Murine Graft-versus-host Disease Research Articles

451 - Protective Effect of Curcumin in Murine Graft Versus Host Disease after Allogeneic Hematopoietic Cell Transplantation

451 - Protective Effect of Curcumin in Murine Graft Versus Host Disease after Allogeneic Hematopoietic Cell Transplantation

Ixazomib suppresses human dendritic cell and modulates murine graft-versus-host disease in a schedule-dependent fashion.

There is an abiding need for innovative approaches to the prevention of graft-versus-host disease (GvHD) following allogeneic hematopoietic stem cell transplantation (HSCT). Interest in prevention of GvHD by dendritic cell (DC) suppression has re-emerged since the introduction of proteasome inhibitors into clinical practice. Ixazomib is an orally bioavailable proteasome inhibitor with a rapid proteasome dissociation rate. We studied the effects of ixazomib on human DC maturation, viability, and cytokine production invitro. We also determined the effects of ixazomib in a murine GvHD model. Although ixazomib suppressed naïve human DC maturation, it had only a limited effect on cell viability. Ixazomib decreased pro-inflammatory cytokine production of resting DCs. This effect was diminished or reversed when DCs were pre-stimulated. Invivo, ixazomib administered post-transplantation on days +1 and+4 or days -1, +2, and +5 ameliorated GvHD in comparison to the GvHD group. Although a fraction of mice treated according to the prolonged schedule died abruptly after the day +5 treatment, both schedules resulted in improved overall survival. When we examined the effects of ixazomib on splenic cells and serum cytokines, we found that ixazomib exerted complex schedule-dependent immunomodulatory effects. Our study provides a rationale for the potential use of ixazomib in the prevention of GvHD.

Third Party Cytokine-Induced Killer Cells Eliminate Highly Activated T-Cells, Leading to Protect from Murine Lethal Graft-Versus-Host Disease

Protecting from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects by adoptive immunotherapy would be an ideal goal in allogeneic hematopoietic stem cell transplantation (HSCT), especially using third party derived cells, because the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. We have demonstrated that third party cytokine-induced killer (CIK) cells protected from murine lethal GVHD with faster hematological recovery, which are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers with strong cytotoxic activity both against autologous and allogeneic tumor cells. However the mechanism responsible for ameliorating acute GVHD by third party CIK cells are still unknown.First, lethally irradiated Balb/c (H-2d) mice were given C3H (H-2k) bone marrow cells with C3H whole splenocytes to induce lethal GVHD with/without third party (C57/BL6 (H-2b)) CIK cells or third party whole splenocytes on day 0 or day4. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes as shown below (p<0.05). On day7 after BMT, the percentage of CD69 on CD8 T-cells in peripheral blood (PB) in the mice receiving third party CIK cells was significantly lower than those in GVHD control mice as shown below (p<0.05), meanwhile donor derived not-activated T-cells were preserved in both CD4 and CD8 T-cells. From these results, we hypothesized that third party CIK cells prevent acute GVHD by selectively depleting highly proliferative alloreactive T-cells stimulated early after transplant, while sparing naïve T-cells. To test this, we performed mixed lymphocyte reaction (MLR) by adding third party CIK cells or third party splenocytes (Stimulator: Balb/c splenocytes, Responder: C3H splenocytes, third party cells: C57/BL6). The percentage of CD69 on responder derived CD8 T-cells in the culture mixture with third party CIK cells was much less compared to that with third party splenocytes as shown below (p<0.05). We have previously shown that allogeneic CIK cells had cytotoxicity against cultured dendritic cells (DCs) by 51Cr release assay. Therefore reduced alloreaction by third party CIK cells might be due in part to the elimination of antigen presenting cells such as DCs. Next, to demonstrate that third party CIK cells could eliminate responder cells directly and then reduce the alloreaction, we performed MLR like experiments using PHA to activate T-cells strongly instead of antigen presenting cells as a stimulator. As shown below, we have demonstrated that the percentage of highly activated T-cells (CD8+CD69+) in third party CIK added plates was significantly lower compared to that in third party splenocytes added plates, indicating that third party CIK cells could have direct effects against highly activated T-cells.To further clarify the mechanisms of how third party CIK cells reduce the highly activated T-cells, we performed MLR as mentioned above with/without the neutralizing antibody against NKG2D, which is an activator receptor expressed on NK cells. Although CIK cells have cytotoxicity against tumor cells via NKG2D signaling pathway in part, adding NKG2D antibody did not show a statistical significant difference in MLR assays. It suggested that third party CIK cells could suppress alloreactive T-cells by several pathways other than NKG2D signaling pathway.Finally, we identified the in vivo kinetics of third party CIK cells after BMT. Third party derived CIK cells could be identified as a rare subpopulation in recipient peripheral blood for some days after CIK infusion, but all of them could not be detected anymore by day 7 after infusion. Whereas third party whole splenocytes expanded in recipient peripheral blood and mice received third party splenocytes with donor BM and splenocytes died until day7 because of GVHD induced by third party splenocytes.In conclusion, infusion of third party CIK cells has strong potential to prevent murine lethal GVHD probably due to selectively depleting highly proliferative alloreactive T-cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Initiation of Acute Graft-Versus-Host Disease By Angiogenesis

