Cell-depleted Bone Marrow Cells Research Articles

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.

Iron Overload and Iron-Induced Microbiome Changes May Affect Lymphoid Levels Post Bone Marrow Transplant and Graft-Versus-Host Disease Survival in Mice

Introduction: Bone Marrow Transplant (BMT) represents the only cure for many malignant and non-malignant hematopoietic disorders. This treatment is often accompanied by multiple red blood cell transfusions, which can lead to iron overload. Iron overload is associated with poorer outcomes following BMT, such as increased risk of infections or Graft-versus-Host Disease (GVHD); however, it is not yet clear whether this correlation is caused by iron itself or is simply confounded by patients with more severe disease requiring more transfusions. Previous studies using mouse models, have shown that transfusional iron overload reduces the population of beneficial bacteria in the gut microbiome or decreases the production of microbiome-produced metabolites (i.e. indoles) that have been associated with BMT outcomes and GVHD risk. Here, we developed a reductionist mouse model of iron overload and BMT to investigate the effect of iron and iron-induced changes to gut microbiota and their metabolites on BMT outcomes and GVHD survival. Methods: Female Balb/C mice were infused twice a week, for two weeks, with a total of 50 mg of iron dextran or saline followed by lethal irradiation (9 Gy). Cohorts of mice (n=10 per group) received 1x10^10 T cell-depleted bone marrow cells with or without 5x10^6 T cells from UBC-GFP C57Bl6 mice to establish allogenic BMT and GHVD models, respectively. Mice were then monitored daily for survival with a formal severity score evaluated twice a week. Analysis of donor red blood cells, platelets, and white blood cells (neutrophils, B and T cells) from peripheral blood was also performed once a week to check for engraftment. To examine the impact of iron-induced microbiota changes, fecal microbiome transplant (FMT) was performed in mice receiving BMT. Briefly, mice were treated with an antibiotic cocktail (ampicillin 1 g/L, kanamycin 1 g/L, metronidazole 1g/L, vancomycin 0.5g/L) by gavage 2 days before the start of FMT. Feces from control and iron overloaded mice were collected freshly and weighed. Stool pellets were then resuspended in PBS (100 mg feces/ml PBS), vortexed and then spun down at 800g for 3 minutes. Mice received 100ul of supernatant by gavage. Additional cohorts of mice were administrated with indole-3 proprionic acid (IPA) in the drinking water at a concentration of 0.1 mg/ml prior to irradiation throughout the experiment and monitored for survival after receipt of allogeneic T cells to induce GVHD. All mice received enrofloxacin (10mg/kg) starting on the day of BMT and for the following 3 days by intraperitoneal injection. Results: There was a significant reduction in overall lymphoid (CD45+ CD172a- CD11b-/low GFP+), B (CD45+ CD172a- CD11b-/low CD19+ GFP+), and T (CD45+ CD172a- CD11b-/low CD3+ GFP+) cells in iron overloaded mice compared to control mice (p<0.0001 at day +28) by flow cytometry analysis in peripheral blood after BMT. Although FMT from control feces into iron overloaded mice did not affect lymphoid numbers, FMT from iron overloaded feces into control mice reduced the number of donor lymphoid and B cells (p<0.05 at day +28; Figure 1A). Iron overload reduced survival compared to control mice in the GHVD mouse model (p<0.05 Mantel-Cox test). Administration of IPA in iron overloaded mice did not significantly affect overall survival (Figure 1B). Conclusions: Our results show that iron overload negatively impacts lymphocyte engraftment in a mouse model of BMT. Furthermore, iron overload also reduces survival in mice with GVHD. Iron overload was previously shown to reduce indole levels in mice. Although we did not observe a significant impact of exogenous indole administration on GVHD survival, FMT from iron overloaded into control mice reduced levels of lymphoid and B cells after BMT implicating gut microbiota in this outcome. Taken together, these results suggest that iron overload is an important factor that negatively affects BMT outcomes. The extent to which iron-induced gut microbiota changes are involved in the mechanisms responsible for this effect requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Indole-3-Carbinol Supplement Reduces GvHD Related Mortality in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Introduction: Following allogeneic hematopoietic stem cell transplant (allo-HCT), donor T cells react against host antigens and cause severe graft versus host disease (GvHD) (Majhail N.S. et al., 2015. Biol Blood Marrow Transplant). Disruption of gut integrity is one of the major contributors to GvHD morbidity and mortality. Dysbiosis due to GvHD decreases host immunity and increases transplant-related mortality (Taur Y. et al., 2014. Blood). Indole, a bacterial metabolite, and indole derivatives are known to ameliorate host immunity and gut epithelial barrier function through modulating goblet cells, innate lymphoid cells (ILCs), dendritic cells (DCs) and T cell subsets (Gutiérrez-Vázquez C. & Quintana F.J., 2018. Immunity; Hubbard T.D. et al., 2015.Sci Rep). Indoles act via the aryl hydrocarbon receptor (AHR) expressed in innate lymphoid cells to generate regulatory T cells (Tregs) while inhibiting Th1 polarization (Gutiérrez-Vázquez C. & Quintana F.J., 2018. Immunity; Hubbard T.D. et al., 2015. Sci Rep). Previously, we showed that enteral administration of indole 3-carboxaldehyde (ICA) to murine allo-transplant recipients reduces GvHD severity and mortality (Swimm A. et al., 2018. Blood). However, since ICA is not available as a GMP-compliant FDA- approved pharmaceutical, our goal is to identify indoles for clinical research that have protective effects against GvHD. Here, we report that indole-3-carbinol (I3C), an indole currently marketed as a nutritional supplement, induces Treg generation and higher survival in allo-HCT. Methods: To study modulation of T cell subsets by indoles, mice were gavaged daily for 14 days with corn-oil suspensions of 50, 100, 200 or 400mg/kg of I3C, 150mg/kg of ICA, or corn oil alone. On day 15, mice were euthanized and splenocytes were isolated to study T cell subsets. To investigate the effect of I3C on GvHD, lethally irradiated (9Gy) B10.BR (H-2Kk) mice received 5 x 106 T cell-depleted bone marrow (TCDBM) cells with or without 2 x 106 T cells from MHC mismatched C57BL/6 (H-2Kb) donors. Allo-transplanted mice received either vehicle or 200mg/kg of I3C daily from day -2 to day 40. Mice were weighed and assessed twice a week for first 30 days and once a week afterwards. Lethally irradiated (9Gy) C57BL/6 mice were received 5 x 106 TCDBM cells with or without 5 x 105 T cells from FVB (H2Kq) donors and 14 days of daily gavage with vehicle or 200 mg/kg of I3C or 150mg/kg of ICA to study gut bacterial translocation into mesenteric lymph nodes (MLN). On the following day, MLNs were collected, immediately homogenized in 0.25ml sterile PBS and cultured (100ul) on Blood Agar plate to determine CFU/g tissue. Results: Percentages of regulatory T cells (Tregs) in the spleen were significantly increased at 14 days in non-transplanted mice treated with 200mg/kg of I3C (11.38%, n=5), 400mg/kg of I3C (11.48%%, n=5) and ICA (11%%, n=5) compared to mice treated with vehicle (6.74%%, n=5) (Figure 1), whereas frequencies of CD8+ T cells were unchanged. Allo-transplanted mice treated with I3C had higher survival (57.14%, n=7) compared to the vehicle-treated control group (33.33%, n=9) (Figure 2). However, weight gain was slower in the I3C-treated mice compared to surviving vehicle-treated mice. Allo-transplanted recipient mice showed trend of reduced bacterial translocation into MLNs of mice that received 200mg/kg of I3C and ICA. Our results indicate I3C supplement enhances rate of survival and decreases gut bacterial translocation into MLNs in allo-HCT recipients. Continuing experiments will study bacterial composition, histological changes, and determine mechanisms for I3C restoration of gut integrity in all-HCT recipients. Conclusion: The results above suggest indoles increase regulatory T cells. Allo-bone marrow transplant recipients treated with indoles had improved survival. We propose I3C as a prospective supplement to treat GvHD in all-HCT patients. Keywords: Allo-HCT, GvHD, Indoles, I3C, Tregs, Survival Acknowledgement: Research reported in this publication was supported in part by the Morningside Center for Innovative and Affordable Medicine through the generous support of donors and Emory University. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Graft-Versus-Host Disease Induces Immune Dysregulation through Irreversible Damage to Specialized Immune-Interacting Fibroblastic Stromal Cells in Secondary Lymphoid Organs

