T-Cell Costimulation and Coinhibition in Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect

Chapter 11

T-Cell Costimulation and Coinhibition in Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect Yongxia Wu1, Claudio Anasetti2 and Xue-Zhong Yu3 Department of Microbiology & Immunology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, United States;

1

Division of Blood and Marrow Transplantation, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, United States; 3Department of

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Microbiology & Immunology, Department of Medicine, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, United States

Chapter Outline Introduction B7/CD28 Superfamily B7/CD28/CTLA4 Costimulation and Coinhibition Antagonistic Monovalent Anti-CD28 Abs B7eH2/ICOS Costimulation PD-L/PD-1 Coinhibition PD-1H Coinhibition B7eH3 Coinhibition B7eH4 Coinhibition HVEM/BTLA Coinhibition TNF-TNFR Super Family CD40L/CD40 Costimulation OX40/OX40L Costimulation

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4-1BBL/4-1BB Costimulation CD30L/CD30 Costimulation CD70/CD27 Costimulation HVEM/LIGHT Pathway Costimulation GITR/GITRL Costimulation Others CD155/DNAM-1 Costimulation Galectine-9/Tim-3 Coinhibition Lag-3 Coinhibition Regulation of GVHD by Costimulation Through Tregs Conclusions References

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INTRODUCTION Allogeneic hematopoietic cell transplantation (HCT) is an effective therapeutic option with a curative potential for many malignant diseases. The therapeutic potential of allogeneic HCT relies on the graft-versus-tumor (GVT) effect to eradicate residual tumor cells by immunologic mechanisms. Under this therapeutic procedure, graft-versus-host disease (GVHD) remains the major complication as it leads to high morbidity and mortality of the patient. GVHD is initiated by mature donor T cells that recognize disparate histocompatibility antigens of the recipient and cause injuries in normal tissues. The pathophysiology of GVHD development consists of several steps [1]: (1) Priming of the immune response through conditioning with irradiation or chemotherapy; (2) T-cell activation and costimulation through alloantigen recognition and ligation of costimulatory molecules; (3) Alloreactive T-cell expansion and differentiation; (4) Activated T-cell trafficking into GVHD target tissues including gut, liver, skin, and lung; and (5) Destruction of the target tissues by effector T cells (Teffs). The central of GVHD development lies in T-cell activation, proliferation, and differentiation. It is a well-established concept that a productive T-cell activation requires two signals: a first signal provided by the interaction of antigenic peptide/major histocompatibility complex (MHC) with the T-cell receptor (TCR) and a second signal derived by the interaction of costimulatory receptors with their cognate ligands. Seminal studies by Jenkins and Schwartz [2]

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demonstrated that TCR signal alone without costimulation resulted in antigen-specific unresponsiveness known as T-cell anergy, which rendered the T cells fail in response to subsequent exposure to antigen even with costimulation. The critical role of costimulation in regulating the immune response has generated tremendous enthusiasm in evaluating costimulation of therapeutic targets aiming toward induction of T-cell tolerance in solid organ as well as HCT [3,4]. Despite substantial progress, further studies in this area have faced unexpected challenges, which hold back the strategies targeting costimulation advancing to clinical applications. One of the first challenges lies on the blockade of B7/ CD28 pathway with CTLA4-Ig. Although CTLA4-Ig can inhibit T-cell response effectively through blocking B7 and CD28 interaction in vitro, treatment with CTLA4-Ig had little effect in suppressing alloresponse in vivo. Subsequent studies indicate that CTLA4-Ig also block CTLA4-signal that negatively regulates T-cell activation and is required for the induction of transplant tolerance. The other unexpected surprise was also found in another highly potent costimulatory pathway: CD40L/CD40. Despite great promise in targeting CD40L/CD40 pathway in rodent models of transplantation, high incidence of thromboembolic events were associated with anti-CD40L treatment in primate and clinical studies. This unexpected complication is likely caused by aggregation of CD40L-expressing platelets through anti-CD40L ligation, which halted the plan for clinical application using anti-CD40L. Subsequently, numerous additional costimulatory molecules have been discovered, which can either positively or negatively regulate T-cell activation and may have redundant or overlapping functions. The same receptor and ligand interactions can have similar or distinct effects on different T-cell subsets. In particular, regulatory T cells (Tregs, CD4þCD25þFoxp3þ) express many of the same costimulatory molecules as activated T effector cells (Teffs). Because Tregs play a critical role in balancing T-cell immunity and many costimulatory molecules also affect the homeostasis and activity of Tregs, a compound impact on both Teffs and Tregs must be taken into consideration for any given strategy that targets costimulation for the control of GVHD. Finally, a close attention must also be paid to the effect of such a strategy on graft-versus-leukemia (GVL) activity. In this article, we review recent progresses in the understanding of T-cell costimulation and coinhibiton in allogeneic HCT and consider the therapeutic implications of modulating costimulatory and coinhibitory pathways in the prevention and treatment of GVHD as well as in the maintenance of GVL effect. While the B7:CD28/CTLA-4 pathway is the bestcharacterized pathway, there are now many additional T-cell costimulatory and coinhibitory pathways. Besides expanded B7/CD28 superfamily, the other large group of costimulatory molecules belongs to TNF/TNFR superfamily. There are excellent review papers on T-cell costimulation and coinhibition published recently [4ae4d, 191]. We will discuss the costimulatory and coinhibitory molecules in these two superfamily members and highlight their roles in regulating the nature of the immune response in consideration of both Teffs and Tregs (Fig. 11.1).

FIGURE 11.1 Costimulatory (A) and coinhibitory (B) pathways in allogeneic BMT. Ligands and receptors in B7/CD28 family members are shown in blue, TNF family members in yellow, and others in purple. * denotes the ligand or receptor with a contradictory role in GVHD. APCs, antigen-presenting cells; BMT, bone marrow transplant; GVHD, graft-versus-host disease.

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B7/CD28 SUPERFAMILY B7/CD28/CTLA4 Costimulation and Coinhibition CD28 and CTLA4 are the most characterized receptors of the immunoglobulin (Ig) superfamily. CD28 receptor is constitutively expressed on naïve CD4þ and CD8þ T cells in mice and humans, except that a proportion of CD8þ memory T cells are CD28- cells in humans. CTLA4 is expressed on activated but not resting T cells. Both of CD28 and CTLA4 form homodimers and bind the ligands B7-1 (CD80) and B7-2 (CD86). However, CTLA-4 binds to B7 ligands with much higher affinity (10- to 20-fold) than CD28, with a preference for B7-1 [5]. B7-1 expression is inducible and can be expressed by APCs and many other cell types including activated T cells. While B7-2 is constitutively expressed by APCs, its levels of expression are upregulated upon activation. Although T-cell activation can occur with a potent TCR signal alone, CD28 costimulation is required in most responses to antigen. Without CD28, TCR often induces a nonresponsive, anergic state or cell death. T cells from CD28-deficient mice show reduced proliferation in response to peptide antigens [6]. By contrast, CTLA4 dampens T-cell responses in a manner that can protect against the development of autoproliferative or autoimmune disease [7]. CTLA4-deficient mice show a profound post-thymic, hyperproliferative phenotype of death within 3 weeks of age, which is due to massive tissue infiltration and organ destruction [8,9]. The contribution of B7/CD28/CTLA4 pathways in GVHD has been extensively studied in animal models. Experimental studies have shown that proinflammatory signals generated following the conditioning treatment upregulate B7-1 and B7-2 expression in GVHD target tissues, providing the rationale for the CD28/B7 pathway blockade in the GVHD treatment. One reagent that has been widely used to block CD28 costimulation is CTLA4-Ig, a recombinant fusion protein containing the extracellular domain of CTLA-4 linked to an IgG Fc portion. CTLA4-Ig acts as a competitive antagonist of CD28/B7 interactions as CTLA-4 has a much higher binding affinity to B7 molecules than that of CD28 [5,10]. In in vitro studies using mixed lymphocyte reaction (MLR) assay, CTLA4-Ig could completely block T-cell proliferation and effector function in response to alloantigen stimulation [11]. In in vivo studies using rodent bone marrow transplant (BMT) models, treatment with CTLA4-Ig was able to alleviate GVHD, but the inhibition of GVHD was far from complete [12]. Similar results were also obtained using blocking monoclonal antibodies (mAbs) against both B7-1 and B7-2 [13e15]. Those studies also showed that expression of B7-1 on donor CD4þ T cells was critical for GVHD development, and hence the treatment with anti-B7 mAbs also contributed to reduce GVHD not only by targeting B7 expression on the APCs but also by a direct effect on CD4þ T cells. It is clear that B7/CD28 interactions are required for optimal GVL activity because B7-1 expression on leukemic cells greatly enhanced the generation of leukemia reactive cytotoxic T lymphocytes (CTLs) in vitro and B7 blockade in vivo significantly reduced GVL activity medicated by delayed lymphocyte infusion (DLI) [14]. One other study showed that treatment with anti-B7 mAbs maintained the GVL effect in a sublethally irradiated transplantation model [16], which was likely due to incomplete inhibition of alloresponse. In contrast, selective blockade of B7/CTLA4 interactions significantly enhances the GVL activity mediated by DLI [17]. Further evidence shows that donor CTLA4 genotype also affects the GVL activity in clinic after allogeneic HCT [18]. Application of human anti-CTLA4 mAb, ipilimumab, led to tumor regression without exacerbated clinical GVHD in patients after HCT [19]. However, a majority of these patients suffered from autoimmune-related adverse events, sometimes symptoms of acute GVHD (aGVHD). Furthermore, CTLA4-CD28 chimera (CTC28) gene-modified T cells, in which the intracellular signaling domain of CTLA4 was replaced with CD28 counterpart, had increased antitumor capacity in syngeneic as well as in allogeneic T-cell therapy models [20,21]. In addition, CD28 costimulation signaling has also been applied for constructing chimeric antigen receptor (CAR). Allogeneic CD28-costimulated CD19 CAR T cells are capable to medicate antilymphoma activity without causing a significant increase of GVHD [22]. Because GVHD treatment based upon B7 blockade was far from satisfaction, more studies focused on CD28. By using CD28-deficient mice, our group and Blazar’s showed that CD28/ T cells had reduced ability in the induction of aGVHD, providing evidence that CD28 contributes to the pathogenesis and the severity of GVHD. Subsequently, both groups clearly demonstrated that CD28 signals amplify GVHD while CTLA4 signals inhibit GVHD, providing evidence that selective targeting of CD28 may be a better therapeutic strategy for inducing immunological tolerance than blocking the ligands for both CD28 and CTLA4 [17,23,24]. In this scenario, preferential blocking or selective targeting of CD28 have been proposed and evaluated. Belatacept, a second-generation CTLA4-Ig, binds CD80 2-fold better and CD86 4-fold better than the parent CTLA4-Ig and has a 10-fold more potent inhibition of T-cell activation in vitro vs. the parent CTLA4-Ig [25]. In clinical trails, Belatacept has shown considerable promise in renal transplantation as part of a maintenance immunosuppression regimen [26]. Thus, the efficacy of Belatacept in the prevention of GVHD waits to be evaluated. To target CD28 receptor specifically while sparing CTLA4-mediated negative costimulation, various anti-CD28

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Abs have been tested in the control of GVHD. Based on their stimulatory activity on T lymphocytes, Abs directed against CD28 can be divided into three classes: (1) Antagonistic Abs bind and block CD28 signaling, (2) Agonistic Abs cross-link CD28 and prompt a costimulatory signal in synergy with a TCR stimulation, (3) Superagonistic Abs induce a nonphysiological CD28 engagement and a full T-cell activation in the absence of TCR stimulation [27].

Antagonistic Monovalent Anti-CD28 Abs Monovalent Fab fragments of anti-CD28 mAb can inhibit CD28/B7 interactions without stimulating CD28. They can be used as true antagonists to inhibit proliferation and cytokine secretion in T lymphocytes [28] and can induce anergy in vitro [11]. In vivo, monovalent antagonist anti-CD28 Abs delayed acute rejection when given as monotherapy and synergized with calcineurin inhibitors to prevent acute and chronic allograft rejection in kidney and heart transplant models in nonhuman primates [25]. However, Fab fragments have very short half-life in vivo, and thus their efficacy in blocking CD28 receptor is limited (our unpublished observations). An alternative approach is to create an intact chimeric Ab in which one Fab fragment is specific for CD28 and the other Fab is nonspecific. Such a chimeric Ab still binds CD28 receptor without cross-linking, but has a long half-life in vivo [29]. In addition, anti-CD28 Abs can be formatted with polyethylene glycol (PEG) to increase their hydrodynamic size and extend their serum half-life [30]. Lulizumab, an antihuman CD28, has completed the phase I studies [31,32] and now is being tested in a phase II clinical trial in lupus and Sjögren’s syndrome. FR104 is another new humanized Fab fragment of antagonist anti-CD28 that was pegylated to prolong its half-life, which has been shown to present a long elimination half-life in monkeys [33]. In contrast to the rapid cytokine secretion and human T-cell activation in response to superagonist and divalent anti-CD28 Abs, FR104 treatment prevented GVHD development in a CTLA-4 dependent manner in humanized NOD/severe combined immunodeficient (SCID) mice that received human PBMCs. Conventional bivalent anti-CD28 Abs. The action of anti-CD28 in bivalent form usually results in CD28 crosslinking and T-cell costimulation, and the degree of cross-linking of CD28 is directly correlated to activation. CD28 transmits a molecular signal through its association with phosphatidylinositol 3-kinase (PI3-kinase) via the cytoplasmic domain and consequently to T-cell activation and proliferation in conjunction with TCR stimulation. The binding of antiCD28 to Fcg receptors (FcgR) reinforces their agonist activity. Therefore, Fc-silent anti-CD28 Abs were designed by introducing mutations into the Fc fragment to reduce or prevent the cross-linking of CD28 through Fc/FcgR interactions. A hamster-mouse chimeric Fc-silent antimouse CD28 mAb (PV1-IgG3) enabled long-term survival of heart allografts in rats by reducing the activation of alloantigen-mediated key signaling events in T cells [34]. FK734, a humanized Fc-silent antihuman CD28 Ab, reduced T-cell-mediated skin allograft rejection in a humanized SCID model [35] and reduced epidermis thinning and HLA-DR-positive lymphocytic infiltrates of human psoriasis plaques transplanted into SCID mice [36]. However, this humanized Fc-silent Ab (FK734) still generated residual agonistic signals leading to T-cell activation and cytokine release. In vitro, it enhanced proliferation and IL-2 and IFN-g secretion of CD4þ or CD8þ T lymphocytes when stimulated with monocytes or human endothelial cells [35], probably as a result of the mechanical cross-linking of CD28 homodimers by this Ab. On the other hand, in the presence of CD86-transfected monocytes, this Ab inhibited proliferation and cytokine secretion in T lymphocytes, a phenomenon that could be attributed to the engagement by CD86 of the negative costimulatory CTLA4 on responding T cells. Although a bivalent anti-CD28 mAb provides a costimulation to T cells in vitro, several mAbs were as immunosuppressive as B7 blockade in rodent models of GVHD [24,37], and therefore behaved like antagonists. These Abs actually induced in vivo a selective depletion of T lymphocytes that recognized alloantigens by an IFN-g-dependent apoptosis mechanism [38]. They could enhance T-cell migration in vitro and induce migration of memory but not naïve T cells to extralymphoid tissue independently of TCR-derived signals or homing receptors [39]. The mouse anti-rat CD28 mAb JJ319 was described to stimulate T lymphocyte activation in vitro, but it downmodulated the CD28 receptor on T-cell surface and acted as an antagonist in vivo. Consequently, JJ319 was functionally antagonist in vivo and efficient in GVHD by blocking expansion of alloreactive T cells and promoting their apoptosis after few divisions [40,41]. Superagonistic anti-CD28 Abs. Inhibition of GVHD with anti-CD28 mAb proved to be superior to the treatment with anti-B7 Abs. To add complexity to the involvement of the CD28 pathway in GVHD, recent studies have also utilized superagonistic anti-CD28 Abs, which were defined by their capacity to cause a strong activation and proliferation of naïve T lymphocytes in the absence of antigenic stimulation [27]. These molecules reduce GVHD mostly by preferential targeting of Tregs over conventional T cells, thus preserving the GVT reaction [42,43]. A combination of CD28 stimulation plus rapamycin was also shown to prevent aGVHD in animal models, confirming previous data suggesting that Tregs require CD28 costimulation to maintain their suppressive functions [44].

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The clinical translation to block the B7/CD28/CTLA-4 pathway for the treatment of GVHD in humans has been scarce, partially because of the complexity of this system and limited availability of clinical grade reagents. One proof-of-concept study demonstrated that pretreatment of donor marrow with CTLA4-Ig during ex vivo culture with irradiated mononuclear cells from the patient reduced the risk of aGVHD after haploidentical BMT [45]. The rationale of this approach was to induce anergy of the alloreactive donor T cells, while preserving the GVT effect. Furthermore, given that both CD28 costimulation and IL-6 signal transduction facilitate T-cell activation after encountering alloantigens, combined inhibition of Aurora kinase A and JAK2 signaling was found to cooperatively suppress alloreactivity of human T cells and prevent xenogeneic GVHD and maintains GVL activity [46].

