Sclerodermatous Graft-versus-host Disease Research Articles

Blockade of CD122 on memory T cells in the skin suppresses sclerodermatous graft-versus-host disease

BackgroundAntigen-stimulated naïve T cells differentiate into effector and memory T cells, of which resident memory T (TRM) cells reside permanently in organ tissues. Involvement of TRM cells has been indicated in pathological conditions of various skin diseases. CD122, which is the β chain subunit of interleukin (IL)− 2 and IL-15 receptors, is expressed on immune cells including TRM cells. ObjectiveTo investigate whether CD122 signaling in skin CD8+ TRM cells mediates the development of mucocutaneous graft-versus-host disease (GVHD). MethodsWe used a genetically modified mouse expressing a membrane-bound form of chicken ovalbumin (OVA) under the control of the keratin 14 promoter, which develops GVHD-like erosive mucocutaneous disease resulting in sclerodermatous disease after transfer of OVA-specific T cell-receptor-transgenic CD8+ OT-I cells. Mice with mucocutaneous GVHD were treated with an anti-CD122 blocking antibody. ResultsAdministration of an anti-CD122 blocking antibody suppresses the development of acute/chronic GVHD-like mucocutaneous disease in our murine model via the reduction of CD122-expressing memory CD8+ T cells, including skin, memory autoaggressive CD8+ T cells. Moreover, blockade of CD122, even after the establishment of acute GVHD, inhibited the development of chronic GVHD-like sclerodermatous disease via the reduction of epidermal and dermal TRM autoaggressive CD8+ T cells. ConclusionSkin memory CD8+ T cells in particular mediate the development of mucocutaneous GVHD, and blockade of CD122 may be an effective treatment strategy, especially for sclerodermatous GVHD.

Donor Pretreatment with Agonistic Anti-DR3 Antibody Prevents Chronic GvHD in a Murine Sclerodermatous Model

Background: DR3 is a cell surface receptor of the TNF superfamily and is expressed preferentially by activated T cells. The agonistic antibody of DR3 (aDR3) can stimulate regulatory T cell (Treg) expansion and ameliorate acute GVHD in a major-mismatch murine model. However, it is still unclear whether the aDR3 is effective in other GVHD models. A murine sclerodermatous GVHD model features skin fibrosis and shares many clinical characteristics with human chronic GVHD, for which currently available therapies have demonstrated limited efficacy. Methods: The αDR3 or isotype control antibody (0.5 mg/kg) was intraperitoneally injected into B10.D2 mice four days before transplantation. To generate a sclerodermatous GVHD model, lethally irradiated BALB/c recipient mice were transplanted with T cell-depleted bone marrow cells from non-treated B10.D2 mice and splenocytes isolated from the αDR3 or isotype-treated B10.D2 mice. Some Balb/c mice were transplanted with only the T cell-depleted bone marrow cells (omitting splenocytes) and served as the non-GVHD control (the control recipients). Recipient mice were monitored for six weeks to assess body weight, GVHD score, and survival or sacrificed to harvest skin, spleen, and colon on 7, 14, and 42 days after transplantation. Bronchoalveolar lavage (BAL) was performed on days 7 and 14. Results: Recipients of the aDR3-treated splenocytes (the aDR3 recipients) experienced significantly less weight loss and showed decreased GVHD scores than recipients of isotype-treated splenocytes (the isotype recipients). The severity of cutaneous GVHD was considerably reduced in the aDR3 recipients than in the isotype recipients. Pathologic examinations such as the ratio of the dermis to subcutaneous tissue in the skin and histologic scores of the colon performed on days 7, 14, and 42 post-transplant confirmed the clinical results. Flow cytometric analysis suggested that the reconstitution of B and T cells in the spleen of isotype recipients was severely disrupted during the later phase of the transplant. Although the number of CD4 T cells and B cells in the control recipients and the aDR3 recipients had been increasing until day 42, those in the isotype recipients slightly increased on day 14 but markedly decreased on day 42. Therefore, the number of CD4 T cells in the isotype recipients was significantly lower than that of the other groups on day 42, and the number of B cells in the isotype showed a significant difference on days 14 and 42. In contrast, the reconstitution of CD11b monocytes was not disrupted in the spleen of the isotype recipients. The reconstitution of splenic Treg showed a similar pattern to CD4 T cells. Interestingly, the number of Treg in the aDR3 recipients was consistently higher than that of the other groups. These findings suggest that GVHD may disrupt lymphocyte reconstitution after hematopoietic cell transplantation but treating donors with αDR3 can prevent it. Next, we performed qPCR analysis to quantitate the mRNA levels of key cytokines and chemokines in the skin and colon. The expression level of TGF-β was remarkably higher in the skin than in the colon, and the reduction of TGF-β in the αDR3 recipients was more pronounced in the skin than in the colon, confirming its primary role in skin fibrosis. In addition, the αDR3 recipients had decreased levels of IFNg, IL-6, and IL-10 in the skin and the colon than the isotype recipients. Moreover, the αDR3 recipients had diminished expression of various chemokines (CCL1, CCL2, CCL3, CCL4, CCL8, CCL17, and CCL22) in the skin compared to the isotype recipients. Chemokine expression profile in the colon was almost identical to the skin except for CCL17 and CCL22. These two cytokines were similarly reduced in the colon of the αDR3 recipients and the isotype recipients. Next, we examined inflammatory cell content in the BAL fluid. The numbers of total cells and macrophages were significantly lower in the αDR3 recipients than in the isotype recipients on days 7 and 14, but the numbers of lymphocytes and neutrophils were different between these two groups only on day 14. The αDR3 recipients had significantly fewer CD4 T cells, B cells, and CD11b monocytes in BAL fluid than the isotype recipients. Conclusions: Treating donors with αDR3 before transplantation could alleviate sclerodermatous GVHD by preventing disruption of hematopoiesis, fostering immune reconstitution, and reducing skin fibrosis and lung inflammation.

