Sclerodermatous cGVHD Research Articles

Ocular Surface and Tear Film Characteristics in a Sclerodermatous Chronic Graft-Versus-Host Disease Mouse Model.

To report the characteristics of the ocular surface in a previously established sclerodermatous chronic graft-versus-host disease (cGVHD) mouse model. The ocular surface features and tear film parameters of the mouse model were assessed by histopathology, immunohistochemistry, electron microscopy, quantitative polymerase chain reaction, and flow cytometry. The mice exhibited loss of body weight and decreased tear secretion (P < 0.001), mimicking the clinical features of patients with cGVHD. Ocular examination demonstrated significant corneal epithelial staining, conjunctival (P < 0.001), and eyelid (P = 0.015) fibrosis compared with the control mice. The density of both goblet cells (P = 0.043) and microvilli was lower (P < 0.001), and the microvilli were shorter (P = 0.007) in the conjunctiva of cGVHD mice than those of the controls. The immunohistochemical studies demonstrated greater expression of CD45, CD4, and CD8 cells in the conjunctiva and eyelid tissues compared with the controls (P < 0.05 for all). In addition, reduced Forkhead box P3 (Foxp3)+ cells were found in both the peripheral blood (P < 0.001) and conjunctiva (P = 0.042) of cGVHD mice compared with the controls. The constellation of these findings suggests that the sclerodermatous cGVHD mouse model well recapitulates ocular manifestations of cGVHD in humans. This model can be used to study the mechanisms involved in the pathogenesis and treatment of chronic ocular graft-versus-host disease.

Tratamiento de la enfermedad de injerto contra huésped crónica esclerodermiforme con imatinib: una perspectiva dermatológica

IntroductionChronic graft-versus-host disease (cGVHD) is the most important cause of late non-relapse mortality after allogeneic hematopoietic stem cell transplantation. Sclerodermatous cGVHD is usually steroid refractory and remains a therapeutic challenge. Activating antibodies against the PDGFR have been reported in patients with sclerodermatous cGVHD. These antibodies induce PDGFR phosphorylation and lead to fibrosis. There is increasing evidence of successful treatment of sclerodermatous cGVHD with imatinib, a tyrosine kinase inhibitor. ObjectiveTo evaluate the response of cutaneous sclerodermatous cGVHD to imatinib. Materials and methodsRetrospective study of 18 patients with sclerodermatous cGVHD refractory to immunosuppressants treated with imatinib in a single center. Evaluation of treatment response was performed by clinicians’ assessment and patients’ subjective response at one, 3, 6, 9, 12 and 18 months after initiation of imatinib. Response was assessed as complete, partial, significant, no change or progression. Tapper off steroids was complete, partial or not possible. ResultsIn our series, 4 (22%) patients achieved complete response, 9 (50%) patients partial response, 2 (11%) patients significant response, 2 (11%) patients had no change and one (6%) patient progressive disease at last follow-up. Mean time from initiation of imatinib to any degree of response was 2,75 months (range 1-9 months). ConclusionsThis study provides further evidence of the role of imatinib for the treatment of steroid refractory sclerodermatous cGVHD.

Pirfenidone ameliorates murine chronic GVHD through inhibition of macrophage infiltration and TGF-β production

AbstractAllogeneic hematopoietic stem cell transplantation is hampered by chronic graft-versus-host disease (cGVHD), resulting in multiorgan fibrosis and diminished function. Fibrosis in lung and skin leads to progressive bronchiolitis obliterans (BO) and scleroderma, respectively, for which new treatments are needed. We evaluated pirfenidone, a Food and Drug Administration (FDA)-approved drug for idiopathic pulmonary fibrosis, for its therapeutic effect in cGVHD mouse models with distinct pathophysiology. In a full major histocompatibility complex (MHC)-mismatched, multiorgan system model with BO, donor T-cell responses that support pathogenic antibody production are required for cGVHD development. Pirfenidone treatment beginning one month post-transplant restored pulmonary function and reversed lung fibrosis, which was associated with reduced macrophage infiltration and transforming growth factor-β production. Pirfenidone dampened splenic germinal center B-cell and T-follicular helper cell frequencies that collaborate to produce antibody. In both a minor histocompatibility antigen–mismatched as well as a MHC-haploidentical model of sclerodermatous cGVHD, pirfenidone significantly reduced macrophages in the skin, although clinical improvement of scleroderma was only seen in one model. In vitro chemotaxis assays demonstrated that pirfenidone impaired macrophage migration to monocyte chemoattractant protein-1 (MCP-1) as well as IL-17A, which has been linked to cGVHD generation. Taken together, our data suggest that pirfenidone is a potential therapeutic agent to ameliorate fibrosis in cGVHD.