Angiogenesis and inflammation are two closely related processes and inhibition of angiogenesis can ameliorate inflammatory diseases by reducing the recruitment of tissue infiltrating leukocytes. However, there is limited evidence on initial mechanisms of both processes and it is unknown if angiogenesis contributes to initiation of inflammation or is a mere consequence of inflammation.Recent research showed that graft-versus-host disease (GVHD) is associated to angiogenesis making the endothelium a potential new therapeutic target during allogeneic stem cell transplantation (bone marrow transplantation, allo-BMT).We studied kinetics and quantity of angiogenesis and leukocyte infiltration in murine acute GVHD models (LP/J→B6, B6→Balb/c, B6→BDF and 129S2/Sv→B6) by assessing CD31, CD4, CD8, CD11b by immune staining and FACS analysis. In GVHD target organs colon, skin and liver, we found significant increased vascular density and endothelial cell (EC) number but no leukocyte infiltration as early as day+2 after BMT (Fig. 1a,b). Leukocyte infiltration occurred secondary to angiogenesis at day+7 or later. No angiogenesis and inflammation occurred in the non-acute GVHD target organs joints, cardiac and skeletal muscle (Fig. 1c) suggesting a previously unrecognized role of the endothelium in GVHD organ tropism. Similar analyses in a standard model for inflammatory bowel disease, the 3% dextran sulfate sodium-induced (DSS) colitis, confirmed that angiogenesis preceded leukocyte infiltration in colon (d+1 vs. d+3) implicating that the significance of our results surpasses the field of transplantation biology.We aimed at identifying pathways that are relevant for initiation of angiogenesis during GVHD and first investigated the Vegfa/VEGFR1+2 axis. Based on our previous observation demonstrating a reduction of GVHD due to inhibition of neovascularization by anti-VE-cadherin antibodies (Penack et al, JNCI 2010), we performed therapeutic interventions with monoclonal blocking antibodies. Anti-VEGFR1+2 (DC101+MFI, Imclone) and anti-Vegfa (B20-4.1.1, Genentech) did not lead to GVHD reduction (Fig. 1d). Furthermore, we found no consistent upregulation of Vegfa and VEGFR2 expression levels in GVHD target organs during initiation of GVHD or later time points (Fig. 1e) suggesting that the Vefga/VEGFR2 pathway is not a major mechanism for initiation of angiogenesis in GVHD target organs.We next assessed known endothelial activation signs and performed qPCR analysis of adhesion molecules Icam1, Vcam1, E- and P-selectin (Fif. 1f) as well as immune staining of MHC-II (Fig. 1g). We confirmed previous knowledge that endothelial cells upregulate adhesion molecules and MHC-II during established GVHD. In sharp contrast, adhesion molecules and MHC-II were downregulated or not changed during the initiation of angiogenesis at day+2. This suggests that alternative pathways are important during initial angiogenesis in early GVHD.To identify alternative pathways during initial angiogenesis in an unbiased approach, we performed microarray analyses of FACS-sorted colon ECs (Fig. 2a) and LC-MS/MS proteome analyses of MACS-isolated liver ECs (Fig. 2c) at day+2 after allo-BMT vs. syn-BMT. Strikingly, we detected substantial metabolic and cytoskeletal changes in ECs of allo-BMT recipients (Fig. 2b,d). Recently, metabolic processes have been shown to be associated with cytoskeletal remodeling during EC migration and angiogenesis (Uebelhoer et al., J Vasc Res 2016). Accordingly, measuring single-cell mechanical characteristics with Real-Time deformabiliy cytometry (Otto et al., Nature Methods 2015) revealed profoundly higher deformation (D = 0.0547 ± 0.0011) and overall softening of ECs from allo-BMT recipients (Fig. 2e) indicating to enhanced EC migration and proliferation.We demonstrate that angiogenesis initiates GVHD in target organs and is restricted to target organs. Our findings implicate that endothelial biology plays a major role in GVHD organ tropism and disease development. We revealed novel genes and proteins regulating migration and proliferation of EC in initial angiogenesis during GVHD. Our study amends the knowledge on the interplay between the vasculature and inflammation opening a new window to develop therapeutic strategies targeting the endothelium. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Host Interleukin 6 and Indoleamine 2,3 Dioxygenase Regulate Inflammation in the Brain during Graft Versus Host Disease