Multi-Omics Profiling of Skin Biopsies of Patients with Sclerodermatous Graft-Vs-Host Disease Suggests Therapeutic Potential of Targeting Don't Eat Me Signals

Introduction: Chronic graft-vs-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT). This debilitating, chronic condition is characterized by inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is a highly morbid form of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy. Understanding the underlying pathophysiology of sclGVHD and identifying the keyfibroticpathways involved iscriticalfor translation into targeted therapies that improve patient care. Methods: To further our understanding of the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on 5 fresh patient biopsy specimens and healthy controls. Human studies were carried out using: i) Patient skin biopsy samples (n =45) and matching them with 5 normal matched skin biopsy controls. These patient tissue samples were used to perform spatial gene expression analyses (10X Vizium) and identify the unique molecular profiles in each SclGVHD sample. ii) ATAC-sequencing analyses was performed on primary dermal fibroblasts from patients with SclGVHD to assess chromatin accessibility patterns genome wide in SclGVHD iii) TMA and tissues from patients were used to validate targets using immunofluorescent staining. iv) Animal studies were conducted to evaluate the therapeutic potential of an immune therapy-based treatment approach with a combined anti-fibrotic/anti-inflammatory strategy, and effectiveness of the treatment was determined with CyTOF studies. Results: To investigate cGVHD immunopathogenesis, normal-appearing skin, lesioned skin, and circulating immune cells from chronic patients were analyzed via single-cell RNA sequencing (figure A). A distinct transcriptional signature, the upregulation of clusters 11, 3, 1 and 17 in Scl cGVHD and the downregulation of clusters 6, 5, 9, 8 was observed. To validate these results in the context of tissue architecture we integrated spatial RNA sequencing on a tissue microarray assembled with 45 skin biopsies of SclGVHD patients to demonstrate a tight correlation between JUN activation and immune checkpoint upregulation (figure B).Hierarchical clustering of ATAC-seq signals revealed that the chromatin landscape was greatly remodeled after JUN deletion (figure C).We blocked CD47 and IL6 in the SclGVHD mouse model to evaluate therapeutic efficacy. We found a significant decrease of dermal thickness and collagen deposition in SclGVHD mice treated with blocking antibodies against IL6 and CD47 but not in untreated mice (figure D). We also detected a noticeable decrease in the expression of CD47, pJUN and IL6 in the treated group (figure E). Analyses of skin tissues from sclerotic lesions in mice using CyTOF revealed different cell populations in the skin clustered in spatially distinct groups. Finally, an increase in total leukocyte as well as a significant decrease in the fibroblast populations was observed (figure F). Conclusion: Our findings are significant because we profile skin biopsies matched with peripheral blood from patients with Scl GVHD with a multiomics approach and identify a unique molecular signature in patients with SclGVHD thus providing a rationale for subsequent combined interventions targeting innate immunity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Gut Bacteria Dysbiosis Contributes to Chronic Graft-Versus-Host Disease Associated With a Treg/Th1 Ratio Imbalance.

IntroductionDysbiosis of gut bacteria has been discovered in a large number of autoimmune diseases. However, the influence of the gut bacteria in the mice model of chronic sclerodermatous graft-versus-host disease (Scl-GVHD), a disease that resembles an autoimmune disease characterized by chronic inflammation of multiple organs, such as skin, remains elusive. Here, we explore the role of gut bacteria in an Scl-cGVHD mice model.MethodsWe established a mouse model of Scl-cGVHD, collected fecal flora, analyzed the composition, and diversity of intestinal flora using 16S rDNA amplicon sequencing, and detected the proportion of Treg and Th1 cells in splenocytes of Scl-cGVHD mice. To verify the immunoregulatory effect of Scl-cGVHD intestinal flora, we prepared bacterial extracts, co-cultured with splenocytes in vitro, and used flow cytometry to detect T cell differentiation and cytokine secretion.ResultsBy examining T-cell differentiation in splenocytes of cGVHD mice, we found that Treg cells were significantly reduced (15.27 ± 0.23 vs. 12.23 ± 0.47, p = 0.0045) and Th1 cells were increased (1.54 ± 0.18 vs. 6.68 ± 0.80, p = 0.0034) in cGVHD mice. Significant differences were observed in the composition and diversity of the gut bacteria in mice with Scl-cGVHD versus without GVHD. Analysis of mice fecal bacteria samples (n = 10, 5 Scl-cGVHD and 5 Non-GVHD) showed significant separation [R = 0.732, p = 0.015, non-parametric analysis (ANOSIM)] in Scl-cGVHD and non-GVHD mice. The abundance of the family and genus Ruminococcaceae bacteria decreased and the family Lachnospiraceae and limited to the species Lachnospiraceae_bacterium_DW17 increased in Scl-cGVHD mice. In vitro results of the cellular level study suggest that the bacteria extracts of gut microbiota from Scl-cGVHD mice modulated the splenic T cells toward differentiation into CD4+IFN-γ+ Th1 cells (14.37 ± 0.32 vs. 10.40 ± 2.19, p = 0.036), and the percentage of CD4+CD25+Foxp3+ Tregs decreased (6.36 ± 0.39 vs. 8.66 ± 0.07, p = 0.001) compared with the non-GVHD mice. In addition, the secretion of proinflammatory interferon- γ (IFN-γ) cytokine in the supplement of cellular culture was increased (4,898.58 ± 235.82 vs. 4,347.87 ± 220.02 pg/ml, p = 0.042) in the mice model of the Scl-cGVHD group, but anti-inflammatory interleukin (IL)-10 decreased (7,636.57 ± 608.05 vs. 9,563.56 ± 603.34 pg/ml, p = 0.018).ConclusionOur data showed the different composition and diversity of gut bacteria in the Scl-cGVHD mice. The dysbiosis of gut bacteria may regulate the differentiation ratio of Treg and Th1 cells, which was associated with Scl-cGVHD.