Sclerodermatous GVHD after Allogeneic Bone Marrow Transplant: a Review

Chronic graft versus host disease (cGVHD) is the leading cause of non-relapse mortality after allogeneic hematopoietic bone marrow transplantation (HCT) for blood malignancy in patients who survive for more than two years. cGVHD can significantly affect quality of life and cause decreased mobility amongst other grave consequences such as end-organ damage, contributing to morbidity and mortality rates for recipients of HCT. Unlike acute GVHD (aGVHD), the chronic variant of graft versus host disease (GVHD) has complex immunopathology involving both humoral and cell immunity. It typically affects the integumentary system, though is known to also affect myofascial, mucocutaneous tissues as well as cause end organ damage ultimately resulting in death. Sclerodermatous cGVHD is a type of cGVHD characterized by involvement of the skin, subcutaneous tissue and fascia without evidence of disease in the viscera. Manifestations of this disease are often evocative of autoimmune disease, which is a self-directed inflammatory reaction to the innate and adaptive immune system in various tissues or multiple organ systems. This inflammatory reaction gives rise to autoantibodies as well as B-cell and T-cell mediated direct toxicity which can cause chronic inflammatory changes of tissues ultimately resulting in tissue scarring and end organ dysfunction. We aim to review the literature on this grave disease and elucidate aspects of the immunopathology of chronic sclerodermatous GVHD in hopes that it may lead to revelations inspiring novel therapies after its diagnosis or preventative measures before stem cell transplantation for malignancy.

Pirfenidone Reverses Murine Chronic Graft Versus Host Disease

Allogeneic hematopoietic stem cell transplantation (aHSCT) is hampered by chronic graft-versus-host disease (cGVHD), which results in multi-organ fibrosis and loss of function. In particular, bronchiolitis obliterans (BO) and scleroderma resulting from fibrotic bronchiolar and cutaneous response, respectively, are two devastating outcomes for cGVHD patients. Fibrotic manifestations often are considered irreversible and progressive. Therefore, new therapies targeting fibrosis are urgently needed.Pirfenidone (5-methyl-1-phenyl-2- (1H)-pyridone) exhibits a well-documented anti-inflammatory and anti-fibrosis function in multiple pre-clinical models and is the first and only FDA-approved drug for idiopathic pulmonary fibrosis. For this study, Pirfenidone was synthesized as a crystalline solid and found to be pure both by melting point and NMR spectroscopy. We evaluated Pirfenidone's anti-fibrosis function in 2 pathophysiologically distinct cGVHD murine models: 1. a major mismatched multi-organ system model (C57BL/6 to B10.BR) that induces BO as a result of a cGVHD-induced germinal center (GC) reaction, antibody deposition and fibrosis in the lung; and 2. a minor antigen mismatched model (B10.D2 to BALB/c) in which severe scleroderma is the major disease manifestation.In the BO model, pulmonary function loss in cGVHD mice (as reflected by increased resistance, elastance and decreased compliance of the lung) was restored by Pirfenidone treatment (400mg/kg) during both the early (day28-56) (Fig A, representative of 3 experiments with 5-8 mice per group) and late stages (day56-84) of the disease. Pathologic changes in the lung, such as collagen deposition and narrowing of bronchioles, were significantly reduced by Pirfenidone. The size and frequency of GCs in the spleen, and the frequency of GC B cells (Fig B, representative of 2 experiments with 5-8 mice per group) and T follicular helper cells were all significantly reduced in Pirfenidone- treated groups. To determine whether GCs were directly affected by Pirfenidone, we evaluated Pirfenidone in C57BL/6 mice immunized with sheep red blood cells (SRBC) to induce GCs. Interestingly, Pirfenidone did not reduce the SRBC-induced GC reaction (Fig C) (comparable frequencies of splenic GC B cells, T follicular helper cells and serum IgG levels were seen between Pirfenidone and vehicle-treated groups). These results suggested that Pirfenidone suppresses the GC reaction through a cGVHD-specific mechanism, rather than through immune regulation. Mechanistically, Pirfenidone administration attenuated the sequestration of pro-fibrogenic F4/80+ macrophages (Fig D, representative of 2 experiments) and TGF-β (Fig E, representative of 2 experiments) production within the lung. These results have led us to elucidate a potential mechanism of cGVHD: antibody deposition in the lung results in the activation of macrophages and TGF-β that drive fibrotic change and tissue damage, resulting in the exposure of auto- and allo- antigens to the immune system that support and sustain pathologic GC reactions.In the B10.D2 to BALB/c sclerodermatous cGVHD model, Pirfenidone treatment (400mg/kg, day21-55) improved clinical signs of scleroderma and reduced macrophage infiltration in the skin (Fig F).In summary, this is the first study evaluating a commercially available anti-fibrosis drug on pathologically distinct pre-clinical cGVHD models. Our data suggests Prifenidone reversed cGVHD in the BO model and, to a lesser extent, in the scleroderma model. Thus, Pirfenidone is a novel therapeutic agent for treating cGVHD patients with fibrosis that have been typically refractory to therapies.A. Resistance of lungs was measured on day56 of transplantation; Elastance and compliance correlated with resistance but were not shown here.B. Flow cytometry analysis of GC B cells of no cGVHD vs cGVHD mice treated with Pirfenidone or vehicle;C. Flow cytometry analysis of GC B cells from SRBC-immunized mice treated with Pirfenidone or vehicle;D and E. Macrophage F4/80 and TGF-β quantification of day56 lungs of no cGVHD vs cGVHD mice treated as indicated;F. Skin GVHD scores were recorded on indicated dates of irradiated BALB/c mice transplanted with B10.D2 donor BM alone or with T cells and treated as indicated. Unpaired student T test was used for statistical analysis. ****:P<0.0001; ***: P<0.001; **: P<0.01; *: P<0.05; ns: not significant. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD

AbstractChronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD.

Pulp Obliteration in a Patient with Sclerodermatous Chronic Graft-versus-Host Disease

Dental pulp calcification is a common finding associated with localized dental trauma, genetic disorders, and systemic inflammatory diseases. Chronic graft-versus-host disease (cGVHD) is a frequent complication after allogeneic hematopoietic cell transplantation (allo-HCT) characterized by immune-mediated injury to the skin, mouth, eyes, liver, and other tissues, resulting in significant disability and reduced quality of life. We report a patient with sclerodermatous cGVHD who presented with general pulp calcification in all teeth 5 years after allo-HCT. A review of full mouth dental radiographs obtained just before allo-HCT revealed normal-appearing pulp chambers. Based on prior reports of generalized pulp calcification associated with progressive systemic sclerosis, we hypothesized that the etiology was likely related to the presence of cGVHD with associated vascular and fibrotic tissue changes within the pulp vasculature. Clinicians should consider cGVHD in the differential diagnosis of generalized pulp calcification.

Ruxolitinib As Sparing Agent for Steroid-Dependent Chronic Graft-Versus-Host Disease (cGVHD)

Ruxolitinib (RUX) is an oral selective Janus-associated kinase 1 (JAK1) and JAK2 inhibitor that is approved by the FDA for the treatment of patients with myeloproliferative neoplasms. JAKs transmit the signaling of ligand binding to inflammatory cytokine receptors into intracellular responses, and inhibition of JAK is effective in the treatment of autoimmune disorders. In MHC-mismatched mouse transplant models, pharmacologic inhibition of IFNγR signaling with RUX reduced graft-versus-host disease (GVHD) and improved survival (Choi et al Blood 2012); and preliminary reports have shown that 13/14 patients with steroid-refractory acute (10) and chronic (4) GVHD responded to RUX therapy. (Sporell et al Blood 2014). Between 09/2014 and 7/2015, 16 patients (12M/4F), with hematological malignancies and recipients of unrelated donor (14), matched sibling (2) blood (15) or marrow (1) transplant developed quiescent (12) or de novo (4) severe (NIH criteria) steroid-dependent cGVHD and received RUX as 2nd (4), 3d (7) 4th (4) or 5th (1) line salvage. 12 had prior grades 1 (3) 2 (6) 3(2) or 4 (1) acute GVHD that affected the skin (12) and gastrointestinal system (GI, 5). Median time of onset of cGVHD was d+130 (range, 90-299), and affected skin (16) eyes (12), mouth (10), GI (8), lungs (4), liver (3) and the musculoskeletal system (3). cGVHD was steroid dependent with recurrences of cGVHD symptoms with steroid tapers that were attempted a median of 4 times(range, 2-10). Median duration of continuous exposure to steroids for cGVHD was 24 months (range, 6-53). RUX was administered at the dose of 5 mg BID. Median weight was 82 kg (range, 56-158). RUX dose was increased to 15 mg/d (4) or 20 mg/d (3) due to physician preference (4), patient weight (1), or flare of cGVHD after initial response due to discontinuation of immunosuppression (1) or temporary perioperative hold of RUX (1). Median duration of RUX therapy was 6 months (range 1-14). All patients were evaluable for response. Complete resolution of clinical manifestations of cGVHD was observed in the following organs: lungs (dyspnea/O2 dependence), mouth (oral ulcerations), skin (non-sclerodermatous erythema), liver (sGOT, sGPT, alkaline phosphatase), musculoskeletal and GI (dysphagia, diarrhea). Subjective improvement was reported in sclerodermatous and ocular cGVHD. Responses were observed early after initiation of RUX (median 14 days). Prednisone was successfully reduced to physiologic doses (n=2) or discontinued (n=10) in 12 (75%), and taper is currently in progress for the 4 patients who started RUX in the past 8 weeks. Median time from initiation of RUX to prednisone discontinuation was 72 days (range, 31-120). With a median follow-up of 4 months (range, 1-12) from prednisone discontinuation/reduction to physiologic doses, 2 patients experienced a transient flare of cGVHD symptoms associated with discontinuation of immunosuppression (1) and temporary hold of RUX. None of the other patients required a restart of prednisone or increased immunosuppression. We conclude that RUX is an effective steroid-sparing agent in steroid-dependent severe cGVHD. Validation of these results in prospective trials is needed. DisclosuresJillella:Seattle Genetics, Inc.: Research Funding.