Graft versus host disease (GVHD) commonly induces pathological damage in peripheral target organs such as the skin, liver and gastrointestinal tract leading to well characterized organ-specific clinical manifestations. A number of studies, however, have shown that patients with GVHD can also have behavioral and mood alterations that can affect overall cognitive function and lead to significant impairments in quality of life. The extent to which GVHD contributes to cognitive dysfunction and induces inflammation within the central nervous system (CNS), however, has not been critically examined. To address this question, we conducted studies using two well-defined murine GVHD models [C57BL/6(H-2b)→Balb/c (H-2d) and B10.BR(H-2k)→B6 (H-2b)]. We observed that there was a significant increase in the number of donor-derived CD4+ and CD8+ T cells in the brains of GVHD recipients early (days 7 -14) and late (day 42) post transplantation compared to BM controls. Histological studies revealed activated microglial cells and CD3+ T cell infiltration in the periventricular regions of brains in GVHD recipients that were not present in BM animals. Real time q-PCR analysis also demonstrated significant increases in IFN-γ, TNF-α, and IL-6 mRNA expression indicative of a proinflammatory state. Notably, GVHD animals exhibited behavioral changes in the forced swim test and elevated plus maze which are validated assays of stress coping and anxiety, respectively. Since IL-6, in particular, plays a pivotal role in GVHD pathogenesis in murine models and humans, we examined whether blockade of IL-6 signaling altered neuroinflammation. Animals treated with an anti-IL-6R antibody had a significant reduction in the number of donor-derived CD4+ and CD8+T cells in the brain compared to isotype control-treated mice. Anti-IL-6R treatment of GVHD mice also resulted in significant reductions in IFN-γ, TNF-α, and IL-6 mRNA and normalized behavior in the forced swim test, indicative of a decreased inflammatory response. Since IL-6 is produced by a wide variety of cells, including microglial and T cells, both donor and recipient cells have the potential to modulate GVHD severity within the CNS. To define whether donor or host IL-6 production was most critical for inducing neuroinflammation, experiments were conducted employing IL-6-/- mice as either donors or recipients. Whereas the absence of IL-6 in donor-derived cells had no impact on the degree of inflammation within the CNS, recipient animals that lacked IL-6 had a significant decrease in the number of donor-derived T cells which accumulated in the brain as well as a marked reduction in inflammatory cytokines, indicating that host IL-6 production was critical. To define the downstream pathways of IL-6-mediated CNS inflammation, we examined the role of indoleamine 2,3-dioxygenase (IDO) since IL-6 has been shown to upregulate IDO-1 expression under inflammatory conditions. We observed that IDO-1 mRNA levels were significantly increased in the brains of GVHD animals, and that blockade of IL-6 signaling resulted in a marked decrease in IDO mRNA levels. Additionally, transplantation studies using IDO-/- mice revealed that host, but not donor, IDO production was required for maximal inflammatory effects. Serotoninergic projections to the prefrontal cortex (PFC), in particular, are sensitive to inflammation and contribute to stress coping behavior. Therefore, to further interrogate this pathway, we performed quantitative mass spectrometry of brain extracts from the PFC. We found that tryptophan and 5HT concentrations were not different between BM and GVHD groups. However, there was an increase in the IDO product, kynurenic acid, in GVHD recipients consistent with an increase in brain IDO expression. To provide additional support for the premise that IL-6 effects were mediated through the IDO pathway, recipient mice were treated with either 1-methyltryptophan (1-MT), a completive inhibitor of IDO, or a vehicle control. GVHD mice treated with 1-MT had decreased accumulation of T cells in the brain and normal behavior in the forced swim test, demonstrating that inhibition of IDO abrogated CNS inflammation and behavioral changes in the presence of intact IL-6 signaling. In summary, these studies demonstrate that host IL-6 and IDO regulate inflammation and adversely impact behavioral function within the brain during GVHD through the tryptophan metabolic pathway. DisclosuresNo relevant conflicts of interest to declare.

