Mouse Model Of Graft-versus-host Disease Research Articles

Human placental mesenchymal stromal cells alleviate intestinal oxidative damage in mice with graft-versus-host disease via CD73/adenosine/PI3K/Akt/GSK-3β axis

BackgroundIntestinal damage is a common and serious complication in patients with graft-versus-host disease (GVHD). Human placental mesenchymal stromal cells (hPMSCs) ameliorate GVHD tissue damage by exerting anti-oxidative effects; however, the underlying mechanisms remain not fully clear. MethodsA GVHD mouse model and tumor necrosis factor-α (TNF-α)-stimulated human colon epithelial cell lines NCM460 and HT-29 cells were used to investigate the mechanisms of hPMSCs alleviating GVHD-induced intestinal oxidative damage. ResultshPMSCs reduced TNF-α concentrations and the number of CD3+TNF-α+ T-cells, which were negatively correlated with the expression of claudin-1, occludin, and ZO-1, through CD73 in the colon tissue of GVHD mice. Meanwhile, hPMSCs reduced the mean fluorescence intensity (MFI) of reactive oxygen species (ROS) and the concentration of malondialdehyde (MDA), promoted superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities, as well as claudin-1, occludin, and ZO-1 expression, in colonic epithelial cells of GVHD mice and TNF-α-stimulated cells via CD73. Moreover, hPMSCs upregulated adenosine (ADO) concentrations in GVHD mice and TNF-α-stimulated cells and mitigated the loss of tight junction proteins via the CD73/ADO/ADO receptors. Further analysis showed that hPMSCs diminished Fyn expression and enhanced Nrf2, GCLC, and HO-1 expression in both TNF-α-stimulated cells and colonic epithelial cells of GVHD mice by activating PI3K/Akt/GSK-3β pathway. ConclusionsThe results suggested that hPMSC-mediated redox metabolism balance and promoted tight junction protein expression were achieved via CD73/ADO/PI3K/Akt/GSK-3β/Fyn/Nrf2 axis, by which alleviating intestinal oxidative injury in GVHD mice.

The effect of ADAMTS13 on graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) can potentially cure malignant blood disorders and benign conditions such as haemoglobinopathies and immunologic diseases. However, allo-HSCT is associated with significant complications. The most common and debilitating among them is graft-versus-host disease (GVHD). In GVHD, donor-derived T cells mount an alloimmune response against the recipient. The alloimmune response involves several steps, including recognition of recipient antigens, activation and proliferation of T cells in secondary lymphoid organs, and homing into GVHD-targeted organs. Adhesion molecules on T cells and endothelial cells mediate homing of T cells into lymphoid and non-lymphoid tissues. In this study, we showed that Von Willebrand factor (VWF), an adhesion molecule secreted by activated endothelial cells, plays an important role in mouse models of GVHD. We investigated the effect of the VWF-cleaving protease ADAMTS13 on GVHD. We found that ADAMTS13 reduced the severity of GVHD after bone marrow transplantation from C57BL6 donor to BALB/C recipient mice. A recombinant VWF-A2 domain peptide also reduced GVHD in mice. We showed that ADAMTS13 and recombinant VWF-A2 reduced the binding of T cells to endothelial cells and VWF invitro, and reduced the number of T cells in lymph nodes, Peyer's patches and GVHD-targeted organs invivo. We identified LFA-1 (αLβ2) as the binding site of VWF on T cells. Our results showed that blocking T-cell homing by ADAMTS13 or VWF-A2 peptide reduced the severity of the GVHD after allo-HSCT, a potentially novel method for treating and preventing GVHD.

CEACAM1-engineered MSCs have a broad spectrum of immunomodulatory functions and therapeutic potential via cell-to-cell interaction

Mesenchymal stem cells (MSCs) have garnered attention for their regenerative and immunomodulatory capabilities in clinical trials for various diseases. However, the effectiveness of MSC-based therapies, especially for conditions like graft-versus-host disease (GvHD), remains uncertain. The cytokine interferon (IFN)-γ has been known to enhance the immunosuppressive properties of MSCs through cell-to-cell interactions and soluble factors. In this study, we observed that IFN-γ-treated MSCs upregulated the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), associated with immune evasion through the inhibition of natural killer (NK) cell cytotoxicity. To co-opt this immunomodulatory function, we generated MSCs overexpressing CEACAM1 and found that CEACAM1-engineered MSCs significantly reduced NK cell activation and cytotoxicity via cell-to-cell interaction, independent of NKG2D ligand regulation. Furthermore, CEACAM1-engineered MSCs effectively inhibited the proliferation and activation of T cells along with the inflammatory responses of monocytes. In a humanized GvHD mouse model, CEACAM1-MSCs, particularly CEACAM1-4S-MSCs, demonstrated therapeutic potential by improving survival and alleviating symptoms. These findings suggest that CEACAM1 expression on MSCs contributes to MSC-mediated regulation of immune responses and that CEACAM1-engineered MSC could have therapeutic potential in conditions involving immune dysregulation.

Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFβ and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

Abstract 2794: Consumption of dietary fiber correlates with a significant increase in a-diversity in the intestinal Microbiome of allo-HCT recipients and with lower GVHD-lethality in pre-clinical models

Abstract Background: Graft-versus-host disease (GVHD) is associated with a decrease in intestinal microbiota and beneficial short-chain fatty acids (SCFAs). Dietary fiber can modulate the intestinal microbiome and SCFA production which effect intestinal epithelium health and immunity. Therefore, we hypothesized that a high-fiber diet in recipients of an allogeneic hematopoietic cell transplantation (allo-HCT) could result in an increase of beneficial commensals, concentrations of SCFAs, and decreased risk of GVHD. Methods: We evaluated dietary data and fecal samples of allo-HCT patients receiving unmodified grafts (n=95) at MSKCC (day -10 to day +30 relative to allo-HCT) and correlated microbial α-diversity (16s rRNA, n=568 samples) with fiber intake and acute GVHD in No-GVHD (n=30), Non-LGI-GVHD (GVHD except lower gastrointestinal tract, n=41), and LGI-GVHD patients (lower gastrointestinal tract GVHD, n=24).We tested concentrations of cellulose fiber (0%, 6%, 12% and 40%) in a GVHD mouse model (C57BL/6J into BALB/c) to assess survival, microbial composition (shotgun sequencing n=180; n=30 per group), cecal SCFAs concentrations (GC-MS n=24, n=8 per group), and lastly, T cell subsets (Th1/Th17/Tregs) and colonic epithelial MHC-II expression (flow cytometry n=54, n=18 per group). Results: In allo-HCT patients, fiber intake correlated positively with microbial α-diversity (R=0.3, p=0.04), especially in the LGI-GVHD group (p=0.007). Additionally, patients with high fiber intake had higher microbial α-diversity compared to the low fiber cohort (p<0.0001).GVHD mice receiving a fiber-rich diet (12%), compared to 0% and 6%, had a higher microbial α-diversity (p=0.01); reduced GVHD lethality (p=0.02); and a decrease in the Enterococcus sp. (p=0.004) and Akkermansia sp. (p=0.01) abundance; taxa which are associated with worse overall survival and increased risk of lethal GVHD. In contrast, compared to 12%, the 40% fiber diet resulted in worse survival (p=0.002), lower α-diversity (p=0.05) and a higher abundance of Bifidobacterium sp. (p<0.0001).Cecal butyrate in GVHD mice was higher in response to the 12% vs. the 0% (p=0.01) and 6% (p=0.05) fiber diets. Mice treated with12% fiber displayed higher number of Tregs (CD4+CD25+FOXP3+), than those with 0% (p=0.001) and 6% (p=0.02); and a lower expression of MHC-II in epithelial cells (EpCAM+CD45-) vs. 0% fiber (p=0.02). Conclusions: We demonstrated that a higher fiber intake in allo-HCT patients is associated with an increase in α-diversity; especially in LGI-GVHD patients. In a pre-clinical GVHD-model, optimal fiber consumption (12% cellulose) leads to an increase in α-diversity, a decrease in pathogen relative abundance, increased T-regulatory cells, and a decrease in GVHD lethality. These results suggest that dietary fiber could be utilized to prevent GVHD. Citation Format: Jenny Paredes, Anqi Dai, Ruben J. Faustino Ramos, Peter Adintori, Teng Fei, Chi Nguyen, Harold Elias, Kristen Victor, Romina Ghale, Charlotte Pohl, Ana Gradissimo, Justin Cross, Oriana Miltiadous, Jonathan Peled, Marina Burgos da Silva, Marcel RM van den Brink. Consumption of dietary fiber correlates with a significant increase in a-diversity in the intestinal Microbiome of allo-HCT recipients and with lower GVHD-lethality in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2794.

Post-Transplant Cyclophosphamide Combined with Brilliant Blue G Reduces Graft-versus-Host Disease without Compromising Graft-versus-Leukaemia Immunity in Humanised Mice.