Colchicine Can be an Effective Adjunctive Treatment for Extensive Refractory Chronic-Graft-Versus-Host Disease

Chronic extensive, severe graft-versus-host-disease (GVHD) is a major cause of morbidity and mortality following an allogeneic hematopoietic stem cell transplant (HSCT). Patients with progressive chronic GVHD (cGVHD) following standard treatments have limited options. Patients with extensive refractory cGVHD have a decreased performance status (PS) and an increased non-relapsed mortality. Extensive sclerodermatous cGVHD responds poorly to standard treatments. Colchicine is a well-known anti-inflammatory drug that has been used in the treatment of many inflammatory disorders (1). We report a series of 8 patients with extensive cGVHD who were refractory to multiple lines of therapy and then were treated with colchicine. All patients had improvements in ECOG PS, skin GVHD and/or oral GVHD after starting colchicine. All patients were able to decrease their immunosuppressive therapies (IST) after starting colchicine.We retrospectively examined 8 patients with refractory extensive cGVHD, treated with colchicine (0.6 mg QD or BID). IST for cGVHD was defined by the following: steroids, extracorporeal photopheresis (ECP), imatinib, mycophenolate mofetil (MMF), tacrolimus, bortezomib, montelukast, cyclophosphamide (Cy), pentostatin, and rituximab. National Institutes of Health 2014 cGVHD consensus criteria (2) were used to grade the cGVHD prior to colchicine and at the time of best response after colchicine was started.Baseline patient characteristics are listed in Table 1. Conditioning regimen for allogeneic HSCT consisted of PPT (ECP, pentostatin, TBI) (4/8), busulfan/Cy (2/8), Cy-TLI (1/8), or Cy/TBI (1/8). GVHD prophylaxis involved CSA/MTX (7/8), tacrolimus/MTX (1/8) and ATG in addition to CSA/MTX (1/8). Median number of organs involved in cGVHD was 4.5 (range 2-5). All patients had extensive cGVHD with skin (8/8), oral (8/8), eye (8/8), GI (7/8), lung (6/8), liver (2/8), and/or genital (2/8) involvement. Median number of failed IST was 3.5 (range 1-6). Patients failed steroids (7/8), ECP (6/8), MMF (5/8), imatinib (3/8), cyclophosphamide (2/8) and bortezomib (1/8). Median number of IST immediately before colchicine was 2.5 (range 1-6). Most patients required twice daily dosing of colchicine (5/8). Median follow-up since beginning colchicine was 11 weeks (range 3-67 weeks).At best response with colchicine, all patients had clinical improvement of chronic GVHD (Figure 1). Patients with extensive cGVHD of the skin, including all patients with extensive sclerodermatous changes, responded to the addition of colchicine, and had steroid and MMF doses lowered, and/or were stopped on IST. Median number of IST fell to 2 (range 0-5). Four patients had a reduction in the number of IST. One patient was taken off 4 IST (ECP, imatinib, Cy and MMF). Another patient was able to stop ECP. Five patients had a reduction in prednisone with median reduction of 5mg (range 5-40 mg), while 2 had reductions in the total daily dose of MMF. Adverse events were uncommon and included 2 patients with grade 1-2 diarrhea. The most severe adverse event was grade 2 diarrhea in one patient that led to eventual discontinuation of colchicine. No hematologic toxicity was observed. The rest (7/8) continued on colchicine therapy.In conclusion, patients with extensive cGVHD involving the skin and mouth, refractory to multiple lines of IST, experienced clinical improvement on colchicine. Toxicity was limited to mild GI side effects. These observations need to be validated in a prospective clinical trial.References(1) Slobodnick et al. Am J Med. 2015 May;128(5):461-70.(2) Jagasia et al. Biol Blood Marrow Transplant. 2015 Mar;21(3):389-401.e1. Epub 2014 Dec 18.Table 1Baseline CharacteristicsVariableFrequency (%)Age, median (range), yr57 (30-65)Donor/recipient genderSex-mismatched female/male1 (13%)Sex-matched7 (88%)RaceWhite/Caucasian7 (88%)Disease categoryAcute myeloid leukemia4 (50%)Myelofibrosis2 (25%)Aplastic anemia1 (13%)Lymphoblastic lymphoma1 (13%)Remission status at transplantationCR8 (100%)Graft sourceBM1 (13%)PBSC7 (88%)Donor typeMatched sibling4 (50%)Matched unrelated3 (38%)Mismatched1 (13%)GVHD typeProgressive3 (38%)Overlap syndrome2 (25%)De novo3 (38%) [Display omitted] DisclosuresOff Label Use: Colchicine will be discussed as treatment for GVHD. Comenzo:Karyopharm: Research Funding; Prothena: Research Funding; Takeda Millennium: Research Funding; Janssen: Research Funding; Takeda Millennium: Membership on an entity's Board of Directors or advisory committees; Prothena: Membership on an entity's Board of Directors or advisory committees. Roberts:Millenium: Speakers Bureau. Miller:Biogen Idec: Consultancy; AbbVie: Speakers Bureau; Millennium: Speakers Bureau; Onynx: Speakers Bureau.

