Murine GVHD Model Research Articles

Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease

Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n= 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B.ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B.ovatus.

Sex differences in donor T cell targeting of host splenocyte subpopulations in acute and chronic murine graft-vs.-host disease: implications for lupus-like autoimmunity.

This study sought to compare in vivo sex differences in either a Th1-dominant CTL response or a Tfh-mediated lupus-like antibody response using the parent-into F1 murine model of acute or chronic GVHD respectively. In acute GVHD we observed no significant sex differences in the hierarchy of donor CD8 CTL elimination of splenocyte subsets. B cells were the most sensitive to elimination in both sexes; however, the male response was significantly stronger. Sex differences in chronic GVHD were more widespread; females exhibited significantly greater numbers of total splenocytes and host CD4 Tfh cells, B cells and CD8 T cells consistent with reports of greater female autoantibody production in this model. The more potent male CTL response in acute GVHD conflicts with reports of greater female CTL responses following infections or vaccines and may reflect the absence of exogenous innate immune stimuli in this model.

Corticosteroids impair epithelial regeneration in immune-mediated intestinal damage.

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-γ. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue-targeted therapies with immunosuppression.

Mesenchymal stromal cells ameliorate chronic GVHD by boosting thymic regeneration in a CCR9-dependent manner in mice.

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Mature donor T cells within the graft contribute to severe damage of thymic epithelial cells (TECs), which are known as key mediators in the continuum of acute GVHD (aGVHD) and cGVHD pathology. Mesenchymal stromal cells (MSCs) are reportedly effective in the prevention and treatment of cGVHD. In our previous pilot clinical trial in patients with refractory aGVHD, the incidence and severity of cGVHD were decreased, along with an increase in levels of blood signal joint T-cell receptor excision DNA circles after MSCs treatment, which indicated an improvement in thymus function of patients with GVHD, but the mechanisms leading to these effects remain unknown. Here, we show in a murine GVHD model that MSCs promoted the quantity and maturity of TECs as well as elevated the proportion of Aire-positive medullary TECs, improving both CD4+CD8+ double-positive thymocytes and thymic regulatory T cells, balancing the CD4:CD8 ratio in the blood. In addition, CCL25-CCR9 signaling axis was found to play an important role in guiding MSC homing to the thymus. These studies reveal mechanisms through which MSCs ameliorate cGVHD by boosting thymic regeneration and offer innovative strategies for improving thymus function in patients with 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.

DRAK2 is a promising target to inhibit GvHD without negatively impacting graft reconstitution after bone marrow transplant

Abstract Graft versus host disease (GvHD) is the most common adverse event after allogeneic bone marrow transplant (BMT). GvHD and the immunosuppression required to treat it limit the desired graft versus leukemic effect and are associated with significant morbidity and mortality. A treatment that selectively controls the harmful immune response during GvHD, without compromising the response to pathogens or tumors, would be transformative. Previously, we demonstrated that mice deficient in Drak2 (Drak2−/−) are resistant to disease in multiple models of T cell-mediated autoimmunity but maintain effective responses to pathogens and tumors. Since similar immune pathologies mediate GvHD and autoimmunity, we sought to investigate whether loss of Drak2 reduced GvHD, while maintaining effective immunity to pathogens. Thus, we utilized a murine model of acute GvHD (aGvHD) in which lethally irradiated mice received MHC-mismatched bone marrow and purified T cells that were either Drak2+/+ or Drak2−/−. We found that mice given MHC-mismatched bone marrow along with Drak2−/− T cells had less severe aGvHD compared to mice receiving Drak2+/+ T cells, as demonstrated by lower clinical scores. Interestingly, mice given Drak2−/− T cells along with MHC-mismatched bone marrow had increased proportions of regulatory T cells and decreased effector T cells compared to mice given wildtype T cells. These data suggest that Drak2 expression within T cells contributes to GvHD development, and therefore is a promising target in the treatment of GvHD. Ongoing studies are investigating whether the absence of Drak2 influences the effective repopulation of a functional immune system following BMT. Funding provided by ALSAC and the St. Jude Graduate School of Biomedial Sciences

Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

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.

295 - RNA and TCR Sequencing Shed Light on Mechanisms of Treg Suppression in a Murine Model of Acute GvHD

295 - RNA and TCR Sequencing Shed Light on Mechanisms of Treg Suppression in a Murine Model of Acute GvHD

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.

