mevalonate. This provides promising clinical relevance of enhancing the differentiation, development, and function of Th2 cells in GVHD patients. Th17: RORct, STAT3, AhR, and IRF4 The development of Th17 cells is mainly dependent on the transcription factors RORct, RORa, STAT3, and IRF4 (Ivanov et al. 2006; Yang et al. 2008c). RORct together with RORa synergistically directs the differentiation program of Th17 lineage and their absence completely aborts Th17 development (Yang et al. 2008c). Fulton et al. (2012) have evaluated the role of Th17 cells in murine aGVHD by infusing donor T cells lacking Rorc. Their data suggest that Rorc deficient T cells have reduced ability to induce aGVHD. However, our study demonstrated that (Yu et al. 2011) RORct alone plays little role on donor T cells in the induction of aGVHD, but RORct significantly controls the T-cell pathogenesis in aGVHD in the absence of T-bet. Thus, targeting both T-bet and RORct can effectively prevent GVHD without offsetting GVL activity in murine models of allo-BMT (Yu et al. 2011). As one of GVHD-specific signaling pathways, STAT3 plays an important role in the differentiation process by activating downstream IL-6, IL-21, and IL-23 signaling. It can also induce RORct expression by binding to IL-17 and IL-17F promoters (Chen et al. 2006). Deficiency of STAT3 causes enhanced expression of T-bet and Foxp3, which are involved in the development of opposing Th1 and Treg lineages, respectively (Yang et al. 2007). Ma et al. (2011c) reported that STAT3 activation in the spleen correlated with high levels of IL-6 and IL-10. The marked change in IL-6/ IL-10 ratio during the development of GVHD could suggest that STAT3 may act as a promoter of inflammation during the early priming and induction phase of GVHD (high IL-6/ IL-10 ratio) and may mediate anti-inflammatory signals at later time points (low IL-6/IL-10 ratio). A study from Lu et al. (2008) also provides evidence that STAT3 and ERK1/2 phosphorylations are critical for T-cell alloactivation and GVHD. Furthermore, by using conditional STAT3 knockout mice as donors, Radojcic et al. (2010) demonstrated that STAT3 is critical for CD4-driven cGVHD.

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Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with important physiological roles in biological responses, such as controlling cell proliferation and inflammation. Studies suggest that AhR promotes Th17 differentiation through the inhibition of STAT1 and STAT5. However, the absence of AhR did not completely abort Th17 differentiation, but was associated with the inability to produce IL-22 (Kimura et al. 2008; Veldhoen et al. 2008a). Activation of AhR by its prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces potent suppression of aGVHD in mice. Suppression is associated with development of a regulatory population of donor CD4?CD25? T cells, but is independent of the CTLA4-IFN-c-IDO pathway (Rohlman et al. 2013). BATF/IRF4 complexes are also involved in the transcriptional network to control Th17 differentiation. Cooperatively bound BATF and IRF4 contribute to the initial chromatin accessibility and play a central role in RORct-mediated activation of Th17 molecular signatures (Ciofani et al. 2012). IRF4-/- mice failed to enhance expression of RORct and subsequently did not develop EAE as a result of impaired Th17 response (Brustle et al. 2007). However, the role of IRF4 in the pathogenesis of GVHD needs to be further investigated. Tregs: Foxp3 Studies in animal model systems clearly indicate that regulatory CD4? T cells play a vital role in attenuating GVHD and are critically important for the maintenance of tolerance. Tregs appear to uniquely express the Foxp3 transcriptional repressor. Foxp3 expression negatively correlated with the severity of GVHD in patients (Miura et al. 2004). Clinical studies on recipients receiving BMT suggest that antigen-specific CD4?CD25?Foxp3? T cells play a critical role in the regulatory control of GVH reactions mediated by both alloreactive and autoreactive lymphocytes (Hess 2006). As an important subset of Th cells, roles of Tregs in GVHD have been widely investigated by our group and others (Brunstein et al. 2011; Hippen et al. 2011; Semple et al. 2011; Veerapathran et al. 2013). Given the role of Tregs in GVHD was summarized (Pidala and Anasetti 2010; Blazar et al. 2012; Josefowicz et al. 2012) by several outstanding papers published lately, we will not discuss in further details here. Effector Cytokines of Distinct T-Cell Subsets: Contribution to GVHD Th1: IFN- c, TNF-a, and IL-2 Acute GVHD has been proposed to be induced by Th1 cells based on the predominant increased production of