Allogeneic haematopoietic stem cell transplantation (HSCT) leads to the establishment of graft-versus-leukaemia (GVL) immunity, but in many cases also results in the development of graft-versus-host disease (GVHD). This study aimed to determine if P2X7 antagonism using Brilliant Blue G (BBG) could improve the beneficial effects of post-transplant cyclophosphamide (PTCy) in a humanised mouse model of GVHD, without comprising GVL immunity. NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice were injected with human peripheral blood mononuclear cells (PBMCs) (Day 0), then with cyclophosphamide (33 mg/kg) on Days 3 and 4, and with BBG (50 mg/kg) (or saline) on Days 0-10. PTCy with BBG reduced clinical GVHD development like that of PTCy alone. However, histological analysis revealed that the combined treatment reduced liver GVHD to a greater extent than PTCy alone. Flow cytometric analyses revealed that this reduction in liver GVHD by PTCy with BBG corresponded to an increase in human splenic CD39+ Tregs and a decrease in human serum interferon-γ concentrations. In additional experiments, humanised NSG mice, following combined treatment, were injected with human THP-1 acute myeloid leukaemia cells on Day 14. Flow cytometric analyses of liver CD33+ THP-1 cells showed that PTCy with BBG did not mitigate GVL immunity. In summary, PTCy combined with BBG can reduce GVHD without compromising GVL immunity. Future studies investigating P2X7 antagonism in combination with PTCy may lead to the development of novel treatments that more effectively reduce GVHD in allogeneic HSCT patients without promoting leukaemia relapse.

HPMSCs Regulate the Level of TNF-α and IL-10 in Th1 Cells and Improve Hepatic Injury in a GVHD Mouse Model via CD73/ADO/Fyn/Nrf2 Axis.

Mesenchymal stem cells (MSCs) ameliorate graft-versus-host disease (GVHD)-induced tissue damage by exerting immunosuppressive effects. However, the related mechanism remains unclear. Here, we explored the therapeutic effect and mechanism of action of human placental-derived MSCs (hPMSCs) on GVHD-induced mouse liver tissue damage, which shows association with inflammatory responses, fibrosis accompanied by hepatocyte tight junction protein loss, the upregulation of Bax, and the downregulation of Bcl-2. It was observed in GVHD miceand Th1 celldifferentiation system that hPMSCs treatment increased IL-10 levels and decreased TNF-α levels in the Th1 subsets via CD73. Moreover, hPMSCs treatment reducedtight junction proteins loss and inhibited hepatocyte apoptosis in the livers of GVHD mice via CD73. ADO level analysis in GVHD mice and the Th1 celldifferentiation system showed that hPMSCs could also upregulate ADO levels via CD73. Moreover, hPMSCs enhanced Nrf2 expression and diminished Fyn expression via the CD73/ADO pathway in Th1, TNF-α+, and IL-10+ cells. These results indicated that hPMSCs promoted and inhibited the secretion of IL-10 and TNF-α, respectively, during Th1 cell differentiation through the CD73/ADO/Fyn/Nrf2 axis signaling pathway, thereby alleviating liver tissue injury in GVHD mice.

Murine models of graft versus host disease (GVHD): Focus on ocular GVHD

Graft versus host disease (GVHD) remains a major and serious complication of allogeneic hematopoietic stem cell transplantation. Based on the time of onset, clinical phenotypes, progression kinetics, and pathophysiology, GVHD is stratified into acute, chronic, and overlapping types. The eyes are among the most commonly affected organs in GVHD. Mouse models have played an important role in understanding the several key elements of GVHD pathobiology. The current review discusses the immunology, pathology, and key phenotypic features of mouse models of systemic GVHD. Furthermore, a critical appraisal of mouse models of ocular GVHD (oGVHD) is provided. The disease mechanisms underlying the ocular surface, meibomian gland, and lacrimal gland injury in these models are reviewed, and the relevance of oGVHD murine models to clinical oGVHD is also included.

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties

BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine–cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Human placental mesenchymal stromal cells modulate IFN-γ and IL-10 secretion by CD4+T cells via CD73, and alleviate intestinal damage in mice with graft-versus-host disease

BackgroundIntestinal inflammatory damage is an important factor in the development of graft-versus-host disease (GVHD). IFN-γ and IL-10 play key roles in gastrointestinal inflammation, and human placental mesenchymal stromal cells (hPMSCs) can alleviate inflammatory damage during GVHD. CD73 is highly expressed by hPMSCs. We aimed to study whether hPMSCs could alleviate intestinal damage in GVHD mice by modulating IFN-γ and IL-10 in CD4+T cells by CD73. MethodsA GVHD mouse model was induced using 8-week-old C57BL/6J and BALB/c mice, which were treated with regular hPMSCs (hPMSCs) or hPMSCs expressing low level of CD73 (shCD73). Then, the levels of IFN-γ and IL-10 in CD4+T cells were determined using flow cytometry. Transmission electron microscopy, western blotting, and morphological staining were employed to observe the intestinal damage. ResultshPMSCs ameliorated pathological damage and inhibited the reduction of the tight junction molecules occludin and ZO-1. They also downregulated IFN-γ and upregulated IL-10 secretion in CD4+T cells via CD73. Moreover, IL-10 mitigated the inhibitory effects of IFN-γ on the expression of occludin in both Caco-2 and NCM460 cells in vitro, but did not affect ZO-1. In addition, hPMSCs upregulated the level of AMPK phosphorylation in CD4+T cells by CD73, which is positively associated with the proportion of CD4+IFN-γ+IL-10+T, and CD4+IFN-γ-IL-10+T cells. ConclusionsOur findings suggested that hPMSCs may balance the levels of IFN-γ and IL-10 in CD4+T cells by promoting the phosphorylation of AMPK via CD73, which alleviates the loss of occludin and ZO-1 in intestinal epithelial cells and, in turn, reduces inflammatory injury in GVHD mice.

Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse.

T cells are the central drivers of many inflammatory diseases, but the repertoire of tissue-resident T cells at sites of pathology in human organs remains poorly understood. We examined the site-specificity of T cell receptor (TCR) repertoires across tissues (5 to 18 tissues per patient) in prospectively collected autopsies of patients with and without graft-versus-host disease (GVHD), a potentially lethal tissue-targeting complication of allogeneic hematopoietic cell transplantation, and in mouse models of GVHD. Anatomic similarity between tissues was a key determinant of TCR repertoire composition within patients, independent of disease or transplant status. The T cells recovered from peripheral blood and spleens in patients and mice captured a limited portion of the TCR repertoire detected in tissues. Whereas few T cell clones were shared across patients, motif-based clustering revealed shared repertoire signatures across patients in a tissue-specific fashion. T cells at disease sites had a tissue-resident phenotype and were of donor origin based on single-cell chimerism analysis. These data demonstrate the complex composition of T cell populations that persist in human tissues at the end stage of an inflammatory disorder after lymphocyte-directed therapy. These findings also underscore the importance of studying T cell in tissues rather than blood for tissue-based pathologies and suggest the tissue-specific nature of both the endogenous and posttransplant T cell landscape.

Improved efficacy of mesenchymal stromal cells stably expressing CXCR4 and IL-10 in a xenogeneic graft versus host disease mouse model.

Previous clinical trials have shown that mesenchymal stromal cells (MSCs) can modulate graft versus host disease (GvHD) after allogeneic hematopoietic transplantation, although with variable efficacy. To improve the anti-GvHD effect of these cells, adipose tissue derived-human MSCs (Ad-MSCs) were transduced with a lentiviral vector conferring stable expression of CXCR4, a molecule involved in cell migration to inflamed sites, and IL-10, a cytokine with potent anti-inflammatory properties. In vitro experiments showed that the expression of these molecules in Ad-MSCs (named CXCR4-IL10-MSCs) efficiently enhanced their migration towards SDF-1α and also improved their immunomodulatory properties compared to unmodified Ad-MSCs (WT-MSCs). Moreover, using a humanized GvHD mouse model, CXCR4-IL10-MSCs showed improved therapeutic effects, which were confirmed by histopathologic analysis in the target organs. Additionally, compared to WT-MSCs, CXCR4-IL10-MSCs induced a more marked reduction in the number of pro-inflammatory Th1 and Th17 cells, a higher polarization towards an anti-inflammatory T cell profile (CD3+-IL10+ cells), and increased the number of regulatory T and B cells. Our in vitro and in vivo studies strongly suggest that CXCR4-IL10-MSCs should constitute an important new generation of MSCs for the treatment of GvHD in patients transplanted with allogeneic hematopoietic grafts.

Blocking common γ chain cytokine signaling ameliorates T cell-mediated pathogenesis in disease models.

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.

EPC infusion ameliorates acute graft-versus-host disease-related endothelial injury after allogeneic bone marrow transplantation.