B7eH2/ICOS Costimulation The inducible costimulator (ICOS; CD278) is a CD28 homolog and expressed in activated CD4þ and CD8þ T cells [47,48]. The ICOS ligand, known as B7eH2, CD275, or B7RP-1, is structurally related to B7 molecules but does not bind to either CD28 or CTLA-4. B7eH2 is expressed at low levels on B cells, dendritic cells (DCs), macrophages, and parenchymal cells including vascular endothelial cells, but its expression is rapidly upregulated by inflammatory cytokines such as IFNg and TNFa and by CD28 costimulation [48]. Analogous to the YMNM motif on CD28, ICOS also contains a YMFM motif that is phosphorylated upon ligation and activates PI3K [49]. Recent study provides evidence that ICOS can execute its function beyond activation of PI3K [50]. Engagement of ICOS by B7eH2 enhances T-cell proliferation and cytokine production preferentially in Th2 polarization [51]. CD28 and ICOS play distinct roles in T-cell differentiation, with the CD28 signal responsible for T-cell activation and the ICOS signal responsible for certain effector functions [52,53]. Unlike CD28, ICOS does not play a significant role in the generation, maintenance, or function of Tregs [54]. In allogeneic BMT settings, initial study using a parent-into-F1 nonirradiated mode showed that blocking ICOS interaction with an ICOS-specific Ab reduces chronic GVHD (cGVHD) by selectively suppressing Th2 cytokines [15]. Using myeloablative full MHC-mismatched BMT models, Taylor et al. found that ICOS blockade, achieved with ICOS/ mice or anti-ICOS mAb administration, resulted in significant inhibition of GVHD by reducing the number of alloantigenspecific effector cells [55]. Results from a separate group indicated that ICOS blockade reduced GVHD while largely sparing GVL activity by skewing toward Th2 differentiation, without affecting T-cell activation, proliferation, cytotoxicity, and target organ infiltration [56]. The studies by our own group indicate that ICOS deficiency or blockade results in significantly less GVHD morbidity and delayed mortality [23,57]. The effect of ICOS is predominately on CD4þ T cells, and the deficiency of ICOS had no impact on their expansion but significantly reduced their effector functions in terms of expression in FasL and production of IFNg and TNFa. Although ICOS blockade significantly reduces aGVHD mediated by CD4þ or total T cells, we observed that ICOS/ CD8þ T cells exhibited enhanced GVHD morbidity and accelerated mortality associated with elevated expansion and Tc1 cytokine production of CD8þ T cells, urging caution in blocking ICOS in CD8-mediated GVHD [57]. An antihuman ICOS mAb is capable of restricting human T-cell expansion and alleviating GVHD severity in a xenogeneic GVHD model [58]. ICOS/B7eH2 signaling is also involved in the pathogenesis of cGVHD. Since ICOS and B7eH2 ligation is required and quintessential throughout the development of follicular helper T cells (Tfh) [59], Our recent study demonstrated that ICOS deficiency in the donor graft alleviated cGVHD severity by reducing Tfh, Th17, germinal center B-cell, and plasma cell differentiation, coupled with lower antibody production in recipients after allogeneic BMT. In addition, we and others also found that blockade of ICOS hindered germinal center response and cGVHD development in mice [59a,60]. Because of interactions between the B7eH2/ICOS and the B7/CD28/CTLA-4 pathways and the nonredundant role of CD28 and ICOS in T-cell costimulation, strategies combining blockade of ICOS and CD28 have been proposed [61]. Nanji et al. showed that combinational therapy with anti-ICOS and CTLA4-Ig prolonged islet allograft survival more significantly than either therapy alone [62]. In allogeneic BMT settings, our recent study provides direct evidence to support the blocking of CD28 and ICOS signals with sparing of CTLA4 signals as an effective approach to prevent GVHD through manipulation of the CD28 family of costimulatory molecules in vivo [23]. More selective CD28 blockade, rather than a B7 blockade (e.g., belatacept and nonactivating CD28-specific antibodies), has been produced [25,63], and a fully humanized Ab against human ICOS has been generated [64]. These reagents can be tested in the translation of the research findings into clinical practice in allogeneic HCT.

PD-L/PD-1 Coinhibition Programmed death-1 (PD-1, CD279) belongs to the CD28 family and shares w 20% homology with CTLA4. PD-1 is expressed on activated CD4þ and CD8þ T cells as well as on activated NK, B cells, and myeloid cells. Two ligands

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interact with PD-1: PD-L1 (B7eH1; CD274), a member of the B7 family, is expressed on DCs and activated T cells; PDL2 (B7-DC; CD273) is expressed on resting monocytes. Both ligands are expressed in some nonhematopoietic tissues, including heart, lung, kidney, pancreas, placenta, and endothelial cells [65]. PD-1 engagement inhibited proliferation and cytokine production by CD4þ and CD8þ T cells after antigen stimulation in vitro, and the inhibitory effect depends on the strength of TCR stimulation, CD28 costimulation, or IL-2 [66]. Furthermore, mice deficient for PD-1 display an autoimmune phenotype with lupuslike glomerulonephritis and progressive arthritis [67], indicating that PD-1 negatively regulates immune responses. However, in comparison with CTLA-4/ mice, the autoimmune phenotype observed in PD-1/ mice is delayed and limited in certain strain of mice [68]. More recent studies showed that PD-L1 also binds B7-1, delivering an inhibitory signal [65]. PD-L1 and PD-1 ligation facilitates apoptosis in alloreactive T cells by increasing ROS in a process dependent upon the oxidation of fat [69]. On the other hand, PD-L1/PD-1 axis also promotes the differentiation and homeostasis of regulatory T cells (Tregs) in HCT [70,71]. In MHC-mismatched murine BMT models, T cells deficient for PD-1 had enhanced the ability to cause aGVHD, and PD-1 blockade with anti-PD-1 mAbs or soluble PD-L1-Fc fusion protein resulted in accelerated GVHD and enhanced mortality in an IFNg-dependent manner [72]. While blocking PD-L1 did not affect in vivo proliferation of CD4þ or CD8þ T cells regardless of CD28 costimulation, blocking PD-L2 resulted in a marked increase in CD8 response in vivo. The effect of PD-L2 on CD8þ T-cell proliferation is regulated by CD28 costimulation and by CD4þ T cells [73]. Another study demonstrated that PD-L1 was expressed in hematopoietic cells, endothelial cells, and stroma cells, whereas PD-L2 expression was limited to hematopoietic cells in GVHD mice. In contrast with the previous study, they found PD-L1, but not PD-L2, blockade markedly accelerated GVHD lethality, suggesting a dominant role of host PD-L1 in regulating GVHD severity [74]. Consistently, PD-L1 expressed on recipient parenchymal cells limits expansion of infiltrated T cells and thus protects target organ from persistent injury [75]. PD-L1 expression in host tissues also controls cGVHD development by suppressing the expansion of IL-17þIFNgþ T cells [76]. Interestingly, interaction of PD-L1 with PD-1 in GVHD-target organs cause CD8 T-cell exhaustion, apoptosis, and GVHD alleviation, whereas interaction of PD-L1 with CD80 in lymphoid tissues promoted CD8 T-cell survival, expansion, and GVL preservation [77]. In contrast to the negative role of PD-L1 in host cells, upregulation of PD-L1 on CD4 and CD8 T cells in vivo was necessary for their survival, proliferation, and effector function in GVHD induction, but not in the GVL effect [77]. Thus, PD-L1/PD-1 axis plays critical but complicated roles in GVHD development through regulating both host and donor cells. PD-1 engagement on donor T cells also constrains GVL effects [78]. An excellent work revealed a differential expression of PD-1 ligand in GVHD target organs (low expression) versus lymphoid tissues (high expression), which cause CD8 T-cell had a distinct killing activity in different tissues, being highest in liver (GVHD target organ) and lowest in lymph nodes. Therefore, weak CTL activity in lymph nodes promoted local tumor escape but could be reversed by anti-PD-L1 treatment [79]. PD-L1 blockade can effectively restore GVL effects mediated by T cells specific for leukemiaassociated antigen [80]. Blocking PD-1/PD-1 ligands in patients with Hodgkin lymphoma, including 78% that had failed autologous HCT, produced response a rate of w80% [81]. Although with high response rate in Hodgkin’s lymphoma patients, caution should be taken for PD-1 blockade treatment because of high frequency (55%) of treatment-emergent GVHD and fetal and steroid-refractory GVHD reported [82e84]. Taken together, the PD-1/PD-L pathway plays an important role in the regulation of GVHD and GVL effect. While agonistic mAbs to PD-1 may represent a novel strategy for preventing GVHD, blockade of PD-1/PD-L may be applied to promote GVL effect mediated by T cells that do not recognize recipient alloantigens.

PD-1H Coinhibition PD-1 homolog (PD-1H; VISTA) is a recently identified coinhibitory molecule of the B7-CD28 family. The phylogenetic analysis of the full PD-1H molecule shares the similarities with PD-1, CD28, and CTLA4, with the highest identity with PD-1 [85]. In mice, PD-1H is constitutively expressed in a majority of hematopoietic cells, including T cells, NK cells, macrophages, and DCs, but not on resting B cells [86,87]. In T cells, CD4 T cells express higher levels of PD-1H than CD8 T cells. Within the CD4 T-cell compartment, PD-1H expression is highest in naïve cells and Tregs, but lower in memory CD4 T cells. Human PD-1H expression was found highest in myeloid cells, including patrolling and inflammatory monocytes, and lymphoid and myeloid DCs. Similar extent of PD-1H was found in human CD4 and CD8 T cells. PD-1H expression is increased on T cells and myeloid cell population upon activation and immunization. PD-1H plays dual roles as a ligand and a receptor. When expressed on APCs, PD-1H negatively regulates T-cell response by acting as a ligand that interacts with an unknown receptor on T cells [88]. When expressed on T cells, engagement of PD-1H by agonist Ab can directly induce T-cell suppression. Administration of PD-1H agonist mAb

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suppressed CD4 T cell-mediated inflammation in a murine acute hepatitis model, whereas deficiency of PD-1H led to CD4 T-cell dependent inhibition of tumor growth in a murine brain glioma model [89]. Similarly, PD-1H/ mice developed more severe experimental autoimmune encephalomyelitis (EAE) compared to control mice, and PD-1H on both T cells and APCs contribute to exacerbated EAE [90]. In allogeneic BMT, a single dose of PD-1H agonist (MH5A) efficiently prevented GVHD lethality in recipient mice [85]. MH5A inhibited both CD4 and CD8 T-cell expansion and infiltration in recipient spleen and GVHD target organs. They further demonstrated that activation of PD-1H coinhibitory signaling in T cells potently arrests donor alloreactive T cells from activation and expansion in the initial phase. In addition, PD-1H promotes the expansion of Tregs to maintain long-term tolerance and GVHD suppression [91]. However, how PD-1H activation affects T-cellmediated GVL activity has not been reported. In fact, PD-1H is considered as an immune checkpoint, and multiple preclinical studies have verified the role of PD-1H in medicating an immunosuppressive tumor microenvironment [86]. Therefore, PD-1H may serve as a therapeutic target for controlling GVHD in patients with low risk of tumor relapse after HCT.

B7eH3 Coinhibition B7 homolog 3 protein (B7eH3; CD276) is another member that belongs to the B7-CD28 family that discovered in 2001 [92]. The B7eH3 mRNA is broadly expressed in lymphoid and nonlymphoid organs, and the B7eH3 protein is constitutively expressed in nonhematopoietic cells at low level, such as fibroblasts and endothelial cells [87,93]. In immune cells, B7eH3 is constitutively expressed on murine APCs, but can be induced for expression on human T cells, NK cells, DCs, macrophages, and monocytes. Increased expression of B7eH3 is correlated with tumor growth and infection. Decreased expression of miR-29 was found highly correlated with elevated B7eH3 levels in tumor tissue, suggesting a microRNA regulator mechanism is involved in B7eH3 expression [94]. The binding partners of B7eH7 have not been identified. TLT-2 (TREML2) was suggested as a receptor of B7eH3 [95], which has not been verified by others [96]. Since B7eH3 is predominantly a T-cell coinhibitory molecule, mAbs against B7eH3, including MGA271, 8H9, and DS-5573a, have been shown to inhibit tumor growth through an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism [97e99]. The function of B7eH3 has been controversial. Human B7eH3 acts as costimulatory molecular and augments TCRmediated T-cell proliferation, IFNg production, and generation of CTLs in vitro [92]. In contrast, mouse B7eH3 acts as a coinhibitor and suppresses Th1-mediated immune response in airway inflammation and in EAE development [100]. B7eH3 also negatively regulates NK cellemediated tumor lysis [101]. In allogeneic BMT, B7eH3 plays a negative role in GVHD pathogenesis [102]. Elevated B7eH3 was observed in the colon, liver, and lung in mice, and in the intestine of GVHD patients. B7eH3 deficiency either in recipients or in donor T cells accelerated GVHD lethality and pathological damage in the colon by promoting T-effector function. T cells lacking B7eH3 are capable of mediating GVL effect in DLI models. Therefore, new approaches may be developed to activate host B7eH3 early after BMT to prevent GVHD, and to block B7eH3 signal later after transplant to facilitate DLI-mediated GVL.

B7eH4 Coinhibition B7eH4 (B7S1, B7, and Vtcn1) is another member of the B7 family that was discovered in 2003 [103]. B7eH4 transcripts are ubiquitously expressed in lymphoid and nonlymphoid tissues, but B7eH4 protein expression is rarely detected on normal tissues. B7eH4 expression can be induced in monocytes, macrophages, and myeloid DCs by IL-6 and IL-10, and inhibited by GM-CSF and IL-4. Studies have shown that B7eH4 interacts with an unknown receptor on T cells and negative regulates T-cell activation, proliferation, and effector function [87]. B7eH4/ mice developed severe disease due to the expansion of Th1 and Th17 cells during EAE induction [104]. B7eH4 agonist mAb has been shown to suppress autoimmune diseases in mice [105]. Aberrant expression of B7eH4 has been detected in many cancers and its expression is associated with poor prognosis in humans [106]. Therefore, B7eH4 is also a therapeutic target for cancer control. B7eH4 expression was found abundantly on human BMederived mesenchymal stem cells (hBMSCs). Blocking B7eH4 decreases the secretion of TGFb of activated T cells cocultured with hBMSCs and significantly attenuated the suppressive effect of hBMSCs in vitro [107]. Therefore, B7eH4 expression is required for the inhibitory role of hBMSCs in controlling T-cell activation and proliferation in vitro. How B7eH4 regulates host and donor cells in GVHD development is not yet elucidated.

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HVEM/BTLA Coinhibition B and T lymphocyte attenuator (BTLA; CD272) is a member of the CD28 family expressed on activated T cells, B cells, and APCs [108]. Herpes virus entry mediator (HVEM), the primary ligand for BTLA, is a TNFR family member and expressed on activated T, B, and NK cells [108]. This is an example of costimulatory interaction between Ig and TNFR superfamily members. Engagement of BTLA by HVEM delivers an inhibitory signal that suppresses T-cell activation and differentiation in vitro. The inhibitory role of BTLA was evidenced in mice genetically deficient for BTLA, which exhibit amplified immune responses in vivo [109]. CD160 is another receptor that binds HVEM, and HVEM/CD160 interaction also delivers a negative signal to inhibit T-cell activation [110]. CD160 is a glycosylphosphatidylinositol-anchored molecule of the Ig superfamily and primarily expressed on CD8þ T cells. A subset of resting CD4þ T cells also expresses CD160, which is upregulated upon activation but in turn limits T-cell proliferation [110]. Thus, HVEM can trigger a negative regulation outcome depending on engaging BTLA or CD160. Unexpectedly, in allogeneic BMT models, sustained GVHD could not be induced if BTLA was blocked either using BTLA-deficient T cells or administration of an antagonistic anti-BTLA Ab in a nonirradiated parental-into-F1 model due to impaired survival of donor T cells [111]. Using a myeloablative haplotype BMT model, the same group showed that BTLA deficiency in donor or host had no significant effect on GVHD, suggesting that BTLA does not normally regulate GVHD [112]. However, a single administration of a nondepleting mAb specific for BTLA at the time of BMT permanently prevented GVHD independently of its ligand HVEM. Furthermore, anti-BTLA treatment led to the reduced expansion of Teffs relevant to naturally occurring donor-derived Tregs and allowed for GVL effects as well as robust responses to pathogens [112]. In contrast, by applying an agonistic anti-BTLA mAb that stimulates BTLA signal without blocking BTLeHVEM interaction, Sakoda et al. revealed that engagement of BTLA on T cells inhibited donor antihost T-cell responses and ameliorated GVHD after allogeneic BMT [113]. Interestingly, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand that delivers HVEM prosurvival signal in donor T cells. Furthermore, human B-cell lymphoma with aberrations in factor receptor superfamily 14 (TNFRSF14), which encodes HVEM, had reduced HVEM expression and acquired greater alloantigen-presenting capability than their control counterparts in vitro. The increased immune-stimulatory capacity of lymphoma B cells with TNFRSF14 aberrations is associated with higher incidence of GVHD in patients undergoing HCT, suggesting HVEM expressing on B cells may provide a coinhibitory signal to T cells through BTLA [114]. Collectively, these studies suggest that to harness the function of BTLA signals may be an important approach in the prevention of GVHD while maintaining GVL responses and immunity to infection.

TNF-TNFR SUPER FAMILY CD40L/CD40 Costimulation CD40 and CD40L (CD154) belong to the TNFR and TNF families, respectively. CD40 is constitutively expressed at low levels on APCs including B cells, DCs, and monocytes, and its expression can be significantly increased upon activation. CD40L is expressed on activated CD4þ T cells, a small subset of CD8þ T cells, NK cells, and in come cases platelets. Ligation of CD40 upregulates the expression of costimulatory molecules on APCs (i.e., B7s) and contributes to an effective activation of T cells. In addition, CD40/CD40L interactions enhance the production of proinflammatory cytokines including IL-2, IL-12, and IFNg that further promote activation of T and NK cells [115]. Blockade of CD4þ T cells using anti-CD40L mAb induces tolerance of CD4þ T cells to host alloantigens ex vivo, and those tolerized T cells are incapable to cause GVHD in vivo [116]. Tolerance induction via ex vivo blockade of CD40/CD40 L interaction resulted from incomplete activation of Teffs as well as the generation of potent Tregs [117]. The role of CD40/CD40L pathway in GVHD was first evaluated in nonirradiated parent-into-F1 GVHD models, where the administration of anti-CD40L mAb (gp39) was able to reduce the occurrence of aGVHD and cGVHD [118]. In sublethally irradiated BMT models, administration of anti-CD40L mAb improved the GVHD caused by CD4þ T-cells, but not by CD8þ T cells, through inhibiting CD4 T-cell expansion and function [119]. However, mechanistic studies revealed that treatment with anti-CD40L not only blocks T cell costimulation, but also selectively deplete activated T cells [120]. Interestingly, several studies have shown that blockade of CD40/CD40L pathway induces Tregs that may contribute to the GVHD inhibition [117,121]. The role of CD40 investigated the effect of an agonistic anti-CD40 mAb on cGVHD in a nonirradiated parent-into-F1 model. Administration of the mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. Ironically, the inhibition of cGVHD was due to the prevention of donor CD8þ T-cell anergy through CD40 engagement, by which activated CD8þ T cells deleted host CD4þ T cells and B cells involved

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in autoantibody production. In addition, these activated donor CD8þ T cells facilitated full engraftment of donor hematopoietic cells and mediated GVL effect without causing severe aGVHD [122]. These results suggest that in vivo engagement of CD40 and possibly other costimulatory molecules may be used as a GVHD prophylaxis in the nonirradiated parent-into-F1 HCT settings. In many murine models of solid organ transplantation, blockade of CD40/CD154 pathway using an anti-CD154 mAb (MR1) has proved to be highly effective in prolonging survival of heart, kidney, and islet allografts [123]. While the potency of CD40/CD154 blockade in rodent models held great promise for translating this strategy toward the clinic, the disappointment laid on the high incidence of thromboses associated with anti-CD154 treatment in both primate studies and phase I clinical trials [124]. The unexpected complication is likely due to the expression of CD154 on platelets, which promotes platelet aggregation when bound by anti-CD154 mAb [125]. Thus, plans for future development of anti-CD154 mAbs in the clinic have been replaced with the development of anti-CD40 mAbs in attempts to circumvent problems associated with anti-CD154 [126]. In the comparison of the blockade of CD40 L versus CD28 pathway, studies have shown that anti-CD40L was similar to or more effective than anti-B7 in the reduction GVHD. However, anti-CD40L had a deleterious effect on the GVL activity in contrast to anti-B7 mAbs blocking CD28. The inhibition of the GVL effect was likely due to the inhibitory effects on Th1 and cytotoxic T cells [16]. Given the predominant roles of CD40/CD40 L and B7/CD28 pathways in T-cell costimulation, additional studies aimed to evaluate combinational effects of two pathways in BMT. GVHD induced by CD28-deficient T cells was significantly improved by treatment of anti-CD40L [127]. Coadministration of CTLA4-Ig and anti-CD40L had more advantages than either treatment alone in the reduction of GVHD severity [121,128]. Furthermore, the coblockage of B7 and CD40 pathways together with sirolimus significantly improved GVHD after haploidentical BMT in rhesus macaques [129]. These studies provide evidence to support nonredundant roles of these two pathways in T-cell pathogenesis under allogeneic BMT in mice.