Aloocetra-OTS for graft versus host (GVHD) prevention and successful transplantation

Background & Aim Allocetra-OTS was developed as a cell-based therapeutic composed of unmatched donor mononuclear cells induced to an early apoptotic state. Allocetra-OTS, as do naturally occurring apoptotic cells within the body, signals to the macrophages and dendritic cells to react less aggressively to immunogenic agents, thereby resetting the system towards immuno-homeostasis. Three types of experiments showed safety and efficacy in pre-clinical studies. A single dose of Allocetra-OTS in a murine model of GvHD had a beneficial effect on GvHD prevention. Allocetra-OTS was also successful as an ancillary treatment to the standard of care for the prevention of GvHD. Finally, in a mice model with co-existing leukemia, Allocetra-OTS was effective against GvHD without interfering with the graft-versus-leukemia (GvL) effect. Methods, Results & Conclusion The clinical development program of Allocetra-OTS for GvHD consists of 2 clinical studies, a completed Phase 1/2a study (TLX-APO-101IL) for related donors (Allocetra), and a planned Phase 2/3 study (ENX-CL-01-002) for unrelated donors. In addition, Allocetra-OTS is being evaluated for sepsis in an ongoing Phase 1b open-label study. Overall, 21 subjects were exposed to at least 1 dose of Allocetra or Allocetra-OTS and no safety concerns were detected. The first GvHD study included dose-escalation of four sequential cohorts. Thirteen eligible subjects with hematologic malignancies, who received conventional myeloablative conditioning were enrolled. The results of this study suggested that a single infusion of donor early apoptotic cells (Allocetra) as prophylaxis for GvHD in myeloablative HSCT is safe and potentially effective and led to 0% (0/6) of acute high grade II-IV GvHD in the two higher dosages compared to 52% in matched historical control. The planned Phase 2/3 multi-center, open-label, 2-arm study (ENX-CL-01-002), in Israel and Germany, will evaluate the efficacy and safety of Allocetra-OTS (140 × 106 cells/kg) with or without anti-thymocyte globulin (ATG) for the prevention of GvHD in subjects undergoing HLA-matched HSCT from an unrelated donor.

Interleukin-27 enhances the function of regulatory T cells to prevent Graft-versus-host diseases

Abstract Graft-versus-host diseases (GvHD) remain life-threatening complications after allogeneic stem cell transplantation. Foxp3+Tregs can inhibit GvHD, while preserving graft-versus-tumor activity, making Tregs a promising candidate for GvHD treatment. Yet, the clinical application is hampered by the limited numbers and ineffective functions during treatment. Conventional CD4 T cells acquire Foxp3 expression and regulatory functions by stimulating them with IL-2 and TGFb (iTregs). Unlike thymus-derived Tregs (tTreg), ineffective regulatory functions and phenotypic instability of iTregs remain areas of improvement. We reported that IL-27 is able to enhance Treg functions to reduce autoimmune and allergic inflammation. Herein, we sought to test if IL-27-stimulation in Tregs improves therapeutic efficacy of Tregs in a murine model of MHC-mismatched allogeneic GvHD. IL-27-pretreated tTregs efficiently reduced acute GvHD-associated mortality, whereas the efficacy of IL-27-pretreated polyclonal iTregs to prevent GvHD was marginal. We hypothesized that elevating antigen specificity in iTregs would increase the therapeutic functions of iTregs. Indeed, IL-27-pretreated allogeneic iTregs exhibited highly improved therapeutic function, equivalent to that of tTregs. IL-27-induced iTreg functions were also tested using human cells. IL-27 prestimulated third party human iTregs partially rescued mice from xenogeneic GvHD. Our data suggest that IL-27 stimulation in Tregs may play an important role in regulating Treg functions during GvHD, raising the hypothesis that defects in Treg functions (possibly via IL-27) may underlie different susceptibility of allogeneic bone marrow transplant patients who develop GvHD or not.