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially life-saving treatment for patients with leukemia and other hematological disorders. However, its success is limited by graft-versus-host disease (GVHD), a life-threatening syndrome caused by alloreactive donor T cells that induce damage in normal host tissues. Many patients who survive transplantation develop chronic GVHD which can be severe and has few effective therapeutic options. Chronic GVHD is characterized both by immune hyperreactivity, reminiscent of autoimmunity, and defective immune responses, including poor responses to vaccination and infectious challenges. The mechanisms underlying the complex immune dysregulation of chronic GVHD remain incompletely understood. Previous work indicates that pathogenic alloreactive T cells can damage lymph node fibroblastic stromal cells, a group of specialized cells increasingly recognized to regulate multiple aspects of immune homeostasis. Using mouse models of allo-HCT, we sought to identify the populations of specialized fibroblastic stromal cells and other stromal elements that were most sensitive to GVHD-mediated injury and to assess the functional impact of this damage during chronic GVHD. We used a sublethal allo-HCT model with a low T cell dose that allowed for long-term survival over 100 days after transplantation (B6 into lethally irradiated B6xBALB/c F1 mice, 2x 5.5 Gy). Unlike allo-HCT recipients of T cell-depleted bone marrow (no GVHD control), recipients of T cell-depleted bone marrow plus allo-T cells (1-5x106 splenocytes) developed low-grade systemic GVHD with mild reversible weight loss. At day 30-60 after transplantation, allo-T cell recipients showed modestly decreased thymopoiesis, consistent with the thymus being a sensitive target organ of GVHD, as previously reported. In contrast to this mild injury, peripheral lymph nodes (pLN) showed profound long-lasting depletion of CD31-/podoplanin+ fibroblastic stromal cells as assessed by flow cytometry. Among these cells, MAdCAM-hi marginal reticular cells (MRCs) and CD157-hi fibroblastic reticular cells were most severely affected, including most cells lineage-traced by a Ccl19-Cre transgene. Total blood endothelial cells and lymphatic endothelial cells (LECs) were only modestly decreased by GVHD at day 30, but the LEC subset characterized by high MAdCAM expression consistent with a floor LEC phenotype virtually disappeared. Thus, MRCs and floor LECs lining the marginal sinus were severely affected by GVHD, suggesting that antigen uptake and processing could be defective in these pLN. Single cell RNA-seq revealed a near complete loss of floor LECs, MRCs and Ccl19/Ccl21+ T zone FRCs in GVHD-affected pLNs. Instead, residual fibroblastic stromal cells showed accumulation of collagen-expressing Cxcl14+ fibroblasts with loss of immune-interacting properties. In terms of functional consequences, GVHD pLNs had a markedly decreased abundance of naïve B and T cells, the latter despite relatively preserved naïve T cell production in the thymus. Compared to pLNs, the spleen was less profoundly affected and remained more receptive than pLNs to homing of adoptively transferred T cells. Immune responses to SARS-CoV2 mRNA used as a potent and clinically relevant T-dependent antigen were profoundly impaired in GVHD pLNs. In summary, our data suggest that specialized fibroblastic stromal cells and LECs in secondary lymphoid organs are even more sensitive to GVHD than thymic epithelial cells, with little evidence of functional recovery. We speculate that subclinical damage to pLN stromal compartments may underlie many aspects of immune dysregulation in chronic GVHD, including persistently impaired responses to vaccination and loss of peripheral tolerance.