CpG-Activated Regulatory B-Cell Progenitors Alleviate Murine Graft-Versus-Host-Disease.

Development of Graft Versus Host Disease (GVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). The observation that the presence of bone marrow and circulating hematogones correlated with reduced GVHD risks prompted us to evaluate whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce this allogeneic disorder. In a murine model of GVHD that recapitulates an initial phase of acute GVHD followed by a phase of chronic sclerodermatous GVHD, we found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of critical cytokines such as TGF-β, IL-13 and IL-21. Adoptive transfer of CpG-proBs increased the T follicular regulatory to T follicular helper (Tfr/Tfh) ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. However, CpG-proBs did not improve survival. Altogether, our findings support the notion that adoptively transferred CpG-proBs exert immunomodulating effect that alleviates symptoms of GVHD but require additional anti-inflammatory strategy to improve survival.

Selective Targeting of Immune Modulatory Proteins to Mitigate Fibrosis and Inflammation in Sclerodermatous Graft-Vs-Host Disease

Chronic graft-vs-host disease (cGVHD) is a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (HCT) in patients. This debilitating condition is characterized by chronic inflammation, cell-mediated and humoral immunity, and ultimately tissue fibrosis. There is currently little or no understanding of the molecular pathogenesis of chronic cGVHD resulting in poor effective treatment strategies. Sclerodermatous GVHD (sclGVHD) is one of the more severe forms of cGVHD associated with poor prognosis and low sensitivity to immune suppressive therapy.Methods: To address the current gap in knowledge pertaining to the underlying pathophysiology of sclGVHD we used single cell RNA sequencing analyses on fresh patient biopsy specimens. In vivo studies were carried out by sub lethal irradiation of BALB.k recipients which underwent HCT from miAg-mismatched AKR/J donors. Recipient sclerodermatous-tissues were analyzed using FACS, IHC and IF staining. Human studies were conducted on (i) Primary samples from patients with severe sclGVHD using tissue microarrays (TMA) by Immuno-histofluorescence (IHF) and IF. (ii) Dermal fibroblasts from sclGVHD samples were subjected to ATACseq and ChiPseq CRISPR-Cas9 JUN deletion. (iii) Also, dermal fibroblasts from human scl-GVHD were implanted under the kidney capsule of NSG mice to study the effects of inhibiting pro-fibrotic pathways in vivo.Results: We show for the first time that in a mouse model of sclGVHD (male), recipients of female T-cell replete grafts developed severe scleroderma with massive skin thickening and collagen deposition. Fibroblasts strongly expressed JUN, which is part of AP-1, a transcription factor involved in the acute phase response that regulates gene expression in response to stimuli from cytokines, growth factors and pathogens. We have previously demonstrated JUN as a key player in the molecular pathogenesis of other fibrotic diseases (Wernig G et al. PNAS 2017, Cui et al. Nature comm. 2020, Lerbs et al. JCI i 2020). Likewise, CD47, an immune checkpoint protein that prevents removal of Mϕ, was strongly co-expressed in fibroblasts in sclGVHD - but not in the control mice (Fig A+B). Here we show that (i) In humans, (n = 45 sclGVHD patients), there is a strong expression and activation of JUN and CD47 in dermal fibroblasts which was not observed in control samples. Mixed inflammatory infiltrates were dominated by Mϕ and granulocytes. (ii) Isolated primary fibroblasts from fresh human sclGVHD skin biopsies analyzed for chromatin accessibility across the genome by ATAC-seq showed wide open accessibility to the JUN promoter, IL-6 promoter and CD47 enhancer and promoter indicating that they play a critical role in regulating the pathogenesis of sclGVHD. In contrast, normal fibroblasts displayed only minimal accessibility to the JUN promoter. We further validated our data using CRISPR-Cas9 knock-down studies on sclGVHD fibroblasts and show that the IL-6 promoter, enhancer and promoter of CD47 are regulated by JUN, with JUN deletion resulting in significant decrease in the promoter binding accessibility to IL-6 and CD47 (Fig C). Further, JUN activity appears to regulate key members of the Hh signaling pathway (GLI1, PTCH1 and PTCH2), as their chromatin accessibility was decreased with JUN deletion. These correlative findings were confirmed by JUN ChIP seq, an assay that identifies binding sites of DNA-associated proteins. (iii) To test our findings in vivo we established xenograft models of primary human sclGVHD by implanting cells under the kidney capsule of NSG mice.All treatments (except placebo) resulted in decreased fibrosis (Fig D), presumably by blocking the activation of JUN (pJUN) and its profibrotic downstream pathway members IL-6 and pSTAT3, as assessed by phospho flow. Conclusion: In our studies we demonstrate that in established SclGVHD, combinatorial therapy consisting of anti-CD47 antibody together with IL6 blockade has the highest potential to translate into a therapeutic intervention given its ability to be more effective than currently used antifibrotic and anti-inflammatory agents in clinic. The findings from our study are significant because we show an important mechanism underlying SclGVHD onset, identify a novel genetic signature that can be targeted, describe a new mouse model and a clinical assay that has a high throughput readout and suggest a treatment regimen for patients. [Display omitted] DisclosuresArai: Magenta Therapeutics: Research Funding. Shizuru: Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020; Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board.