Complete Resolution of Extensive Chronic Graft-Versus-Host Disease with Ibrutinib

We herein document a complete response of extensive chronic graft-versus-host disease (cGvHD) to a Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib. A 41 years old female with primary refractory MCL underwent mismatched unrelated donor (MMUD) allogeneic hematopoietic stem cell transplantation in December 2011 (conditioning with CY/TBI and alemtuzumab, graft=6.6x 106/kg CD-34+ cells, tacrolimus alone for GVHD prevention). Following engraftment on day 11, she developed grade III acute GvHD involving the skin and gut on day 17 of transplantation that persisted beyond 100 days post-transplant. Her cGvHD was treated with steroids, but remained active and extensive. Despite persistent cGvHD and 100% donor chimerism she relapsed in July 2012. Treatment with radiation and bendamustine with rituximab failed. By December 2013, the patient had extensive cGvHD manifesting as scleromatous skin thickening, oral ulcers and sclerosis of the buccal mucosa, ocular dryness and diarrhea, and was started on ibrutinib1 560 mg once daily for relapsed MCL. After 8 weeks of therapy, cGvHD had begun to improve. Oral steroids were reduced and ultimately stopped after 26 weeks of ibrutinib; after 30 weeks treatment all cGvHD manifestations resolved completely. A complete remission for MCL was documented at 8 weeks of ibrutinib initiation. Currently she continues to be on 560 mg daily ibrutinib without cGvHD exacerbation or MCL relapse for 22 weeks and 52 weeks, respectively. Chronic graft versus host disease (cGvHD) is mediated donor T cells. The role of B cells in the pathogenesis of cGvHD is increasingly recognized. Two murine studies have explored the role of ibrutinib in cGVHD-like syndromes, one in which there is T cell driven sclerodermatous cGvHD and a second in which there is Ab driven multiorgan system cGvHD that includes bronchiolitis obliterans (BO). Administration of ibrutinib decreased the incidence and severity of sclerodermatous, and improved pre-existing lesions and also improved pulmonary fibrosis and reduced BO. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Our report provides the evidence that BTK inhibition led to complete resolution of cGvHD and supports exploration of its role in future clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease

AbstractNovel therapies for chronic graft-versus-host disease (cGVHD) are needed. Aberrant B-cell activation has been demonstrated in mice and humans with cGVHD. Having previously found that human cGVHD B cells are activated and primed for survival, we sought to further evaluate the role of the spleen tyrosine kinase (Syk) in cGVHD in multiple murine models and human peripheral blood cells. In a murine model of multiorgan system, nonsclerodermatous disease with bronchiolitis obliterans where cGVHD is dependent on antibody and germinal center (GC) B cells, we found that activation of Syk was necessary in donor B cells, but not T cells, for disease progression. Bone marrow–specific Syk deletion in vivo was effective in treating established cGVHD, as was a small-molecule inhibitor of Syk, fostamatinib, which normalized GC formation and decreased activated CD80/86+ dendritic cells. In multiple distinct models of sclerodermatous cGVHD, clinical and pathological disease manifestations were not eliminated when mice were therapeutically treated with fostamatinib, though both clinical and immunologic effects could be observed in one of these scleroderma models. We further demonstrated that Syk inhibition was effective at inducing apoptosis of human cGVHD B cells. Together, these data demonstrate a therapeutic potential of targeting B-cell Syk signaling in cGVHD.