Th1-type cytokines, including IFN-c, TNF-a, and IL-2, which are detected in serum or donor T cells during aGVHD in both humans and mice (Ferrara and Krenger 1998; Lu et al. 2001). Paradoxically, the principal Th1 cytokine IFN-c appears to be unnecessary for GVHD induction and it can even inhibit the development of GVHD in lethally irradiated allo-HSCT recipients (Brok et al. 1993; Robb and Hill 2012; Welniak et al. 2000; Yang et al. 2002). One study (Welniak et al. 2000) showed that CD4? T cells from IFN-c-/- mice resulted in accelerated GVHD after lethal total body irradiation (TBI) using a single major histocompatibility class II mismatch model. In sharp contrast, the use of these same IFN-c-/- CD4? donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. Administration of anti-IFN-c antibodies to sublethally irradiated recipients of WT donor cells confirmed the role of IFN-c neutralization in CD4? T cell-mediated GVHD. Thus, opposite roles of IFN-c on CD4-mediated GVHD were shown to depend on different irradiation conditions. The absence of IFN-c is protective against GVHD in models using sublethal TBI, whereas deleterious GVHD occurs in lethal TBI conditioning. Further studies indicate that IFN-c plays a protective role in lung, but a detrimental role for gut tissues (Sun et al. 2012; Yi et al. 2009). IFN-c specifically suppresses lung GVHD through IFN-cR signaling in lung parenchymal cells (Burman et al. 2007; Mauermann et al. 2008). TNF-a has been implicated to be involved in the pathophysiology of GVHD at several steps. It participates in the initiating stage and amplifies the disease progression once established. A series of clinical experiments demonstrated the strong correlation between TNFR1 levels and GVHD, supporting the importance of TNF-a in this process (Levine 2011). Cooke et al. (1998) found that donor resistance to lipopolysaccharide significantly reduces the severity of aGVHD after allogeneic BMT and that attenuation of early intestinal toxicity mediated by TNF-a is responsible for this effect. TNF-a has both direct and indirect effect on GVHD: TNF directly induces apoptosis on target tissues and indirectly activates T-cell proliferation pathways. TNF-a has been used as a therapeutic target in preclinical GVHD models with promising results. Soluble TNF receptors or monoclonal antibodies can be used to inhibit TNF-a pharmacologically. Korngold et al. (2003) have found that a chimeric rat/mouse anti-TNF-a mAb could reduce the development of GVHD, but preserve the GVL responses in murine models, with a more significant effect in CD4- rather than CD8-mediated disease. Infliximab is a genetically constructed IgG1 murine–human chimeric monoclonal antibody against TNF-a. Binding of infliximab to the soluble subunit of TNF-a blocks the interaction of TNF-a with its receptors and causes cell lysis. Infliximab was proven to be well tolerated and active for the treatment

of steroid-resistant aGVHD in patients (Couriel et al. 2004). Further investigations are ongoing to define the optimal dose and duration of TNF-a inhibition for GVHD prevention and treatment. IL-2 was initially described as a T-cell growth factor. Given this knowledge, IL-2 treatment was initially used as a strategy to augment immune responses in cancer. Despite high doses of IL-2, this treatment only induced modest antitumor responses; it also caused substantial toxicities in patients with metastatic melanoma (Atkins et al. 1999). Because IL-2 is a stimulant in the immune environment, it initially has been expected to exacerbate GVHD rather than relieve it. But earlier studies found that IL-2 inhibits GVHD-promoting activity of CD4? cells while preserving CD4- and CD8-mediated GVL effects in mice (Sykes et al. 1994). Two recent clinical studies (Koreth et al. 2011; Saadoun et al. 2011) using much lower doses of IL-2 in different immune-mediated diseases, suggesting that IL-2 may have a dominant role in immunosuppression rather than in immune stimulation. It may also explain the equivocal effects of IL-2 treatment seen in earlier clinical trials. Low dose of IL-2 administration in patients after allo-HSCT suppressed the occurrence of cGVHD (Koreth et al. 2011) and improved vasculitis arising from chronic HCV infection (Saadoun et al. 2011). Increased numbers of Tregs were observed after IL-2 treatment in both studies. Given that Tregs have a high affinity for IL-2, meaning they bind especially well to IL-2 at low concentrations, researchers theorized that low doses of IL-2 could activate Tregs to a far greater extent than conventional effector T cells, thereby reducing the immune response causing cGVHD (Koreth et al. 2011). Another preclinical study showed the synergistic effects of rapamycin and IL-2, displayed by increased expansion of donor-derived Tregs and reduced lethal aGVHD in mice (Shin et al. 2011). Given these findings, IL-2 represents the complex dynamic state of the immune system involving both suppression and activation. Th2: IL-4 and IL-13 The production of pro-inflammatory cytokines has been associated with GVHD development; however, the functional contributions of specific cytokines are always difficult to establish due to their seemingly paradoxical results, such as the role of IFN-c in GVHD as discussed above. Similarly, although early studies indicated a role for Th2 cells in organ-specific GVHD in mice (Nikolic et al. 2000), the adoptive transfer of Th2-type cells can actually attenuate murine GVHD pathology (Fowler et al. 1994). Also, Rigby et al. (2003) initially found that a nonmyeloablative regimen increased IL-4 secreting host natural killer T cells, which causes the differentiation of