Graft-versus-host disease (GVHD) damages vascular endothelium. Endothelial progenitor cell (EPC) can differentiate to endothelial cell and promote angiogenesis, but its role in endothelial damage in GVHD is unclear. In this study, we intend to assess whether EPC infusion promotes the repair of endothelial injury in GVHD mouse model. Male BALB/c mice were randomly divided into 5 groups: control group, total body irradiation group (TBI group), allogeneic bone marrow transplantation group (Allo-BMT group), acute graft versus host disease group (GVHD group), EPC infusion group (GVHD+EPC group) followed by analysis of mice survival, acute GVHD (aGVHD) score, T cell infiltration by immunofluorescence, as well as continuity of vascular endothelium in liver. Compared with Allo-BMT group, the clinical and pathological score of aGVHD mice were higher. On day 21 after transplantation, a large number of mononuclear cell infiltrations were seen in the target tissues of aGVHD mice and mice died within 30 days. In addition, aGVHD group also presented increased subendothelial infiltration of CD3+ T cells in the liver, decreased VE-cadherin expression and elevated major histocompatibility complex (MHC) II molecule expression in the endothelium. Moreover, expression of MHC-II molecule increased in endothelial cell after irradiation injury and LPS stimulation, indicating abnormally activated endothelial cell with antigen-presenting function. Interestingly, infusion of EPC reduced the clinical and pathological score of aGVHD, decreased infiltration of mononuclear cells, improved survival as well as upregulated VE-cadherin and downregulated MHC-II molecule. EPC infusion can mobilize to affected endothelium to decrease the infiltration of T cells and pathological endothelial activation contributing to ameliorating the damage of endothelium. EPC infusion combined with bone marrow transplantation might be a perspective strategy for the prevention and treatment of aGVHD.

Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Acute graft-versus-host disease (GVHD) causes apoptosis of intestinal stem cells (ISCs) that are key targets in the gastrointestinal (GI) tract (Zeiser and Blazar, NEJM 2917; 377:2167). The receptor interactive protein kinase 1 (RIP1) is an important mediator of apoptosis and we investigated the role of the (RIP1) pathway in GI tract and ISC damage in both human and mouse GVHD models. Immunohistochemical staining of human GI biopsies from GVHD patients showed strong phospho-RIP1 staining in biopsies with Lerner grades 2/3 compared to grades 0/1 (12/17 v 0/8, p < 0.01). We then established in vitro models of ISC apoptosis (at least 50% loss) by adding 10 ng/ml of both IFNg and TNFa to organoid cultures from both ileum and colon (organoids cultures with either IFNg or TNFa alone showed no change). Addition to cultures of 0.1 mcg/ml GNE684, a well-characterized small molecule RIP inhibitor (Patel et al, Cell Death Diff 2020; 27:161), dramatically reversed ISC loss in both human and mouse ileal organoids as measured by cross sectional area (Fig 1). WE observed an identical pattern in colon cultures where GNE684 reversed loss of human ISCs (70.0 + 7.7 % v 29.3 + 2.5 %, p < 0.001) and mouse ISCs (100 + 6.6 % v 41 + 6.6 % p < 0.001). In vivo, 14 daily ip injections of GNE684 prevented mortality in a GVHD mouse model (C3H.SW → B6) in a dose dependent fashion (Fig 2). Systemic GNE administration also improved survival in a second model (Balb/c → B6: day 30 survival 7/10 v 0/10, p < 0.001). In the C3H.SW → B6 model there were dramatic reductions in CD4+ lamina propria effector lymphocytes secreting either IFNg (56 + 11 v 9 + 2, p < 0.001) or IL-17 ( 22 + 10 v 7 + 4, p < 0.01) and in CD8+ effector cells secreting IFNg (39 + 11 v 8 + 3, p < 0.001) or IL-17 ( 19 + 8 v 7 + 2, p < 0.001) on day 10 after HCT. Importantly, GVHD was dramatically reduced when administration of GNE684 was used as treatment for GVHD beginning on day +7 after GVHD was established (day 40 survival: 8/12 v 2/12, p = 0.02). Additionally, survival dramatically improved in RIP1 kinase-dead recipients compared to wild-type B6 controls in both the C3H.SW → B6 model (13/16 v 4/16, p < 0.001) and in the Balb/c →B6 model (13/20 v 1/20, p < 0.001). IFNg secreting CD4+ and CD8+ effector lymphocytes that infiltrated the lamina propria were significantly reduced in RIP1 kinase-dead recipients compared to wild-type controls (58 + 13 v 11 + 7 and 39 + 11 v 14 + 4, respectively [p < 0.001 for both]). Infiltrating lymphocytes secreting IL-17 were also reduced four fold. Importantly, administration of GNE684 preserved beneficial GVL effects when lethal doses of myeloid tumor C1498 cells that caused 100% mortality by day 28 in syngeneic BMT recipients were added to the donor inoculum (day 42 survival: 7/10 v 4/13, p = 0.03). We conclude that RIP1 is a key mediator of GI damage during GVHD. In vitro RIP1 inhibition prevents ISC apoptosis in both mouse and human models of GI GVHD. RIP1 inhibition, either through genetic alteration of the RIP1 kinase domain in transplant recipients or through systemic administration of the RIP1 inhibitor GNE684, protects the GI tract from acute GVHD damage as measured both clinically and histologically in preclinical models while preserving beneficial GVL effects. These data suggest that GNE684 a clinical trial of GNE684 is warranted in patients with gastrointestinal GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunomodulatory Microbial Metabolites Identified in Allo-SCT Patients Protect from GvHD By Sting Dependent Modulation of Intestinal Tissue Homeostasis