Selective ROCK2 inhibitor reverses experimental chronic graft-versus-host disease via concurrent regulation of STAT3/STAT5 phosphorylation (TRAN1P.933)

Abstract Rho-associated kinase 2 (ROCK2) was recently shown to be implicated in regulation of IL-21 and IL-17 secretion in mice and humans. Here, we report that the potent and highly selective ROCK2 inhibitor KD025 effectively reverses an established chronic graft-versus-host (cGVHD) murine model driven by IL-21 and characterized by an aggressive multiorgan system fibrotic disease. KD025 treatment leads to a dose dependent decrease in the development of pathogenic pulmonary function which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. The spleens of mice treated with KD025 had a decrease in the frequency of germinal centers associated with a reduction in STAT3 and concurrent increase in STAT5 phosphorylation, supporting the molecular mechanism of targeted ROCK2 inhibition in humans. The critical role of STAT3 activation in this cGVHD model was further confirmed by data showing that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls. Finally, KD025 effectively blocked the progression of sclerodermatous cGVHD model characterized by improvements in weight loss, posture and skin integrity of treated animals. Together, these data demonstrate the importance of ROCK2 signaling in STAT3-dependent regulation of cytokine secretion and highlight the potential of targeted ROCK2 inhibition for cGVHD therapy.

Essential Role of Interleukin-12/23p40 in the Development of Graft-versus-Host Disease in Mice

Graft-versus-host disease (GVHD), in both its acute (aGVHD) and chronic (cGVHD) forms, remains a major obstacle impeding successful allogeneic hematopoietic stem cell transplantation (allo-HSCT). T cells, in particular pathogenic T helper (Th) 1 and Th17 subsets, are a driving force for the induction of GVHD. IL-12 and IL-23 cytokines share a common p40 subunit and play a critical role in driving Th1 differentiation and in stabilizing the Th17 phenotype, respectively. In our current study, we hypothesized that p40 is an essential cytokine in the development of GVHD. By using p40-deficient mice, we found that both donor- and host-derived p40 contribute to the development of aGVHD. Neutralization of p40 with an anti-p40 mAb inhibited Th1- and Th17-polarization in vitro. Furthermore, anti-p40 treatment reduced aGVHD severity while preserving the graft-versus-leukemia (GVL) activity. Alleviation of aGVHD was associated with an increase of Th2 differentiation and a decrease of Th1 and Th17 effector T cells in the GVHD target organs. In addition, anti-p40 treatment attenuated the severity of sclerodermatous cGVHD. These results provide a strong rationale that blockade of p40 may represent a promising therapeutic strategy in preventing and treating aGVHD and cGVHD while sparing the GVL effect after allo-HSCT.

Prediction of Chronic Lung Graft-Versus-Host Disease By Angiopoietin-2

Background: Severe chronic graft versus host disease (cGVHD) of the lung is a rare but often fatal complication of allogeneic stem cell transplantation (SCT). In order to identify patients at high risk of lung cGVHD prior to transplant, the aim of the present study was to test systematically candidate biomarkers for this purpose, thereby focusing on endothelial risk factors previously shown to be associated with the risk of refractory acute GVHD. These factors included angiopoietin-2 (ANG2), serum nitrates and asymmetric dimethylarginine (ADMA), as well as single nucleotide polymorphisms (SNPs) in the thrombomodulin gene (THBD).Methods: Patients were eligible if they were allo-grafted between June 2002 and December 2011 at our institution, and if their blood samples were available for nitrate, ANG2 and ADMA measurement at different landmarks (collected immediately before conditioning and on day +100 after allogeneic SCT). Concentrations of ANG2, ADMA and serum nitrates were quantified in patients’ sera by the multiplex protein array technology (Luminex). THBD SNP genotyping was performed using KASPar SNP Genotyping System v2.0 of K Bioscience in 384-well format. Cumulative incidence analysis of cause-specific hazards was performed. The occurrence of cGVHD was evaluated retrospectively by chart review applying clinical and histological criteria developed by the National Institute of Health’s consensus project (Filipovich et al., 2005).Results: Of a total sample of 329 eligible patients, 14 (4%) fulfilled the criteria for lung cGVHD. 19 out of 329 patients (6%) developed severe gastrointestinal cGVHD and 15 out of 329 patients (4%) developed sclerodermatous cGVHD. Elevated pre-transplant levels of ANG2 (> 1000 pg/ml) correlated with the incidence of lung cGVHD (p=0.037). In contrast, there was no association between pre-transplant levels of ANG2 and severe gastrointestinal cGVHD (p=0.684) or sclerodermatous cGVHD (p=0.242). Similarly, high ANG2 levels (> 4000 pg/ml) on day +100 after allogeneic SCT predicted lung cGVHD (p=0.009). Again, this effect was specific for lung cGVHD, as there was no association between high ANG2 levels on day +100 and sclerodermatous cGVHD (p=0.300) or severe gastrointestinal cGVHD (p=0.702). There was no correlation between lung cGVHD and antecedent acute GVHD (p=0.796). Moreover, no significant correlations between serum nitrates, ADMA and thrombomodulin-(THBD)-SNPs and the risk of lung cGVHD or any other manifestation of severe cGVHD could be identified.Conclusion: In contrast to other endothelial markers, elevated pretransplant and d +100 post-transplant ANG2 levels may be predictors of a high risk of lung cGVHD but not of gastrointestinal or sclerodermatous cGVHD. These preliminary results warrant validation by further studies. Moreover, this data suggests a different role of the endothelial component in the pathogenesis of acute vs chronic GVHD. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Effects of MLN4924 on Sclerodermatous Chronic Graft Versus Host Disease