Tracking T Cell Activation By OX40 Immuno-PET: A Novel Strategy for Imaging of Graft Versus Host Disease

BACKGROUNDGraft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior non-invasive imaging strategies specifically detecting early GvHD are critically needed to improve clinical care of HCT recipients. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F-FDG) have largely been confounding, mainly due to their low specificity. Monitoring T cell activation and expansion using T-cell targeted PET tracers seems a more promising approach (Ronald et al., Cancer Res 77(11) 2893, 2017). We recently reported a novel immuno-PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive imaging of activated murine T cells expressing the cell surface activation marker OX40 (Alam et al., JCI 128(6) 2569, 2018). In the present work, we evaluated the utility of this immuno-PET strategy to image activated T cells in a major MHC-mismatch mouse model of acute GvHD.METHODSBalb/C (H-2Kd) recipients were irradiated with 8.8 Gy and on the same day received intravenously (i.v.) 5 x 10e6 T-cell depleted bone marrow (BM) cells with or without 1 x 10e6 CD4 and CD8 T cells positively selected from C57BL/6 (H-2Kb) mice. Severity of GvHD was assessed by clinical GvHD scoring. Flow cytometry of lymphoid organs from BM control and GvHD mice was performed at day 7 after HCT to determine OX40 protein expression on immune cells. For imaging studies, anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (an average ratio of 1.4 DOTAs per mAb was obtained) and subsequently radiolabeled with 64CuCl2 (final specific activity 10-15μCi/μg and radiochemical purity >99%). Mice were tail-vein injected with 64Cu-DOTA-mAbOX40 (100 µCi, i.v.) at day 7 after HCT and PET-CT imaging performed 24 hours after injection. Immediately following PET-CT mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter.RESULTSFlow cytometry analysis of OX40 expression in lymphoid organs isolated at day 7 after HCT revealed significantly higher proportions and absolute numbers of OX40 expressing cells in the spleen and cervical lymph nodes (LN) isolated from mice that received BM + T cells (GvHD group) compared with mice having received BM cells alone (p<0.05). In vivo OX40-ImmunoPET performed at day 8 after HCT revealed increased radiotracer uptake in spleen (p < 0.0001), mesenteric LN (p < 0.01) and the abdominal region (p < 0.001) of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64Cu-DOTA-mAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Biodistribution analysis using gamma counting of tissues confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01) and GvHD target organs e.g. small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). Importantly, outcome analysis of GvHD mice receiving tracer doses of OX40 mAb at day 7 after HCT did not reveal any significant worsening of GvHD in terms of survival, body weight loss or GvHD score, compared with mice receiving the appropriate isotype control, supporting the safety of this OX40-targeted imaging approach.CONCLUSIONThe OX40 immuno-PET tracer enabled specific imaging of alloreactive OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64Cu-DOTA-mAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting.FIGURE 1. 64Cu-DOTA-AbOX40 PET-CT imaging in a mouse model of acute GvHD. (A) Representative day 8 64Cu-DOTA-AbOX40 PET-CT images in BM controls or GvHD group. H, heart (including cardiac muscle and blood); Li, liver; Sp, spleen; Bl, blood vessels and venous sinuses; BM, bone marrow; Ab, abdomen. (B) Quantitative region of interest PET image analysis of indicated organs in BM controls (n=12, blue filled boxes) or GvHD mice (n=12, red filled boxes). Outliers are represented as dots. [Mann-Whitney test , ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05]. [Display omitted] DisclosuresGambhir:CellSight Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

A Critical Role of Host-Derived Semaphorin-4A for Regulating T Cell Immune Responses in Acute Graft Versus Host Disease