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donor CD4 T cells toward Th2 and away from pathogenic Th1 cells. Foley et al. (2005) previously found that prevention of GVHD was dependent on Th2 cell production of IL-4, which was responsible for type 2 cytokine skewing. They further evaluated whether Th2 adoptive cell therapy could be used as a treatment, as an alternative to prophylactic therapy, for 14-day established murine GVHD and found that cell therapy was also dependent on IL-4 (Foley et al. 2008). Th2 cell infusion during established GVHD resulted in a relatively dramatic alteration in the Th1/Th2 cytokine balance post-BMT; it was characterized by a marked reduction in allospecific IFN-c secretion and a concomitant increase in allospecific IL-4 and IL-10 secretion. Recipients of Th2 cells deficient in either IL-4 or IL10 had a post-BMT increase in allospecific IFN-c, an increase in GVHD by histology analysis, and reduced postBMT survival. Their results are the first to demonstrate that Th2 cell secreting IL-4 can be used as a therapy of established GVHD. Interestingly, it seems like IL-4 deficient donor T cells can cause severe GVHD in some (Foley et al. 2008; Tawara et al. 2008), but not all (Murphy et al. 1998) studies, which may be due to different experimental settings and mice models used by investigators. Whether and how to regulate these Th2-related cytokines or transcription factors to provide a clinically translatable strategy for GVHD attenuation still need to be elucidated. IL-13 is a typical Th2-type cytokine that is produced by a wide variety of cells including T cells (McKenzie et al. 1993). IL-13 production by donor T cells responding to host antigens in vitro was previously shown to correlate with the severity of clinical aGVHD (Jordan et al. 2004). However, by using an established mouse model, Hildebrandt et al. (2008) showed that the systemic cytokine milieu following allo-BMT with IL-13 deficient donors is characterized by decreases of Th2 cytokines and an increase of TNF-a in recipient serum and ultimately correlated with higher mortality. In vitro studies further demonstrated that donor-derived IL-13 downregulates TNF-a production following allo-BMT and augment the secretion of IL-4, IL-5 and other immunomodulatory proteins like TGF-b (Lee et al. 2001). All these effects combined may contribute to a protective role of IL-13 in aGVHD pathophysiology. Since alloreactive T cells are a significant source of IL-13, the correlation mentioned in clinical aGVHD studies may simply reflect the overall alloreactive status of these cells rather that a direct link between IL-13 and GVHD severity. Th17: IL-17, TGF-b, IL-21, IL-23, and IL-6 Among investigators, a conclusive role of IL-17 in GVHD development has not yet reached a consensus. Adoptive transfer of highly purified ex vivo polarized murine Th17

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cells results in a more aggressive disease that is dependent on IL-17 (Carlson et al. 2009; Iclozan et al. 2010). However, IL-17-/- donor CD4? T cells can attenuate, exacerbate, or have no effect on GVHD depending on the model and knockout mouse strains used (Kappel et al. 2009; Yi et al. 2008, 2009). Kappel et al. (2009) found that IL-17-/- total T cells induced similar GVHD responses as WT cells, although when purified CD4?IL-17-/- T cells were given, there were improved outcomes regarding the prolonged median survival. They conclude that IL-17 contributes to the early development of CD4? T cellmediated GVHD by promoting the production of proinflammatory cytokines; however, it is dispensable for GVHD and GVT activity by unfractionated T cells. Yi et al. (2008) obtained substantially different results using a similar murine BMT model. IL-17-/- T cells were found to exacerbate GVHD and to accelerate recipient mortality compared with WT T cells. Administration of exogenous IL-17 or neutralizing IFN-c could prevent the deleterious outcomes seen with IL-17-/- cells. This leads the investigators to conclude that GVHD is exacerbated due to the augmentation of Th1 differentiation. TGF-b plays critical roles in two antagonistically related Th cell subsets, Th17 and iTreg cells. TGF-b in conjunction with IL-6 is the initial driver of Th17 specification. Hill’s group demonstrated that (Banovic et al. 2005) neutralization of TGF-b after allo-BMT significantly increased aGVHD severity in mice and the concurrent prevention of IL-10 production further exaggerated this effect. Early after allo-BMT, donor T cells were the predominant source of TGF-b and were able to alleviate aGVHD in a TGF-bdependent manner. Although the neutralization of TGF-b augmented the proliferation and expansion of donor T cells after allo-BMT, it paradoxically impaired cellular cytotoxicity to host antigens and associated GVL effects. Different from the protective effects in aGVHD, TGF-b contributed to the severity of cGVHD after allo-BMT. IL-21 is produced by CD4? T cells, especially Th17 cells (Coghill et al. 2011) and NKT cells (Parrish-Novak et al. 2000) which signal through the IL-2Rcc and IL-21R complex. Recent reports show that inhibiting IL-21 decreases disease severity in murine models of GVHD (Bucher et al. 2009; Hanash et al. 2011). Genetically disrupting IL-21 signaling or by using neutralizing mAbs could reduce transplant-related weight loss, tissue pathology, and mortality. Disease amelioration correlated with decreased numbers of CD4? T cells secreting IFN-c and a concomitant increase in the proportion of CD4? T cells expressing Foxp3, which is due to the conversion of CD4?CD25- T cells into iTregs rather than preferential expansion of nTregs themselves (Bucher et al. 2009). Critical differences exist in how IL-21 functions in mouse and humans. IL-21 can act as the second signal required for