▪Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for the treatment of a variety of hematological diseases. However, high incidences of graft versus host disease (GVHD) and tumor relapse remain as major hurdles to this approach. While recent advances in transplant preparation and prophylactic regimens have lowered the incidence of acute GVHD, chronic GVHD (cGVHD) still remains a major cause of long-term morbidity and mortality. Recent studies have suggested that post-translation modifications (PTMs) may serve as therapeutic targets during GVHD. PTMs of proteins are important pathways that confer biochemical alterations, such as acetylation, phosphorylation, and ubiquitination. Targeting PTMs by using small molecular inhibitors has demonstrated potent anti-tumor effects, as well as immune modulatory properties. We have previously shown that bortezomib, an inhibitor of PTMs pathways, can successfully ameliorate sclerodermatous cGVHD responses in both preclinical and clinical studies. Here we reported a new small molecular targeting agent, MLN4924, a selective inhibitor of NEDD8-activating enzyme, on the treatment of cGVHD.MLN4924 is a selective inhibitor of NEDD8-activating enzyme and has been shown to be well tolerated at higher doses and exhibit potent anti-tumor activity in murine models and in early clinical trials. Furthermore, MLN4924 can suppress dendritic cell functions, as well as their production of proinflammatory cytokines during allogeneic responses. Due to these findings, we sought to test the treatment effects of MLN4924 on cGVHD responses. To investigate this, we utilized a minor MHC mismatch murine model by transferring donor B10.D2 (H-2d) cells into BALB/c (H-2d) recipients. MLN4924 or vehicle control (10% 2-hydroxy-propyl-beta cyclodextrin) was injected intraperitoneally on day 20, the point at which skin sclerodermatous cGVHD is initiated. Compared with the vehicle groups, animals that received either bortezomib (0.1 mg/kg) or MLN4924 (20 mg/kg) developed significantly less cGVHD, as determined by clinical scores (2.34±0.34 versus 1.03±0.37 versus 0.8±0.14, respectively; P <0.01). Surprisingly, a preventive approach started from either day 0 or day 10 post-HSCT resulted in higher cGVHD scores compared to early therapeutic interventions, started from Day 20, (3.3 and 2.37± 0.18 versus 0.8±0.14, respectively). The temporally dependent efficacy of MLN4924 suggests that PTMs may play a suppressive role in the early development of cGVHD pathogenesis while worsening cGVHD response as a prevention treatment. Flow cytometric analysis revealed a significant reduction in the total number of macrophages and dendritic cells with MLN4924 treatment starting on day 20, while no significant changes were noticed in T cell subpopulations. Similarly, MLN4924 can successfully inhibit mixed lymphocyte reactions (MLR) in vitro. Importantly, a reduction in the total number of plasma cells, a major source of autoantibody production, was also observed. Together these data suggest that inhibition of neddylation with small molecules, such as MLN4924, can be used as a new approach for the treatment of patients with sclerodermatous cGVHD, while potentially maintaining graft versus tumor effects through preserving T cell populations. DisclosuresNo relevant conflicts of interest to declare.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.

CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease.

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.

Skin ulcers related to chronic graft-versus-host disease: clinical findings and associated morbidity

According to the National Institutes of Health classification of chronic graft-versus-host disease (cGVHD), skin ulcers after allogeneic haematopoietic stem-cell transplantation (HSCT) are recorded as having the maximal severity score but published data are scarce. To describe skin ulcers related to cGVHD with an emphasis on clinical findings, associated morbidity, management and evolution. A multicentre retrospective analysis was carried out of patients with a diagnosis of cGVHD skin ulcers. All 25 patients included in the study had sclerotic skin cGVHD and 21 had lichenoid skin lesions associated with the sclerotic skin lesions. Thirteen patients had severe cGVHD without considering the skin, because of the involvement of an extracutaneous organ by cGVHD. The median time from HSCT to the onset of ulcers was 44 months. In addition to scleroderma, initial skin lesions at the site of ulcers were bullous erosive lichen in 21 patients and bullous erosive morphoea in four patients. Fifteen patients had an inaugural oedema. Ulcers were mostly bilateral with a predilection for the lower limbs. They were frequently colonized but few infections occurred. Four patients died during a median follow-up period of 55 months. Chronic graft-versus-host disease skin ulcers occur in patients with sclerodermatous skin cGVHD, are associated with severe cGVHD, often start with bullous lichenoid lesions or bullous morphoea and seem to cause more morbidity than mortality, given the low rate of mortality observed in our series of patients.