OX40/OX40L Costimulation OX40 (CD134) is a member of the TNFR family and is abundantly expressed on activated but not resting T cells. Its ligand, OX40L, is expressed preferentially on APCs such as B cells, DCs, and macrophages and other cell types including langerhans cells, endothelial cells, mast cells, and NK cells, suggesting that OX40/OX40L interactions can be involved in many aspects of physiological responses between T cells and lymphoid and nonlymphoid cells [130]. Costimulatory signals from OX40 to conventional T cells promote division and survival and augment the clonal expansion of effector and memory populations as they are being generated to the antigen [131]. Signaling through OX40 promotes T-cell proliferation and cytokine secretion and induces Th1 and Th2 differentiation and development into memory cells [132]. In line with these important modulatory functions, OX40/OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic [133]. An early study using a rat haploidentical BMT model showed that increased numbers of OX40þ-activated CD4þ T cells were detected in peripheral blood, spleen, and other target organs during aGVHD [134]. Clinical studies in allogeneic HCT also showed that OX40 was preferentially upregulated in activated T cells from patients with aGVHD as well as cGVHD [132,135]. These published results suggest that OX40 might be a particularly useful surface marker to assess T-cell activation in GVHD. The increased presence of OX40þ T cells in GVHD models leads to studying how OX40/OX40L blockade could affect GVHD. Using OX40/ mice as donors, OX40L/ mice as recipients, or the administration of a blocking anti-OX40L mAb similarly reduced GVHD lethality independent of CD28 in a myeloablative MHC-mismatched BMT model [136]. In contrast, agonistic anti-OX40 antibody markedly increased the mortality in the same model. These results are consistent with the data showing that OX40/OX40L interactions clearly accelerate the GVHD when it is preferentially mediated by CD4þ T cells. In preclinical study, in vitro depletion of alloreactive OX40þ T cells resulted in a T-cell population that has reduced alloantigen-specific reactivity while retaining T-cell specificity against third-party antigens including virus (CMV) and tumor antigens (WT1) in vitro [137]. Collectively, all the preclinical data suggest that interrupting the OX40/OX40L pathway may be an interesting strategy to modulate GVHD in patients undergoing allogeneic HCT.

4-1BBL/4-1BB Costimulation 4-1BB (CD137) is a member of the TNFR family and is rapidly expressed on activated CD8þ T cells and also on activated CD4þ T cells with lower levels. Its ligand, 4-1BBL, is a TNF family member and expressed on APCs such as mature DCs,

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activated B cells, and macrophages [138]. When signaling through the TCR is strong, 4-1BB engagement by 4-1BBL provides costimulatory signals to T cells independently of CD28. Although 4-1BB can activate both CD4þ and CD8þ T cells in vitro, 4-1BBL/4-1BB interaction preferentially stimulates CD8þ T cells leading to expansion of CD8þ T cells, and increase in survival, cytolytic function, and IL-2 and IFNg production [139]. By using 4-1BB/ mice as donors or 4-1BBL/ mice as recipients, GVHD lethality was largely reduced compared to WT groups. In contrast, treatment of recipients with anti-4-1BB mAb augmented GVHD lethality induced by either CD4þ or CD8þ T cells. Ligation of 4-1BB with its specific mAb led to enhanced cytolytic function of CD8þ T cells and CD8mediated GVL activity in vivo [140,141]. Subsequently, other groups have explored the potential effect of 4-1BB blockade in the prevention of GVHD. The administration of anti-CD137 mAb at the time of GVHD induction ameliorated the lethality of aGVHD, but accelerated cGVHD [142]. The treatment efficiently inhibited expansion, IFNgproduction, and cytotoxic activity of CD8þ T cells in both GVHD models. However, in cGVHD, the number of CD4þ T cells producing IL-4 was enhanced after treatment, which might contribute to the exacerbation of cGVHD [143]. Consistently, treatment of donor T-cells with anti-4-1BBL in vitro reduced GVHD and improved survival over a standard cyclosporin A þ methotrexate combination in an MHC disparate BMT model and in an irradiated haploidentical BMT model [144,145]. Taken together, these data indicate that 4-1BBL/4-1BB pathway can contribute to the development of GVHD significantly. Interestingly, a single injection of an agonistic anti-CD137 mAb at onset of chronic cutaneous GVHD reversed skin fibrosis, ulceration, and alopecia, ultimately improving general health conditions [146]. The reversal is associated with markedly reduced CD4þ T-cell cytokines and increased apoptosis of donor CD4þ T cells in a Fas-dependent manner. These data indicate that the anti-CD137 mAb has a therapeutic effect on cutaneous GVHD by removing donor CD4þ T cells that cause cutaneous GVHD. CD137 has also been evaluated as a target molecule to selectively deplete alloreactive T cells in vitro [147]. Compared with other activation-induced antigens, CD137 showed a superior performance based on a consistently low baseline expression and a rapid upregulation following alloantigen stimulation. The frequency of alloreactive CD8 T cells can be reduced to a median of 9.5% compared with undepleted control populations. The allo-depleted T-cell subsets maintained significant antitumor and antiviral CD8 responses, proposing a promising approach that infusion of leukemia/tumorreactive CD8 T-cell lines followed by CD137 depletion may confer, strengthened GVL reactivity without causing GVHD in HLA haploidentical and single allele mismatch settings.

CD30L/CD30 Costimulation CD30, a member of the TNFR family, is expressed on activated T cells, NK cells, and B cells. Its ligand, CD30L (CD153), is expressed on activated T cells, resting B cells, and macrophages. Early in vitro studies led to the classification of CD30 as a T-cell “costimulatory receptor” based on observations that immobilized CD30-specific mAb or CD30L-transfected cells enhance the proliferation of human T cells in response to suboptimal stimulation via the TCR [148]. The physiological relevance of these early findings is not clear because antihuman or antimouse CD30L mAb do not appear to block antigen-presenting cell-dependent T-cell proliferation and/or function in a variety of in vitro systems. As CD30 is expressed on T cells rather late after in vitro activation, it is possible that CD30/CD30L interactions occurring relatively late after antigen encounter promote T-cell survival and/or establishment of strong memory responses [149]. CD30 signaling regulates peripheral T-cell responses, controlling T-cell survival, and downregulating cytolytic capacity. Although initial analysis suggested a role for CD30 on Th2 development, studies have shown a role of CD30 as a T-cell costimulatory molecule involved in Th1, Th2, and Th17 immune responses [150,151]. The role of the CD30/CD153 interaction in GVHD has been demonstrated in murine BMT models using a neutralizing anti-CD153 mAb, CD30/ donor mice, and CD153/ recipient mice. Recipients receiving CD30/ donor T cells had longer survival compared to those receiving WT T cells in an MHC class II disparate BMT model. Similarly, administration of a blocking anti-CD30L mAb improved survival in mice after receiving MHC disparate CD4þ T cells [152]. Thus, blockade of the CD30/CD153 pathway represents a new approach for preventing CD4þ T cellemediated GVHD. CD30-expressing lymphocytes were found in the intestine and skin of GVHD patients [153,154]. CD30 expression on CD8 T cells and plasma level of soluble CD30 are increased in patients with aGVHD after HCT [153], suggesting CD30 may be a potential biomarker for GVHD in the clinic.

CD70/CD27 Costimulation CD70 and CD27 have a similar expression pattern in mice and humans. The CD27, a member of the TNFR family, is constitutively expressed on thymocytes, naïve T cells, B cells, and NK cells. Upon T-cell activation, the expression of

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CD27 is upregulated and then downregulated after several rounds of division and differentiation toward effector cells. Its ligand, CD70, is a type II transmembrane glycoprotein belonging to the TNF family. CD70 is expressed on APCs and is rapidly induced on both T and B cells upon cellular activation [155]. Antigen stimulation on T and B cells and Toll-like receptor triggering on B cells and DCs induces CD70 expression, which is further enhanced by CD40 triggering [156,157]. Although CD27 is expressed on thymocytes and naïve T cells, CD27 seems not to play a critical role in T-cell generation and maintenance, as CD27-deficient mice have normal numbers of T cells in the thymus as well as peripheral lymphoid organs. However, more detailed analyses on CD27/ mice revealed that CD27 deficiency has a more profound impact on CD8þ, rather than CD4þ and T cells [158]. CD27 is not required for entry into cell cycle upon T-cell activation or for the differentiation toward IFNg-producing or cytolytic effector T cells. Instead, CD27 promotes survival of activated T cells throughout successive rounds of division and thereby contributes to the accumulation of effector T cells [159]. It has also been found that CD27 on CD8þ T cells can induce proliferation in the absence of IL-2, which does not lead to T-cell differentiation [160]. These studies indicate that CD27 triggering alone is neither required nor sufficient to induce effector cell formation, but that CD27 contributes to accumulation of effector T cells by inducing proliferation and survival. Further studies showed that CD27 expressed on CD4þ T cells promotes both the primary CD8þ T-cell response and the secondary expansion of memory CD8þ T cells, and thus CD27 instructs CD4þ T cells to provide help to CD8þ T cells through Th1-cytokines such as IL-2- and IFNg [161]. The role of CD27 in the alloimmune response has been studied in a mouse cardiac transplant model. Blockade of CD70/CD27 interaction prolonged allograft survival in WT recipients and induced long-term graft survival in the absence of CD28 costimulation. CD70 blockade had little effect on CD4þ-mediated allograft rejection, but prevented CD8þmediated rejection by inhibiting proliferation and effector function of CD8þ T cells and reducing CD8þ memory T cells in vivo [162]. Thus, the CD27/CD70 pathway appears to be critical for CD8þ T-cell activation, especially in the absence of CD28/B7 costimulation. In a clinical study, expression of CD27 on peripheral CD4þ T cells correlates with the development of severe aGVHD after allogeneic HCT [163]. In contrast, a recent study showed the protective role of host-derived CD70 in suppressing GVHD by limiting donor T-cell expansion and effector function [164]. In this study, anti-CD70 treatment on day 4 and 7 posttransplant increased GVHD severity in an MHC-mismatched murine BMT model. Using genetic KO mice, they found the expression of CD70 on host, but not on donor T cells or BM, controlled donor T-cell apoptosis and expansion, and thus GVHD induction. Therefore, the CD70/CD27 pathway can function to promote as well as inhibit T-cell responses in allogeneic BMT.

HVEM/LIGHT Pathway Costimulation HVEM is a member of the TNFR family and acts as both ligand and receptor. As a ligand, HVEM engages the members of the Ig superfamily, BTLA and CD160 (discussed above). As a receptor, HVEM binds the canonical TNF-related ligands, lymphotoxin-a (LT-a) and LIGHT, both being TNF family members. LT-a is expressed on stromal cells and macrophages but does not provide a costimulatory signal. In contrast, LIGHT is expressed on activated T cells, NK cells, and also immature DCs and provides a costimulatory signal on T cells independent of the CD28 pathway [165]. Costimulation mediated by LIGHT enhances T-cell proliferation and Th1 cytokine secretion [166]. In addition to its effects on T cells, LIGHT contributes to DC maturation, enhanced by CD40L, resulting in further T-cell activation [167]. As a costimulatory molecule, LIGHT has been found to play an important role in alloreactivity and GVHD. Blockade of LIGHT by its soluble receptors inhibits the induction of DC-mediated primary allogeneic T-cell response in vitro. Engagement of LIGHT costimulates human T-cell proliferation, amplifies the NF-kB signaling pathway, and preferentially induces the production of IFNg, not IL-4 [166]. Using LIGHT- or HVEM-deficient (KO) T cells, in vivo studies showed that antihost CTL activity of donor T cells was completely abrogated, and survival of the recipient mice was significantly prolonged [168]. In the absence of LIGHT-HVEM costimulation, alloreactive donor T cells undergo vigorous apoptosis while their proliferative potential remains intact. Furthermore, blockade of LIGHT with a soluble receptor was able to reduce GVHD and improve survival in preclinical mouse BMT models [169]. However, this protective effect was only detected in CD8- but not in CD4-mediated GVHD. The investigators further tested the blockade of LIGHT combined with CD40 L blockade in a murine GVHD model [170] and found that coblockade starting at the day of BMT completely prevented aGVHD and improved survival. However, given that alloantigen-specific T cells were rendered tolerant, the GVL effect was also impaired.

GITR/GITRL Costimulation GITR, a glucocorticoid-induced TNF receptor family related gene, was originally identified by a differential display strategy as a gene induced by stimulation of the TCR in the presence of the glucocorticoid dexamethasone [171]. GITR is

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predominantly expressed on activated Teffs, B cells, NK cells, and macrophages [171]. One exception is Foxp3þ Tregs, which constitutively express GITR [172]. Its ligand, GITRL, is widely expressed in the immune system and can be detected at basal level APCs such as DCs, B cells, monocytes, and macrophages, with higher expression following cellular activation, particularly on plasmacytoid DCs (pDCs). GITRL is also expressed on endothelial cells as well as on activated T cells. Engagement of GITR on T cells enhances both CD4þ and CD8þ T-cell proliferation to suboptimal TCR stimulation and augments production of cytokines [173]. GITR lowers the threshold of CD28 costimulated in CD8þ T cells and is required for NF-kB activation and Bcl-xL upregulation downstream of CD28 [174]. One interesting feature of GITR is that its cytoplasmic tail can also activate signaling pathways that trigger apoptotic cell death, suggesting that manipulating the GITR/GITRL pathway could be applied in tumor immunotherapy and in autoimmune disease [175]. The role of the GITR/GITRL pathway in GVHD has not been extensively studied. Using an MHC-disparate murine BMT model, Muriglan et al. showed that GITR stimulation in vitro and in vivo enhances alloreactive CD8þ T-cell proliferation, whereas it decreases alloreactive CD4þ T-cell proliferation by increasing apoptosis through the Fas-FasL pathway. Furthermore, treatment with agonistic anti-GITR mAb increased GVHD-mediated CD8þ T cells but decreased GVHD mediated by CD4þ T cells, indicating that GITR has opposite effects on the regulation of alloreactive CD4 versus CD8 T cells [176]. A separate group showed that treatment with a single dose of agonistic anti-GITR mAb converted cGVHD to aGVHD by activating donor CD8þ T cells that otherwise become anergic [177]. Given that GITR signaling regulates both Teffs and Tregs, the effect of GITR on GVHD development through both populations of T cells is warranted to be further defined.

OTHERS CD155/DNAM-1 Costimulation DNAX accessory molecule 1 (DNAM-1; CD226) is a costimulatory molecule belonging to the immunoglobulin superfamily. DNAM-1 is primarily expressed on NK and CD8 T cells, which has been shown to enhance the cytotoxic effector function of NK cells and T cells against tumor- and virus-infected cells [178e180]. CD155 and its related family member CD112, the known ligands for DNAM-1, broadly expressed on hematopoietic, epithelial, and endothelial cells [181,182]. T-cell immunoglobulin and ITIM domain (TIGIT) is a coinhibitory molecule that can also bind with CD155 and CD112 and counterregulates the function of DNAM-1 [183]. Dysregulation of DNAM-1 is associated with susceptibility to juvenile idiopathic arthritis, type 1 diabetes (T1D), lupus, and rheumatoid arthritis in patients [184]. Deficiency of DNAM-1 in donor splenocytes reduced CD8 T cells producing IFNg in response to alloantigens and further alleviated aGVHD severity in a murine model with sublethal conditioning. An anti-DNAM-1 Ab was effective in the prevention and also treatment of GVHD in mice [185]. Using multiple models of GVHD, Hill’s group reported that CD4 Tregs, but not CD8 T cells, were responsible for the attenuated GVHD severity in mice receiving DNAM-1/ donor cells [186]. They found DNAM-1 and TIGIT expression were increased both on Tregs and Teffs. DNAM-1/ Tregs exhibited increased expansion and suppressive ability than WT Tregs after BMT. In addition, DNAM-1 is dispensable for the GVL activity of T cells in controlling leukemia regardless of CD155 expression. In a retrospective clinical study, high serum level of soluble DNAM-1 (30 p.m.) in the 7 days before allo-HSCT was associated with increased incidence of all grade (grade IeIV) and grade IIeIV aGVHD in patients, suggesting the potential role of DNAM-1 as a predictive biomarker for the development of GVHD [187]. In contrast, absence of CD155 in the recipient mice paradoxically increased GVHD mortality [188], suggesting the potential protective role of TIGIT signal may require CD155 expression on the host. Future studies are required to evaluate the role of CD112 and TIGIT in GVHD pathogenesis and GVL effect.

Galectine-9/Tim-3 Coinhibition The Tim-3 belongs to Tim (T-cell immunoglobulin domain and mucin domain) family genes that were first identified in 2002 [189]. In mice, there are eight TIM genes, among which, Tim-3 is the most widely studied member of the family [190,191]. However, only Tim-1, Tim-3, and Tim-4 are expressed in humans. Tim-3 protein is not expressed by naïve T cells, but is upregulated as they differentiated into IFNg-producing Th1 or Tc1 cells. In addition, Tim-3 was also found expressed on Tregs, DCs, NK cells, and monocytes. The C-type lectin galectin-9 is the first identified ligand of Tim-3 [192]. Besides galectine-9, high-mobility group protein B1 (HMGB-1), phosphatidyl serine (PtdSer), and Ceacam-1 can also bind to Tim-3. Tim-3 signaling is critically involved in autoimmune diseases, chronic infection, tumor immunity, and transplantation. Tim-3 pathway negatively regulates Th1 pathogenicity in auto- and alloimmune response. Blockade of Tim-3 pathway with anti-Tim-3 Ab accelerated autoimmune diabetes and EAE in mice [189,193]. Engagement of Tim-3

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by mouse recombinant galectin-9 suppresses allograft rejection and improves survival of skin allografts and cardiac allografts [194,195]. Tim-3 also marks dysfunctional or exhausted CD8 T cells in cancer and chronic virus infection. In addition, Tim-3 and PD-1 have nonredundant and synergistic functions in inhibiting effector T-cell response in antivirus and antitumor activity [196,197]. In allogeneic BMT, Tim-3 expression is upregulated on splenic and hepatic donor T cells, especially on CD8 T cells, during the development of aGVHD in mice [198]. The administration of anti-Tim-3 Ab increased IFNg-producing cells in spleen and liver and accelerated GVHD severity. Consistently, recombinant human galectin-9 significantly induces Ca2þ influx and apoptosis of T cells in MLR in vitro and reduces GVHD severity in mice [199]. Further study using anti-Tim-3 Ab, Tim-3 KO donor T cells, or galectin-9 transgenic recipients demonstrated the protective role of Tim-3/galectin-9 pathway in the development of GVHD. Paradoxically, blockade of Tim-3/galectin-9 pathway in the absence of donor Tregs inhibits, rather than augments, GVHD lethality [200]. This result is explained by elevated IFNg production and increased activation-induced cell death (AICD) in Tim-3 KO CD25- T cells. Furthermore, studies have demonstrated the predictive value of serum soluble Tim-3 level in GVHD patients in the clinic. Higher plasma Tim-3 concentration was found in patients with severe midgut GVHD, compared with those with upper gut GVHD and patients without GVHD [201]. Plasma Tim-3 level can be used to predict the development of peak grade 3e4 GVHD in patients [202,203].