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS/STING can promote the integrity of the intestinal barrier and limit GVHD. However, the signals that drive these protective IFN-I responses are poorly understood. Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling and (ii) predict mortality in ASCT patients. We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels (Submitted as a separate abstract to ASH 2022 (Orberg & Meedt et al.)). We showed that patients with high metabolite expression are less likely to develop GVHD. In this study, we translated our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoid models to uncover the molecular mechanisms of protective metabolites during ASCT. Methods: Stool samples from ASCT patients were obtained in Munich and Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry. Next, we tested metabolites detected in patients in vitro in murine and human intestinal crypt-derived organoids and organoid/T cell coculture assays. To obtain a mechanistic understanding, we stimulated WT or IFN-I-signaling-impaired organoids or used small molecule inhibitors and analysed them regarding growth performance, cell composition and by scRNAseq. Finally, Metabolites were applied as treatment in preclinical intestinal damage models and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. Results: Here, we present expression profiles of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids. Following ASCT, and especially at the early time-points, metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced. Metabolite stimulation of mouse small intestinal organoids promoted organoid growth and required intact STING signaling. Additionally, organoids showed an increased stemness and resistance to T cell mediated damage. Both healthy donor and GvHD patient derived human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151. Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly better outcomes in barrier integrity and organoid based intestinal regeneration assays. Additionally, metabolite treatment was associated with an increased regulatory T cell infiltration. These effects were abrogated in STING-/- recipients, but not in IFNaR-/- recipients. Finally, metabolite treatment was protective in major mismatch GVHD mouse models. Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia may reduce occurrence of GVHD in ASCT patients.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, Protected Mice from Inflammatory and Autoimmune Diseases

Pathogenic immune cell responses in inflammatory and autoimmune diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the roles of γc cytokines in driving inflammatory and autoimmune diseases, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic immune cell responses in mouse models of graft-versus-host disease (GVHD), immune aplastic anemia and multiple sclerosis (MS). We first evaluated the efficacy of REGN7257 in xenogeneic and allogeneic mouse models of GVHD. In the xenogeneic model, immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). In the allogeneic GVHD model, irradiated BALB/c mice were engrafted with bone marrow cells and splenocytes from Il2rghu/hu mice. In the xenogeneic GVHD mouse model, we also analyzed the effects of γc cytokine signaling blockade with REGN7257 on inflammation and immune cell infiltration into tissues (e.g. liver, bone marrow), as well as liver fibrosis. Finally, we assessed the effects of IL2RG blockade on pathogenic immune cell responses in an experimental autoimmune encephalomyelitis (EAE) model of MS, looking at immune cell infiltration into the central nervous system (CNS) and associated demyelination. In both xenogeneic and allogeneic models of GVHD, prophylactic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Similarly, in the xenogeneic GVHD model, dosing with REGN7257 starting 3 weeks after huPBMC administration effectively protected mice from weight loss and death. Consistent with the classic pathology of GVHD, tissues of control mice were highly infiltrated by immune cells, including CD4+ and CD8+ T-cells, while γc cytokine signaling blockade strongly reduced immune cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Finally, in an EAE mouse model of MS, inhibition of γc cytokine signaling with REGN7257 alleviated disease progression and significantly prolonged survival. This was associated with reduced inflammation and infiltration by immune cells (CD4+ T-cells, CD8+ T-cells, B-cells and monocytic cells) in the CNS, as well as decreases in myelin oligodendrocyte glycoprotein-specific antibody production. We showed that REGN7257 treatment efficiently protected the spinal cord from demyelination, which was associated with decreased oligodendrocyte injury and neuron protection. Blockade of γc cytokine signaling with REGN7257 protected mice against immune cell-mediated pathology in multiple disease models: an EAE model of MS, an allogeneic GVHD model, and a xenogeneic GVHD model that uniquely presents hallmarks of both acute and chronic GVHD as well as immune aplastic anemia. These data provide evidence of γc cytokines as key drivers of pathogenic immune cell responses, offering a potentially novel strategy for the management of inflammatory and autoimmune diseases, such as GVHD, immune aplastic anemia and MS.