Semaphorins, originally identified as repulsive axon-guidance factors that participate in neuronal development. Several semaphorins are involved in various phases of immune responses, as has been shown recently. Semaphorin4A (Sema4A), a class IV transmembrane semaphorin, is preferentially expressed in dendritic cells (DCs) and Th1 cells. Previous studies suggested that Sema4A is involved in Ag-specific T cell priming, and in Th1 cell and Th17 cell differentiation. Additionally, Sema4A is required for the stability and function of Tregs. However, the role of Sema4A in alloimmune responses remains to be elucidated. In this study, we examined the contribution of Sema4A to T cell immune responses in the context of allogeneic hematopoietic stem cell transplantation (allo-HCT).To test the role of Sema4A in T cell proliferation, we performed in vitro T cell proliferation assay using dendritic cells harvested from wild-type or Sema4A-KO mice. Conventional CD4+T cells cocultured with DCs harvested from wild-type and Sema4A-KO mice proliferated equally in the presence of anti-CD3 antibody. In contrast, anti-CD3-induced proliferation of Tregs cocultured with DCs harvested from Sema4A-KO mice was attenuated compared to Tregs cocultured with DCs harvested from wild-type mice, suggesting that Sema4A is required for maximum proliferation of Tregs.We next investigated the effects of Sema4A deficiency in acute GVHD. We employed a murine acute GVHD model (B6→BALB/c) and monitored every day for survival. Body weight was assessed 2-3 times per week. First, to investigate the role of host Sema4A, lethally irradiated wild-type and Sema4A-KO mice were injected with T cell-depleted bone marrow cells and T cells isolated from wild-type B6. Sema4A-KO host mice developed significantly higher mortality compared to wild-type host mice (p<0.001) (Figure). On day 7 after allo-HCT, the proportion of Tregs in the spleen were significantly decreased in Sema4A-KO host mice compared to wild-type host mice (p<0.05). Both donor-derived preexisting natural Tregs and inducible Tregs in Sema4A-KO host mice were significantly decreased compared to wild-type host mice, although relatively less in inducible Tregs. We investigated the production of proinflammatory cytokines (IFN-g, IL-17, IL-4 and IL-13) of T cells in the spleen by intracellular cytokine staining. The proportion of IL-17+CD4+ T cells in Sema4A-KO host was slightly but not significantly decreased compared to wild-type host, whereas similar proportion of IFN-g+CD4+T cells was observed in Sema4A-KO and wild-type host mice. In contrast, the proportion of IL-4+CD4+ T cells in Sema4A-KO host was significantly increased in Sema4A-KO host mice compared to wild-type host mice, suggesting Sema4A deficiency skewed cytokine polarization of T cells after allo-HCT.Subsequently, we used Sema4A-KO and wild-type B6 mice donor to investigate the role of donor-derived Sema4A. Host mice transplanted from Sema4A-KO donor developed comparable mortality with host mice transplanted with wild-type donor, suggesting that donor-derived Sema4A does not play an important role in controlling graft versus host reactions in this model.Together, these results suggest that Sema4A contributes to Treg maintenance and the regulation of T cell responses after allo-HCT, which may have clinical implications for the novel approach in the treatment and protection of GVHD. [Display omitted] DisclosuresIto:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; Mundipharma: Honoraria; Celgene: Honoraria.

Bilirubin Nanoparticle as an anti-inflammatory therapy for graft versus host disease

Abstract Graft versus host disease (GvHD) caused by alloreactive donor lymphocytes is a fatal complication of hematopoietic stem cell transplant (HSCT). Conditioning regimen consisting of chemotherapy and/or radiation given prior to HSCT can cause the initial tissue damage, which triggers the cross-presentation of alloantigens to the donor immune cells. Previous studies have shown that water-soluble PEGylated bilirubin nanoparticles (BRNP) selectively accumulate at the site of inflammation and prevent further tissue damage through scavenging reactive oxygen species. Therefore we, hypothesized that BRNP treatment after conditioning regimen can reduce GvHD by abating the initial tissue damage in HSCT, and investigated therapeutic efficacy of BRNP using murine GVHD model. Sublethally irradiated recipient mice (Balb/c) were infused with 4×106 bone marrow and 1×106 splenic T cells from donor mice (C57/B6) on day 1, and with or without BRNP (10mg/kg) on days 0, 2, and 4. GvHD symptoms were monitored for 60 days, and scored for changes in fur, skin, posture, activity, and weight. Untreated recipient mice (n=9) developed significantly worse GVHD (mean GVHD score=4.667) compared to BRNP treated recipient mice (n=9, mean GVHD score=1.556) (p=0.0028). This translated into the significantly better survival of BRNP treated mice with day 60 survival of 65% as compared to the untreated recipient mice with day 60 survival of 11% (p=0.03). In summary, we show that BRNP treatment can ameliorate clinical GvHD symptoms and improve survival in murine GVHD model. In the future, we plan to investigate the role of reactive oxygen species in GVHD and mechanism of action on anti-GVHD effects by BRNP.