Th17 polarization in humans (Yang et al. 2008a), whereas only IL-6 but not IL-21 has this function in mice (Bettelli et al. 2006). Blazar’s group established a human xenogeneic GVHD model (Hippen et al. 2012) and found that the treatment of neutralizing mAb of human IL-21 led to significantly reduced GVHD-associated weight loss, mortality, increased Tregs, and a decrease in T cells secreting IFN-c or granzyme B in recipient mice. Based on these findings, anti-IL-21 mAb could be considered for GVHD prevention in clinic. By using genetic and antibody-based strategies, Das et al. (2010) examined the effect of blocking of IL-23 signaling on GVH and GVL activity after allo-BMT in mice. They demonstrated that IL-23 blockade selectively protects the colon, specifically by attenuating gut injury, while still preserving the GVL effect. Interestingly, the colon did not serve as a sanctuary site for subsequent systemic relapse in GVHD-protected mice since a potent GVL response could be mounted in this organ under conditions where tumor cells migrated to this site. Thus, blockade of IL-23 signaling is an effective strategy for separating GVH and GVL responses. IL-23 could be a potential therapeutic target for the regulation of alloresponses in humans. As a pivotal inflammatory cytokine in immune system, IL-6 plays important roles in altering the balance between the effector and regulatory arms. It also drives a proinflammatory phenotype that defines GVHD characteristics. Studies show that inhibition of the IL-6 signaling pathway, by antibody-mediated blockade of the IL-6 receptor (IL6R), markedly reduces GVHD-caused pathologic damage in mice (Chen et al. 2009). This is accompanied by a significant increase in the absolute number of Tregs and a significant reduction of Th1 and Th17 cells in GVHD target organs. This demonstrates that blockade of IL-6 signaling decreases the ratio of proinflammatory T cells to Tregs. However, another group (Tawara et al. 2011) showed that a brief duration of IL-6 inhibition in recipient mice did not increase the absolute numbers of mature donor Tregs. This could be a consequence of the several key differences between the models, including the dose of radiation, the infusion of unsorted splenocytes, and the duration of IL-6 blockade. Thus, blockade of IL-6 with anti-IL-6R mAb therapy may be testable in clinical trials to prevent GVHD in allo-BMT. Reciprocal Differentiation of Th1, Th2, and Th17 Cells in GVHD In addition to the well-established balance between regulatory and effector T cells, the balance among Th1, Th2, and Th17 effector subsets also plays an important role in regulating T-cell immune response. In aGVHD, naı¨ve

donor CD4? T cells recognize alloantigens on host APCs and differentiate into Th subsets, but the role of Th subsets in GVHD pathogenesis was incompletely characterized until 4 years ago when Yi et al. (2009) published their study in which they discussed the reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in GVHD. They found that in an MHC-mismatched murine model (C57BL/6 ? BALB/c), donor CD4? WT T cells predominantly differentiated into Th1 cells and preferentially mediated tissue damage in the gut and liver. However, deficiency of IFN-c in CD4? T cells enhanced Th2 and Th17 differentiation and exacerbated tissue damage in the lung and skin. Absence of both IL-4 and IFN-c augmented Th17 differentiation and caused preferential skin damage. Deficiency of both IFN-c and IL-17 led to further augmentation of Th2 differentiation and idiopathic pneumonia. The tissue-specific GVHD pathology elicited by Th1, Th2, and Th17 cells was partially associated with their tissue-specific migration, which is dependent upon differential expression of chemokine receptors. Several studies have described the increased expression of chemokines and chemokine receptors in GVHD (Mapara et al. 2006; New et al. 2002). The profile of chemokine and chemokine receptor expression is diverse in different target organs of GVHD. The results obtained by Yi et al. (2009) indicate that donor CD4? T cells can reciprocally differentiate into Th1, Th2, and Th17 cells, and each Th subset contributes to specific GVHD target organ tissue damage. This study further enriches the content of the standard model for Th differentiation, especially in aGVHD settings. Although Th2 cells may still be pathogenic to certain tissues (e.g., lung), our recent work demonstrated that Th1 and Th17 subsets are the primary driving force for inducing aGVHD in mice, thus targeting these two subsets, while preserving or promoting Tregs may represent an effective strategy to control aGVHD (Yu et al. 2011). Role of miRNA in T-Cell Differentiation in GVHD MiRNAs are endogenous RNAs that play important regulatory roles in animals and plants by targeting miRNAs for cleavage or translational repression (Bartel 2004). Approximately, 400 miRNAs have been identified in humans and more than half of mammalian transcripts are predicted to be targets of miRNAs. Many reports indicate that miRNAs play essential roles in regulating hematopoiesis and the development and function of immune cells, including T cells (Baltimore et al. 2008). For T cells, the expression patterns vary among various T-cell subsets and stages of development. This indicates that the unique expression patterns of miRNAs may contribute to the identity of the cell subset or their functional state (Monticelli et al. 2005). Dicer and Drosha are the RNase III