Lag-3 Coinhibition Lymphocyte activation gene-3 (Lag-3; CD223) is a cell surface molecule expressed on activated T cells, NK cells, B cells, and pDCs [191]. Lag-3 was also found expressed in intracellular compartments in close association with the microtubuleorganizing center, which potentially facilitates a rapid transit to the T-cell surface during cell activation [204]. Lag-3 structurally resembles CD4 coreceptor and binds to MHC-II with a higher affinity than CD4. LSECtin is another ligand of Lag-3, which regulates the function of Lag-3 expressing CD8 T cells and NK cells [205]. Lag-3 is a coinhibitory receptor that modulates CD4 T-cell homeostasis, proliferation, and activation. Absence of Lag-3 resulted in accelerated autoimmune disease [191]. Furthermore, increased Lag-3 expression on CD8 T cells marks their dysfunction in antitumor activity. Therefore, Lag-3 has become a major target for checkpoint blockade in cancer immunotherapy. In allogeneic BMT, genetic absence of Lag-3 on donor CD4, but not CD8 conventional T cells, resulted in increased activation and proliferation of T cells and more severe GVHD [206]. Although Lag-3 is required for the suppressive function of Tregs reported by others, Lag-3/ Tregs were functionally intact in controlling GVHD. An agonist of Lag-3 is needed for evaluating the translational potential of modulating Lag-3 in alleviating GVHD.

REGULATION OF GVHD BY COSTIMULATION THROUGH TREGS While many types of T cells with regulatory function have been described before, CD4þCD25þFoxp3þ Tregs are extensively studied and best defined, in a large part, because they are a distinct T-cell subset naturally developed in thymus and play a key role in maintaining peripheral tolerance and protecting from autoimmunity in the immune system [207]. Besides maintaining self-tolerance, Tregs also play an indispensable role in the induction and maintenance of allograft tolerance [208]. In BMT, freshly isolated or ex vivoeexpanded donor CD4þCD25þ cells present in the marrow inoculum significantly delay or prevent GVHD in multiple donor-recipient strain combinations although with a low efficiency [209e212]. While controlling GVHD, recipient-specific Tregs have also been shown to favor immune reconstitution [213]. Furthermore, freshly isolated Tregs could control GVHD but still permit GVT activity against leukemic cells, presumably by preserving the perforin-mediated killing pathway [214]. We and others have demonstrated that ex vivo foxp3transduced, antigen-specific Tregs effectively prevent GVHD [215]. In addition to the wealth of data from experimental models, several groups have shown that a counterpart of mouse CD4þCD25þ cells, in terms of phenotype and activity in vitro, is present in human peripheral blood [216e218]. Expression of foxp3 in CD4 T cells correlates with their ability to function as Tregs. CD4þCD25þ T cells, generated as a consequence of stimulation of CD4þCD25- human T cells, also express foxp3 and acquire Treg function [219]. Furthermore, expression of foxp3 is negatively correlated with the severity of GVHD in the patient [220], indicating that increasing foxp3 expression and presumably increasing a number of Tregs over time was associated with the absence of GVHD, while the absence of foxp3 expression and presumably low numbers of Tregs was associated with the persistence of GVHD. Taken together, Tregs play a significant role in the control of GVHD development. Contributions of costimulatory pathways have been originally focused on Teffs. However, it has become clear that costimulation is also critical for the development, maintenance, and function of Tregs [221]. Significant differences in the biology of Teffs and Tregs are reflected by differences in the roles played by some of the costimulatory molecules on these

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two cell types. The initial study showing that CD28 deficiency resulted in aggressive autoimmunity in the nonobese diabetic (NOD) mouse due to a defect in Tregs opened up a new field of costimulatory biology on Tregs. Many costimulatory receptors or ligands that were previously defined as positive regulators for Teffs such as CD28, ICOS, OX40, CD40L, and 4-1BB are now implicated in negative regulation. Likewise, those negative regulators such as PD-1 and CTLA-4 for Teffs are now considered as a basis for Treg function [221]. In this review, we focus on the studies from many laboratories, including ours, which demonstrated that although CD28 and CTLA-4 are expressed on both regulatory and effector T cells, the B7/CD28:CTLA-4 pathways play a central role in the biology of regulatory T cells. Thus, a strategy that targets costimulatory signals for Teff suppression may also cause negative effect on Treg function in the induction of transplant tolerance. The first evidence indicating the role of CD28 in the homeostasis of Tregs is observed in the autoimmune-prone NOD mouse [222]. Whereas mice deficient for CD28 or B7 generally display defective immune responses, NOD mice deficient for CD28 or B7 surprisingly developed diabetes at 8e10 weeks of age. Exacerbation of disease was accompanied by a dramatic decrease in the percentage of CD4þCD25þ Tregs in those deficient NOD mice, and the infusion of Tregs from WT mice could control diabetes in NOD-CD28KO mice. These studies clearly indicate that CD28-B7/CD28 interaction is critical for maintaining normal levels of Tregs in the periphery and consequently for prevention of autoimmune diseases. The critical role of B7/CD28 interactions for Tregs has been observed in murine models of GVHD, where CD28 costimulatory signals were shown to be indispensable for the generation of Tregs that suppressed GVHD-induced allogeneic lymphocyte infusion [223]. Impaired homeostasis of Tregs in CD28-deficient mice is resulted from defects in Treg development in thymus and Treg maintenance peripherally [224]. The potential effects of CD28 Treg homeostasis are likely on survival, proliferation, and IL-2 production. Although CD28 mediates Bcl-XL upregulation through PI3K-signaling that promotes survival of Teffs [225], the same activity seems not necessary for CD28-mediated Foxp3 stabilization and Treg development [226], suggesting that Bcl-XL is not involved in the CD28 control of the Treg population. Tregs can vigorously proliferate, particularly in vivo [227,228], and blocking CD28 completely prevents the spontaneous proliferation of Tregs [224]. Furthermore, CD28 also affects the tissue distribution of Tregs that impacts tolerance versus immunity [229]. A third major mechanism by which CD28 regulates Treg homeostasis could be an indirect influence of IL-2 and IL-2 signaling, given that IL-2 and its receptor are critically important for the maintenance of the Treg population in the periphery [230,231]. However, the function of CD28 and IL-2 in Treg homeostasis only partially overlaps, and the additional role of CD28 directly supporting Treg homeostasis remains to be further defined. Application of CD28 blockade in the control of GVHD has been aimed to suppress Teffs, but more attention should be paid to its effects on Tregs as well in allogeneic HCT. In fact, Beyersdorf et al. showed that engagement of CD28 by an agonistic conventional anti-CD28 mAb protected GVHD by increasing frequencies of Tregs among total CD4þ T cells [37]. The same group also demonstrated that in vivo pretreatment of donor mice or short-term in vitro culture of donor lymph node cells with a superagonistic anti-CD28 mAb efficiently protected recipient mice from aGVHD [42]. The protection strongly relied on the presence of activated Tregs in the donor T-cell inoculum. By using tamoxifen-inducible CD28 KO mice, Tregs deficient for CD28 showed reduced survival in vivo and failed to mediate long-term protection of recipient mice from aGVHD [232]. Furthermore, CD28 signaling in host Tregs is responsible for controlling the fibrosis damage in skin and internal organs in recipient mice during cGVHD development [233]. Selective activation of CD28 in human Tregs can be achieved by using a low-dose CD28 superagonist without significantly activating conventional T cells to produce inflammatory cytokines [234]. In addition, single CD28 superagonist stimulation significantly increased human Treg expansion in vitro compared with standard anti-CD3/CD28 bead stimulation [235]. Taken together, because of the critical role of CD28 on Tregs, caution must be taken to apply CD28 blockade in combination with Treg therapy in GVHD. Besides naturally derived Tregs (nTregs), CD4þCD25þFoxp3þ cells can also be generated or differentiated from naïve CD4þ T cells in the peripheral and are termed as induced Tregs (iTregs). Several groups including ours showed that CD28but not ICOS signal is required for the differentiation of iTregs from naïve CD4þCD25- T cells. The requirement of CD28 for Treg differentiation was mediated by IL-2 but not other g-chain cytokines because neutralization of IL-2 with its specific mAb blocked Treg differentiation from WT T cells and addition of IL-2 restored Treg differentiation from CD28/ T cells [54]. However, using a series of transgenic mice on a CD28-deficient background that bears either WT or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K, or Itk, we found that strong CD28 costimulation suppresses iTreg induction through Lck signaling independent of IL-2 production. Furthermore, suppression of iTreg generation through CD28-mediated Lck signal contributes to T-cell pathogenicity in the induction of GVHD [236]. As observed previously, Tregs constitutively express high levels of CTLA-4, suggesting that CTLA4 may be involved in their suppressive function. Early evidence that supports the role of CTLA4 in regulating Tregs came from the observation that Tregs were required to control lymphoproliferative disease caused by CTLA4 KO T cells [237]. More recent

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studies confirmed a dual role of CTLA4 on Teffs and Tregs and further showed that CTLA4 on Tregs was critical to prevent activation of autoreactive Teffs in lymphoid tissues but CTLA4 on Teffs prevented infiltration and destruction of peripheral tissues [238,239]. Importantly, Wing et al. showed that a fatal lymphoproliferative disease still develops in mice with conditional deletion of CTLA4 in Foxp3-expressing Tregs despite slower kinetics, clearly demonstrating a dual role of CTLA4 on Teff and Tregs in maintaining T-cell homeostasis and tolerance. However, CTLA4 is dispensable for the development, survival, and homeostasis of Tregs, as normal numbers and percentages of CD4þCD25þFoxp3þ Tregs are found in the absence of CTLA4 [240]. The mechanisms by which Tregs induce immune suppression through CTLA4 is that ligation of B7 on APCs by CTLA4 on Tregs induces indoleamine 2,3-dioxygenase, an enzyme that catabolizes tryptophan in the extracellular milieu, which inhibits T-cell proliferation [241,242]. Recent data suggest that CTLA4 expressed by Tregs may inhibit APCs by stripping off B7 molecules, thereby inhibiting CD28-mediated costimulation to Teffs [240]. Importantly, the downmodulation of B7 expression required direct contacts between Tregs and APCs and was dependent on the presence of CTLA4 on Tregs [240,243]. The role of CTLA4 in GVHD through direct effect on Tregs has not been formerly tested, but CTLA4 should not be blocked in GVHD prevention given its dominant negative role in regulating Teffs but requirement for Treg suppressive function. Using WT, CD28, ICOS, or CD28/ICOS-double KO mice, we found that CD28 plays a major whereas ICOS plays a minor role in the development of and homeostasis of nTregs [54]. We and others also found that ICOS is dispensable for the generation and function of induced Tregs in vitro and in vivo [54,244]. However, in the NOD model of T1D, blockade or genetic deficiency of ICOS in NOD mice exacerbates T1D via abrogating Treg-mediated function and T1D protection [245,246]. ICOSL/ICOS pathway is required for pDC-mediated and mesenchymal stem cellemediated Treg induction [247,248]. Furthermore, abrogation of the ICOS pathway also blocks the ability of Tregs to produce IL-10 and prevent pulmonary inflammation and asthma [249]. Other studies indicate that ICOS costimulation promotes activation and expansion of Tregs, and thus ICOSþ Tregs have advantages in survival and proliferation as well as suppressive activity [250,251]. Engagement of the ICOS with ICOSL represents a key event in a process that culminates in Bcl-6 expression and acquisition of the follicular regulatory T-cell (Tfr) phenotypes, which are critical for regulating germinal center response [50,252]. Using mice with conditional deletion of ICOS in Tregs, we found that ICOS is required not only for the survival and homeostasis of iTregs, but also for the differentiation of Tfrs in recipients during the development of cGVHD [59a]. In patients with oral and cutaneous lichenoid cGVHD, Tregs expressed high level of ICOS and CD39 in GVHD target tissues than in blood, suggesting ICOS represents the functional marker of Tregs in cGVHD [253]. Overall, ICOS may contribute to the fitness of Tregs under certain circumstances. Previous studies have shown that ligation of PD-1 by either PD-L1 or PD-L2 attenuates Teff proliferation, cytokine secretion, and survival. As both nTregs and iTregs express PD-1 and PD-L1, the expression of ligand and receptor on the same cell has some interesting implications. PD-L1 expression on the APC or a newly encountered Treg has the potential to drive the differentiation of a naïve T cell toward an iTreg. In fact, substantial evidence supports that PD-1 ligation with PD-L1 increases in the de novo generation of iTregs from naïve CD4þ T cells [65] and enhances Foxp3 expression and suppressive function of established iTregs. In mechanistic studies, the PD-1 signal was shown to induce iTreg generation by attenuation of Akt-mTOR signaling that is inhibitory to the development of iTregs [254]. In chronic virus infection, PD1 was found upregulated on Tregs, and its interaction with PD-L1 on effector CD8 T cells enhanced the suppressive ability of Tregs in controlling antivirus CD8 T-cell response [255]. In contrast, expression of PD-1 marks dysfunctional and exhausted Tregs that are enriched in tumor infiltrated in malignant glioma patients [256]. PD-1 and PD-L1 interaction inhibits generation and function of Tfr cells. PD-1/ or PD-L1/ mice had greater abundance of Tfr cells after immunization with myelin-oligodendrocyte glycoprotein, and those Tfr cells had enhanced suppressive ability in controlling germinal center response [257]. In HCT, PD-L1/PD-1 axis has been involved in promoting Treg differentiation and homeostasis. During low-dose IL-2 treatment, central memory (CD44þCD62Lþ) Tregs especially had enhanced PD-1 expression [70]. Deficiency of PD-1 on Tregs diminished the effect of low-dose IL-2 treatment on Treg expansion due to increased apoptosis in PD-1/ Tregs. PD-L1 and PD-1 interaction promotes conversion of human Th1 cells into Tregs and thereby preventing GVHD in a xenogeneic model [71]. PD-1H also plays a role in controlling the differentiation, stability, and function of Tregs. PD-1H blockade impaired the induction of iTregs as well as the suppressive function of nTregs in mice [258]. PD-1H-Ig fusion protein promotes the induction of iTregs from human naïve T cells [258]. PD-1H deficiency impaired iTreg, but not nTreg, accumulation in lymphoid organs in mice [259]. In addition, PD-1H/- iTregs were less stable and easy to convert to Th1 or Th17 cells in the inflammatory environment. Consistently, injection of PD-1H agonistic mAb alleviated GVHD severity in mice. Expansion of donor Tregs is one of the major mechanisms in the long-term tolerance maintenance in these mice [91]. Besides the important role of CD28, CTLA4, ICOSL/ICOS, PD-L1/PD-1, and PD-1H in regulating Treg generation and function, some other coinhibitors in CD28/B7 family, including B7eH3, B7eH4, and BTLA, are also involved in

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Treg-mediated DC tolerance and also in DC-mediated Treg generation. Study shows that Treg-exposed DCs upregulated B7eH3, reduced numbers of MHC-peptide complex, and impaired T-cell stimulatory function, further suggesting the immunosuppressive role of B7eH3 pathway [260]. Tregs trigger the expression of IL-10 in APCs, which stimulates B7eH4 expression on APCs and further renders APCs immunosuppressive [261]. Furthermore, BTLA expression on DCs is required for iTreg induction by DEC205þCD8þ DCs. The ligation of BTLA and HVEM is required for CD5 upregulation in T cells and iTreg induction [262]. Further studies are needed to evaluate how these coinhibitors regulate iTreg differentiation and function in allogeneic HCT. New lines of evidence support that several other costimulatory molecules may also affect Treg function and/or differentiation. For example, OX40 is also expressed on Tregs (both nTregs and iTregs), but OX40 has diametrically opposite effects on Tregs and Teffs. While providing a positive costimulation on Teffs, OX40 is a potent negative regulator of Tregs [263e265]. Engagement of OX40 on Tregs using an agonist anti-OX40 mAb or OX40L-transgenic APCs consistently abolished their suppressive activity in vitro and in vivo. Furthermore, OX40 costimulation on Teffs completely inhibited the generation of iTregs from CD4þ naïve T cells with TGF-b and blocked nTreg suppressive function [266]. OX40 is dispensable for Treg homeostasis, as Treg number and function are intact in OX40-deficient mice [263]. Blockade of OX40L/OX40 interaction enhanced skin allograft survival by controlling alloreactive effector T-cell response but supporting Treg survival [267]. These findings suggest that unlike the CD28/B7 pathway, blockade of OX40 might promote tolerance by both inhibiting Teffs and by promoting Treg activity, which makes OX40 blockade a promising target for the prevention of GVHD. A subset of Tregs constitutively expresses GITR [172,268]. GITR/ mice have slightly decreased Tregs, whereas GITRL transgenic mice have threefold higher Tregs, suggesting GITR promotes Treg development [269]. In vitro study indicated that ligation of GITR abrogates Treg suppressive function [270]. Removal of GITRþ T cells or administration of a mAb to GITR produced organ-specific autoimmune disease in otherwise normal mice. Thus, GITR plays a key role in dominant immunological self-tolerance maintained by Tregs and could be a suitable molecular target for preventing or treating autoimmune disease. In a transplant model, GITR ligation compromised Treg-dependent tolerance but had no effect on the response of T cells alone [271]. Furthermore, engagement of GITR on effector T cells by its ligand mediates resistance to suppression by Tregs [272]. Taken together, the GITR signal appears to augment the responses of Teffs but inhibit Treg suppressive function, and therefore blockade of GITR should promote GVHD prevention. However, how GITR affects GVHD development through Tregs has not yet been evaluated. CD30-deficient mice display elevated numbers of thymocytes due to a gross defect in negative selection, but have a normal number and phenotype of mature T cells in the periphery and lack obvious immunologic abnormalities [273]. Other studies suggest that CD30þ T cells, present at sites of inflammation in autoimmune diseases such as rheumatoid arthritis, may serve a regulatory role [274]. Development and homeostasis of nTregs are not impaired in CD30/ mice, and CD30/ nTregs have intact suppressive function in vitro [275]. However, CD30 was found to be critical for CD4þCD25þ Tregs to suppress allograft rejection induced by CD8þ memory cells in vivo. Consistently, Zhang et al. described CD30 as a unique marker on antigen-induced T cells with regulatory properties that prevented allograft rejection by inducing apoptosis of activated T cells [276]. CD30 can also serve as a marker to distinguish suppressive Foxp3þ Tregs among nonsuppressive Foxp3þ Teffs after in vitro activation of human CD4þ T cells [277]. In allogeneic BMT, Zeiser et al. investigated the role of CD30 signaling in Treg cell function during aGVHD. They found that nTregs derived from CD30/ donor mice were significantly less effective in preventing GVHD lethality. Early blockade of the CD153/CD30 pathway with a neutralizing anti-CD153 mAb reduced Treg expansion and Treg-mediated protection from proinflammatory cytokine accumulation and donor T-cell apoptosis. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after allogeneic BMT [278]. Another TNFR family member involved in the modulation of Treg suppression is 4-1BB. Unlike resting CD4þ and CD8þ T cells, Tregs constitutively express 4-1BB that can be further increased by TCR or IL-2 stimulation. After stimulation of human PBMC with alloantigens, 4-1BB was selectively upregulated on Tregs and more than 90% of 41BBþCD40L cells were Foxp3þ cells. Given a reciprocal expression of 4-1BB and CD40L on Tregs compared with Teffs (4-1BBCD40Lþ), human alloantigen-reactive nTregs could be directly sorted using 4-1BBþCD40L markers [279]. Engagement of 4-1BB on Tregs increases their expansion without affecting their function [280,281]. Interestingly, 4-1BBL/ mice have a small but significant decrease in the numbers of Tregs [138]. However, in a study using a mixed culture of 4-1BB/ Teffs and 4-1BBþ/þ Tregs, 4-1BB ligation with anti-4-1BB on the Tregs suppressed their function in vitro without inducing their proliferation. Moreover, delivery of anti-4-1BB and CD25þ 4-1BBþ/þ Tregs accelerated GVHD mediated by 4-1BB-deficient effectors in vivo [282]. Provision of 4-1BB ligand abrogates Treg suppression in standard coculture assays, and this is largely believed to reflect a role for 4-1BB signaling in rendering Teffs resistant to suppression rather than impairing Treg function [280,283].