Blocking Two Signals of Immuno-Activated T Cells from Antigen Presenting Cells Can Prevent Chronic GVHD of Murine

To explore the effects of blocking TCR-CD3 and B7-CD28 signals on immune function of mice with chronic GVHD by using TJU103 and CTLA4-Ig. On the basis of foregoing murine model of chronic GVHD, according to interference modes after infusion 6×107 spleen cells of donor mice, the recipients were divided into 5 groups: blank control, cGVHD, TJU103 interference, CTLA4-Ig interference and TJU103+CTLA4-Ig interference groups. The score of clinical manifestation and tissue histopathology were used to evaluate the effects of all the interferences on chronic GVHD. TJU103 and CTLA4-Ig could not influence the formation of the mouse chimera. The analysis of Kaplan survival curve of mice with chronic GVHD showed that the CTLA4-Ig and TJU103+CTLA4-Ig reduced the incidence of chronic GVHD, the TJU103 could delay the occurrence of chronic GVHD, but all the interference factors could not change the severity of chronic GVHD. TJU103 can delay the onset time of chronic GVHD, and the CTLA4-Ig can reduce the incidences of cGVHD, the combining use of TJU103 and CTLA4-Ig can significantly reduce the incidence of chronic GVHD, but can not change the severity of chronic GVHD.

Chitinase 3-Like-1-Deficient Splenocytes Deteriorated the Pathogenesis of Acute Graft-Versus-Host Disease via Regulating Differentiation of Tfh Cells.

Acute graft-versus-host disease (aGVHD) is an intractable complication in transplant patients, limiting the efficacy of this therapy. Chitinase 3-like-1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, plays a critical role in a variety of inflammatory diseases. Here, we investigated the in vitro and in vivo effects of CHI3L1 on the development of aGVHD. In this study, mixed lymphocyte reactions (MLR) in vitro showed that CHI3L1 deficiency in CD4+ T cell promoted the production of interferon (IFN)-γ and T follicular helper (Tfh)-related cytokines such as interleukin-6 (IL-6) and interleukin-21 (IL-21). Meanwhile, the inducible Tfh cell population increased remarkably in CHI3L1-KO CD4+ T cells' induction group, compared with WT group. Then, in the murine acute GVHD model, we found that CHI3L1 deficiency in donor splenocytes dramatically increased the severity of aGVHD through enhancing Tfh cell differentiation. Moreover, at mRNA and protein levels, we defined several molecules that may account for the enhanced ability of CHI3L1-KO splenocytes to migrate into target organs and produce IFN-γ and Tfh-related cytokines and chemokines, such as IL-6, IL-21, and CXCL13. Expression of inducible co-stimulator (ICOS) and B cell lymphoma 6 (Bcl6) increased in the skin, the intestine, the lung, and the liver from CHI3L1-KO splenocyte-treated aGVHD mice. Therefore, these results strongly imply that CHI3L1 levels in donor cells may be related to the risk of aGVHD and targeting CHI3L1 represents a novel therapeutic strategy for controlling aGVHD progression.

Busulfan-Cyclophosphamide Worsens Peripheral Gvhd While Fludarabine-Busulfan Results in Wide Bone Marrow Involvement in a Murine Model of Acute Gvhd

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and A proper conditioning regimen is vital to stop the development of aGVHD. To provide a platform for the study of aGVHD and evaluate the impact of different conditioning regimens, we established a murine aGVHD model that simulates the clinical situation and can be conditioned with Busulfan-Cyclophosphamide (BU-Cy) and Fludarabine-BU (Flu-BU). In our study, BALB/c mice were conditioned with BU-Cy or Flu-BU and transplanted with 2×107 bone marrow cells and 2×107splenocytes from either allogeneic (C57BL/6) or syngeneic (BALB/c) donors. The allogeneic recipients conditioned with BU-Cy had shorter survival (P<0.05) and more severe hepatic and intestinal clinical manifestations and pathological changes associated with increased INF-γ expression and diminished IL-4 expression in serum compared to allogeneic recipients conditioned with Flu-BU. Meanwhile increased donor-derived T-cell infiltration and impaired bone marrow B-cell development could be seen in the aGVHD mice conditioned with Flu-BU. Our study showed that the conditioning regimen with BU-Cy resulted in more severe peripheral aGVHD, while the Flu-BU regimen was associated with aGVHD with wide bone marrow involvement. DisclosuresNo relevant conflicts of interest to declare.