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enzymes critical for generating mature miRNA (Carmell and Hannon 2004). Deletion of Dicer specifically in T cells results in more IFN-c?CD4? T cells in mice (Muljo et al. 2005). Deletion of Drosha in T-cell compartment results in spontaneous inflammatory disease due to the presence of more IL-17?CD4? T cells and/or IFN-c?CD4? T cells (Chong et al. 2008). Among miRNAs, miRNA-155 has been studied first in GVHD. Garzon’s group found that miR-155 expression was upregulated in T cells from mice developing aGVHD after allo-HSCT (Ranganathan et al. 2012). Mice receiving miR-155-deficient donor T cells had markedly reduced lethal aGVHD, whereas the recipients of miR-155 overexpressing T cells developed accelerated aGVHD. Blocking miR-155 using a synthetic anti-miR-155 after allo-HSCT could attenuate aGVHD and prolong survival in mice. Furthermore, patients with intestinal aGVHD presented augmented expression of miR-155 in their pathologic specimen. These data suggest that miR-155 plays a role in the regulation of GVHD and may serve as a novel target for therapeutic intervention after allo-BMT. Several miRNAs have been found to regulate T-cell differentiation and function in either mice or humans. For example, miR-181a and miR-182 are important in modulating T-cell development (Li et al. 2007; Stittrich et al. 2010). Also, miR-146a suppresses regulatory T-cell function (Lu et al. 2010), whereas miR-326 can regulate the differentiation and function of Th17 cells (Du et al. 2009). MiR-17-92 cluster was identified as a multifaceted and indispensible positive regulator of CD4 T-cell antigen responses in mice by Jiang et al. (2011), particularly in the context of Th1-mediated tumor rejection, suggesting that global inhibition of miR-17-92 is likely to subvert tumor immune responses. Furthermore, functional divergence among individual miRNAs in this cluster was demonstrated. MiR-19b and miR-17 account almost entirely for the pro-Th1 influence of miR-17-92, whereas miR-18a acts as an internal antagonist of the cluster’s function. MiR-142 is specifically expressed in hematopoietic cells and regulates immune responses. A critical role for miR-142 is regulating DC responses to lipopolysaccharide (Sun et al. 2011). One study published recently by the same group further reveals the direct cis-acting sequences of miR-142 specific promoter and that transcription factor PU.1 is necessary for its exclusive expression in hematopoietic cells and regulation of IL-6 in mice (Sun et al. 2013). Two papers published recently showed that the miR-29 suppresses IFN-c production, although different mechanisms of action were reported. Steiner et al. (2011) showed that the 30 UTRs (untranslated regions) of Tbx21 and Eomes (but not that of Ifng) are directly responsive to miR-29 in CD4? T cells. Furthermore, miR-29 was shown to contribute to the regulation of T-bet expression in both

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CD4? and CD8? T cells in a murine model of virus infection. The regulation of IFN-c production by miR-29 involves the simultaneous targeting of both T-bet and Eomes. Ma et al. (2011a) found that miR-29 directly targets the 30 UTR of Ifng mRNA in HEK293T cells. They generated NK cells, CD4? T cells, polarized Th1 cells, and CD8? T cells from GS29 mice (mir-29 downregulated) and found that those cells produce more IFN-c than cells from control mice following activation. However, the expression levels of T-bet and Eomes were unaffected in CD4? or CD8? T cells from GS29 mice. The roles of miRNAs in T-cell differentiation have been gradually explored and proven to be critical. Both the regulation of miRNA expression by master transcription factors and the involvement of miRNAs in modulating master transcription factors are very important in understanding Th cell differentiation in GVHD.