Allogeneic MHC-matched T-cell receptor α/β-depleted bone marrow transplants in SHIV-infected, ART-suppressed Mauritian cynomolgus macaques

Allogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay. Thus, we infected four MCMs with CCR5-tropic SHIV162P3 and started them on ART 6–16 weeks post-infection (p.i.), maintaining continuous ART during myeloablative conditioning. To prevent graft-versus-host disease (GvHD), we transplanted allogeneic MHC-matched α/β T cell-depleted bone marrow cells and prophylactically treated the MCMs with cyclophosphamide and tacrolimus. The transplants produced ~ 85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their blood post-transplant. However, SHIV-harboring cells persisted in various tissues, with detectable viral DNA in lymph nodes and tissues between 38 and 62 days post-transplant. Further, removing one MCM from ART at 63 days post-transplant resulted in SHIV rapidly rebounding within 7 days of treatment withdrawal. In conclusion, transplanting SHIV-infected MCMs with allogeneic MHC-matched α/β T cell-depleted bone marrow cells prevented high-grade GvHD and decreased SHIV-harboring cells in the blood post-transplant but did not eliminate viral reservoirs in tissues.

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microrna-191 Regulates T-Cell Clonal Expansion during Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation is a potentially curative treatment choice for a wide variety of hematological malignancies. However, graft-versus-host disease (GVHD), which is mediated by donor alloreactive T cells, limits the success of this procedure. Previous studies have demonstrated that several microRNAs (miRs) modulate graft-versus-host disease. miR-191 was previously reported to be able to support T cell survival after TCR stimulation. We hypothesize that miR191 regulates T cell response during GVHD. To test this hypothesis, we first studied miR-191 expression in alloreactive T cells. The result demonstrated that miR-191 was up-regulated in donor T cells isolated from murine GVHD recipients, suggesting that miR-191 may play a role in GVHD induction. We further studied the role of miR-191in GVHD using miR-191 deficient T cells (KO). Lethally irradiated (8.5 Gy) BALB/c mice were injected intravenously with 1×107 T cell-depleted bone marrow (TCDBM) cells along with 1×106 purified T cells from wild-type (WT) or KO mice, which are in C57BL/6 background. Interestingly, all recipients in the WT group died within 35 days after transplantation, while only one out of ten animals died in the KO group during an observation period of 56 days. Body weights and clinical scores were also improved in KO T cell recipients when compared with the WT controls. Similar results were also observed in a second GVHD model (C57BL/6→C3H/HeJ). To understand the mechanism by which miR-191 KO T cells have decreased ability to mediate GVHD, we first measured the ability of KO T cells to respond to alloantigens in vitro in a mixed lymphocytes reaction assay. Dramatically decreased alloresponse was observed with KO T cells as compared with WT T cells. Similarly, decreased clonal expansion was observed in KO T cells in vivo upon challenge with alloantigens as measured by bioluminescent imaging (Figure 1A). These results were further supported by data from a co-transfer experiment, in which equal numbers of WT and KO T cells were transplanted into the same GVHD recipient. At day7 after transplantation, KO T cells showed significantly reduced expansion in the spleen and liver compared with WT T cells. Reduced alloresponses mediated by KO T cells may not due to decreased proliferative capability directly as an in vivo carboxyfluorescein succinimidyl ester (CFSE) assay showed a comparable cell division between WT and KO T cells upon challenge with alloantigens. Rather, increased cell death is responsible for decreased alloresponse observed in KO T cells because dramatically increased number of dead cells was observed in KO group compared with WT group upon response to alloantigens in vitro and vivo. To determine the genes that are regulated by miR-191, we did a screening based on the prediction. Humans and mice share more than 100 predicted targets for miR-191. We chose top 20 of these targets for RT-qPCR screening. The result demonstrated that Taf5 was a target gene of miR-191. Expression of TAF5 protein was down-regulated in activated KO T cells when compared with the WT T cells. Finally, we investigated whether miR-191 KO T cells preserve graft-versus-leukemia effects. 1×106 T cells from WT or KO mice were transplanted into lethally irradiated BALB/c mice along with 1×107 TCDBM cells and 1×105 host-type BCL-1 cells. While all recipients that received only TCDBM and tumor cells developed lethal leukemia/lymphoma, none of WT and KO T cells recipients developed tumor. In conclusion, our findings reveal a critical role of miR-191 during GVHD process and demonstrate that miR-191 is a novel therapeutic target for GVHD. Figure 1 Disclosures No relevant conflicts of interest to declare.

Baricitinib Prevents and Treats GvHD through Both Immune Modulation and Enhancement of Tissue Repair