CD73-Expressing Microbiota-Reactive DP8α Regulatory T Cells Are Lacking in Acute GvHD Patients and Prevent Disease Development in a Pre-Clinical In Vivo Humanized Mouse Model of GvHD

Allogeneic stem cell transplantation (allo-HSCT) to treat hematological malignancies can induce life-threatening complications, such as graft-versus-host disease (GvHD). Steroids remain the first-line treatment for acute GvHD (aGvHD). Nevertheless, new treatments are needed to, not only, treat steroid-resistant patients and improve allo-HSCT outcomes, but also reduce steroid complications. Increasing evidences strongly suggest that gut microbiota composition and the activity of regulatory T cells (Tregs) could be involved in GvHD prevention. We have identified a novel FoxP3-negative IL-10-secreting Treg subset, named DP8α, enriched in the colonic mucosa and present in blood. These cells display a TCR-specificity for the gut ClostridiumIV bacterium Faecalibacterium prausnitzii. Sizable fractions of these cells also expressed the membrane-bound ectonucleotidases CD39 and CD73, which are directly involved in their suppressive activity in vitro. In vivo, these enzymes play key regulatory roles by both degrading proinflammatory extracellular ATP and inducing the production of immunosuppressive adenosine. An alteration in this adenosine/purinergic pathway has also been linked to GvHD occurrence. Altogether, these data prompted us to hypothesize that F. prausnitzii-reactive DP8α Tregs, whose suppressive activity is driven by the purinergic pathway, could bridge microbiota dysbiosis and GvHD incidence in allo-HSCT patients. Moreover, decreased levels of Clostridia, especially Faecalibacteriumspp, in patient's stools, have been associated with aGvHD risk, and mice gavaged with Treg-inducing Clostridia displayed milder GvHD and improved survival. Therefore, we addressed a potential role for DP8α Tregs in the prevention of aGvHD. We first analyzed cells from 63 patients with hematological malignancies, who received allo-HSCT, among whom a third developed aGvHD. We quantified their circulating DP8α Tregs and their CD39 and CD73 expression pre- and post-allo-HSCT. While the percentage of CD73-expressing DP8α Tregs within CD3+ T cells before HSCT was not different, a strikingly deficiency of CD73-expressing DP8α Tregs was strongly associated with aGvHD development (p<.001) at 1-month post-transplant, as compared to aGvHD-free patients (Fig.1). Importantly, CD73 expression was not affected on any other T cell subset analyzed. CD73 decrease on DP8α Tregs did not result from corticotherapy since it was 1/ equally observed before and after treatment in aGvHD patients and 2/ not observed following in vitro treatment of PBMCs with methylprednisolone. Moreover, the decrease of CD73-expressing DP8α Tregs in aGvHD patients was observed across all treatment conditions, which were heterogeneous, and therefore appears relevant and likely related to the aGvHD condition itself. Altogether, these data strongly suggest that a CD73-dependent functional alteration of DP8α Tregs could, at least in part, be involved in aGvHD occurrence and/or development. We next evaluated the therapeutic efficacy of CD73+ DP8α Tregs in a pre-clinical model of acute xeno-GvHD induced through human PBMC injection in irradiated NSG recipient mice. In vitro-expanded CD73+ DP8α Treg injected i.v. drastically protected mice against xeno-GvHD (Log-rank, p<.0001; Fig.2) without preventing the development of human chimerism. Moreover, these in vivo experiments, repeated 4 times using different PBMC donors, all confirmed the potent protective role of CD73+ DP8α Tegs in this model. Accordingly, serum inflammatory human cytokines and tissue infiltration by human cells were significantly lower in mice treated with CD73+ DP8α Tregs, as compared to untreated mice. In addition, the colon was rapidly populated with the injected DP8α Tregs and well-preserved in treated mice, while clearly damaged in untreated mice. Altogether, these results strongly support a role for CD73+ DP8α Tregs in aGVHD prevention and advocate for the use of these cells to both predict aGvHD risks and give rise to the development of innovative therapeutic strategies to preclude GvHD-related inflammation. Such therapeutic approaches would be based on the infusion of DP8α Tregs and/or their in vivo stimulation, e.g., with F. prausnitzii-derived antigens or probiotics. Of note, DP8α Tregs display an uniquely high proliferating potential and stable immunosuppressive functions in vitro, a key feature to implement such therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Chemical Series of Anti-ST2 Small Molecules Suppress Allogeneic T Cells Proliferation While Increasing Treg Cells and Improve Gvhd and Survival In Vivo