Helper T-Cell Subsets in GVHD: Clinical Strategies Current Findings of the Role of T-Cell Subsets in Clinical GVHD The pathogenesis of GVHD involves various Th-cell subsets. Each individual disease manifestation appears to have a distinct T-cell milieu, which is determined by target structure and inflammatory environment. Several aspects contribute to the understanding of T-cell subset function in humans including cytokine quantifications in peripheral blood, lesion tissue biopsies, or from single nucleotide polymorphisms (SNPs) of cytokine genes and their association with GVHD (Cavet et al. 2001; Ritchie et al. 2005; Roy et al. 1995). Much of the information about the role of T-cell subsets in clinical GVHD is confusing and contradictory, which indicates that the picture of GVHD etiology is likely more complex in humans. Despite the perceived skewing of GVHD toward a Th1 process, no study has found increased serum levels of IL12 in patients after allo-HSCT compared with healthy donors (Bonnotte et al. 1996). Consistently, investigators have found increased expression of IL-6, TNF-a, and IL1b, which correlated with either disease severity (Carayol et al. 1997; Tanaka et al. 1993) or disease onset (Ritchie et al. 2005). Zhao et al. (2011) found that, donor-derived Th17 cells in the allografts emerged as the independent risk factor during the course of aGVHD in humans, which suggests that IL-17-producing cells might be a type of initiating cell in aGVHD. Recently, the Japan Donor Marrow Program analyzed rs2275913, an SNP within the promoter region of the IL-17 gene, and found that patients with this SNP had increased IL-17 secretion increasing their risk of aGVHD (Espinoza et al. 2011). G-CSF


treatment in vivo reduced the occurrence of aGVHD through decreasing IL-17 production by T cells. In accordance with the study reported by Zhao et al. (2011), Dander et al. (2009) found that the peripheral blood of patients with ongoing GVHD showed an increased number of IL17-secreting T cells after transplantation, and not surprisingly this increase was associated with the clinical course of aGVHD. In contrast, Ratajczak et al. (2010) demonstrated that Th17 cells in human GVHD was not associated with any evidence of severe tissue damage at the onset of disease, but the Th17/Treg ratio detected in lesion biopsies from gastrointestinal tract and skin is positively correlated with the severity of aGVHD and serves as a sensitive and specific marker of human GVHD. In addition, they detected significantly more IFN-c-producing T cells in biopsies from affected areas. These data suggest that detection of individual T-cell subsets might not be sufficient to reflect the complicated immune processes in human GVHD. New Approaches to Target T-Cell Differentiation for Controlling GVHD in Clinic Tocilizumab, a humanized anti-IL-6R mAb, has been approved in several countries for the treatment of rheumatoid arthritis and other inflammatory diseases. Administration of tocilizumab has been reported to effectively reduce GVHD severity in the gastrointestinal tract as determined by a marked reduction in the volume of diarrhea (Gergis et al. 2010). A small-scale clinical study in GVHD patients indicates that tocilizumab has activity in the treatment of steroid refractory GVHD (Drobyski et al. 2011). Tocilizumab is currently under phase I clinical trial and the expected effects are to decrease Th17 differentiation while increasing Treg induction in GVHD patients (Blazar et al. 2012). However, Betts et al. (2011) did not observe an effect of tocilizumab on human monocytederived dendritic cells maturation and activation, alloreactive T-cell proliferation, Treg expansion, or allogeneic Th1/Th17 responses in vitro, despite its on-target suppression of IL-6R signaling (Fig. 3). Therefore, the efficacy of tocilizumab on GVHD regulation warrants further evaluation in clinic. Ustekinumab (Stelara) is a humanized monoclonal antibody that directly binds to p40, a subunit shared by IL12 and IL-23. As a treatment utilized to regulate the immune-mediated inflammatory disorders such as psoriasis, ustekinumab had not been previously used for GVHD until Pidala et al. (2012) observed its activity in a patient with advanced refractory aGVHD. Ustekinumab was successful in inducing clinical remission of aGVHD in that patient and the correlative data on peripheral blood serum cytokine production indicated the skewing of CD4? T-cell repertoire from Th1 to Th2 and Tregs. Given that IL-12