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the major curative immunotherapy for hematologic malignancies. The therapeutic benefits of allo-HSCT are primarily derived from an anti-leukemia effect or graft-versus-leukemia (GvL) that is mediated by mature T cells in donor grafts. Unfortunately, these T cells could react against recipient tissues resulting in graft-versus-host disease (GvHD), a life-threatening complication post allo-HSCT. Due to this strong association between GvL and GvHD, developing a more effective therapeutic strategy to selectively suppress GvHD while preserving or enhancing the GvL effects remains a fundamental yet unmet medical need.We have previously published that co-blockade of interferon gamma receptor (IFNGR) and interleukin-6 receptor (IL6R) results in a complete prevention of GvHD (Choi et al., Leukemia, 2018). Moreover, in vivo administration of baricitinib, an inhibitor of Janus kinases 1 and 2 (JAK1/JAK2) which are downstream of IFNGR and IL6R, also prevents GvHD while preserving GvL in our preclinical murine model. The possible mechanistic pathways by which baricitinib mitigates GvHD include robust upregulation of regulatory T cells (Tregs), inhibition of GvHD-inducing Th1/Th2 cell differentiation, an altered T cell trafficking away from GvHD organs, and reduction of antigen presenting cells' allo-reactivity.Given the potent modulatory effect of baricitinib on GvHD and GvL, we next sought to determine the minimum duration of baricitinib treatment for the complete prevention of GvHD. Since we have found that baricitinib significantly increases Tregs during the first two weeks of the treatment, we hypothesized that two weeks of baricitinib administration is sufficient to completely prevent GvHD. We performed allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with pan T cells (5x105) were intravenously injected into lethally irradiated Balb/c mice (H-2d). Baricitinib was subcutaneously administered to the recipient mice once a day (5 days/week) for a period of one, two, three, or four weeks following allo-HSCT. Overall survival rates, clinical GvHD scores, complete blood counts, immunophenotyping of blood cells and histopathological scores were assessed to determine whether the GvHD was prevented. We have found that two to four-week treatment groups demonstrate significantly better overall survival rates, blood count recovery and histopathological scores than vehicle control or the one-week treatment groups, while no such significant differences were observed among the other groups (Fig. A). All of these data suggest that two weeks of baricitinib treatment is sufficient for complete prevention of GvHD.Lastly, we have previously reported that baricitinib reverses established GvHD (Choi et al., Leukemia, 2018) and we have recently found that both JAK1/JAK2 inhibitors, ruxolitinib and baricitinib, significantly enhance human intestinal organoid growth in vitro (Fig. B). Of note, the intestines are frequently diagnosed and severely damaged GvHD target organs in both human patients and our mouse models of GvHD. Thus, we hypothesized that baricitinib not only modulates immune cell functions as described above, but also promotes restoration of tissues damaged by GvHD. To test this hypothesis, we delayed baricitinib administration until mice developed clinically apparent GvHD and administered baricitinib starting on days 10, 17, or 31 after allo-HSCT for a period of two weeks. Despite this latency, the day 10 group showed 100% overall survival rate (5% in control, p<0.0001) while 70% and 60% in the days 17 and 31 groups, respectively (Fig. C). According to histopathological analysis of the three major GvHD target organs (GI, skin, liver) the day 10 group also demonstrated a significantly lower total grade than the vehicle control (Fig. D) whereas only a trend towards lower GvHD scores were observed in the rest of the groups. Hence, our data suggest that baricitinib reverses established GvHD by promoting tissue regeneration in GvHD damaged organs.In conclusion, our studies demonstrate that baricitinib-induced blockade of IFNGR and IL6R represents a promising therapeutic strategy for both prevention and treatment of GvHD while preserving GvL. Molecular mechanisms underlying baricitinib-mediated reversal of established GvHD are being currently investigated and will be presented. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Signal Transducing Adaptor Protein (STAP) Family Accelerates Gut and Thymic Graft-Versus-Host-Disease in Murine Model

Graft-versus-host-disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most frequent complication and one of the major causes of non-relapse mortality. However, its pathogenesis has not yet been fully understood. We cloned signal-transducing adaptor protein (STAP)-2 as a c-fms binding protein from a fetal liver library in 2003. The family that contains STAP-1 and STAP-2 has a pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains, suggesting that this adapter protein functions as an immune and inflammatory regulator. Indeed, STAP-2 regulates adhesion and chemotaxis in T cells (Sekine et al., J Immunol. 2009). In this study, we aimed to elucidate the roles of STAP family in GVHD.First, we examined the expression of STAP-1 and STAP-2 mRNA in various human hematopoietic subsets, including CD34+ CD38- hematopoietic stem cells (HSCs), CD34+ CD38+ hematopoietic progenitor cells (HPCs), CD19+ CD27- naïve B cells, CD19+ CD27+ memory B cells, CD3+ CD4+ helper T-cells, and CD3+ CD8+ cytotoxic T lymphocytes, using real-time PCR. As a result, STAP-1 and STAP-2 were expressed in lymphoid cells, as well as HSCs and HPCs. STAP-2 mRNA was highly expressed in T cells. Next, to investigate the role of STAPs in GVHD, we made an experimental murine model. To study the pathogenesis of immune reconstitution and tolerance after allo-HSCT, lethally irradiated BALB/c mice were injected with T and B cell-depleted bone marrow cells (5×106 cells) derived from syngeneic BALB/c or allogeneic C57BL/6 mice on day 0. Co-transplantation of splenocytes was not adapted in this model. Survival and clinical degree of GVHD were assessed by a scoring system that sums changes in 5 clinical parameters: body weight (BW) loss, posture, activity, fur texture, and skin integrity. Recipients transplanted from allogenic wild type (WT) C57BL/6 donor survived without suffering from severe GVHD symptoms, owing to development of immune tolerance against allogeneic antigens. However, compared to syngeneic transplanted mice, these recipients started to show gradual BW loss and GVHD score was increased approximately 28 days after allo-HSCT, indicating the existence of an allogeneic immune reaction. To evaluate the role of STAPs in GVHD, we generated transgenic mice (Tg) that overexpress STAP under the control of Em enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from the common lymphoid progenitor (CLP) stage. When STAP-2 Tg marrow was used as a donor source, we found that the overall survival of STAP-2 Tg recipients was significantly lower than that of WT recipients (22.2% and 91.7%, respectively; p<0.001) on day 60. STAP-2 Tg recipients showed decreased BW and had a higher clinical GVHD score with statistical significance compared to control WT recipients. The overexpression of STAP-1 also exacerbated the severity of GVHD. At day 42, BW was decreased by 16.3% in WT recipients. In contrast, recipients of STAP-1 Tg and the STAP-2 Tg donor showed more severe BW loss along with diarrhea (23.3% and 29.8%, respectively). STAP-1 as well as STAP-2 Tg recipients showed significantly worsened GVHD scores, and this lasted until day 90 at the end of follow-up. In histologic examination of both STAP Tg recipients, inflammatory damages with lymphocyte infiltration were most notably observed in the colon. Interestingly, we found that thymus was atrophic or indistinguishable and the cortico-medullary junction disappeared. Moreover, compared to control WT recipients, the number of CD4+ CD25+ regulatory T (Treg) cells in the peripheral blood was significantly low in STAP-2 Tg recipients on day 60 (WT vs STAP-2 Tg; 44.0 /μL vs 18.2 /μL; p<0.05).In this study, we show that STAPs in reconstituted lymphocytes after allo-HSCT regulate the pathogenesis of GVHD. Our results suggest that STAP activation in lymphocytes during immune reconstitution accelerates gut and thymic GVHD. Severe thymic damage induced by STAP overexpression might contribute to impairment of immune tolerance such as a decreased number of Treg cells as well as dysfunction of thymic negative selection of host-reactive T cells after allo-HSCT, which is involved in persistence of GVHD. Future study should further elucidate the detailed molecular mechanisms involved. DisclosuresIchii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Third-party regulatory T cells prevent murine acute graft-versus-host disease.