Elevation of soluble Stimulation-2 (sST2) is considered a prognostic biomarker in several disease progression, such as cardiac allo-rejection, graft-versus-host disease (GVHD), and is associated with the response to tissue damage that secrets Interleukin-33 (IL-33), an alarmin. sST2 is an alternatively spliced isoform of the Interleukin 1 Receptor-Like 1 (IL1RL1) gene that also encodes the major membrane-bound ST2 (ST2) isoform expressed on tolerogenic immune cells including Foxp3+ regulatory T cells (Tregs), type 2 innate lymphoid cells (ILC2), type 2 helper T cells (Th2), monocytes, M2 macrophages, mast cells, eosinophils and neutrophils. IL-33 is the only known ligand that binds to both sST2 and ST2. Binding of IL-33 to ST2 activates intracellular signaling in ST2 expressing cells whereas sST2 functions as a decoy receptor to sequester the secreted free IL-33. After allogeneic hematopoietic cell transplantation (HCT), elevated sST2 levels are associated with patients with GVHD resistant to steroid therapy and with subsequent non-relapse related death. We hypothesized that therapeutic intervention to release IL-33 from sST2 may modulate the T cells responses for treating GVHD. As proof-of-concept, we have reported studies using either ST2 antibodies (Zhang et. al., Sci. Trans. Med. 2015) or small-molecule ST2 inhibitors (iST2-1 in Ramadan et. al., JCI Insight, 2018) in the murine GVHD models. Herein, we report a novel series of small-molecule ST2 inhibitors from our follow-up development of iST2-1. We show a representative compound, XY-52, effectively reduced sST2 levels, alleviated GVHD and improved survival in a murine GVHD model. Based on our previously discovered iST2-1, we recently developed 1-(furan-2ylmethyl)pyrrolidine-based ST2 Inhibitors and constructed the structure-activity relationship (SAR) of compounds against ST2/IL-33 binding (Yuan et. al., Bioorg. Med. Chem., 2022). That study led to identify 14e (Fig. 1A) that has an IC50 value of 7.77 mM against ST2/IL33 binding determined by the AlphaLISA assay. Despite the improvement of the IC50 value as compared to iST2-1, 14e has deficiency in solubility and metabolic stability (including a similar in vivo half-life as iST2-1) that required further optimization. To improve 14e, we first focused on replacing the furan group (C-ring) of 14e with the goal of eliminating the potential metabolic issue of the furan group. We found replacement of furan by either a pyrrole (ST2-J-100) or an alkylated pyrrole group (ST2-J-106, 107) retained the activity of the inhibitors. We then investigated the replacement of the chemical groups on A and B rings of 14e aiming to improve the solubility of the inhibitors while maintaining their activities. These modifications led to identify a new series of ST2 inhibitors represented by XY-52. In the mixed lymphocyte reaction (MLR) assay, we found XY-52 and analogs (XY-125, XY-146, XY-148, XY-150) inhibited CD4+, CD8+ T cell proliferation while increased Tregs (Fig. 1B). XY-52 has improved in vitro (t1/2= >60m in mouse, 41m in human liver microsome) and in vivo metabolic stability. In vivo PK study showed that XY-52 has higher tissue distribution including spleen, lung, skin, small intestine and liver than iST2-1 or Ruxolitinib (Fig. 1C). XY-52 was then selected to compare with 3c (previously reported) in a clinically relevant minor-mismatched GVHD mouse model (C57BL/6→C3H.SW). Using a 21-day prophylactic regimen of 40mg/kg/day (BID), we found that 3c reduced GVHD score (P=0.02) and extended survival (HR 3.90, P=0.02) in mice. Mice receiving XY-52 treatment had a more profound reduction in GVHD score (P<0.0001) and improvement in survival (HR, 31.22, P<0.0001) (Fig. 1D). Both 3c and XY-52 yielded lower plasma concentrations of sST2 and IFNg compared with the DMSO control (Fig. 1E) at Day 7 and 21 post-HCT. Ex vivo analysis of T cells in the gastrointestinal tract (GI) at day 21 post-HCT showed that XY-52 was more effective than 3c at reducing CD4+ IFNg+, CD8+ IFNγ+, CD4+ IL-17A+ T cells and increasing CD4+ Foxp3+ Tregs frequencies. In summary, XY-52 represents a promising ST2 inhibitor that reduces the plasma sST2 levels, CD4+, CD8+ effector T cells and increases CD4+ Foxp3+ Tregs frequencies in in vitro and in vivo GVHD models. Further improvement in the potency and the PK profile of XY-52 will allow us to develop effective ST2 inhibitors for use as a prophylactic or therapeutic regimen for GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Construction of HGF-Displaying Yeast by Cell Surface Engineering.

Hepatocyte growth factor (HGF) has been investigated as a regulator for immune reactions caused by transplantation and autoimmune diseases and other biological functions. Previous studies demonstrated that cDNA-encoding HGF administration could inhibit acute graft-versus-host disease (GVHD) after treatment via hematopoietic stem cell transplantation. This study aimed to show the preparation of HGF protein on yeast cell surfaces to develop a tool for the oral administration of HGF to a GVHD mouse model. In this study, full-length HGF and the heavy chain of HGF were genetically fused with α-agglutinin and were successfully displayed on the yeast cell surface. This study suggested that yeast cell surface display engineering could provide a novel administration route for HGF.