and IL-23 are key drivers of the differentiation of Th1 and Th17 cells responsible for GVHD (Williamson et al. 1997; Das et al. 2009), the use of ustekinumab in targeting against both Th1 and Th17 may provide a powerful novel approach for GVHD prevention (Fig. 3). A comparative trial, led by Dr. Pidala, is currently ongoing to assess the biologic and clinical activity of ustekinumab in GVHD patients when given in concert with the established regimen of sirolimus/tacrolimus. Rapamycin (Sirolimus), a macrolide antibiotic produced by Streptomyces hygroscopicus, is the prototypical inhibitor of the molecular target of rapamycin (mTOR). It has been used as a prophylaxis and treatment in a number of immune disorders including GVHD (Antin et al. 2003; Johnston et al. 2005). Pidala et al. (2009) provided preliminary evidence for the efficacy of sirolimus as a sole primary therapy in the treatment of aGVHD. In a series of ten patients at high risk for corticosteroid toxicity or leukemia relapse who developed grade II–III aGVHD after allo-HSCT, it was reported that primary therapy with sirolimus resulted in durable complete remission of aGVHD in five patients (50 %) without requirement for glucocorticoids. The projected overall survival at 18 months is 79 % and projected relapse-free survival at 15 months is 70 %. Several studies recently have reported the use of gene targeting to disrupt mTOR function in lymphocytes, confirming an essential role of mTOR signaling in T-cell differentiation (Peter et al. 2010). Complete deficiency of mTOR signaling (both mTORC1 and mTORC2) abrogated the differentiation of Th1, Th2, and Th17 lineages, and instead T cells were diverted into a Foxp3? regulatory phenotype. This was associated with reduced levels of activation of STAT transcription factors and subsequent failure to induce expression of the Th1, Th2, and Th17 master regulators T-bet, GATA3, and RORct (Delgoffe et al. 2009). Different groups report distinct roles for mTORC1 and mTORC2 in T cells, although with discrepancies (Delgoffe et al. 2009; Lee et al. 2010). Rapamycin induces in vitro expansion of nTreg (Battaglia et al. 2005), which has also been used recently to establish good manufacturing practice for expansion protocols for human CD4?CD25? T cells (Tresoldi et al. 2011). Rapamycin and IL-2 enable the expansion of donor-derived nTreg and conversion of CD25- T cells to iTreg, which potently inhibits GVHD development in vivo (Shin et al. 2011). Thus, the application of rapamycin in combination with IL-2 to get higher yields of nTreg and iTreg represents a promising strategy of pharmacologic Treg modification to treat or prevent GVHD (Fig. 3). The main oral alternative to sirolimus is everolimus with slightly improved bioavailability and the use of everolimus in refractory cGVHD has been reported last year by Lutz et al. during the 38th Annual Meeting of the European Group for Blood

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Fig. 3 Therapeutic strategies to control GVHD by targeting of T-cell differentiation. (1) Ustekinumab, anti-p40 mAb, binds to p40 subunit of IL-12 and IL-23 preventing their interaction with IL-12Rb1 on the T-cell surface, thus, inhibiting IL-12 and IL-23 signaling. As a consequence, ustekinumab is expected to reduce the differentiation to Th1 and Th17, while promoting the differentiation toward Th2 and Tregs. (2) Tocilizumab, anti-IL-6R mAb, interferes with IL-6 binding to IL-6R in either membrane bound or soluble form, thus preventing the dimerization of signal transducer gp130 and its interaction with the IL-6/IL-6R complex. Treatment is expected to result in blocking Th17 differentiation and enhancing Treg generation. (3) Rapamycin inhibits mTOR activation. In conventional T cells, diverse signals determine mTOR activity, including ligated costimulatory molecule CD28, TCR, the cytokine IL-2, and its receptor. Among the complex networks, signals are transduced through PI3K-AKT-mediated

pathway to activate mTOR. Upon activation, mTOR promotes Th1, Th2, and Th17 differentiation, but reduces Treg differentiation by regulating their lineage-specific transcription factors. mTOR functions as part of the complexes mTORC1 and mTORC2. Rapamycin in complex with the immunophilin FK506-binding protein 1A, 12 kDa (FKBP12), inhibits mTORC1 and decreases the T-cell differentiation toward Th1, Th2, and Th17 while enhancing Tregs. (4) Statins, HMGCoA reductase inhibitor, blocks the conversion of HMG-CoA to mevalonate and thereby prevents the formation of cholesterol and isoprenoids, which leads to the inhibition of isoprenylation of small GTPases such as Ras, Rho, and Rac. This influences multiple intracellular signaling pathways and cellular functions including proliferation and differentiation. Statins cause preferential development of Th2 cells, as well as Tregs, and inhibition of Th1-driven responses

and Marrow Transplantation in Switzerland in 2012. Sirolimus and everolimus are currently under phase II/III or phase I clinical trials, respectively, and the expected effects are to decrease Th1 cell differentiation and retain or increase Treg cells in GVHD patients (Blazar et al. 2012). HMG-CoA reductase inhibitors (statins) are prescribed worldwide for the prevention and treatment of dyslipidemia and atherosclerosis (Liao and Laufs 2005). Statins can bind to HMG-CoA reductase and displace its natural substrate HMG-CoA, leading to inhibition of mevalonate and cholesterol biosynthesis. Cholesterol metabolism has been indicated to play an important role in modulating T-cell proliferation and differentiation through liver X receptor

signaling (Bensinger et al. 2008; Cui et al. 2011). Statins can also indirectly inhibit several posttranslational modification proteins such as Ras, Rho, and Rac, through reduced mevalonate production (Liao and Laufs 2005). Thus, statins can further affect multiple intracellular signaling pathways and cellular functions including differentiation, motility, secretion, and proliferation (Greenwood et al. 2006). Atorvastatin is the prototypic statin drug that has a proven safety track record (Wenger et al. 2007). Inhibiting Ras leads to preferential development of Th2 cells and inhibition of proinflammatory Th1driven responses (Greenwood et al. 2006). This change in intracellular signaling may also effect the development of