Background/AimsAdoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation.MethodsTo induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 105 T cell-depleted bone marrow cells and 5 × 105 CD4+CD25– splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 105 cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1).ResultsSystemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups.ConclusionsOur findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

Establishment of Acute GVHD Mouse Model of Haplo identical Hematopoietic Stem Cell Transplantation and Its Pathogenesis

To establish the acute graft-versus-host disease(GVHD) mouse model based on haplo identical hematopoietic stem cell transplantation (HSCT) and to further investigate the pathogenesis of GVHD. Recipient mice ([C57BL/6×CBA/Ca]F1(H-2b×k)) received lethal irradiation (11 Gy) of γ ray (137Cs), followed by transplantation with donor-derived [C57BL/6(H-2b)] T cell-depleted bone marrow cells (TD-BM) with or without donor-derived T cells. Recipients were randomly divided into 4 groups, including irradiation group, TD-BM group, TD-BM+T cell group 1 and TD-BM+T cell group 2. Survival rate and weight change were detected after HSCT. Pathological scoring were performed on the collected organs from recipients on day 14. Moreover, bone marrow chimerism was detected on days 14 and 18 after HSCT. Additionally, differentiation of donor-derived T cells towards Th1 cells in vivo was analyzed by flow cytometry. Furthermore, proliferation of donor-derived T cells in vivo was tested using CFSE. The average survival in TD-BM, irradiation, TD-BM+T cell group 1 and TD-BM+T cell groups 2 were 60, 13.29±5.50, 33±2.3 and 29.14±1.77 days, respectively. On day 14 after transplantation, recipients of TD-BM + T cell group 1 displayed significantly more severe pathology in liver, colon, lung and skin. On days 14 and 28 after HSCT, bone marrow chimerism in recipients of both TD-BM group and TD-BM+ T cell groups 1 was over 94%. On day 14 after HSCT, serum cytokines IFN-γ, TNF-α and IL-1 levels in TD-BM+T cell group 1 were significantly higher than those in TD-BM group. Percentage of H-2b+H-2k-CD4+IFN-γ+ in spleen cells of recipients in TD-BM+T cell group 1 was higher than that of TD-BM group, with (0.5240±0.08447)% vs (7.912±0.6087)% (P<0.05). On days 14 after HSCT, donor-derived T cells displayed obvious proliferation in vivo. C57BL/6(H-2b)→［C57BL/6×CBA/Ca］F1(H-2b×k) HSCT model can be used as a aGVHD model of haploidentical HSCT. Additionally, Th1 may play an important role in the pathogenesis of aGVHD resulting from haplo-identical HSCT.

Delayed Donor Bone Marrow Infusion Induces Liver Transplant Tolerance

Nonmyeloablative conditioning followed by donor bone marrow infusion (BMI) to induce tolerance has not been robustly tested in liver transplantation (LT) and may be unsafe at the time of LT. We hypothesized T cell-depleted BMI is effective in inducing tolerance when delayed after LT, resulting in potentially safer future clinical applications. Nonimmunosuppressed syngeneic (Lewis to Lewis) and allogeneic (ACI to Lewis) rat LT transplants were initially performed as controls. Three experimental allogeneic LT groups were treated with tacrolimus (TAC) for 3 to 4 weeks and then underwent: (1) TAC withdrawal alone; (2) nonmyeloablative conditioning (anti-αβTCR mAb + total body irradiation [300 cGy]) followed by TAC withdrawal; (3) Nonmyeloablative conditioning + donor BMI (100 × 10 T cell-depleted bone marrow cells) followed by TAC withdrawal. All group 1 recipients developed chronic rejection. Group 2 had long-term survival but impaired liver function and high donor-specific antibody (DSA) levels. In contrast, group 3 (conditioning + BMI) had long-term TAC-free survival with preserved liver function and histology, high mixed chimerism and blood/liver/spleen CD4 + CD25 + Foxp3+ regulatory T cells, and low DSA titers, similar to syngeneic grafts. While donor-specific tolerance was observed post-BMI, graft-versus-host disease was not. These results support that donor-specific tolerance can be achieved with BMI even when delayed after LT and this tolerance correlates with increased mixed chimerism, regulatory T cell generation, and diminished DSA.

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.

GM-CSF Therapy Expands Regulatory T Cells and Protects Against Chronic Graft Versus Host Disease