Adoptively transferred donor IL-17-producing CD4+ T cells augment, but IL-17 alleviates, acute graft-versus-host disease.

The role of IL-17 and IL-17-producing CD4+ T cells in acute graft-versus-host disease (GVHD) has been controversial in recent mouse and human studies. We carried out studies in a murine acute GVHD model of fully major histocompatibility complex-mismatched myeloablative bone marrow transplantation. We showed that donor wild-type CD4+ T cells exacerbated acute GVHD compared with IL-17-/- CD4+ T cells, while IL-17 reduced the severity of acute GVHD. The augmentation of acute GVHD by transferred donor IL-17-producing CD4+ T cells was associated with increased Th1 responses, while IL-17 decreased the percentages of Th1 cells in the GVHD target organs. Furthermore, IL-17 reduced the infiltration of macrophages into the GVHD tissues. In vitro study showed that IL-17 could downregulate Th1 responses, possibly through inhibiting IL-12 production by donor macrophages. Depletion of macrophages in vivo diminished the protective effect of IL-17. Our results demonstrated the differential roles of adoptively transferred donor IL-17-producing CD4+ T cells and IL-17 in the same acute GVHD model.

IL-22 promoted CD3 + T cell infiltration by IL-22R induced STAT3 phosphorylation in murine acute graft versus host disease target organs after allogeneic bone marrow transplantation

Graft versus host disease (GVHD) is a life threatening complication of bone marrow stem cell transplantation, in which considerable numbers of proinflammatory cytokines secreted by allo-reactive donor T cells are involved. We and other previous studies have found that interleukin-22 (IL-22) was able to aggravate the target organs damage of GVHD. However, the mechanism and the signal pathway of IL-22 in murine acute GVHD was not clear. Here, we observed that compared with GVHD group, more serious pathological damage and more CD3+ T cells infiltrated in GVHD target organs were detected in the mice injected with IL-22. Meanwhile, transcription factor T-bet, RORγt and AhR respectively associated with Th1, Th17 and Th22 cells changed in varying degrees in different GVHD target organs. Furthermore, the increased expression of IL-22R and its downstream protein P-STAT3 were detected in GVHD mice with IL-22 treated. These results suggested that the pathological role of IL-22 in GVHD target organs contribute to exogenous injected IL-22 as well as secreted IL-22 from the infiltrated allo-reactive effector T cells. In addition, the IL-22R-STAT3 pathway may play important role in GVHD tissue injury and target this way may yield new approaches for reduction of GVHD.

TNF-α priming enhances CD4+FoxP3+ regulatory T-cell suppressive function in murine GVHD prevention and treatment

AbstractCD4+CD25+FoxP3+ regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4+FoxP3− T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α–primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α–primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the β2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4β7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of β2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice.

Intestinal bacteria may modulate the risk of infection and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients often develop neutropenic fever, which is treated with antibiotics that may target anaerobic bacteria in the gut. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam antibiotics was associated with increased GVHD-related mortality at 5 years (21.5% for imipenem-cilastatin-treated patients versus 13.1% for untreated patients, P = 0.025; 19.8% for piperacillin-tazobactam-treated patients versus 11.9% for untreated patients, P = 0.007). However, two other antibiotics also used to treat neutropenic fever, aztreonam and cefepime, were not associated with GVHD-related mortality (P = 0.78 and P = 0.98, respectively). Analysis of stool specimens from allo-HSCT recipients showed that piperacillin-tazobactam administration was associated with perturbation of gut microbial composition. Studies in mice demonstrated aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactam compared to aztreonam (P < 0.01 and P < 0.05, respectively). We found pathological evidence for increased GVHD in the colon of imipenem-cilastatin-treated mice (P < 0.05), but no difference in the concentration of short-chain fatty acids or numbers of regulatory T cells. Notably, imipenem-cilastatin treatment of mice with GVHD led to loss of the protective mucus lining of the colon (P < 0.01) and the compromising of intestinal barrier function (P < 0.05). Sequencing of mouse stool specimens showed an increase in Akkermansia muciniphila (P < 0.001), a commensal bacterium with mucus-degrading capabilities, raising the possibility that mucus degradation may contribute to murine GVHD. We demonstrate an underappreciated risk for the treatment of allo-HSCT recipients with antibiotics that may exacerbate GVHD in the colon.