LPSlow-Macrophages Alleviate the Outcome of Graft-Versus-Host Disease Without Aggravating Lymphoma Growth in Mice.

Despite significant therapeutic advances, graft-versus-host disease (GvHD) remains the main life-threatening complication following allogeneic hematopoietic stem cell transplantation. The pathogenesis of GvHD is dominated by a dysregulated allogeneic immune response that drives fibrosis and autoimmunity in chronic forms. A multitude of cell therapy approaches, including infusion of myeloid cells, has been proposed to prevent GvHD through tolerance induction but yielded variable results. Myeloid cells like macrophages can be reprogrammed to develop adaptive-like features following antigenic challenge to reinforce or inhibit a subsequent immune response; a phenomenon termed ‘trained immunity’. Here we report that, whereas LPSlow-trained macrophages elicit a suppressor effect on allogeneic T cell proliferation and function in vitro in an IL-10-dependent manner, Bacille Calmette et Guérin (BCG)-trained macrophages exert an opposite effect. In a murine model of sclerodermatous chronic GvHD, LPSlow-trained macrophages attenuate clinical signs of GvHD with significant effects on T cell phenotype and function, autoantibodies production, and tissue fibrosis. Furthermore, infusion of LPSlow-macrophages significantly improves survival in mice with acute GvHD. Importantly, we also provide evidence that LPSlow-macrophages do not accelerate A20-lymphoma tumor growth, which is significantly reduced upon transfer of BCG-macrophages. Collectively, these data indicate that macrophages can be trained to significantly inhibit in vitro and in vivo allo-reactive T cell proliferation without exhibiting pro-tumoral effect, thereby opening the way to promising clinical applications.

Prophylactic Effects of Recombinant Thrombomodulin Against Sclerodermatous Chronic Graft-Versus-Host Disease in a Rodent Model

Graft-versus-host disease (GVHD) is the one of the most serious complications of the allogenic stem cell transplantation (SCT). Especially, chronic GVHD has complex pathologies including the aspects of autoimmune diseases and microangiopathies. Recombinant thrombomoduin (rTM), which shows anticoagulant activity independent of antithrombin through binding to thrombin to activate protein C, has been used for disseminated intravascular coagulation (DIC). Recent studies have also demonstrated that rTM suppresses inflammatory response through various pathways including activated protein C (APC), degradation of HMGB1, inhibiting inflammatory cytokine and increasing regulatory T-cells (Treg), leading to attenuate tissue damages in animal models such as acute respiratory distress syndrome and liver injury by heatstroke. In acute GVHD mice model, rTM is reported to have therapeutic effect with contribution of Treg and blockade of HMGB1. In addition, rTM protects endothelial cells from calcineurin inhibitor-induced cytotoxicity in vitro using human umbilical vein endothelial cells. Thus, rTM is expected to show beneficial effects for post transplant complications. Actually, rTM has been sometimes used for severe veno-occlusive disease, DIC and thrombotic microangiopathy after SCT in clinical settings, but it is not clear whether rTM could have impact on acute or chronic GVHD themselves. In this study, we investigated the role of rTM especially focusing on preventing chronic GVHD in a rodent model.Bone marrow and splenocyte from donor mice (B10.D2) were transplanted into irradiated host mice (Balb/c) to induce sclerodermatous chronic GVHD (Scl-cGVHD). First, to demonstrate whether rTM could ameliorate the Scl-cGVHD, irradiated host mice were given splenocytes with rTM. Compared with the control group, rTM significantly improved chronic skin GVHD. However, rTM did not improve survival rate when increased dose of splenocytes was given to induce lethal intestinal GVHD together with Scl-cGVHD. In addition using another mice strain combination mimicking acute GVHD, rTM did not show any impact on ameliorating GVHD. Collectively, these results suggested that rTM had a beneficial effect on skin sclerodermatous lesions, not systemically. Next, to test whether rTM has therapeutic effects against skin lesions, rTM was started from 3 weeks after transplantation when Scl-cGVHD had already appeared. rTM did not improved Scl-cGVHD anymore. Next, to clarify whether the prophylactic mechanism of rTM to Scl-cGVHD is due to anticoagulant activity alone and/or complicated immure regulations by APC and rTM, Scl-GVHD mice was given dalteparin, APC and rTM, respectively. As shown in figure below, only rTM attenuated Scl-cGVHD with retaining normal skin structure and less inflammatory cell invasion compared to other groups, suggesting that rTM related immune regulation was a significant contributor to skin prophylactic effect, not anticoagulant activity. Though skin lesions were dramatically different among groups, in splenocytes there were no significant difference of cell subpopulation, T-cell activation and inflammatory cytokines (TNFα, IFNγ, IL17A). There was also no significant difference in regulatory B-cell, which was known to have a suppressive role in Scl-cGVHD model, and Treg. Moreover to confirm that rTM dose not influence these properties in vivo and in vitro, we analyzed splenocytes after repetitive administration of rTM to non-treated mice and short-term culture of splenocytes mixed with rTM. As expected, there was no significant difference by adding rTM in cell subpopulation including Treg, Breg and cytokine production in vivo and in vitro. From these findings, prophylactic mechanism of rTM in Scl-cGVHD was not due to anticoagulant activity nor APC related anti-inflammatory effects, but more likely to rTM related immune regulation. HMGB1 is one of the targets that rTM acts on as an immune modulator, but a previous report demonstrated that HMGB1 blockade attenuated GVHD systemically including skin, intestine and liver. In contrast to our findings that rTM worked mainly at skin lesions, unknown mechanisms of immune regulation by rTM might exist. In conclusion, rTM would be a promising drug for preventing sclerodermatous skin GVHD in addition to improving various complications associated with coagulation abnormalities. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preclinical evaluation of JAK1/2 inhibition by ruxolitinib in a murine model of chronic graft-versus-host disease