Regulatory T cells (Tregs) possess the ability to suppress chronic graft-versus-host disease (cGVHD). Hence, the in vivo expansion of Tregs can be used as therapy against cGvHD. In addition to IL-2, Tregs require TCR and costimulatory signals from antigen presenting cells such as dendritic cells (DCs) for their optimal proliferation. Both fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony stimulation factor (GM-CSF) induce the development of DCs and promote the proliferation of Tregs in a DC-dependent manner. GM-CSF preferentially increases CD11c+CD8a- DCs, whereas FLT3L more equally supports the development of many DC subsets. However, it is unknown whether GM-CSF-mediated CD11c+CD8a- DC expansion leads to the proliferation of Tregs and contributes to the inhibition of alloimmune responses against host antigens.To test whether the injection of GM-CSF augments Tregs and ameliorates cGVHD, we used a MHC-matched mouse cGVHD model (B10.D2 → Balb/c). Balb/c mice were lethally irradiated (850 cGy) and transplanted with 8 × 106 T cell-depleted bone marrow cells and 3.5 × 106 CD4+ T cells from either syngeneic or B10.D2 mice. Host mice were treated with vehicle or GM-CSF (in the form of immune complexes; GM-CSF ICs) for 3 days (days 17-19) and monitored for skin GVHD score and mortality. We used GM-CSF ICs because the injection of GM-CSF ICs but not GM-CSF itself increased splenic CD11c+CD8a- DCs and Tregs. The administration of GM-CSF to allogeneic host mice significantly protected against GVHD-induced skin diseases (p<0.001) (Fig 1). Similar results were obtained when GM-CSF ICs were administrated at a later stage (days 27-29). Although CD11c+CD8- DCs were decreased in mice transplanted from the allogeneic donor compared with mice transplanted from the syngeneic donor, the administration of GM-CSF increased the CD11c+CD8a-/CD11c+CD8a+ DC ratio. However, the expansion of macrophages was not observed in mice administered GM-CSF. Expectedly, the administration of GM-CSF increased Tregs in the peripheral blood and the peripheral lymph nodes (PLNs) (P<0.05) (Fig 2). We investigated the production of proinflammatory cytokines (IFN-g, IL-17) of CD4+ T cells in the spleen, PLNs, and the skin by intracellular cytokine staining, as these cytokines are important for cGVHD pathogenesis in this model. The proportion of IFN-g+CD4+ T cells in the spleen and PLNs was slightly but not significantly decreased in GM-CSF-administered mice. The proportion of IL-17+CD4+ T cells in the skin was decreased in GM-CSF-administered mice compared to vehicle-administered mice. We next investigated the production of IL-2 and IL-10, as these cytokines are associated with survival and function of Tregs. Skin infiltrating Tregs were not increased in GM-CSF-administered mice compared to vehicle-administered mice; however, the proportion of IL-10+ Tregs was increased in GM-CSF-administered mice. The proportion of IL-2+CD4+ T cells was comparable in the allogeneic host with or without GM-CSF, indicating that GM-CSF-induced Treg expansion did not result from an increase in IL-2 production by CD4+ T cells. Together, these data suggest that GM-CSF induces the proliferation of Tregs by expanding CD11c+CD8a- DCs, and can regulate alloimmune responses in a cGVHD mouse model. Our findings indicate the potential of GM-CSF as a therapeutic strategy to ameliorate cGVHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacologic Co-Blockade of IFNγR and IL6R Pathways to Prevent and Treat GvHD

The therapeutic benefits of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematologic malignancies are primarily derived from an anti-leukemia effect that is mediated by T cells in donor grafts. Unfortunately, these T cells also mediate graft-versus-host disease (GvHD), the major complication of allo-HSCT.We have previously published that in vivo administration of JAK1/JAK2 inhibitors to murine allo-HSCT recipients of interferon gamma receptor deficient (IFNγR-/-) T cells results in 100% survival in a fully MHC-mismatched B6 to Balb/c allo-HSCT model (Choi et al., 2014, PLoS ONE). Since the infusion of IFNγR-/- T cells alone is associated with only ~70% survival, we hypothesize that JAK1/JAK2 inhibitors have either additional off-target effects or are inhibiting other non-IFNγR signaling pathways which are themselves dependent on JAK1/JAK2. The major other cytokine receptor signaling pathway mediated via both JAK1 and JAK2 is the interleukin 6 receptor (IL6R) signaling pathway. Thus, it is possible that JAK1/JAK2 inhibitors also block signaling through IL6R in addition to IFNγR. In addition, Alam et al. have recently reported that single nucleotide polymorphisms of donor IFNγ and IL6 are closely linked to gastrointestinal GvHD in patients (2015, BMT). Therefore, we examined if blockade of both IFNγR and IL6R signaling results in complete elimination of GvHD after a fully MHC-mismatched allo-HSCT in which B6 (H-2b) T cell-depleted bone marrow cells (5x106) along with B6 pan T cells (5x105) are intravenously injected into lethally irradiated Balb/c mice (H-2d). As shown in Fig. 1, we have found that blocking both IFNγR (IFNγR-/- T cells) and IL6R (α-IL6R Ab) signaling dramatically reduces GvHD and results in >95% survival. In addition, we found that blocking both IFNγR and IL6R signaling significantly increased regulatory T cells (Tregs) in peripheral blood (23.2% Foxp3+ in CD4+ T cells (n=17) vs 2.5% in WT T cell control (n=16) at day 27 after allo-HSCT), suggesting that increase in Tregs might be a potential mechanism underlying the reduced GvHD after dual blockade of IFNγR and IL6R signaling.Baricitinib is a potent and balanced JAK1/JAK2 inhibitor currently being clinically developed by Eli Lilly for the treatment of inflammatory diseases. We hypothesize that baricitinib will optimally block both IFNγR and IL6R signaling pathways and prevent GvHD. We found that that baricitinib is a potent suppressor of GvHD in B6 to Balb/c allo-HSCT models (100% survival), superior to ruxolitinib and similar to blockade of both IFNγR and IL6R signaling (Fig. 2A). Baricitinib increases Tregs in vivo (Fig. 2B) and reduces the ratio of IL5 (Th2 cytokine) to IL2 (cytokine for Treg induction) in plasma (p=0.0046), a potential diagnostic marker for GvHD (Fujii et al., 2006, Int J Mol Med), significantly better than ruxolitinib. Lastly, we found that baricitinib inhibits the expression of T-bet (Fig. 2C), which is the master transcription factor of Th1 cells, that are primary effector T cells in inducing GvHD. These data suggest that the suppression of GvHD by baricitinib results from increased Tregs and decreased Th1 and Th2 cells.We next examined if the prevention of GvHD by baricitinib is dependent on natural regulatory T cells (Tregs) in donor grafts. Tregs were depleted from donor pan T cells before allo-HSCT (B6 to Balb/c). We found that in vivo administration of baricitinib resulted in 70% survival (0% control, p<0.0001; 100% Treg-replete T cells + baricitinib. In addition, based on clinical GvHD score when recipients of Treg-replete T cells were compared with those of Treg-deplete T cells, the beneficial effect of Tregs in the donor grafts for the prevention of GvHD was observed only for the first two weeks after allo-HSCT (p≤0.01).Lastly, we examined whether baricitinib can cure ongoing GvHD by administering baricitinib starting at day 10 after allo-HSCT when GvHD is established (B6 to Balb/c). We found that baricitinib treatment results in a significant reduction of GvHD and 100% survival (10% control, p<0.0001).All of these data suggest that pharmacologic co-blockade of IFNγR and IL6R pathways is a promising therapeutic strategy to prevent and effectively treat established GvHD. The inhibitory effect of baricitinib, ruxolitinib, and blockade of IFNγR and IL6R on JAK-STAT signaling using JAK/STAT phosphorylation antibody arrays is currently being investigated and will be presented. [Display omitted] [Display omitted] DisclosuresDiPersio:Incyte Corporation: Research Funding.