Dendritic polyglycerol sulfate attenuates murine graft-versus-host disease.

Graft-versus-host disease (GvHD) is a severe immune reaction commonly occurring after hematopoietic stem cell transplantation. The outcome of patients who do not respond to the currently used immunosuppressive drugs is poor, thus there is an urgent need for the evaluation of new therapies. Heparin has a well-known anti-inflammatory effect and heparin analogues with a low anticoagulant effect are interesting candidates as new anti-inflammatory drugs. We explored the therapeutic potential of dendritic polyglycerol sulfates (dPGS), a novel class of heparin derivatives, on murine acute GvHD in vivo. The therapeutic effect of dPGS on murine GvHD was more intense after intravenous application compared to subcutaneous injection. An increased survival rate and improved clinical scores were observed in mice treated with 5 mg/kg once a week. In these animals, there was a reduction in the percentage of CD4(+) and CD8(+) T cells, which are the main effectors of GvHD. In addition, dPGS treatment decreased the number of tumor necrosis factor alpha (TNFα)-producing T cells. Increasing the dose of dPGS reversed the positive effect on survival as well as the clinical score, which indicates a small therapeutic range. Here, we report for the first time that dPGS have a significant immunosuppressive in vivo effect in a mouse model of severe acute GvHD. Therefore, we propose to study dPGS as promising candidates for the development of potential new drugs in the treatment of steroid-refractory GvHD patients first in larger animals and later in humans.

Transcriptome analysis of GVHD reveals aurora kinase A as a targetable pathway for disease prevention.

Graft-versus-host disease (GVHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate treatment strategies. To address this, we measured the gene expression profile of nonhuman primate (NHP) T cells during acute GVHD. Utilizing microarray technology, we measured the expression profiles of CD3(+) T cells from five cohorts: allogeneic transplant recipients receiving (i) no immunoprophylaxis (No Rx), (ii) sirolimus monotherapy (Siro), (iii) tacrolimus-methotrexate (Tac-Mtx), as well as (iv) autologous transplant recipients (Auto) and (v) healthy controls (HC). This comparison allowed us to identify transcriptomic signatures specific for alloreactive T cells and determine the impact of both mTOR (mechanistic target of rapamycin) and calcineurin inhibition on GVHD. We found that the transcriptional profile of unprophylaxed GVHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function, and cytokine synthesis. Within these pathways, we discovered potentially druggable targets not previously implicated in GVHD, prominently including aurora kinase A (AURKA). Utilizing a murine GVHD model, we demonstrated that pharmacologic inhibition of AURKA could improve survival. Moreover, we found enrichment of AURKA transcripts both in allo-proliferating T cells and in sorted T cells from patients with clinical GVHD. These data provide a comprehensive elucidation of the T cell transcriptome in primate acute GVHD and suggest that AURKA should be considered a target for preventing GVHD, which, given the many available AURKA inhibitors in clinical development, could be quickly deployed for the prevention of GVHD.

Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow-derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow-derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee-mandated, cool standard temperatures (∼ 22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼ 30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor-deficient (β2-AR(-/-)) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting.