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CD27/CD70 axis regulates Treg generation and function. Treg number in thymus and peripheral lymphoid organs was reduced in mice with genetic depletion of CD27 or CD70 [284]. CD70 expressed on both thymic epithelial cells and DCs in the thymic medulla contributed to Treg development. CD27-CD70 signaling promoted the positive selection of Tregs within thymus and prevented Treg apoptosis. Although CD70 signal is essential for optimal virus-specific CD8 T-cell generation during the early phase of virus infection, CD70/ mice had heighted cytokine response due to impaired generation and suppressive function of Tregs in these mice [285]. Similarly, CD27-CD70 signal is required for the suppressive function of Tregs in constraining Th1 response. In a CD27-dependent manner, thymus-derived Tregs directly downregulated CD70 expression on DCs by inducing endocytosis of CD27 and CD70 into DCs [286]. Therefore, modulating Treg generation and function could be another mechanism in the suppressive role of host-derived CD70 in GVHD development [164]. In addition to the role in regulating IFNg-producing Th1 and Tc1 cells, Tim-3 also plays a role in regulating the function of Tregs cells. In a model of allograft rejection, Tim-3þ Tregs are prominent among graft-infiltrating Tregs. Tim3þ Tregs, which coexpress PD-1 as well, had increased expression of immune-suppressive molecules, such as IL-10, CD39, CD73, and enhanced effector function in vitro. However, these Tim-3þ Tregs appear to be short-lived in vivo [287]. Compared with Tim-3- Tregs, human Tim-3þ Tregs are highly effective in suppressing Th1 and Th17 cells in vitro [288]. Tim3 signal in Tregs promotes their homeostasis and regulatory function at the site of tissue inflammation [289,290]. Interestingly, Tim-3/ donor T cells accelerated GVHD, while Tim-3/ CD25- T cells alleviated GVHD compared with their WT counterparts [200]. The controversial results were explained as Tim-3/ CD25- T cells caused increased level of IFNg and AICD in donor T cells in the early phase of GVHD. However, no difference was observed in the ability of Tim-3/ Tregs to suppress Teff division in vitro compared with WT Tregs. The study of costimulatory molecules on Tregs is in its infancy. It is of critical importance to target costimulatory molecules that will specifically inhibit effector T cells without inhibiting Treg homeostasis or suppressor function. An ideal costimulatory target will be the one that specifically augments Treg number and/or suppressor function without boosting Teff generation (e.g., unlike CD28 signaling). Although conventional T cells can be converted to Foxp3þ Tregs in vitro by activation in the presence of TGF-b, spontaneous conversion in allogeneic BMT in vivo is extremely rare [54]. Although it has been hoped that costimulatory blockade would promote this process, in vivo studies have not shown clear-cut evidence that anti-CD154 or CTLA-4-Ig enhance conversion of non-Tregs into Foxp3þ Tregs. In the meantime, several other promising approaches to enhance Tregs to promote tolerance induction are now emerging. For example, extrapolating from studies of OX40 costimulation discussed above, this pathway is a particularly attractive target for therapeutic blockade. OX40 blockade may inhibit T-effector cells and simultaneously augment Treg function. There may be strategies that might specifically promote Treg homeostatic or antigen-induced proliferation, resulting in preferential expansion of Tregs over Teffector cells in vivo. The fact that homeostatic proliferation of Tregs is regulated differently than that of conventional T cells and that at least some costimulatory molecules exhibit distinct functions on Tregs compared with conventional T cells suggests that research in this area will be fruitful. It is also possible that agents targeting costimulatory pathways can be combined, for example, by use of agents that potently inhibit T-effector cells with other agents that promote Tregs. This combination may also achieve the goal of tipping the balance of Teffs and Tregs toward active immune regulation to create transplant tolerance. However, a particular caution must be paid to the potential effect on the GVL activity of any strategy in favor of immune regulation and tolerance.

CONCLUSIONS T-cell costimulation and coinhibition has been one of the focused areas in GVHD research for many years. It is clear that costimulatory and coinhibitory pathways are central to regulating T-cell immunity, and thereby targeting these pathways represents one of the most promising approaches for the prevention and treatment of GVHD. In addition to the extensively studied B7/CD28:CTLA4 pathway, other costimulatory pathways have been ultimately implicated in GVHD. In particular, several members of the TNFR family such as CD40, OX-40, and 4-1BB have been demonstrated to contribute to T-cell activation and the development of aGVHD. Targeting costimulatory receptors or ligands using genetically modified mice or various mAbs specific for these molecules have been substantially studied in preclinical GVHD models with promising results. However, modulating costimulation to control GVHD in clinic will undoubtedly give rise to new challenges. We have realized that costimulatory pathways regulate multiple aspects of the alloimmune responses on Teffs and Tregs, and thus when targeting costimulation to inhibit Teffs, preservation of Tregs should be considered. Given the potential overlapping functions of costimulatory molecules, multiple positive costimulators may have to be blocked to control GVHD. On the other hand, better understanding of the mechanism of coinhibitory molecules involved in counterregulation of T-cell activation (i.e., CTLA4 and PD-1) makes these molecules attractive targets for GVHD therapy. However, the

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agonists for most coinhibitory receptors are not yet available. It is likely that a CTLA4 fusion protein will be tested in patients with acute GVHD soon, as it is already used in patients with autoimmune diseases. In allogeneic HCT settings, a critical issue is to develop therapeutic strategies that, while blocking alloreactivity, do not compromise the GVT effect. Finally, researchers and clinicians will need to collaborate with the industry to develop druggable reagents to promote translation of knowledge learned in animals toward clinical application.

REFERENCES [1] Socie G, Blazar BR. Acute graft-versus-host disease: from the bench to the bedside. Blood 2009;114(20):4327e36. [2] Schwartz RH. A cell culture model for T lymphocyte clonal anergy. Science 1990;248(4961):1349e56. [3] Li XC, Rothstein DM, Sayegh MH. Costimulatory pathways in transplantation: challenges and new developments. Immunol Rev 2009;229(1):271e93. [4] Briones J, Novelli S, Sierra J. T-cell costimulatory molecules in acute-graft-versus host disease: therapeutic implications. Bone Marrow Res 2011;2011:976793. [4a] Kean LS, Turka LA, Blazar BR. Advances in targeting co-inhibitory and co-stimulatory pathways in transplantation settings: the Yin to the Yang of cancer immunotherapy. Immunological reviews 2017;276(1):192e212. [4b] Ford ML. T Cell Cosignaling Molecules in Transplantation. Immunity 2016;44(5):1020e33. [4c] Ward-Kavanagh LK, Lin WW, Sedy JR, Ware CF. The TNF Receptor Superfamily in Co-stimulating and Co-inhibitory Responses. Immunity 2016;44(5):1005e19. [4d] Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA. CD28 Costimulation: From Mechanism to Therapy. Immunity 2016;44(5):973e88. [5] Bluestone JA, St Clair EW, Turka LA. CTLA4Ig: bridging the basic immunology with clinical application. Immunity 2006;24(3):233e8. [6] Shahinian A, Pfeffer K, Kelvin P, Kundig T, Kishihara K, Wakeham A, Kawai K, Ohashi P, Thompson C, Mak T. Differential T cell costimulatory requirements in CD28-deficient mice. Science 1993;261(609):12. [7] Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994;1(5):405e13. [8] Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995;3(5):541e7. [9] Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 1995;270(5238):985e8. [10] Thompson CB, Allison JP. The emerging role of CTLA-4 as an immune attenuator. Immunity 1997;7(4):445e50. [11] Tan P, Anasetti C, Hansen JA, Melrose J, Brunvand M, Bradshaw J, Ledbetter JA, Linsley PS. Induction of alloantigen-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7/BB1. J Exp Med 1993;177(1):165e73. [12] Blazar BR, Taylor PA, Linsley PS, Vallera DA. In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice. Blood 1994;83(12):3815e25. [13] Blazar BR, Sharpe AH, Taylor PA, Panoskaltsis-Mortari A, Gray GS, Korngold R, Vallera DA. Infusion of anti-B7.1 (CD80) and anti-B7.2 (CD86) monoclonal antibodies inhibits murine graft-versus-host disease lethality in part via direct effects on CD4þ and CD8þ T cells. J Immunol 1996;157(8):3250e9. [14] Blazar BR, Taylor PA, Boyer MW, Panoskaltsis-Mortari A, Allison JP, Vallera DA. CD28/B7 interactions are required for sustaining the graftversus-leukemia effect of delayed post-bone marrow transplantation splenocyte infusion in murine recipients of myeloid or lymphoid leukemia cells. J Immunol 1997;159(7):3460e73. [15] Ogawa S, Nagamatsu G, Watanabe M, Watanabe S, Hayashi T, Horita S, Nitta K, Nihei H, Tezuka K, Abe R. Opposing effects of anti-activationinducible lymphocyte-immunomodulatory molecule/inducible costimulator antibody on the development of acute versus chronic graft-versus-host disease. J Immunol 2001;167(10):5741e8. [16] Ohata J, Sakurai J, Saito K, Tani K, Asano S, Azuma M. Differential graft-versus-leukaemia effect by CD28 and CD40 co-stimulatory blockade after graft-versus-host disease prophylaxis. Clin Exp Immunol 2002;129(1):61e8. [17] Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Sharpe AH, Vallera DA. Opposing roles of CD28:B7 and CTLA-4:B7 pathways in regulating in vivo alloresponses in murine recipients of MHC disparate T cells. J Immunol 1999;162(11):6368e77. [18] Bosch-Vizcaya A, Perez-Garcia A, Brunet S, Solano C, Buno I, Guillem V, Martinez-Laperche C, Sanz G, Barrenetxea C, Martinez C, et al. Donor CTLA-4 genotype influences clinical outcome after T cell-depleted allogeneic hematopoietic stem cell transplantation from HLA-identical sibling donors. Biol Blood Marrow Transplant 2012;18(1):100e5. [19] Bashey A, Medina B, Corringham S, Pasek M, Carrier E, Vrooman L, Lowy I, Solomon SR, Morris LE, Holland HK, et al. CTLA4 blockade with ipilimumab to treat relapse of malignancy after allogeneic hematopoietic cell transplantation. Blood 2009;113(7):1581e8. [20] Park HB, Lee JE, Oh YM, Lee SJ, Eom HS, Choi K. CTLA4-CD28 chimera gene modification of T cells enhances the therapeutic efficacy of donor lymphocyte infusion for hematological malignancy. Exp Mol Med 2017;49(7):e360. [21] Shin JH, Park HB, Oh YM, Lim DP, Lee JE, Seo HH, Lee SJ, Eom HS, Kim IH, Lee SH, et al. Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models. Blood 2012;119(24):5678e87. [22] Ghosh A, Smith M, James SE, Davila ML, Velardi E, Argyropoulos KV, Gunset G, Perna F, Kreines FM, Levy ER, et al. Donor CD19 CAR T cells exert potent graft-versus-lymphoma activity with diminished graft-versus-host activity. Nat Med 2017;23(2):242e9.

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185

[23] Li J, Semple K, Suh WK, Liu C, Chen F, Blazar BR, Yu XZ. Roles of CD28, CTLA4, and inducible costimulator in acute graft-versus-host disease in mice. Biol Blood Marrow Transplant 2011;17(7):962e9. [24] Yu XZ, Bidwell SJ, Martin PJ, Anasetti C. CD28-specific antibody prevents graft-versus-host disease in mice. J Immunol 2000;164(9):4564e8. [25] Poirier N, Azimzadeh AM, Zhang T, Dilek N, Mary C, Nguyen B, Tillou X, Wu G, Reneaudin K, Hervouet J, et al. Inducing CTLA-4-dependent immune regulation by selective CD28 blockade promotes regulatory T cells in organ transplantation. Sci Transl Med 2010;2(17):17ra0. [26] Vincenti F, Dritselis A, Kirkpatrick P. Belatacept. Nat Rev Drug Discov 2011;10(9):655e6. [27] Luhder F, Huang Y, Dennehy KM, Guntermann C, Muller I, Winkler E, Kerkau T, Ikemizu S, Davis SJ, Hanke T, et al. Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists. J Exp Med 2003;197(8):955e66. [28] Vanhove B, Laflamme G, Coulon F, Mougin M, Vusio P, Haspot F, Tiollier J, Soulillou JP. Selective blockade of CD28 and not CTLA-4 with a single-chain Fv-alpha1-antitrypsin fusion antibody. Blood 2003;102(2):564e70. [29] Tan P, Mitchell DA, Buss TN, Holmes MA, Anasetti C, Foote J. “Superhumanized” antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28. J Immunol 2002;169(2):1119e25. [30] Suchard SJ, Davis PM, Kansal S, Stetsko DK, Brosius R, Tamura J, Schneeweis L, Bryson J, Salcedo T, Wang H, et al. A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety. J Immunol 2013;191(9):4599e610. [31] Shi R, Honczarenko M, Zhang S, Fleener C, Mora J, Lee SK, Wang R, Liu X, Shevell DE, Yang Z, et al. Pharmacokinetic, pharmacodynamic, and safety profile of a novel anti-CD28 domain antibody antagonist in healthy subjects. J Clin Pharmacol 2017;57(2):161e72. [32] Yang Z, Wang H, Salcedo TW, Suchard SJ, Xie JH, Schneeweis LA, Fleener CA, Calore JD, Shi R, Zhang SX, et al. Integrated pharmacokinetic/ pharmacodynamic analysis for determining the minimal anticipated biological effect level of a novel anti-CD28 receptor antagonist BMS-931699. J Pharmacol Exp Therapeut 2015;355(3):506e15. [33] Poirier N, Mary C, Dilek N, Hervouet J, Minault D, Blancho G, Vanhove B. Preclinical efficacy and immunological safety of FR104, an antagonist anti-CD28 monovalent Fab’ antibody. Am J Transplant 2012;12(10):2630e40. [34] Jang MS, Pan F, Erickson LM, Fisniku O, Crews G, Wynn C, Hong IC, Tamura K, Kobayashi M, Jiang H. A blocking anti-CD28-specific antibody induces long-term heart allograft survival by suppression of the PKC theta-JNK signal pathway. Transplantation 2008;85(7):1051e5. [35] Shiao SL, McNiff JM, Masunaga T, Tamura K, Kubo K, Pober JS. Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities. Transplantation 2007;83(3):304e13. [36] Raychaudhuri SP, Kundu-Raychaudhuri S, Tamura K, Masunaga T, Kubo K, Hanaoka K, Jiang WY, Herzenberg LA. FR255734, a humanized, Fc-Silent, Anti-CD28 antibody, improves psoriasis in the SCID mouse-psoriasis xenograft model. J Invest Dermatol 2008;128(8):1969e76. [37] Beyersdorf N, Ding X, Blank G, Dennehy KM, Kerkau T, Hunig T. Protection from graft-versus-host disease with a novel B7 binding site-specific mouse anti-mouse CD28 monoclonal antibody. Blood 2008;112(10):4328e36. [38] Yu XZ, Albert MH, Martin PJ, Anasetti C. CD28 ligation induces transplantation tolerance by IFN-gamma-dependent depletion of T cells that recognize alloantigens. J Clin Invest 2004;113(11):1624e30. [39] Mirenda V, Jarmin SJ, David R, Dyson J, Scott D, Gu Y, Lechler RI, Okkenhaug K, Marelli-Berg FM. Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28. Blood 2007;109(7):2968e77. [40] Dengler TJ, Szabo G, Sido B, Nottmeyer W, Zimmerman R, Vahl CF, Hunig T, Meuer SC. Prolonged allograft survival but no tolerance induction by modulating CD28 antibody JJ319 after high-responder rat heart transplantation. Transplantation 1999;67(3):392e8. [41] Haspot F, Villemain F, Laflamme G, Coulon F, Olive D, Tiollier J, Soulilou J-P, Vanhove B. Differential effect of CD28 versus B7 blockade on direct pathway of allorecognition and self-restricted responses. Blood 2002;99:2228e34. [42] Beyersdorf N, Ding X, Hunig T, Kerkau T. Superagonistic CD28 stimulation of allogeneic T cells protects from acute graft-versus-host disease. Blood 2009;114(20):4575e82. [43] Kitazawa Y, Fujino M, Li XK, Xie L, Ichimaru N, Okumi M, Nonomura N, Tsujimura A, Isaka Y, Kimura H, et al. Superagonist CD28 antibody preferentially expanded Foxp3-expressing nTreg cells and prevented graft-versus-host diseases. Cell Transplant 2009;18(5):627e37. [44] Albert MH, Yu XZ, Martin PJ, Anasetti C. Prevention of lethal acute GVHD with an agonistic CD28 antibody and rapamycin. Blood 2005;105(3):1355e61. [45] Guinan EC, Boussiotis VA, Neuberg D, Brennan LL, Hirano N, Nadler LM, Gribben JG. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med 1999;340(22):1704e14. [46] Betts BC, Veerapathran A, Pidala J, Yang H, Horna P, Walton K, Cubitt CL, Gunawan S, Lawrence HR, Lawrence NJ, et al. Targeting Aurora kinase A and JAK2 prevents GVHD while maintaining Treg and antitumor CTL function. Sci Transl Med 2017;9(372). [47] Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I, Kroczek RA. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 1999;397(6716):263e6. [48] Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, Horan T, Shih G, Zhang M, Coccia MA, Kohno T, et al. T-cell co-stimulation through B7RP-1 and ICOS. Nature 1999;402(6763):827e32. [49] Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, Manning S, Nguyen T, Burwell T, Schneider H, Gonzalo JA, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000;13(1):95e105. [50] Gigoux M, Shang J, Pak Y, Xu M, Choe J, Mak TW, Suh WK. Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase. Proc Natl Acad Sci USA 2009;106(48):20371e6. [51] Dong C, Juedes AE, Temann UA, Shresta S, Allison JP, Ruddle NH, Flavell RA. ICOS co-stimulatory receptor is essential for T-cell activation and function. Nature 2001;409(6816):97e101.