Obese mice exhibit accelerated immune and myeloid reconstitution after congenic hematopoietic stem cell transplantation

Abstract The obesity rate is increasing in the United States with more than a third of U.S. adults being obese. Obesity is a form of sterile chronic inflammation, termed “meta-inflammation”, which is local, chronic and triggered by metabolical pathways. Therefore, there is a need to uncover the potential role of obesity in the development of immune cell subsets post-HSCT. In this study, we wanted to assess the impact of obesity on immune reconstitution following congenic HSCT in lean and DIO (diet-induced obese) recipients. C57BL/6 mice received lethal radiation dose at 1100cGy and 5 million T cell-depleted bone marrow cells from H2-matched B6.SJL mice. Our data demonstrated that DIO recipients displayed a more rapid and robust reconstitution affecting both myeloid and lymphoid compartments. DIO recipients had increased neutrophils, platelets and total lymphocytes post-HSCT compared to control mice. Colony forming unit-granulocyte macrophage (CFU-GM) assays showed significantly higher numbers in DIO recipients at day 14 post-HSCT. Organ cellularity from thymus, spleen and bone marrow were increased in DIO recipients compared to lean mice at day 14 and 21 post-HSCT. Higher numbers of CD4+CD8+ thymocytes, as well as higher number of NK cells and B cells in the spleen and lymph nodes were also observed. Splenocytes from DIO recipients had greater proliferative ability either after stimulation with concanavalin A or in mixed lymphocyte reaction compared to lean controls. Taken together; these results indicated that obesity results in acceleration of engraftment and donor immune reconstitution.

Intermediate Filament (IF) Protein Vimentin Regulates T Cell Mediated Immune Response in Gvhd

Graft-versus-host-disease (GVHD) is an alloimmune response complicating allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T lymphocytes and recipient antigen presenting cells (APCs) are the primarily mediators of GVHD, the molecular and cellular basis are not well understood. Recent published studies investigated T cell migration and homing in GVHD, especially effects of adhesion molecules including chemokines and integrins. Intermediate filaments (IFs) are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support. As the most abundant IF protein and the only known IF protein in leukocytes, vimentin plays an important role in stabilizing intracellular architecture, maintaining cellular integrity and providing resistance against stress. Vimentin-deficient (Vim-/-) mice are viable with impaired wound healing, and defects in PMN and lymphocyte adhesion to endothelial cells. In addition, Vimentin has been reported to be involved in colitis, Crohn's disease and allograft rejection. Herein, we report that vimentin regulates the organization of proteins in cell adhesion, migration and signaling, all of which are important for T cell activation in GVHD.Using homotypic aggregation assay, we found there was significantly reduced aggregation of primary T cells isolated from Vim-/- mice compared to WT control. However, the expression of both LFA-1 and ICAM-1 are similar between WT and Vim-/- T cells, thus the reduced T cell adhesion is not attributed to LFA-1 expression. We further investigated whether vimentin regulates mouse primary T cell proliferation upon activation in mixed lymphocyte reactions (MLR), and demonstrated there was a significant reduction of both CD4+ and CD8+ T cell proliferation in the absence of vimentin (Vim-/-) compared to WT control. Moreover, the frequency of IFN-γ (Th1) and IL-17 (Th17) producing CD4+ cells was significantly reduced in T cells isolated from Vim-/- mice compared to WT control. To investigate the role of vimentin in regulating TCR-induced activation, we examined the immune synapse formation in mouse CD8+ T cells, and found vimentin was enriched proximal to the membrane and associated with the prominent CD3/LFA-1 cluster upon TCR stimulation. In the control without any TCR stimulation, the predominant pattern was different with vimentin evenly distributed beneath the cell membrane. In the absence of vimentin (Vim-/-), immune synapse failed to form in mouse CD8+ T cells upon TCR stimulation. The data demonstrate that vimentin can regulate immunological synapse function and signal transduction in T cell activation and proliferation.To determine whether vimentin expression in T cells plays a role in GVHD, the MHC class I and II disparate model, C57BL/6 (H-2b) to BALB/c (H-2d) transplantation, was used to establish GVHD. T cells from WT or Vim-/- (C57BL/6 background) mice were used as donors and Balb/c mice as recipients. Irradiated BALB/c mice received 5x106 T cell-depleted bone marrow cells (WT) and 10×106 T cells (WT or Vim-/-). In comparison to WT control, mice received Vim-/- T cells showed a lower mortality rate. Within 8 weeks post-transplantation, about 65% of mice received Vim-/- T cells survived, compared with only less than 10% of control mice received WT T cells (P=0.036; n=15 in each group). Control recipients had severe GVHD in the skin, intestine and liver. Mice received Vim-/- T cells exhibited only mild changes in these organs, reflected in their significantly lower GVHD scores. There were significantly reduced donor-derived CD4+ and CD8+ T cells in secondary lymphoid organs. Thus, the reduced homing and proliferation of Vim-/- T cells in vivo led to the reduced mortality and morbidity associated with GVHD.In summary, we have shown that there are significantly reduced T cell adhesion, proliferation and Th1/17 polarization in Vim-/- T cells, and vimentin participates in TCR clustering and immunological synapse formation in CD8+ T cells. Furthermore, vimentin plays an important role in GVHD through regulating donor T cell adhesion, proliferation and activation. Our data will not only significantly advance our knowledge of GVHD, but also define a new function of vimentin and IF proteins in health and diseases, and thus provide a rationale for using vimentin inhibitors as potential novel therapeutic interventions for GVHD. DisclosuresNo relevant conflicts of interest to declare.