Significance of Th1/Th17 Imbalance in Mice with Acute Graft-versus-Host Disease

To investigate the effects of Th1/Th17 cell imbalance on the pathogenesis of acute graft-versus-host disease (GVHD) in mice. In a murine GVHD model of C57BL/6 (H-2(b)), a low dose of halofuginone (HF) was applied for treating the recipients in order to result in Th1/Th17 imbalance. Rechipient mice were divided into GVHD group (without HF intervention) and GVHD plus HF group (treated by HF). The recipients were monitored for survival rate, clinical scores of acute GVHD, contents of circulatory Th1 and Th17 cells, Th1/Th17 ratio and serum level of IFN-γ and IL-17A. Expression levels of IFN-γ and IL-17A in target organs were analyzed by using real-time PCR, and the target organs were delivered for histological examinations. Recipients treated with HF showed that all the mortality, circulatory Th1/Th17 ratio and clinical score were higher than those in the mice without HF intervention (P < 0.05). Circulatory Th1/Th17 ratio positively correlates with clinical score (P < 0.001). HF administration reduces the expression level of intestinal IL-17A and increases intrahepatic and intestinal IFN-γ level (P < 0.05), HF treatment aggravates GVHD in liver and small intestine with augmented hepatic and intestinal inflammation. Th1/Th17 imbalance contributes to the pathogenesis of acute GVHD.

Differential Effects of Gut-Homing Molecules CC Chemokine Receptor 9 and Integrin-β7 during Acute Graft-versus-Host Disease of the Liver.

Homing of allogeneic donor T cells to recipient tissue is imperative for the development of acute graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this study we show that alteration of T cell homing due to integrin-β7 deficiency on T cells or its ligand MAdCAM-1 in BMT recipients contributes to the pathophysiology of experimental GVHD. In contrast, lack of CC chemokine receptor 9 on donor T cells alters tissue homing but does not impact GVHD survival. We further demonstrate that MAdCAM-1 is aberrantly expressed in hepatic murine GVHD as well as in patients with active liver GVHD. However, infiltration of donor T cells in gut but not liver was dependent of MAdCAM-1 expression, indicating, that homing and/or retention of donor T cells rests on divergent molecular pathways depending on the GVHD target tissue.

In vitro-generated MDSCs prevent murine GVHD by inducing type 2 T cells without disabling antitumor cytotoxicity

AbstractMyeloid-derived suppressor cells (MDSCs) inhibit T-cell expansion and functions by versatile mechanisms such as nutrient depletion, nitrosylation, or apoptosis. Since graft-versus-host disease (GVHD) is characterized by the expansion of donor-derived T cells destroying recipient tissue, we analyzed whether MDSCs can be used for GVHD prevention in murine allogeneic bone marrow transplantation models. Transplantation of MDSCs, generated from bone marrow cells by granulocyte-macrophage colony-stimulating factor (GM-CSF)/G-CSF in vitro, inhibited GVHD-induced death and attenuated histologic GVHD, whereas antitumor cytotoxicity of alloantigen-specific T cells was maintained. MDSCs expanded in vivo and invaded lymphatic and GVHD target organs. Major histocompatibility complex class I expression on MDSCs was dispensable for their suppressive capacity. Inhibition of GVHD required the presence of MDSCs during T-cell priming, whereas allogeneic T-cell numbers and homing in lymphoid and GVHD target organs were not considerably affected in MDSC-treated mice. However, MDSCs skewed allogeneic T cells toward type 2 T cells upregulating T helper 2 (Th2)-specific cytokines. Type 2 T-cell induction was indispensable for GVHD prevention since MDSC treatment failed to prevent GVHD when allogeneic STAT6-deficient T cells, which are unable to differentiate into Th2 cells, were transplanted. MDSC-induced Th2 induction might be applicable for GVHD treatment in clinical settings.

Pharmacologic Inhibition of JAK1/JAK2 Signaling Reduces Experimental Murine Acute GVHD While Preserving GVT Effects.

Immune-mediated graft-versus-tumor (GVT) effects can occur after allogeneic hematopoietic stem cell transplantation (HSCT), but GVT is tightly linked to its main complication, graft-versus-host disease (GVHD). Strategies aimed at modulating GVHD, while maintaining the GVT effect, are needed to improve the cure rate of transplant. Given the emerging role of Janus-activated kinase (JAK) signaling in lymphoproliferative and myeloproliferative diseases and its established function at dictating T-cell differentiation, we postulated that JAKs might be potential therapeutic targets through a pharmacologic approach. We examined the effect of JAK1/JAK2 modulation by ruxolitinib in a mouse model of fully MHC mismatched bone marrow transplant comprising in vivo tumor inoculation. JAK1/JAK2 inhibition by ruxolitinib improved both overall survival (P = 0.03) and acute GVHD pathologic score at target organs (P ≤ 0.001) of treated mice. In addition, treatment with ruxolitinib was associated with a preserved GVT effect, as evidenced by reduction of tumor burden (P = 0.001) and increase of survival time (P = 0.01). JAK1/JAK2 inhibition did not impair the in vivo acquisition of donor T-cell alloreactivity; this observation may account, at least in part, to the preserved GVT effect. Rather, JAK1/JAK2 inhibition of GVHD was associated with the modulation of chemokine receptor expression, which may have been one factor in the reduced infiltration of donor T cells in GVHD target organs. These data provide further evidence that JAK inhibition represents a new and potentially clinically relevant approach to GVHD prevention.