The objective of this study was to examine the therapeutic effect of ruxolitinib, an orally administered selective Janus kinase (JAK) 1/2 inhibitor, on chronic graft-versus-host disease (cGVHD) using a murine model of sclerodermatous GVHD (scl-GVHD). Compared with scl-GVHD controls, ruxolitinib-treated recipients had scl-GVHD of significantly attenuated clinical and pathological severity in the skin and decreased frequencies of effector cells, CD4+ T cells, and CD11b+ macrophage/monocytes. Regulatory CD4+ Foxp3+ T cells were expanded whereas interferon-γ (IFN-γ)-producing CD4+ T cells were significantly decreased in ruxolitinib-treated recipients. Ruxolitinib suppressed not only the production of IFN-γ from CD4+ T cells and monocyte chemoattractant protein 1 (MCP-1) from CD11b+ macrophage/monocytes, but also the proliferation of these cells in vitro. Levels of both cytokines (IFN-γ and MCP-1) were also reduced in the spleen and skin of ruxolitinib-treated recipients in vivo. IFN-γ-induced MCP-1 production and migration of RAW 264.7 cells, a macrophage cell line, were inhibited by ruxolitinib. However, supplementation with MCP-1 restored this effect of ruxolitinib. In addition, blocking JAK-STAT signaling using ruxolitinib reduced the activation of STAT1 in stimulated immune effector cells. Taken together, these results suggest that ruxolitinib can prevent scl-GVHD by suppressing IFN-γ produced by T cells and MCP-1 expression in macrophage/monocytes via inhibition of JAK-STAT signaling.

Signaling through the type 2 cannabinoid receptor regulates the severity of acute and chronic graft-versus-host disease

Graft-versus-host disease (GVHD) pathophysiology is a complex interplay between cells that comprise the adaptive and innate arms of the immune system. Effective prophylactic strategies are therefore contingent upon approaches that address contributions from both immune cell compartments. In the current study, we examined the role of the type 2 cannabinoid receptor (CB2R), which is expressed on nearly all immune cells, and demonstrated that absence of the CB2R on donor CD4+ or CD8+ T cells or administration of a selective CB2R pharmacological antagonist exacerbated acute GVHD lethality. This was accompanied primarily by the expansion of proinflammatory CD8+ T cells, indicating that constitutive CB2R expression on T cells preferentially regulated CD8+ T-cell alloreactivity. Using a novel CB2ReGFP reporter mouse, we observed significant loss of CB2R expression on T cells, but not macrophages, during acute GVHD, indicative of differential alterations in receptor expression under inflammatory conditions. Therapeutic targeting of the CB2R with the agonists Δ9-tetrahydrocannabinol (THC) and JWH-133 revealed that only THC mitigated lethal T cell–mediated acute GVHD. Conversely, only JWH-133 was effective in a sclerodermatous chronic GVHD model where macrophages contributed to disease biology. In vitro, both THC and JWH-133 induced arrestin recruitment and extracellular regulated kinase phosphorylation via CB2R, but THC had no effect on CB2R-mediated inhibition of adenylyl cyclase. This study shows that the CB2R plays a critical role in the regulation of GVHD and suggests that effective therapeutic targeting is dependent upon agonist signaling characteristics and receptor selectivity in conjunction with the composition of pathogenic immune effector cells.