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Treg cells, as exposure of human CD4? T cells to AT in vitro increased expression of Foxp3. Furthermore, patients with hyperlipidemia who took statins up to 8 weeks had more circulating Foxp3? Treg cells (MausnerFainberg et al. 2008). In addition to the direct effects on T cells, statins inhibit IFN-c-induced expression of MHC-II and block the upregulation of a variety of costimulatory molecules and cytokines in APCs (Greenwood et al. 2006). Hamadani et al. (2008) performed a retrospective review of 67 individuals who had received allo-HSCT containing T cells. They found that the rate of grade II–IV aGVHD in patients receiving statins was 10 %, compared with 40 % in the non-treated group. Also, the progression-free survival at 3 years in acute myeloid leukemia patients with or without statin use were 54 and 28 %, respectively, which indicates the preserved GVL effect in patients using statins at the time of allografting. Extensive preclinical and retrospective clinical data suggest that statins can prevent aGVHD without decreasing GVL effects. To prospectively validate these observations, phase-II clinical trials evaluating the role of AT in preventing aGVHD is currently ongoing. The expected effects are to inhibit development of Th1 cells and enhance the induction of Th2 and Treg cells in GVHD patients (Blazar et al. 2012; Hamadani et al. 2011).

Conclusions In this review, we summarize the identifications, generations, functions, characteristics, and relationships among Th cells. We focus on the preclinical evaluations of Th subsets in aGVHD, including the networks of transcription factors involved in differentiation, the cytokine and signaling requirements for development, the reciprocal differentiation features, and the effect of miRNAs on T-cell differentiation. We also discuss the current findings of the role of T-cell subsets in clinical GVHD and the new approaches regarding T-cell differentiation being used in clinical trials. We believe that understanding the immunobiology of T-cell differentiation in GVHD will enhance treatment options for patients undergoing allo-HSCT. Acknowledgments We thank the current and past members of Dr. Yu’s laboratory for their intellectual input and hard work. The research in Dr. Yu’s laboratory has been supported in part by grants from the National Institutes of Health (R01s CA11816, CA143812, AI 082685, and CA169116).

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Outcomes of lung transplantation after allogeneic hematopoietic stem cell transplantation.

Successful fertility restoration after allogeneic hematopoietic stem cell transplantation.

Late-Onset Cerebral Toxoplasmosis After Allogeneic Hematopoietic Stem Cell Transplantation.

Bone Marrow GvHD after Allogeneic Hematopoietic Stem Cell Transplantation.

Aspergillus Thyroiditis after Allogeneic Hematopoietic Stem Cell Transplantation.

Invasive pneumococcal disease following adult allogeneic hematopoietic stem cell transplantation.

Complications of allogeneic hematopoietic stem cell transplantation.

ABO-Mismatched Allogeneic Hematopoietic Stem Cell Transplantation.

Potential of glycosylation research in graft versus host disease after allogeneic hematopoietic stem cell transplantation.

Late-onset noninfectious interstitial lung disease after allogeneic hematopoietic stem cell transplantation.

HLA-C Incompatibilities in Allogeneic Unrelated Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation in mycosis fungoides.

Surveillance cultures in pediatric allogeneic hematopoietic stem cell transplantation.

Persistent parvovirus B19 infection resulting in red cell aplasia after allogeneic hematopoietic stem cell transplantation.

Allogeneic donor availability for hematopoietic stem cell transplantation in children with sickle cell disease.

Early Clostridium difficile infection during allogeneic hematopoietic stem cell transplantation.

Effect of Total Nucleated and CD34(+) Cell Dose on Outcome after Allogeneic Hematopoietic Stem Cell Transplantation.

Progenitor Cell Transplantation.

T-cell characterization using multicolor flow cytometry after allogeneic hematopoietic stem-cell transplantation.

Engraftment syndrome after allogeneic hematopoietic cell transplantation in adults.

Incidence and risk factors for early-onset hypertension after allogeneic hematopoietic stem cell transplantation in children.

BK Virus-Associated Hemorrhagic Cystitis After Allogeneic Hematopoietic Stem Cell Transplantation in the Pediatric Population.

Central nervous system relapse in adults with acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation.

Bendamustine combined with donor lymphocytes infusion in Hodgkin's lymphoma relapsing after allogeneic hematopoietic stem cell transplantation.