186 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

[52] Coyle AJ, Gutierrez-Ramos JC. The expanding B7 superfamily: increasing complexity in costimulatory signals regulating T cell function. Nat Immunol 2001;2(3):203e9. [53] Ozkaynak E, Gao W, Shemmeri N, Wang C, Gutierrez-Ramos JC, Amaral J, Qin S, Rottman JB, Coyle AJ, Hancock WW. Importance of ICOSB7RP-1 costimulation in acute and chronic allograft rejection. Nat Immunol 2001;2(7):591e6. [54] Guo F, Iclozan C, Suh WK, Anasetti C, Yu XZ. CD28 controls differentiation of regulatory T cells from naive CD4 T cells. J Immunol 2008;181(4):2285e91. [55] Taylor PA, Panoskaltsis-Mortari A, Freeman GJ, Sharpe AH, Noelle RJ, Rudensky AY, Mak TW, Serody JS, Blazar BR. Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells down-regulates graft-versus-host disease (GVHD) and faciliates engraftment of allogeneic bone marrow (BM). Blood 2005;105(8):3372e80. [56] Hubbard VM, Eng JM, Ramirez-Montagut T, Tjoe KH, Muriglan SJ, Kochman AA, Terwey TH, Willis LM, Schiro R, Heller G, et al. Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation. Blood 2005;106(9):3285e92. [57] Yu XZ, Liang Y, Nurieva RI, Guo F, Anasetti C, Dong C. Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells. J Immunol 2006;176(12):7394e401. [58] Burlion A, Brunel S, Petit NY, Olive D, Marodon G. Targeting the human T-cell inducible COStimulator molecule with a monoclonal antibody prevents graft-vs-host disease and preserves graft vs leukemia in a xenograft murine model. Front Immunol 2017;8(756). [59] Mak TW, Shahinian A, Yoshinaga SK, Wakeham A, Boucher LM, Pintilie M, Duncan G, Gajewska BU, Gronski M, Eriksson U, et al. Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses. Nat Immunol 2003;4(8):765e72. [59a] Zhang M, Wu Y, Bastian D, et al. Inducible T-Cell Co-Stimulator Impacts Chronic Graft-Versus-Host Disease by Regulating Both Pathogenic and Regulatory T Cells. Front Immunol. 2018;9(1461). [60] Flynn R, Du J, Veenstra RG, Reichenbach DK, Panoskaltsis-Mortari A, Taylor PA, Freeman GJ, Serody JS, Murphy WJ, Munn DH, et al. Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans. Blood 2014;123(25):3988e98. [61] Suh WK, Tafuri A, Berg-Brown NN, Shahinian A, Plyte S, Duncan GS, Okada H, Wakeham A, Odermatt B, Ohashi PS, et al. The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28. J Immunol 2004;172(10):5917e23. [62] Nanji SA, Hancock WW, Anderson CC, Adams AB, Luo B, Schur CD, Pawlick RL, Wang L, Coyle AJ, Larsen CP, et al. Multiple combination therapies involving blockade of ICOS/B7RP-1 costimulation facilitate long-term islet allograft survival. Am J Transplant 2004;4(4):526e36. [63] Poirier N, Blancho G, Vanhove B. A more selective costimulatory blockade of the CD28-B7 pathway. Transpl Int 2011;24(1):2e11. [64] Tajima N, Tezuka K, Tanimoto A, Miyai A, Tanimoto M, Maruhashi J, Watanabe Y. JTA-009, a fully human antibody against human AILIM/ ICOS, ameliorates graft-vs-host reaction in SCID mice grafted with human PBMCs. Exp Hematol 2008;36(11):1514e23. [65] Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev 2010;236:219e42. [66] Brown JA, Dorfman DM, Ma FR, Sullivan EL, Munoz O, Wood CR, Greenfield EA, Freeman GJ. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 2003;170(3):1257e66. [67] Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11(2):141e51. [68] Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science 2001;291(5502):319e22. [69] Deng R, Cassady K, Li X, Yao S, Zhang M, Racine J, Lin J, Chen L, Zeng D. B7H1/CD80 interaction augments PD-1-dependent T cell apoptosis and ameliorates graft-versus-host disease. J Immunol 2015;194(2):560e74. [70] Asano T, Meguri Y, Yoshioka T, Kishi Y, Iwamoto M, Nakamura M, Sando Y, Yagita H, Koreth J, Kim HT, et al. PD-1 modulates regulatory Tcell homeostasis during low-dose interleukin-2 therapy. Blood 2017;129(15):2186e97. [71] Amarnath S, Mangus CW, Wang JC, Wei F, He A, Kapoor V, Foley JE, Massey PR, Felizardo TC, Riley JL, et al. The PDL1-PD1 axis converts human TH1 cells into regulatory T cells. Sci Transl Med 2011;3(111):111ra20. [72] Blazar BR, Carreno BM, Panoskaltsis-Mortari A, Carter L, Iwai Y, Yagita H, Nishimura H, Taylor PA. Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-gamma-dependent mechanism. J Immunol 2003;171(3):1272e7. [73] Habicht A, Kewalaramani R, Vu MD, Demirci G, Blazar BR, Sayegh MH, Li XC. Striking dichotomy of PD-L1 and PD-L2 pathways in regulating alloreactive CD4(þ) and CD8(þ) T cells in vivo. Am J Transplant 2007;7(12):2683e92. [74] Saha A, Aoyama K, Taylor PA, Koehn BH, Veenstra RG, Panoskaltsis-Mortari A, Munn DH, Murphy WJ, Azuma M, Yagita H, et al. Host programmed death ligand 1 is dominant over programmed death ligand 2 expression in regulating graft-versus-host disease lethality. Blood 2013;122(17):3062e73. [75] Li X, Deng R, He W, Liu C, Wang M, Young J, Meng Z, Du C, Huang W, Chen L, et al. Loss of B7-H1 expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8þ T cells and persistence of graft-versus-host disease. J Immunol 2012;188(2):724e34. [76] Fujiwara H, Maeda Y, Kobayashi K, Nishimori H, Matsuoka K, Fujii N, Kondo E, Tanaka T, Chen L, Azuma M, et al. Programmed death-1 pathway in host tissues ameliorates Th17/Th1-mediated experimental chronic graft-versus-host disease. J Immunol 2014;193(5):2565e73. [77] Ni X, Song Q, Cassady K, Deng R, Jin H, Zhang M, Dong H, Forman S, Martin PJ, Chen YZ, et al. PD-L1 interacts with CD80 to regulate graftversus-leukemia activity of donor CD8þ T cells. J Clin Invest 2017;127(5):1960e77. [78] Asakura S, Hashimoto D, Takashima S, Sugiyama H, Maeda Y, Akashi K, Tanimoto M, Teshima T. Alloantigen expression on non-hematopoietic cells reduces graft-versus-leukemia effects in mice. J Clin Invest 2010;120(7):2370e8.

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[79] Michonneau D, Sagoo P, Breart B, Garcia Z, Celli S, Bousso P. The PD-1 axis enforces an anatomical segregation of CTL activity that creates tumor Niches after allogeneic hematopoietic stem cell transplantation. Immunity 2016;44(1):143e54. [80] Koestner W, Hapke M, Herbst J, Klein C, Welte K, Fruehauf J, Flatley A, Vignali DA, Hardtke-Wolenski M, Jaeckel E, et al. PD-L1 blockade effectively restores strong graft-versus-leukemia effects without graft-versus-host disease after delayed adoptive transfer of T-cell receptor geneengineered allogeneic CD8þ T cells. Blood 2011;117(3):1030e41. [81] Ansell SM, Lesokhin AM, Borrello I, Halwani A, Scott EC, Gutierrez M, Schuster SJ, Millenson MM, Cattry D, Freeman GJ, et al. PD-1 blockade with nivolumab in relapsed or refractory Hodgkin’s lymphoma. N Engl J Med 2015;372(4):311e9. [82] Haverkos BM, Abbott D, Hamadani M, Armand P, Flowers ME, Merryman R, Kamdar M, Kanate AS, Saad A, Mehta A, et al. PD-1 blockade for relapsed lymphoma post-allogeneic hematopoietic cell transplant: high response rate but frequent GVHD. Blood 2017;130(2):221e8. [83] Singh AK, Porrata LF, Aljitawi O, Lin T, Shune L, Ganguly S, McGuirk JP, Abhyankar S. Fatal GvHD induced by PD-1 inhibitor pembrolizumab in a patient with Hodgkin’s lymphoma. Bone Marrow Transplant 2016;51(9):1268e70. [84] McDuffee E, Aue G, Cook L, Ramos-Delgado C, Shalabi R, Worthy T, Vo P, Childs RW. Tumor regression concomitant with steroid-refractory GvHD highlights the pitfalls of PD-1 blockade following allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2017;52(5):759e61. [85] Flies DB, Wang S, Xu H, Chen L. Cutting edge: a monoclonal antibody specific for the programmed death-1 homolog prevents graft-versus-host disease in mouse models. J Immunol 2011;187(4):1537e41. [86] Nowak EC, Lines JL, Varn FS, Deng J, Sarde A, Mabaera R, Kuta A, Le Mercier I, Cheng C, Noelle RJ. Immunoregulatory functions of VISTA. Immunol Rev 2017;276(1):66e79. [87] Schildberg FA, Klein SR, Freeman GJ, Sharpe AH. Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 2016;44(5):955e72. [88] Wang L, Rubinstein R, Lines JL, Wasiuk A, Ahonen C, Guo Y, Lu LF, Gondek D, Wang Y, Fava RA, et al. VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J Exp Med 2011;208(3):577e92. [89] Flies DB, Han X, Higuchi T, Zheng L, Sun J, Ye JJ, Chen L. Coinhibitory receptor PD-1H preferentially suppresses CD4(þ) T cell-mediated immunity. J Clin Invest 2014;124(5):1966e75. [90] Wang L, Le Mercier I, Putra J, Chen W, Liu J, Schenk AD, Nowak EC, Suriawinata AA, Li J, Noelle RJ. Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity. Proc Natl Acad Sci USA 2014;111(41):14846e51. [91] Flies DB, Higuchi T, Chen L. Mechanistic assessment of PD-1H coinhibitory receptor-induced T cell tolerance to allogeneic antigens. J Immunol 2015;194(11):5294e304. [92] Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, Dong H, Sica GL, Zhu G, Tamada K, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production. Nat Immunol 2001;2(3):269e74. [93] Janakiram M, Shah UA, Liu W, Zhao A, Schoenberg MP, Zang X. The third group of the B7-CD28 immune checkpoint family: HHLA2, TMIGD2, B7x, and B7-H3. Immunol Rev 2017;276(1):26e39. [94] Xu H, Cheung IY, Guo HF, Cheung NK. MicroRNA miR-29 modulates expression of immunoinhibitory molecule B7-H3: potential implications for immune based therapy of human solid tumors. Cancer Res 2009;69(15):6275e81. [95] Hashiguchi M, Kobori H, Ritprajak P, Kamimura Y, Kozono H, Azuma M. Triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a counter-receptor for B7-H3 and enhances T cell responses. Proc Natl Acad Sci USA 2008;105(30):10495e500. [96] Leitner J, Klauser C, Pickl WF, Stockl J, Majdic O, Bardet AF, Kreil DP, Dong C, Yamazaki T, Zlabinger G, et al. B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction. Eur J Immunol 2009;39(7):1754e64. [97] Loo D, Alderson RF, Chen FZ, Huang L, Zhang W, Gorlatov S, Burke S, Ciccarone V, Li H, Yang Y, et al. Development of an Fc-enhanced antiB7-H3 monoclonal antibody with potent antitumor activity. Clin Cancer Res 2012;18(14):3834e45. [98] Nagase-Zembutsu A, Hirotani K, Yamato M, Yamaguchi J, Takata T, Yoshida M, Fukuchi K, Yazawa M, Takahashi S, Agatsuma T. Development of DS-5573a: a novel afucosylated mAb directed at B7-H3 with potent antitumor activity. Canc Sci 2016;107(5):674e81. [99] Ahmed M, Cheng M, Zhao Q, Goldgur Y, Cheal SM, Guo HF, Larson SM, Cheung NK. Humanized affinity-matured monoclonal antibody 8H9 has potent antitumor activity and binds to FG loop of tumor antigen B7-H3. J Biol Chem 2015;290(50):30018e29. [100] Suh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, Dawicki W, Duncan GS, Bukczynski J, Plyte S, Elia A, et al. The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses. Nat Immunol 2003;4(9):899e906. [101] Castriconi R, Dondero A, Augugliaro R, Cantoni C, Carnemolla B, Sementa AR, Negri F, Conte R, Corrias MV, Moretta L, et al. Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell-mediated lysis. Proc Natl Acad Sci USA 2004;101(34):12640e5. [102] Veenstra RG, Flynn R, Kreymborg K, McDonald-Hyman C, Saha A, Taylor PA, Osborn MJ, Panoskaltsis-Mortari A, Schmitt-Graeff A, Lieberknect E, et al. B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality. Blood 2015;125(21):3335e46. [103] Sica GL, Choi IH, Zhu G, Tamada K, Wang SD, Tamura H, Chapoval AI, Flies DB, Bajorath J, Chen L. B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 2003;18(6):849e61. [104] Wei J, Loke P, Zang X, Allison JP. Tissue-specific expression of B7x protects from CD4 T cell-mediated autoimmunity. J Exp Med 2011;208(8):1683e94. [105] Wang X, Hao J, Metzger DL, Mui A, Ao Z, Akhoundsadegh N, Langermann S, Liu L, Chen L, Ou D, et al. Early treatment of NOD mice with B7H4 reduces the incidence of autoimmune diabetes. Diabetes 2011;60(12):3246e55.

188 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

[106] Ni L, Dong C. New B7 family checkpoints in human cancers. Mol Canc Therapeut 2017;16(7):1203e11. [107] Xue Q, Luan XY, Gu YZ, Wu HY, Zhang GB, Yu GH, Zhu HT, Wang M, Dong W, Geng YJ, et al. The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity. Stem Cells and Development 2010;19(1):27e38. [108] Murphy KM, Nelson CA, Sedy JR. Balancing co-stimulation and inhibition with BTLA and HVEM. Nat Rev Immunol 2006;6(9):671e81. [109] Watanabe N, Gavrieli M, Sedy JR, Yang J, Fallarino F, Loftin SK, Hurchla MA, Zimmerman N, Sim J, Zang X, et al. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol 2003;4(7):670e9. [110] Cai G, Anumanthan A, Brown JA, Greenfield EA, Zhu B, Freeman GJ. CD160 inhibits activation of human CD4þ T cells through interaction with herpesvirus entry mediator. Nat Immunol 2008;9(2):176e85. [111] Hurchla MA, Sedy JR, Murphy KM. Unexpected role of B and T lymphocyte attenuator in sustaining cell survival during chronic allostimulation. J Immunol 2007;178(10):6073e82. [112] Albring JC, Sandau MM, Rapaport AS, Edelson BT, Satpathy A, Mashayekhi M, Lathrop SK, Hsieh CS, Stelljes M, Colonna M, et al. Targeting of B and T lymphocyte associated (BTLA) prevents graft-versus-host disease without global immunosuppression. J Exp Med 2010;207(12):2551e9. [113] Sakoda Y, Park JJ, Zhao Y, Kuramasu A, Geng D, Liu Y, Davila E, Tamada K. Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway. Blood 2011;117(8):2506e14. [114] Kotsiou E, Okosun J, Besley C, Iqbal S, Matthews J, Fitzgibbon J, Gribben JG, Davies JK. TNFRSF14 aberrations in follicular lymphoma increase clinically significant allogeneic T-cell responses. Blood 2016;128(1):72e81. [115] Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 2009;229(1):152e72. [116] Blazar BR, Taylor PA, Noelle RJ, Vallera DA. CD4(þ) T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L: CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses. J Clin Invest 1998;102(3):473e82. [117] Taylor PA, Friedman TM, Korngold R, Noelle RJ, Blazar BR. Tolerance induction of alloreactive T cells via ex vivo blockade of the CD40:CD40L costimulatory pathway results in the generation of a potent immune regulatory cell. Blood 2002;99(12):4601e9. [118] Durie FH, Aruffo A, Ledbetter J, Crassi KM, Green WR, Fast LD, Noelle RJ. Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-vs-host disease. J Clin Invest 1994;94(3):1333e8. [119] Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Buhlman J, Xu J, Flavell RA, Korngold R, Noelle R, Vallera DA. Blockade of CD40 ligand-CD40 interaction impairs CD4þ T cell-mediated alloreactivity by inhibiting mature donor T cell expansion and function after bone marrow transplantation. J Immunol 1997;158(1):29e39. [120] Hargreaves RE, Monk NJ, Jurcevic S. Selective depletion of activated T cells: the CD40L-specific antibody experience. Trends Mol Med 2004;10(3):130e5. [121] Verbinnen B, Billiau AD, Vermeiren J, Galicia G, Bullens DM, Boon L, Cadot P, Hens G, Dewolf-Peeters C, Van Gool SW, et al. Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade. J Immunol 2008;181(2):1034e42. [122] Kim J, Park K, Kim HJ, Kim HA, Jung D, Choi HJ, Choi SY, Seo KW, Cho HR, Kwon B. Breaking of CD8þ T cell tolerance through in vivo ligation of CD40 results in inhibition of chronic graft-versus-host disease and complete donor cell engraftment. J Immunol 2008;181(10):7380e9. [123] Quezada SA, Jarvinen LZ, Lind EF, Noelle RJ. CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol 2004;22:307e28. [124] Kawai T, Andrews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 2000;6(2):114. [125] Xu H, Zhang X, Mannon RB, Kirk AD. Platelet-derived or soluble CD154 induces vascularized allograft rejection independent of cell-bound CD154. J Clin Invest 2006;116(3):769e74. [126] Adams AB, Shirasugi N, Jones TR, Durham MM, Strobert EA, Cowan S, Rees P, Hendrix R, Price K, Kenyon NS, et al. Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival. J Immunol 2005;174(1):542e50. [127] Saito K, Sakurai J, Ohata J, Kohsaka T, Hashimoto H, Okumura K, Abe R, Azuma M. Involvement of CD40 ligand-CD40 and CTLA4-B7 pathways in murine acute graft-versus-host disease induced by allogeneic T cells lacking CD28. J Immunol 1998;160(9):4225e31. [128] Vogel I, Verbinnen B, Van Gool S, Ceuppens JL. Regulatory T cell-dependent and -independent mechanisms of immune suppression by CD28/B7 and CD40/CD40L costimulation blockade. J Immunol 2016;197(2):533e40. [129] Miller WP, Srinivasan S, Panoskaltsis-Mortari A, Singh K, Sen S, Hamby K, Deane T, Stempora L, Beus J, Turner A, et al. GVHD after haploidentical transplantation: a novel, MHC-defined rhesus macaque model identifies CD28 CD8þ T cells as a reservoir of breakthrough T-cell proliferation during costimulation blockade and sirolimus-based immunosuppression. Blood 2010;116(24):5403e18. [130] Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev 2009;229(1):173e91. [131] Gramaglia I, Weinberg AD, Lemon M, Croft M. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 1998;161(12):6510e7. [132] Lane P. Role of OX40 signals in coordinating CD4 T cell selection, migration, and cytokine differentiation in T helper (Th)1 and Th2 cells. J Exp Med 2000;191(2):201e6. [133] Sugamura K, Ishii N, Weinberg AD. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat Rev Immunol 2004;4(6):420e31.