CCL1 blockade alleviates human mesenchymal stem cell (hMSC)-induced pulmonary fibrosis in a murine sclerodermatous graft-versus-host disease (Scl-GVHD) model

BackgroundHuman chronic graft-versus-host disease (CGVHD) shares clinical characteristics with a murine sclerodermatous GVHD (Scl-GVHD, B10.D2 → BALB/c) model that is characterized by skin and lung fibrosis. In this study, bone marrow- or adipose tissue-derived human mesenchymal stem cells (hMSCs) were injected into the Scl-GVHD mice to address their therapeutic effect on CGVHD.MethodsLethally irradiated BALB/c mice were transplanted with B10.D2 T cell-depleted bone marrow with or without spleen cells to generate Scl-GVHD. hMSCs were intravenously treated on days 3, 5, and 7 post-transplantation, and the control antibody or CCL1 blocking antibody was subcutaneously injected according to the same schedule as the hMSCs. Fourteen days after transplantation, the recipient mice were sacrificed, and their skin and lungs were analyzed.ResultsAfter the early injection of hMSCs after transplantation, the clinical and pathological severity of Scl-GVHD in the skin was significantly attenuated, whereas the pathological score was exacerbated in the lungs. hMSCs had migrated into the lungs, but not into the skin. CD11b monocyte/macrophages and CD4 T cells were markedly decreased in skin tissues, whereas there was an early recruitment of CD11b cells, and subsequently increased infiltration of CD4 T cells, in the lungs. Importantly, hMSCs persistently upregulated the expression of CCL1 in the lungs, but not in the skin. Concurrent treatment of hMSCs with a CCL1-blocking antibody alleviated the severity of the lung histopathology score and fibrosis with the preservation of the cutaneous protective effect against CGVHD. Infiltration of CD3 T cells and CD68 macrophages and upregulation of chemokines were also decreased in lung tissues, along with the recruitment of eosinophils and tissue IgE expression. In the skin, chemokine expression was further reduced after CCL1 blockade.ConclusionsThese data demonstrate that despite a protective effect against Scl-GVHD in the skin, administration of hMSCs exacerbated lung fibrosis associated with eosinophilia and airway inflammation through persistent CCL1 upregulation. CCL1 blockade offers a potential treatment of pulmonary complications induced after treatment with hMSCs.

Recombinant Adenosine Deaminase Ameliorates Inflammation, Vascular Disease, and Fibrosis in Preclinical Models of Systemic Sclerosis.

Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease, and inflammation. Adenosine signaling plays a central role in fibroblast activation. We undertook this study to evaluate the therapeutic effects of adenosine depletion with PEGylated adenosine deaminase (PEG-ADA) in preclinical models of SSc. The effects of PEG-ADA on inflammation, vascular remodeling, and tissue fibrosis were analyzed in Fra-2 mice and in a B10.D2→BALB/c (H-2d ) model of sclerodermatous chronic graft-versus-host disease (GVHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness skin model. PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary fibrosis by 34.3% (with decreased collagen expression) (P = 0.0079; n = 6), dermal fibrosis by 51.8% (P = 0.0006; n = 6), and intestinal fibrosis by 17.7% (P = 0.0228; n = 6) in Fra-2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in sclerodermatous chronic GVHD (reduced by 38.4%) (P = 0.0063; n = 8), and in a human full-thickness skin model. PEG-ADA treatment decreased inflammation and corrected the M2/Th2/group 2 innate lymphoid cell 2 bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (reduced by 40.5%) (P < 0.0001; n = 6), and prevented thickening of the vessel walls (reduced by 39.6%) (P = 0.0028; n = 6) and occlusions of pulmonary arteries (reduced by 63.9%) (P = 0.0147; n = 6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (reduced by 65.4%) (P = 0.0001; n = 6) and blunted the capillary rarefication (reduced by 32.5%) (P = 0.0199; n = 6). RNA sequencing demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy, and inflammation in Fra-2 mice. Treatment with PEG-ADA ameliorates the 3 cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications, as PEG-ADA has already been approved by the Food and Drug Administration for the treatment of patients with ADA-deficient severe combined immunodeficiency disease.

Hedgehog Pathway Inhibitor for Treatment of Steroid-Refractory Sclerodermatous Chronic Graft-Versus-Host Disease (GVHD)

Background: Sclerodermatous chronic GVHD (scGVHD) is a unique form of cutaneous GVHD that predominantly involves the skin, subcutaneous tissue, fascia and joints. Steroid-refractory scGVHD is especially challenging to manage since it is usually a progressive condition with devastating effects on the quality of life of affected patients. Inhibition of hedgehog signaling has been shown to be effective in the treatment of murine scGVHD. Here, we report a retrospective review of patients with scGVHD who were treated with hedgehog pathway inhibitor, Vismodegib, at Stanford University.Results: A total of n=8 patients with steroid-refractory scGVHD were treated with Vismodegib 150 mg daily as an off-label use since 2016. The male:female ratio is 5:3 and median age was 49 (range 39-62) at time of start of Vismodegib. The median time from transplant to diagnosis of scGVHD was 26.9 months (range 8.9-37.5) and the median time from diagnosis of scGVHD to Vismodegib treatment was 14.4 months (range 2.7-45.2). All patients had GVHD involvement of other organ besides scGVHD and have received multiple lines of therapy prior to Vismodegib.Hair thinning/alopecia and muscle cramps were noted in 100% of the patients. 75% (6/8) had dysgeusia, 88% (7/8) had fatigue (gr 2) and 1 patient had an asymptomatic elevated lipase level. All patients required dose reduction or drug holiday due to side effects (mainly muscle cramps). Four patients (50%) stopped the drug due to side effects (2/4) or lack of response (2/4). All Vismodegib-related side effects resolved after discontinuation of the drug. The median duration of Vismodegib treatment was 6.1 months (range 2.3-17.1) for the entire group. One patient received multiple courses of Vismodegib with benefit noted with each course.Five patients (63%) had partial response including 1 patient responded to multiple courses of Vismodegib and 1 patient had subjective symptom improvement. The median time to clinical response was 103 days (range 24-145) and the duration of response was 7.8 months (range 1.6-13.4). Three patients had experienced prolonged clinical response off the Vismodegib. At the last follow-up visit, all patients were alive and 4 patients continued to take Vismodegib including 1 patient who had been tapered off systemic steroid.Conclusion: Steroid-refractory scGVHD is an extremely difficult disease to manage with a high rate of morbidity. Our experience with hedgehog pathway inhibitor, Vismodegib, is very encouraging. Additional study to evaluate the optimal dosing, side effects and its efficacy is under way. DisclosuresNo relevant conflicts of interest to declare.