A Calcineurin Inhibitor Does Not Affect the Post-Transplantation Cyclophosphamide -Induced Regulatory T Cell Expansion after Bone Marrow Transplantation in a Murine Chronic Gvhd Model

Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Prolonged administration of a calcineurin inhibitor, such as cyclosporin A (CsA), does not decrease the risk of chronic GVHD. Indeed, we and others have reported that long-term use of CsA impairs reconstitution of bone marrow-derived regulatory T cells (Tregs) and increases susceptibility to chronic GVHD. Recently, the administration of post-transplantation cyclophosphamide (PTCY) has been used widely in GVHD prophylaxis and lower incidences of acute and chronic GVHD have been reported. Here, we evaluated the effects of PTCY and CsA on Tregs and cytokine production in effector T cells using the well-defined chronic GVHD mouse model of B10.D2 (H2d) cells into BALB/c (H2d) mice. Sublethally-irradiated recipient BALB/c mice were transplanted with 8.0 × 106 T cell-depleted bone marrow cells and 2.0 × 106 T cell-replete spleen cells from donor B10.D2 mice. PTCY was administered by intraperitoneal injection at days 3 and 4 post bone marrow transplantation (BMT). CsA was injected peritoneally once daily, starting on day 0 of BMT until the day of analysis. Administration of PTCY, with or without CsA, improved the clinical chronic GVHD score significantly at day 50 versus the controls and the CsA-alone group (controls: 2.8 ± 0.34, PTCY: 0.5 ± 0.0, p < 0.01; CsA alone: 1.8 ± 0.73, PTCY-CsA: 0.5 ± 0.0, p < 0.01). There was no significant difference in the GVHD score between the PTCY and PTCY-CsA groups. Histopathological examinations of the skin at day 50 after BMT also showed significantly reduced chronic GVHD damage in the PTCY group versus the controls and the CsA-alone group (controls vs. PTCY, p < 0.001; CsA alone vs. PTCY, p < 0.01). Flow cytometry analysis of the cells isolated from the peripheral lymph nodes 28 days after BMT revealed that the proportions of Tregs to CD4+ T cells in the PTCY (25.8 ± 2.1%) and PTCY-CsA (29.5 ± 2.2%) groups were remarkably higher than the controls (8.3 ± 2.7%) and CsA-alone (6.7 ± 1.6%) groups (p < 0.05). There was no significant difference in the Treg ratio between the PTCY and PTCY-CsA groups. Intracellular staining showed that IFN-γ single-positive and IFN-γ/IL-17 double-positive CD4 T cells were well suppressed in the PTCY and PTCY-CsA groups versus the controls and CsA-alone group (controls: 38.2 ± 3.5%, PTCY: 11.9 ± 3.1%, p < 0.01; CsA alone: 46.9 ± 2.2%, PTCY-CsA: 17.9%, p < 0.01). Our data suggest that PTCY treatment attenuated chronic GVHD via enhanced reconstitution of donor Tregs and suppression of IFN-γ/IL-17-producing effector T cells. Moreover, CsA does not appear to hinder this suppressive effect of PTCY. This result seems helpful for understanding how PTCY prevents chronic GVHD in clinical settings. DisclosuresMaeda:Mundipharma KK: Research Funding.

Role of IL-26+CD26+CD4 T Cells in Pulmonary Chronic Graft-Versus-Host Disease and Treatment with Caveolin-1-Ig Fc Conjugate.

Obliterative bronchiolitis is the primary noninfectious pulmonary complication after allogeneic hematopoietic cell transplantation and the only pathognomonic manifestation of pulmonary chronic graft-versus-host disease (cGVHD). In our recent study, we identified a novel effect of IL-26, which is absent in rodents, on transplant related-obliterative bronchiolitis. Sublethally irradiated NOD/Shi-scidIL2rγnull mice transplanted with human umbilical cord blood gradually exhibited obliterative bronchiolitis with increased collagen deposition and predominant infiltration with human IL-26+CD26+CD4 T cells. Moreover, we showed that IL-26 increased collagen synthesis in fibroblasts in vitro and that collagen contents were increased in a murine GVHD model using IL26 transgenic mice. In vitro analysis demonstrated a significant increase in IL-26 production by CD4 T cells following CD26 costimulation, while immunoglobulin Fc domain fused with the N-terminal of caveolin-1, the ligand for CD26, (Cav-Ig) effectively inhibited production of IL-26. Administration of Cav-Ig before or after onset of GVHD impeded the development of clinical and histologic features of GVHD without interrupting engraftment of donor-derived human cells, with preservation of the graft-versus-leukemia effect. We concluded that cGVHD of the lungs is caused in part by IL-26+CD26+CD4 T cells, and that treatment with Cav-Ig could be beneficial for cGVHD prevention and therapy.