T-Cell Costimulation and Coinhibition in GVHD and GVL Chapter | 11

189

[134] Tittle TV, Weinberg AD, Steinkeler CN, Maziarz RT. Expression of the T-cell activation antigen, OX-40, identifies alloreactive T cells in acute graft-versus-host disease. Blood 1997;89(12):4652e8. [135] Sanchez J, Casano J, Alvarez MA, Roman-Gomez J, Martin C, Martinez F, Gomez P, Serrano J, Herrera C, Torres A. Kinetic of regulatory CD25high and activated CD134þ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation. Br J Haematol 2004;126(5):697e703. [136] Blazar BR, Sharpe AH, Chen AI, Panoskaltsis-Mortari A, Lees C, Akiba H, Yagita H, Killeen N, Taylor PA. Ligation of OX40 (CD134) regulates graft-versus-host disease (GVHD) and graft rejection in allogeneic bone marrow transplant recipients. Blood 2003;101(9):3741e8. [137] Ge X, Brown J, Sykes M, Boussiotis VA. CD134-allodepletion allows selective elimination of alloreactive human T cells without loss of virusspecific and leukemia-specific effectors. Biol Blood Marrow Transplant 2008;14(5):518e30. [138] Wang C, Lin GH, McPherson AJ, Watts TH. Immune regulation by 4-1BB and 4-1BBL: complexities and challenges. Immunol Rev 2009;229(1):192e215. [139] Cannons JL, Lau P, Ghumman B, DeBenedette MA, Yagita H, Okumura K, Watts TH. 4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy. J Immunol 2001;167(3):1313e24. [140] Blazar BR, Kwon BS, Panoskaltsis-Mortari A, Kwak KB, Peschon JJ, Taylor PA. Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J Immunol 2001;166(5):3174e83. [141] Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, Brown TJ, Emswiler J, Raecho H, Larsen CP, et al. 4-1BB costimulatory signals preferentially induce CD8þ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med 1997;186(1):47e55. [142] Kim W, Kim J, Jung D, Kim H, Choi HJ, Cho HR, Kwon B. Induction of lethal graft-versus-host disease by anti-CD137 monoclonal antibody in mice prone to chronic graft-versus-host disease. Biol Blood Marrow Transplant 2009;15(3):306e14. [143] Nozawa K, Ohata J, Sakurai J, Hashimoto H, Miyajima H, Yagita H, Okumura K, Azuma M. Preferential blockade of CD8(þ) T cell responses by administration of anti-CD137 ligand monoclonal antibody results in differential effect on development of murine acute and chronic graft-versushost diseases. J Immunol 2001;167(9):4981e6. [144] Xu K, Li C, Pan X, Du B. Study of relieving graft-versus-host disease by blocking CD137-CD137 ligand costimulatory pathway in vitro. Int J Hematol 2007;86(1):84e90. [145] Lee SC, Seo KW, Kim HJ, Kang SW, Choi HJ, Kim A, Kwon BS, Cho HR, Kwon B. Depletion of alloreactive T-cells by anti-CD137-saporin immunotoxin. Cell Transplant 2015;24(6):1167e81. [146] Kim J, Kim HJ, Park K, Choi HJ, Yagita H, Nam SH, Cho HR, Kwon B. Costimulatory molecule-targeted immunotherapy of cutaneous graftversus-host disease. Blood 2007;110(2):776e82. [147] Wehler TC, Nonn M, Brandt B, Britten CM, Grone M, Todorova M, Link I, Khan SA, Meyer RG, Huber C, et al. Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines. Blood 2007;109(1):365e73. [148] Smith CA, Gruss HJ, Davis T, Anderson D, Farrah T, Baker E, Sutherland GR, Brannan CI, Copeland NG, Jenkins NA, et al. CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF. Cell 1993;73(7):1349e60. [149] Croft M. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat Rev Immunol 2003;3(8):609e20. [150] Sun X, Yamada H, Shibata K, Muta H, Tani K, Podack ER, Iwakura Y, Yoshikai Y. CD30 ligand is a target for a novel biological therapy against colitis associated with Th17 responses. J Immunol 2010;185(12):7671e80. [151] Pellegrini P, Berghella AM, Contasta I, Adorno D. CD30 antigen: not a physiological marker for TH2 cells but an important costimulator molecule in the regulation of the balance between TH1/TH2 response. Transpl Immunol 2003;12(1):49e61. [152] Blazar BR, Levy RB, Mak TW, Panoskaltsis-Mortari A, Muta H, Jones M, Roskos M, Serody JS, Yagita H, Podack ER, et al. CD30/CD30 ligand (CD153) interaction regulates CD4þ T cell-mediated graft-versus-host disease. J Immunol 2004;173(5):2933e41. [153] Chen YB, McDonough S, Hasserjian R, Chen H, Coughlin E, Illiano C, Park IS, Jagasia M, Spitzer TR, Cutler CS, et al. Expression of CD30 in patients with acute graft-versus-host disease. Blood 2012;120(3):691e6. [154] Amedei A, Pimpinelli N, Grassi A, Bella CD, Niccolai E, Brancati S, Benagiano M, D’Elios S, Bosi A, D’Elios MM. Skin CD30(þ) T cells and circulating levels of soluble CD30 are increased in patients with graft versus host disease. Auto Immun Highlights 2014;5(1):21e6. [155] Nolte MA, van Olffen RW, van Gisbergen KP, van Lier RA. Timing and tuning of CD27-CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology. Immunol Rev 2009;229(1):216e31. [156] Tesselaar K, Xiao Y, Arens R, van Schijndel GM, Schuurhuis DH, Mebius RE, Borst J, van Lier RA. Expression of the murine CD27 ligand CD70 in vitro and in vivo. J Immunol 2003;170(1):33e40. [157] Sanchez PJ, McWilliams JA, Haluszczak C, Yagita H, Kedl RM. Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo. J Immunol 2007;178(3):1564e72. [158] Hendriks J, Gravestein LA, Tesselaar K, van Lier RA, Schumacher TN, Borst J. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 2000;1(5):433e40. [159] Hendriks J, Xiao Y, Borst J. CD27 promotes survival of activated T cells and complements CD28 in generation and establishment of the effector T cell pool. J Exp Med 2003;198(9):1369e80. [160] Carr JM, Carrasco MJ, Thaventhiran JE, Bambrough PJ, Kraman M, Edwards AD, Al-Shamkhani A, Fearon DT. CD27 mediates interleukin-2independent clonal expansion of the CD8þ T cell without effector differentiation. Proc Natl Acad Sci USA 2006;103(51):19454e9.

190 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

[161] Xiao Y, Peperzak V, Keller AM, Borst J. CD27 instructs CD4þ T cells to provide help for the memory CD8þ T cell response after protein immunization. J Immunol 2008;181(2):1071e82. [162] Yamada A, Salama AD, Sho M, Najafian N, Ito T, Forman JP, Kewalramani R, Sandner S, Harada H, Clarkson MR, et al. CD70 signaling is critical for CD28-independent CD8þ T cell-mediated alloimmune responses in vivo. J Immunol 2005;174(3):1357e64. [163] Yoshida H, Maeda T, Ishikawa J, Inoue S, Matsunaga H, Kosugi S, Shiraga M, Oritani K, Kanakura Y, Tomiyama Y. Expression of CD27 on peripheral CD4þ T-lymphocytes correlates with the development of severe acute graft-versus-host disease after allogeneic bone marrow transplantation. Int J Hematol 2006;84(4):367e76. [164] Leigh ND, O’Neill RE, Du W, Chen C, Qiu J, Ashwell JD, McCarthy PL, Chen GL, Cao X. Host-derived CD70 suppresses murine graft-versushost disease by limiting donor T cell expansion and effector function. J Immunol 2017;199(1):336e47. [165] Cai G, Freeman GJ. The CD160, BTLA, LIGHT/HVEM pathway: a bidirectional switch regulating T-cell activation. Immunol Rev 2009;229(1):244e58. [166] Tamada K, Shimozaki K, Chapoval AI, Zhai Y, Su J, Chen SF, Hsieh SL, Nagata S, Ni J, Chen L. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. J Immunol 2000;164(8):4105e10. [167] Morel Y, Truneh A, Sweet RW, Olive D, Costello RT. The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity. J Immunol 2001;167(5):2479e86. [168] Xu Y, Flies AS, Flies DB, Zhu G, Anand S, Flies SJ, Xu H, Anders RA, Hancock WW, Chen L, et al. Selective targeting of the LIGHT-HVEM costimulatory system for the treatment of graft-versus-host disease. Blood 2007;109(9):4097e104. [169] Tamada K, Shimozaki K, Chapoval AI, Zhu G, Sica G, Flies D, Boone T, Hsu H, Fu YX, Nagata S, et al. Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway. Nat Med 2000;6(3):283e9. [170] Tamada K, Tamura H, Flies D, Fu YX, Celis E, Pease LR, Blazar BR, Chen L. Blockade of LIGHT/LTbeta and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease. J Clin Invest 2002;109(4):549e57. [171] Nocentini G, Giunchi L, Ronchetti S, Krausz LT, Bartoli A, Moraca R, Migliorati G, Riccardi C. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc Natl Acad Sci USA 1997;94(12):6216e21. [172] Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S. Stimulation of CD25(þ)CD4(þ) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 2002;3(2):135e42. [173] Snell LM, Lin GH, McPherson AJ, Moraes TJ, Watts TH. T-cell intrinsic effects of GITR and 4-1BB during viral infection and cancer immunotherapy. Immunol Rev 2011;244(1):197e217. [174] Ronchetti S, Nocentini G, Bianchini R, Krausz LT, Migliorati G, Riccardi C. Glucocorticoid-induced TNFR-related protein lowers the threshold of CD28 costimulation in CD8þ T cells. J Immunol 2007;179(9):5916e26. [175] Ramirez-Montagut T, Chow A, Hirschhorn-Cymerman D, Terwey TH, Kochman AA, Lu S, Miles RC, Sakaguchi S, Houghton AN, van den Brink MR. Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity. J Immunol 2006;176(11):6434e42. [176] Muriglan SJ, Ramirez-Montagut T, Alpdogan O, Van Huystee TW, Eng JM, Hubbard VM, Kochman AA, Tjoe KH, Riccardi C, Pandolfi PP, et al. GITR activation induces an opposite effect on alloreactive CD4(þ) and CD8(þ) T cells in graft-versus-host disease. J Exp Med 2004;200(2):149e57. [177] Kim J, Choi WS, Kang H, Kim HJ, Suh JH, Sakaguchi S, Kwon B. Conversion of alloantigen-specific CD8þ T cell anergy to CD8þ T cell priming through in vivo ligation of glucocorticoid-induced TNF receptor. J Immunol 2006;176(9):5223e31. [178] Iguchi-Manaka A, Kai H, Yamashita Y, Shibata K, Tahara-Hanaoka S, Honda S, Yasui T, Kikutani H, Shibuya K, Shibuya A. Accelerated tumor growth in mice deficient in DNAM-1 receptor. J Exp Med 2008;205(13):2959e64. [179] Tahara-Hanaoka S, Shibuya K, Kai H, Miyamoto A, Morikawa Y, Ohkochi N, Honda S, Shibuya A. Tumor rejection by the poliovirus receptor family ligands of the DNAM-1 (CD226) receptor. Blood 2006;107(4):1491e6. [180] Cella M, Presti R, Vermi W, Lavender K, Turnbull E, Ochsenbauer-Jambor C, Kappes JC, Ferrari G, Kessels L, Williams I, et al. Loss of DNAM-1 contributes to CD8þ T-cell exhaustion in chronic HIV-1 infection. Eur J Immunol 2010;40(4):949e54. [181] Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla B, Cantoni C, Grassi J, Marcenaro S, Reymond N, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J Exp Med 2003;198(4):557e67. [182] Tahara-Hanaoka S, Shibuya K, Onoda Y, Zhang H, Yamazaki S, Miyamoto A, Honda S, Lanier LL, Shibuya A. Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and nectin-2 (PRR-2/CD112). Int Immunol 2004;16(4):533e8. [183] Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, Levine Z, Beiman M, Dassa L, Achdout H, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci USA 2009;106(42):17858e63. [184] Hafler JP, Maier LM, Cooper JD, Plagnol V, Hinks A, Simmonds MJ, Stevens HE, Walker NM, Healy B, Howson JM, et al. CD226 Gly307Ser association with multiple autoimmune diseases. Gene Immun 2009;10(1):5e10. [185] Nabekura T, Shibuya K, Takenaka E, Kai H, Shibata K, Yamashita Y, Harada K, Tahara-Hanaoka S, Honda S, Shibuya A. Critical role of DNAX accessory molecule-1 (DNAM-1) in the development of acute graft-versus-host disease in mice. Proc Natl Acad Sci USA 2010;107(43):18593e8. [186] Koyama M, Kuns RD, Olver SD, Lineburg KE, Lor M, Teal BE, Raffelt NC, Leveque L, Chan CJ, Robb RJ, et al. Promoting regulation via the inhibition of DNAM-1 after transplantation. Blood 2013;121(17):3511e20. [187] Kanaya M, Shibuya K, Hirochika R, Kanemoto M, Ohashi K, Okada M, Wagatsuma Y, Cho Y, Kojima H, Teshima T, et al. Soluble DNAM-1, as a predictive biomarker for acute graft-versus-host disease. PLoS One 2016;11(6). e0154173.

T-Cell Costimulation and Coinhibition in GVHD and GVL Chapter | 11

191

[188] Seth S, Ravens I, Lee CW, Glage S, Bleich A, Forster R, Bernhardt G, Koenecke C. Absence of CD155 aggravates acute graft-versus-host disease. Proc Natl Acad Sci USA 2011;108(10):E32e3. [189] Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, Manning S, Greenfield EA, Coyle AJ, Sobel RA, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002;415(6871):536e41. [190] Kuchroo VK, Umetsu DT, DeKruyff RH, Freeman GJ. The TIM gene family: emerging roles in immunity and disease. Nat Rev Immunol 2003;3(6):454e62. [191] Anderson AC, Joller N, Kuchroo VK. Lag-3, Tim-3, and TIGIT: Co-inhibitory receptors with specialized functions in immune regulation. Immunity 2016;44(5):989e1004. [192] Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005;6(12):1245e52. [193] Sanchez-Fueyo A, Tian J, Picarella D, Domenig C, Zheng XX, Sabatos CA, Manlongat N, Bender O, Kamradt T, Kuchroo VK, et al. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nat Immunol 2003;4(11):1093e101. [194] Wang F, He W, Yuan J, Wu K, Zhou H, Zhang W, Chen ZK. Activation of Tim-3-Galectin-9 pathway improves survival of fully allogeneic skin grafts. Transpl Immunol 2008;19(1):12e9. [195] He W, Fang Z, Wang F, Wu K, Xu Y, Zhou H, Du D, Gao Y, Zhang WN, Niki T, et al. Galectin-9 significantly prolongs the survival of fully mismatched cardiac allografts in mice. Transplantation 2009;88(6):782e90. [196] Jin HT, Anderson AC, Tan WG, West EE, Ha SJ, Araki K, Freeman GJ, Kuchroo VK, Ahmed R. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci USA 2010;107(33):14733e8. [197] Fourcade J, Sun Z, Benallaoua M, Guillaume P, Luescher IF, Sander C, Kirkwood JM, Kuchroo V, Zarour HM. Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8þ T cell dysfunction in melanoma patients. J Exp Med 2010;207(10):2175e86. [198] Oikawa T, Kamimura Y, Akiba H, Yagita H, Okumura K, Takahashi H, Zeniya M, Tajiri H, Azuma M. Preferential involvement of Tim-3 in the regulation of hepatic CD8þ T cells in murine acute graft-versus-host disease. J Immunol 2006;177(7):4281e7. [199] Sakai K, Kawata E, Ashihara E, Nakagawa Y, Yamauchi A, Yao H, Nagao R, Tanaka R, Yokota A, Takeuchi M, et al. Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis. Eur J Immunol 2011;41(1):67e75. [200] Veenstra RG, Taylor PA, Zhou Q, Panoskaltsis-Mortari A, Hirashima M, Flynn R, Liu D, Anderson AC, Strom TB, Kuchroo VK, et al. Contrasting acute graft-versus-host disease effects of Tim-3/galectin-9 pathway blockade dependent upon the presence of donor regulatory T cells. Blood 2012;120(3):682e90. [201] Hansen JA, Hanash SM, Tabellini L, Baik C, Lawler RL, Grogan BM, Storer B, Chin A, Johnson M, Wong CH, et al. A novel soluble form of Tim-3 associated with severe graft-versus-host disease. Biol Blood Marrow Transplant 2013;19(9):1323e30. [202] McDonald GB, Tabellini L, Storer BE, Lawler RL, Martin PJ, Hansen JA. Plasma biomarkers of acute GVHD and nonrelapse mortality: predictive value of measurements before GVHD onset and treatment. Blood 2015;126(1):113e20. [203] Ali AM, DiPersio JF, Schroeder MA. The role of biomarkers in the diagnosis and risk stratification of acute graft-versus-host disease: a systematic review. Biol Blood Marrow Transplant 2016;22(9):1552e64. [204] Huard B, Prigent P, Tournier M, Bruniquel D, Triebel F. CD4/major histocompatibility complex class II interaction analyzed with CD4 and lymphocyte activation gene-3 (LAG-3)-Ig fusion proteins. Eur J Immunol 1995;25(9):2718e21. [205] Xu F, Liu J, Liu D, Liu B, Wang M, Hu Z, Du X, Tang L, He F. LSECtin expressed on melanoma cells promotes tumor progression by inhibiting antitumor T-cell responses. Cancer Res 2014;74(13):3418e28. [206] Sega EI, Leveson-Gower DB, Florek M, Schneidawind D, Luong RH, Negrin RS. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation. PLoS One 2014;9(1):e86551. [207] Sakaguchi S. Naturally arising Foxp3-expressing CD25þCD4þ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6(4):345e52. [208] Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003;3(3):199e210. [209] Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL. CD4(þ)CD25(þ) immunoregulatory T Cells: new therapeutics for graft-versus-host disease. J Exp Med 2002;196(3):401e6. [210] Hoffmann P, Ermann J, Edinger M, Fathman CG, Strober S. Donor-type CD4(þ)CD25(þ) regulatory T cells suppress lethal acute graft-versushost disease after allogeneic bone marrow transplantation. J Exp Med 2002;196(3):389e99. [211] Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4(þ)CD25(þ) immune regulatory cells inhibits graft-versushost disease lethality. Blood 2002;99(10):3493e9. [212] Taylor PA, Noelle RJ, Blazar BR. CD4(þ)CD25(þ) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med 2001;193(11):1311e8. [213] Trenado A, Charlotte F, Fisson S, Yagello M, Klatzmann D, Salomon BL, Cohen JL. Recipient-type specific CD4þCD25þ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. J Clin Invest 2003;112(11):1688e96. [214] Edinger M, Hoffmann P, Ermann J, Drago K, Fathman CG, Strober S, Negrin RS. CD4þCD25þ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med 2003;9(9):1144e50. [215] Albert MH, Liu Y, Anasetti C, Yu XZ. Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation. Eur J Immunol 2005;35(9):2598e607. [216] Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. Ex vivo isolation and characterization of CD4(þ)CD25(þ) T cells with regulatory properties from human blood. J Exp Med 2001;193(11):1303e10.