Obesity results in marked increases in gut permeability, inflammatory cytokines, and acute gut graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) remains the most applicable curative option for treating hematologic diseases. In this study, we assessed the impact of obesity on recipient graft-versus-host disease (GVHD) outcome post allogeneic HSCT using a minor histocompatibility antigens (miHA)-mismatched mouse model. Control or diet-induced obese (DIO) BALB/c mice received HSCT from H2-identical B10.D2 mice which is known for resulting in chronic skin GVHD. Strikingly, DIO mice rapidly succumbed to gut GVHD while control mice survived and developed sclerodermatous GVHD much later post-HSCT. The DIO recipient mice had higher expression of pro-inflammatory cytokines (IL-6 and TNF-α) in the gut and serum, as well as higher serum ST2. There were increased numbers of donor CD4+ T cells in the mesenteric lymph nodes of DIO mice compared to control mice. Untreated DIO mice also displayed increased gut permeability and lipopolysaccharide translocation, as well as having less-diverse microbiota which potentially contributed to the severe GVHD post-HSCT. Importantly, administration of anti-IL-6R monoclonal antibody and Enbrel significantly protected DIO mice from lethal gut GVHD, correlating with lower pro-inflammatory cytokine concentrations (IL-6 and TNF-α) and ST2 in the serum. In an initial evaluation, treating DIO mice with broad-spectrum antibiotics 2 weeks before HSCT also ameliorated GVHD. Taken together, these results indicate that obesity exacerbates GVHD, manifested primarily as gut pathology, and therapeutic intervention by blockade of pro-inflammatory cytokine signaling ameliorates GVHD post-HSCT.

Mesenchymal Stem Cells (MSCs) Attenuate Cutaneous Sclerodermatous Graft-Versus-Host Disease (Scl-GVHD) through Inhibition of Immune Cell Infiltration in a Mouse Model

Human chronic graft-versus-host disease (GVHD) shares clinical characteristics with a murine sclerodermatous GVHD model that is characterized by skin thickening and lung fibrosis. A B10.D2 → BALB/c transplant model of sclerodermatous GVHD was used to address the therapeutic effect of mesenchymal stem cells (MSCs) on the development of chronic GVHD. The clinical and pathological severity of cutaneous sclerodermatous GVHD was significantly attenuated in MSC-treated recipients relative to sclerodermatous GVHD control subjects. After MSC treatment, skin collagen production was significantly reduced, with consistent down-regulation of Tgfb expression. Effects of MSCs on molecular markers implicated in persistent transforming growth factor-β signaling and fibrosis, such as PTEN, phosphorylated Smad-2/3, and matrix metalloproteinase-1, were observed in skin tissue. MSCs neither migrate to the skin nor affect the invivo expansion of immune effector cells, but they inhibited the infiltration of immune effector cells into skin via down-regulation of CCR4 and CCR8 expression on CD4+ T cells and CCR1 on CD11b+ monocyte/macrophages. MSCs diminished expression of chemokines such as CCL1, CCL3, CCL8, CCL17, and CCL22 in skin. MSCs were also dependent on stimulated splenocytes to suppress fibroblast proliferation. Our findings indicate that MSCs attenuate the cutaneous sclerodermatous GVHD by selectively blocking immune cell migration and down-regulating chemokines and chemokine receptors.

Obesity predisposes to rapid gastrointestinal graft-versus-host disease lethality after allogeneic hematopoietic stem cell transplantation in mice

Abstract Obesity is a form of chronic inflammation, termed “meta-inflammation”, resulting in the development of many chronic diseases such as diabetes and cancer. In this study, we assessed the impact of obesity on recipient graft-versus-host disease (GVHD) outcome post allogeneic HSCT using mouse models. In a model of sclerodermatous chronic GVHD, lean or diet-induced obese (DIO) BALB/c mice received 800 cGy total body irradiation (TBI), bone marrow cells, and splenocytes from H2-identical but minor MHC-mismatched B10.D2 mice. To mimic major MHC mismatch transplant which results in acute GVHD impacting gut, skin and liver, lean or DIO C57BL/6 mice received 1050 cGy TBI, bone marrow cells, and different doses of purified T cells from BALB/c mice. Strikingly, after the minor mismatch HSCT, all DIO mice succumbed to gut pathology while all lean mice survived and developed sclerodermatous GVHD at week 3 post transplant. In both models, pathological assessment indicated marked gut pathology in DIO mice correlating with lethality. Lean mice in the major MHC mismatch strain combination had significantly increased survival and less severe gut pathology compared to the DIO mice. The DIO mice had higher expression of pro-inflammatory cytokines in the gut, liver, visceral fat, and serum post-HSCT. Flow cytometric analysis demonstrated increased numbers of donor CD8+ T cells in the gut of DIO mice compared to lean mice. In an initial evaluation, untreated DIO mice displayed increased gut permeability as well as having limited diversity in their microbiome which would contribute to severe GVHD post-HSCT. Taken together, these results indicate that obesity exacerbates GVHD after allogeneic HSCT primarily affecting the gastrointestinal tract.