Combination Of Regulatory T Cells and Rapamycin As Treatment For Experimental Chronic Graft-Versus-Host Disease

Background Chronic graft-versus-host disease (cGvHD) is one of the main complications of allogeneic hematopoietic cell transplantation (allo-HCT). Donor CD4+ conventional T cells (Tconv) as well as regulatory T cells (Treg) are the key-players in its pathogenesis. Rapamycin (sirolimus, rapa) is a mTor inhibitor that can suppress activation and proliferation of Tconv without inhibiting Treg. Aims To assess the impact of a combined treatment with Tregand rapa on experimental cGvHD. Methods Lethally irradiated BALB/c mice were injected with 10x106 bone marrow cells and 70x106 splenocytes from B10.D2 donor mice. Mice were divided in four groups on day -1 and treatments were started on day 20 with either PBS, rapa 1 mg/kg/Day, Treg 1.106 cells (D+20), or rapa 1 mg/kg/Day + Treg 1.106 cells (D+20). Treg (CD4+ CD25+ cells) were purified from spleen cells from donor B10.D2 mice using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, GmbH, Germany). Treg purity (defined as CD4+ FoxP3+ cells) was ≥ 70% of total cells and &gt; 92% of CD4+T cells. The severity of sclerodermatous cGvHD was assessed with the following clinical scoring system. Briefly, animals were individually scored every 3 days for five parameters: weight loss (1, 10-20%; 2, &gt; 20%), posture (1, kyphosis only at rest; 2, severe kyphosis when the animal moved), activity (1, moderate activity impairment; 2, no move unless stimulated), skin (1, erythema or scaling tail; 2, open lesion on the body surface) and hair loss (1, &gt; 1 cm2; 2, &gt; 2 cm2). Mice which reached a score of 8 were estimated to have terminal GvHD and were sacrificed. Terminal GvHD-free survivals between the 4 groups were compared using the Log-rank test. Results The effects of the treatments were evaluated 7 days after starting the treatments (D+27 post-transplantation). Numbers of total and central memory and effector memory CD4+ T cells/µL were significantly (p&lt;0.05) decreased in rapa-, Treg-, and Treg + rapa- treated mice compared to PBS mice, while numbers of naïve CD4+ T cells/µL were significantly (p&lt;0.05) decreased in Treg-, and Treg + rapa- treated mice. Further, proliferation of CD4+ T cells (assessed by Ki67 expression) was significantly decreased in rapa- (p&lt;0.05) and Treg + rapa- treated mice (p&lt;0.05). In addition, counts and proliferation of CD8+ T cells/µL were significantly (p&lt;0.05) lower in rapa- and Treg+ rapa- treated mice compared to PBS mice. Importantly, terminal-GvHD-free survival was significantly shorter in PBS mice than in Treg (P=0.03), rapa (P=0.04), and Treg+ rapa mice (P=0.02). Conclusion Our results showed that Treg and rapa administration improved cGvHD in this model of cGvHD. Disclosures: No relevant conflicts of interest to declare.

Nailfold capillary abnormalities in sclerodermatous chronic GVHD

Chronic GVHD (cGVHD) complicating allo-SCT commonly presents as sclerotic skin changes resembling systemic sclerosis (SSc), suggesting a common pathophysiological pathway. Damage to capillaries is considered an early event in the pathogenesis of SSc, and is associated with characteristic nailfold capillary abnormalities. Whether such nailfold capillary abnormalities occur in sclerodermatous cGVHD is unknown. Nailfold videocapillaroscopy (NVC) was used to evaluate capillary morphology, density and loop dimensions in 14 patients with sclerodermatous cGVHD, 14 sex- and age-matched SSc patients, and 14 healthy controls. It was shown that none of the cGVHD patients and controls, whereas all SSc patients showed severe capillary abnormalities. cGVHD patients and controls showed no differences in capillary density (9.05 vs 9.16 loops/mm, respectively, P=0.84), and capillary loop dimensions (total loop width 44.36 vs 45.56 μm, respectively, P=0.84). Compared with cGVHD patients, SSc patients had a reduced capillary density (9.05 vs 5.25 loops/mm, respectively, P<0.001), and an increase in capillary loop dimensions (total loop width 44.36 vs 99.97 μm, respectively, P=<0.001). In conclusion sclerodermatous cGVHD patients do not show the characteristic microvascular abnormalities seen in SSc, suggesting that capillary damage does not contribute to the pathophysiology of sclerodermatous cGVHD, and making NVC unsuitable for early identification.