192 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

[217] Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. Identification and functional characterization of human CD4(þ)CD25(þ) T cells with regulatory properties isolated from peripheral blood. J Exp Med 2001;193(11):1285e94. [218] Ng WF, Duggan PJ, Ponchel F, Matarese G, Lombardi G, Edwards AD, Isaacs JD, Lechler RI. Human CD4(þ)CD25(þ) cells: a naturally occurring population of regulatory T cells. Blood 2001;98(9):2736e44. [219] Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4þCD25- T cells. J Clin Invest 2003;112(9):1437e43. [220] Miura Y, Thoburn CJ, Bright EC, Phelps ML, Shin T, Matsui EC, Matsui WH, Arai S, Fuchs EJ, Vogelsang GB, et al. Association of Foxp3 regulatory gene expression with graft-versus-host disease. Blood 2004;104(7):2187e93. [221] Bour-Jordan H, Bluestone JA. Regulating the regulators: costimulatory signals control the homeostasis and function of regulatory T cells. Immunol Rev 2009;229(1):41e66. [222] Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA. B7/CD28 costimulation is essential for the homeostasis of the CD4þCD25þ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000;12(4):431e40. [223] Johnson BD, Konkol MC, Truitt RL. CD25þ immunoregulatory T-cells of donor origin suppress alloreactivity after BMT. Biol Blood Marrow Transplant 2002;8(10):525e35. [224] Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK, Zheng XX, Strom TB, Bluestone JA. Cutting edge: CD28 controls peripheral homeostasis of CD4þCD25þ regulatory T cells. J Immunol 2003;171(7):3348e52. [225] Burr JS, Savage ND, Messah GE, Kimzey SL, Shaw AS, Arch RH, Green JM. Cutting edge: distinct motifs within CD28 regulate T cell proliferation and induction of Bcl-XL. J Immunol 2001;166(9):5331e5. [226] Tai X, Cowan M, Feigenbaum L, Singer A. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T cell differentiation independently of interleukin 2. Nat Immunol 2005;6(2):152e62. [227] Fisson S, Darrasse-Jeze G, Litvinova E, Septier F, Klatzmann D, Liblau R, Salomon BL. Continuous activation of autoreactive CD4þ CD25þ regulatory T cells in the steady state. J Exp Med 2003;198(5):737e46. [228] Walker LS, Chodos A, Eggena M, Dooms H, Abbas AK. Antigen-dependent proliferation of CD4þ CD25þ regulatory T cells in vivo. J Exp Med 2003;198(2):249e58. [229] Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 2004;199(11):1455e65. [230] D’Cruz LM, Klein L. Development and function of agonist-induced CD25þFoxp3þ regulatory T cells in the absence of interleukin 2 signaling. Nat Immunol 2005;6(11):1152e9. [231] Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(þ) CD25(þ) CD4(þ) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 2005;201(5):723e35. [232] Uri A, Werner S, Luhder F, Hunig T, Kerkau T, Beyersdorf N. Protection of mice from acute graft-versus-host disease requires CD28 Costimulation on donor CD4þ Foxp3þ regulatory T cells. Front Immunol 2017;8(721). [233] Akieda Y, Wakamatsu E, Nakamura T, Ishida Y, Ogawa S, Abe R. Defects in regulatory T cells due to CD28 deficiency induce a qualitative change of allogeneic immune response in chronic graft-versus-host disease. J Immunol 2015;194(9):4162e74. [234] Tabares P, Berr S, Romer PS, Chuvpilo S, Matskevich AA, Tyrsin D, Fedotov Y, Einsele H, Tony HP, Hunig T. Human regulatory T cells are selectively activated by low-dose application of the CD28 superagonist TGN1412/TAB08. Eur J Immunol 2014;44(4):1225e36. [235] He X, Smeets RL, van Rijssen E, Boots AM, Joosten I, Koenen HJ. Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells. Sci Rep 2017;7:43003. [236] Semple K, Nguyen A, Yu Y, Wang H, Anasetti C, Yu XZ. Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling. Blood 2011;117(11):3096e103. [237] Friedline RH, Brown DS, Nguyen H, Kornfeld H, Lee J, Zhang Y, Appleby M, Der SD, Kang J, Chambers CA. CD4þ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance. J Exp Med 2009;206(2):421e34. [238] Ise W, Kohyama M, Nutsch KM, Lee HM, Suri A, Unanue ER, Murphy TL, Murphy KM. CTLA-4 suppresses the pathogenicity of self antigenspecific T cells by cell-intrinsic and cell-extrinsic mechanisms. Nat Immunol 2010;11(2):129e35. [239] Jain N, Nguyen H, Chambers C, Kang J. Dual function of CTLA-4 in regulatory T cells and conventional T cells to prevent multiorgan autoimmunity. Proc Natl Acad Sci USA 2010;107(4):1524e8. [240] Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S. CTLA-4 control over Foxp3þ regulatory T cell function. Science 2008;322(5899):271e5. [241] Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, Marshall B, Chandler P, Antonia SJ, Burgess R, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002;297(5588):1867e70. [242] Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, Candeloro P, Belladonna ML, Bianchi R, Fioretti MC, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002;3(11):1097e101. [243] Onishi Y, Fehervari Z, Yamaguchi T, Sakaguchi S. Foxp3þ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation. Proc Natl Acad Sci USA 2008;105(29):10113e8. [244] Miyamoto K, Kingsley CI, Zhang X, Jabs C, Izikson L, Sobel RA, Weiner HL, Kuchroo VK, Sharpe AH. The ICOS molecule plays a crucial role in the development of mucosal tolerance. J Immunol 2005;175(11):7341e7. [245] Herman AE, Freeman GJ, Mathis D, Benoist C. CD4þCD25þ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004;199(11):1479e89.

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193

[246] Kornete M, Sgouroudis E, Piccirillo CA. ICOS-dependent homeostasis and function of Foxp3þ regulatory T cells in islets of nonobese diabetic mice. J Immunol 2012;188(3):1064e74. [247] Faget J, Bendriss-Vermare N, Gobert M, Durand I, Olive D, Biota C, Bachelot T, Treilleux I, Goddard-Leon S, Lavergne E, et al. ICOS-ligand expression on plasmacytoid dendritic cells supports breast cancer progression by promoting the accumulation of immunosuppressive CD4þ T cells. Cancer Res 2012;72(23):6130e41. [248] Lee HJ, Kim SN, Jeon MS, Yi T, Song SU. ICOSL expression in human bone marrow-derived mesenchymal stem cells promotes induction of regulatory T cells. Sci Rep 2017;7:44486. [249] Akbari O, Freeman GJ, Meyer EH, Greenfield EA, Chang TT, Sharpe AH, Berry G, DeKruyff RH, Umetsu DT. Antigen-specific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat Med 2002;8(9):1024e32. [250] Ito T, Hanabuchi S, Wang YH, Park WR, Arima K, Bover L, Qin FX, Gilliet M, Liu YJ. Two functional subsets of FOXP3þ regulatory T cells in human thymus and periphery. Immunity 2008;28(6):870e80. [251] Chen Y, Shen S, Gorentla BK, Gao J, Zhong XP. Murine regulatory T cells contain hyperproliferative and death-prone subsets with differential ICOS expression. J Immunol 2012;188(4):1698e707. [252] Leavenworth JW, Verbinnen B, Yin J, Huang H, Cantor H. A p85alpha-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nat Immunol 2015;16(1):96e106. [253] Imanguli MM, Cowen EW, Rose J, Dhamala S, Swaim W, Lafond S, Yagi B, Gress RE, Pavletic SZ, Hakim FT. Comparative analysis of FoxP3(þ) regulatory T cells in the target tissues and blood in chronic graft versus host disease. Leukemia 2014;28(10):2016e27. [254] Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009;9(5):324e37. [255] Park HJ, Park JS, Jeong YH, Son J, Ban YH, Lee BH, Chen L, Chang J, Chung DH, Choi I, et al. PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8þ T cell immune response via the interaction with PD-L1 expressed on CD8þ T cells. J Immunol 2015;194(12):5801e11. [256] Lowther DE, Goods BA, Lucca LE, Lerner BA, Raddassi K, van Dijk D, Hernandez AL, Duan X, Gunel M, Coric V, et al. PD-1 marks dysfunctional regulatory T cells in malignant gliomas. JCI Insight 2016;1(5). [257] Sage PT, Francisco LM, Carman CV, Sharpe AH. The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat Immunol 2013;14(2):152e61. [258] Le Mercier I, Chen W, Lines JL, Day M, Li J, Sergent P, Noelle RJ, Wang LVISTA. Regulates the development of protective antitumor immunity. Cancer Res 2014;74(7):1933e44. [259] Wang Q, He J, Flies DB, Luo L, Chen L. Programmed death one homolog maintains the pool size of regulatory T cells by promoting their differentiation and stability. Sci Rep 2017;7(1):6086. [260] Mahnke K, Ring S, Johnson TS, Schallenberg S, Schonfeld K, Storn V, Bedke T, Enk AH. Induction of immunosuppressive functions of dendritic cells in vivo by CD4þCD25þ regulatory T cells: role of B7-H3 expression and antigen presentation. Eur J Immunol 2007;37(8):2117e26. [261] Kryczek I, Wei S, Zou L, Zhu G, Mottram P, Xu H, Chen L, Zou W. Cutting edge: induction of B7-H4 on APCs through IL-10: novel suppressive mode for regulatory T cells. J Immunol 2006;177(1):40e4. [262] Jones A, Bourque J, Kuehm L, Opejin A, Teague RM, Gross C, Hawiger D. Immunomodulatory functions of BTLA and HVEM govern induction of extrathymic regulatory T cells and tolerance by dendritic cells. Immunity 2016;45(5):1066e77. [263] Vu MD, Xiao X, Gao W, Degauque N, Chen M, Kroemer A, Killeen N, Ishii N. Li XC. OX40 costimulation turns off Foxp3þ Tregs. Blood 2007;110(7):2501e10. [264] Valzasina B, Guiducci C, Dislich H, Killeen N, Weinberg AD, Colombo MP. Triggering of OX40 (CD134) on CD4(þ)CD25þ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR. Blood 2005;105(7):2845e51. [265] So T, Croft M. Cutting edge: OX40 inhibits TGF-beta- and antigen-driven conversion of naive CD4 T cells into CD25þFoxp3þ T cells. J Immunol 2007;179(3):1427e30. [266] Voo KS, Bover L, Harline ML, Vien LT, Facchinetti V, Arima K, Kwak LW, Liu YJ. Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function. J Immunol 2013;191(7):3641e50. [267] Kinnear G, Wood KJ, Fallah-Arani F, Jones ND. A diametric role for OX40 in the response of effector/memory CD4þ T cells and regulatory T cells to alloantigen. J Immunol 2013;191(3):1465e75. [268] McHugh RS, Whitters MJ, Piccirillo CA, Young DA, Shevach EM, Collins M, Byrne MC. CD4(þ)CD25(þ) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 2002;16(2):311e23. [269] van Olffen RW, Koning N, van Gisbergen KP, Wensveen FM, Hoek RM, Boon L, Hamann J, van Lier RA, Nolte MA. GITR triggering induces expansion of both effector and regulatory CD4þ T cells in vivo. J Immunol 2009;182(12):7490e500. [270] Ji HB, Liao G, Faubion WA, Abadia-Molina AC, Cozzo C, Laroux FS, Caton A, Terhorst C. Cutting edge: the natural ligand for glucocorticoidinduced TNF receptor-related protein abrogates regulatory T cell suppression. J Immunol 2004;172(10):5823e7. [271] Bushell A, Wood K. GITR ligation blocks allograft protection by induced CD25þCD4þ regulatory T cells without enhancing effector T-cell function. Am J Transplant 2007;7(4):759e68. [272] Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, Carreno BM, Collins M, Shevach EM. Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4þCD25þ T cells. J Immunol 2004;173(8):5008e20. [273] Amakawa R, Hakem A, Kundig TM, Matsuyama T, Simard JJ, Timms E, Wakeham A, Mittruecker HW, Griesser H, Takimoto H, et al. Impaired negative selection of T cells in Hodgkin’s disease antigen CD30-deficient mice. Cell 1996;84(4):551e62.

194 Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation

[274] Gerli R, Pitzalis C, Bistoni O, Falini B, Costantini V, Russano A, Lunardi C. CD30þ T cells in rheumatoid synovitis: mechanisms of recruitment and functional role. J Immunol 2000;164(8):4399e407. [275] Dai Z, Li Q, Wang Y, Gao G, Diggs LS, Tellides G, Lakkis FG. CD4þCD25þ regulatory T cells suppress allograft rejection mediated by memory CD8þ T cells via a CD30-dependent mechanism. J Clin Invest 2004;113(2):310e7. [276] Zhang ZX, Yang L, Young KJ, DuTemple B, Zhang L. Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression. Nat Med 2000;6(7):782e9. [277] de Kleer I, Vercoulen Y, Klein M, Meerding J, Albani S, van der Zee R, Sawitzki B, Hamann A, Kuis W, Prakken B. CD30 discriminates heat shock protein 60-induced FOXP3þ CD4þ T cells with a regulatory phenotype. J Immunol 2010;185(4):2071e9. [278] Zeiser R, Nguyen VH, Hou JZ, Beilhack A, Zambricki E, Buess M, Contag CH, Negrin RS. Early CD30 signaling is critical for adoptively transferred CD4þCD25þ regulatory T cells in prevention of acute graft-versus-host disease. Blood 2007;109(5):2225e33. [279] Schoenbrunn A, Frentsch M, Kohler S, Keye J, Dooms H, Moewes B, Dong J, Loddenkemper C, Sieper J, Wu P, et al. A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigenreactive natural Foxp3þ Treg. J Immunol 2012;189(12):5985e94. [280] Zheng G, Wang B, Chen A. The 4-1BB costimulation augments the proliferation of CD4þCD25þ regulatory T cells. J Immunol 2004;173(4):2428e34. [281] Hippen KL, Harker-Murray P, Porter SB, Merkel SC, Londer A, Taylor DK, Bina M, Panoskaltsis-Mortari A, Rubinstein P, Van Rooijen N, et al. Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells. Blood 2008;112(7):2847e57. [282] Choi BK, Bae JS, Choi EM, Kang WJ, Sakaguchi S, Vinay DS, Kwon BS. 4-1BB-dependent inhibition of immunosuppression by activated CD4þCD25þ T cells. J Leukoc Biol 2004;75(5):785e91. [283] Elpek KG, Yolcu ES, Franke DD, Lacelle C, Schabowsky RH, Shirwan H. Ex vivo expansion of CD4þCD25þFoxP3þ T regulatory cells based on synergy between IL-2 and 4-1BB signaling. J Immunol 2007;179(11):7295e304. [284] Coquet JM, Ribot JC, Babala N, Middendorp S, van der Horst G, Xiao Y, Neves JF, Fonseca-Pereira D, Jacobs H, Pennington DJ, et al. Epithelial and dendritic cells in the thymic medulla promote CD4þFoxp3þ regulatory T cell development via the CD27-CD70 pathway. J Exp Med 2013;210(4):715e28. [285] Allam A, Swiecki M, Vermi W, Ashwell JD, Colonna M. Dual function of CD70 in viral infection: modulator of early cytokine responses and activator of adaptive responses. J Immunol 2014;193(2):871e8. [286] Dhainaut M, Coquerelle C, Uzureau S, Denoeud J, Acolty V, Oldenhove G, Galuppo A, Sparwasser T, Thielemans K, Pays E, et al. Thymusderived regulatory T cells restrain pro-inflammatory Th1 responses by downregulating CD70 on dendritic cells. EMBO J 2015;34(10):1336e48. [287] Gupta S, Thornley TB, Gao W, Larocca R, Turka LA, Kuchroo VK, Strom TB. Allograft rejection is restrained by short-lived TIM-3þPD1þFoxp3þ Tregs. J Clin Invest 2012;122(7):2395e404. [288] Gautron AS, Dominguez-Villar M, de Marcken M, Hafler DA. Enhanced suppressor function of TIM-3þ FoxP3þ regulatory T cells. Eur J Immunol 2014;44(9):2703e11. [289] Song H, Zhou Y, Li G, Bai J. Regulatory T cells contribute to the recovery of acute lung injury by upregulating Tim-3. Inflammation 2015;38(3):1267e72. [290] Ju Y, Shang X, Liu Z, Zhang J, Li Y, Shen Y, Liu Y, Liu C, Liu B, Xu L, et al. The Tim-3/galectin-9 pathway involves in the homeostasis of hepatic Tregs in a mouse model of concanavalin A-induced hepatitis. Mol Immunol 2014;58(1):85e91. [291] Zhang M, Wu Y, Bastian D, et al. Inducible T-Cell Co-Stimulator Impacts Chronic Graft-Versus-Host Disease by Regulating Both Pathogenic and Regulatory T Cells. Front Immunol 2018;9(1461).