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.

Therapeutic Effects of a NEDD8-Activating Enzyme Inhibitor, Pevonedistat, on Sclerodermatous Graft-versus-Host Disease in Mice

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole treatment option for highly malignant hematologic disease; however, the major complication—graft-versus-host disease (GVHD)—still hinders its clinical application. In addition, chronic GVHD remains the major cause of long-term morbidity and mortality after allo-HSCT. Previously we showed that bortezomib, a proteasome inhibitor, can ameliorate the sclerodermatous GVHD response while maintaining graft-versus-tumor (GVT) effects. Here we report that pevonedistat (MLN4924), an inhibitor of the Nedd8-activating enzyme, which functions upstream of the proteasome in the ubiquitin-proteasome pathway, can also show similar protective effects. Recipient mice treated with pevonedistat demonstrated inhibitory effects on sclerodermatous GVHD pathogenesis. The beneficial effect of pevonedistat was observed to be temporally dependent. Whereas treatment given at the time of allo-HSCT administration or before the onset of symptoms worsened the scleroderma response, therapeutic administration starting at 20 days post-transplantation ameliorated the sclerodermatous GVHD. Flow cytometry analysis revealed differential effects on immune subsets, with inhibition of only antigen-presenting cells and not of donor T cells. Finally, pevonedistat preserved GVT effects in a sclerodermatous murine model of B cell lymphoma. Taken together, these data suggest that inhibition of neddylation with pevonedistat can serve as an alternative approach for the treatment of GVHD while maintaining GVT effects in a murine model of sclerodermatous GVHD.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics

ObjectiveUnderstanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets.MethodsGene expression measured in skin from mice with sclerodermatous graft‐versus‐host disease (GVHD), bleomycin‐induced fibrosis, Tsk1/+ or Tsk2/+ mice was mapped to human orthologs and compared to SSc skin biopsy–derived gene expression. Transforming growth factor β (TGFβ) activation was assessed using a responsive signature in mice, and tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) expression was measured in SSc patient and mouse skin.ResultsGene expression in skin from mice with sclerodermatous GVHD and bleomycin‐induced fibrosis corresponded to that in SSc patients in the inflammatory molecular subset. In contrast, Tsk2/+ mice showed gene expression corresponding to the fibroproliferative SSc subset. Enrichment of a TGFβ‐responsive signature was observed in both Tsk2/+ mice and mice with bleomycin‐induced skin fibrosis. Expression of TNFRSF12A (the TWEAK receptor/fibroblast growth factor–inducible 14) was elevated in skin from patients with fibroproliferative SSc and the skin of Tsk2/+ mice.ConclusionThis study reveals similarities in cutaneous gene expression between distinct mouse models of SSc and specific molecular subsets of the disease. Different pathways underlie the intrinsic subsets including TGFβ, interleukin‐13 (IL‐13), and IL‐4. We identify a novel target, Tnfrsf12a, with elevated expression in skin from patients with fibroproliferative SSc and Tsk2/+ mice. These findings will inform mechanistic and translational preclinical studies in SSc.

The effects of mesenchymal stem cells in a murine sclerodermatous model of graft-versus-host disease

Abstract Human chronic graft-versus-host disease (CGVHD) shares clinical characteristics with a murine sclerodermatous GVHD (scl GVHD) model that is characterized by skin and lung fibrosis. We used B10.D2 → BALB/c model of scl GVHD, which differ at minor histocompatibility loci, to address the therapeutic effect of mesenchymal stem cells (MSCs) on the development of CGVHD. MSCs were intravenously treated on days 3, 5 and 7 post-transplantation. The clinical severity of cutaneous scl GVHD was significantly attenuated after the treatment of MSCs. Pathologically, animals treated with MSCs showed less fibrosis and inflammatory cell infiltrations in skin and ear but not in visceral organs including lung and liver. Accordingly, the mRNA expression of collagen type 1 α1, 1 α2 and 3 α1 as well as collagen production were significantly reduced after the MSC treatment only in skin compared to the allogeneic scl GVHD controls. MSCs down-regulated mRNA of proinflammatory cytokines mainly in skin tissues and among them TGF-β expression was consistently reduced 28 days after HSCT. The effects of MSCs on molecular markers implicated in persistent TGF-β signaling and fibrosis, such as phosphatase and tensin homolog (PTEN), p-Smad2/3 and matrix metaloprotease-1 (MMP-1) were observed at the skin tissues. MSCs inhibited infiltration of immune cells into skin through down-regulating CCR4, CCR8 and CCR10 on donor CD4 T cells and CCR1 on CD11b monocytes. Moreover, MSCs diminished the expression of mRNA for chemokines such as CCL1, CCL8, CCL17, CCL22 and CCL3, in skin. In conclusion, treatment of MSCs early after transplant attenuated cutaneous scl GVHD by selectively blocking of immune cell migration and down-regulating the chemokines and chemokine receptors.