IL4RA regulates lymphocytes trafficking in sclerodermatous graft-versus-host disease. (CAM4P.154)

Abstract Scleroderma is an autoimmune disease characterized by inflammation and fibrosis in skin and internal organs. We utilized a murine Graft Versus Host Disease (GVHD) model of Scleroderma (SclGVHD), in which lymphocytes are transferred from B10.D2 mice (graft) into MHC-matched, lymphocyte deficient Balb/c Rag2-/- mice (host, sclGVHD mice). Over the course of 6 weeks, sclGVHD mice develop skin inflammation and fibrosis. Previously, we identified IL13 pathway activation in the skin of sclGVHD mice and scleroderma patients. Accordingly, host mice lacking IL4RA, a functional subunit for the IL13 receptor complex, are protected from sclGVHD. Here, we show that sclGVHD-IL4Ra-/- mice display a defect in lymphocyte trafficking, with more activated T cells in subcutaneous lymph nodes (sLNs) but fewer T cells in lymph, blood and skin compared to control mice. In addition, sphingosine 1-phosphate (S1P) kinase 1 was decreased in the sLNs and in sorted lymphatic endothelial cells (LEC) from sclGVHD-IL4Ra-/- mice. Since S1P is the major regulator of lymphocyte egress from sLNs, these data suggest that IL4Ra promotes sclGVHD by inducing the production of this mediator from LECs, which drives activated T cells into the periphery. These experiments highlight a potential novel role for IL4RA in inflammatory diseases, and indicate that strategies which impede lymphocyte trafficking may be effective therapeutically in the context of GVHD and scleroderma.

Granzyme A Is Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease.

In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS-FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4+CD25+ Tregs from gzmA -/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. GzmA -/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas gzmA -/- Tregs are highly suppressive in vitro, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model.

Regulation of pulmonary graft-versus-host disease by IL-26+CD26+CD4 T lymphocytes.

Obliterative bronchiolitis is a potentially life-threatening noninfectious pulmonary complication after allogeneic hematopoietic stem cell transplantation and the only pathognomonic manifestation of pulmonary chronic graft-versus-host disease (cGVHD). In the current study, we identified a novel effect of IL-26 on transplant-related obliterative bronchiolitis. Sublethally irradiated NOD/Shi-scidIL2rγ(null) mice transplanted with human umbilical cord blood (HuCB mice) gradually developed clinical signs of graft-versus-host disease (GVHD) such as loss of weight, ruffled fur, and alopecia. Histologically, lung of HuCB mice exhibited obliterative bronchiolitis with increased collagen deposition and predominant infiltration with human IL-26(+)CD26(+)CD4 T cells. Concomitantly, skin manifested fat loss and sclerosis of the reticular dermis in the presence of apoptosis of the basilar keratinocytes, whereas the liver exhibited portal fibrosis and cholestasis. Moreover, although IL-26 is absent from rodents, we showed that IL-26 increased collagen synthesis in fibroblasts and promoted lung fibrosis in a murine GVHD model using IL-26 transgenic mice. In vitro analysis demonstrated a significant increase in IL-26 production by HuCB CD4 T cells following CD26 costimulation, whereas Ig Fc domain fused with the N-terminal of caveolin-1 (Cav-Ig), the ligand for CD26, effectively inhibited production of IL-26. Administration of Cav-Ig before or after onset of GVHD impeded the development of clinical and histologic features of GVHD without interrupting engraftment of donor-derived human cells, with preservation of the graft-versus-leukemia effect. These results therefore provide proof of principle that cGVHD of the lungs is caused in part by IL-26(+)CD26(+)CD4 T cells, and that treatment with Cav-Ig could be beneficial for cGVHD prevention and therapy.