Targeting Alpha 4 Integrin (CD49d) to Reduce GvHD

Acute graft versus host disease (aGvHD) is a life-threatening condition that complicates allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells recognize the recipientÕs tissues as foreign causing GvHD, however, these same donor T cells are also responsible for the beneficial graft versus leukemia (GVL). Distinguishing between GvHD and GvL is crucial for the development of safe and effective therapies following allogeneic bone marrow transplantation.We have recently reported that the disruption of alloreactive donor T cell trafficking to the GvHD target organs significantly reduces GvHD in both an MHC fully-mismatched and a minor-mismatched allo-HCT models (Choi et al., Blood 2012). In this study, we examined if inhibition of α4, which is required for transendothelial migration and access to lymphoid system and GvHD target organs, could reduce GVHD. On T cells α4 integrin subunit can form a heterodimer with either β1 to form α4β1 integrin (VLA-4) or β7 to form α4β7 (LPAM-1). To genetically eliminate α4 from allogeneic donor T cells, we generated Tie-2 cre+ α4fl/fl mice (B6, H-2b, CD45.2+). Splenic pan T cells were isolated from these mice and T cell-depleted bone marrow cells (TCD BM) isolated from congenic B6 mice (CD45.1+). 5x106 TCD BM and 5x105 splenic pan T cells were transplanted into lethally irradiated (900 cGy) allogeneic Balb/c recipient mice (H-2d, CD45.2+). We found that α4 deficient T cells significantly reduced GvHD compared to Tie-2 cre+ control T cells (Fig. A). α4 deficient T cells had no significant effect on donor engraftment and achieved complete donor chimerism. In addition, recipients transplanted with α4 deficient T cells had significantly lower histopathology score (median score 3 vs. 6; P =0.0263). Similar results were observed when donor T cells were infused at day 11 post allo-HCT. To test whether α4 deficient T cells maintain GvL we performed bioluminescence imaging (BLI) using a systemic leukemia mouse model. CBRluc-expressing A20 leukemia cells (Balb/c-derived) were transplanted intravenously (1 × 105 cells) along with TCD BM (B6, CD45.1+) into Balb/c recipients at day 0. Pan T cells (B6, CD45.2+) either from Tie-2 cre+ or α4 deficient mice were infused at day 11 to allow sufficient time for the leukemia cells to expand. After weekly BLI, we found α4 deficient T cells were able to control leukemia cells as effectively as Tie-2 cre+ Control T cells (p=0.3748). In addition, we performed BLI to track CBRluc-transduced pan T cells (2 × 106 cells) after allo-HCT in vivo. We found a significant difference in the percentage of BLI signal intensity between control and α4 deficient T cells in spleen and gut at day 14 and 21 post allo-HCT. (Fig. B)While we speculate defective T cell trafficking to GvHD target organs would be the primary reason for the reduced GvHD in the recipients of α4 deficient T cells, we examined other possible mechanisms such as T cell alloreactivity and proliferation. We found that α4 deficient T cells proliferated at the same rate as Tie-2 cre+ T cells in the presence of anti-CD3/CD28 antibody-coated beads. However, α4 deficient CD8 T cells proliferate slower in the presence of allogeneic antigen presenting cell compared to Tie-2 cre+ T cells. Interestingly, α4 deficient T cells significantly upregulate CTLA-4 and GZMB compared to Tie-2 cre+ control T cells. These data suggest that not only altered T cell trafficking to GvHD target organs but also altered T cell functions might be the reason for the reduced GvHD.In conclusion, we propose that α4 represents a promising therapeutic target for future efforts to mitigate GvHD after allo-HCT. In addition, this strategy can be exploited in other diseases besides GvHD such as solid organ transplantation, chronic inflammatory diseases and autoimmune diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P-Glycoprotein Modulation Facilitates the Selective Inhibition of Oxidative Phosphorylation in Alloreactive T Cells to Prevent Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplant

T lymphocytes play a central role in many human immunologic disorders including both autoimmune and alloimmune diseases. In hematopoietic stem cell transplant (HSCT), acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Ex vivo application of phototherapy to selectively deplete GVHD-causing cells prior to transplant may prevent GVHD. However, none of the photosensitive agents in use today have demonstrated selectivity without significant toxicity occurring in resting cells. We have designed the first-in-class, novel photosensitizer 2-Se-Cl with the ability to accumulate in pathogenic T cells in proportion to degree of oxidative phosphorylation (OXPHOS). Unique to 2-Se-Cl is the ability to potently stimulate P-glycoprotein (P-pg). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria, and protects resting lymphocytes essential for antipathogen and antitumor responses. To confirm that 2-Se-Cl selectively depletes antigen-specific immune responses, human lymphocytes were cultured with staphylococcal enterotoxin B (SEB) for 72 hours. Cells were then pulsed with 2-Se-Cl and exposed to 5 J/cm2 of light, which resulted in the impedance of OXPHOS and induction of apoptosis in stimulated T cells. After photodepletion (PD), resting cells remained intact with the ability to respond to stimulation by Toxic Shock Syndrome Toxin-1 (TSST-1) but not SEB. To evaluate the selective depletion of alloimmune responses, a well-established complete mHC antigen-mismatched murine model of HSCT was employed. To prepare the PD - treated primed splenocytes, donor C57BL/6 splenocytes were cocultured for 5 days with irradiated Balb/c splenocytes, and then photodepleted. In vitro analysis of PD - treated primed splenocytes demonstrated selective depletion of antihost immune responses by CFSE dilution, with complete retention of third-party responses (C3H/HeJ mice). On the day of HSCT, recipient Balb/c mice were irradiated with 900 cGy, and received 5x106 T cell-depleted bone marrow cells accompanied by 5x106 PD - treated (treatment group) or untreated (control group) primed splenocytes, and then monitored for signs of GVHD according to an established mouse GVHD grading system. Five animals underwent HSCT in each group in 3 independent experiments. All mice died of acute GVHD after the addition of 5x106 untreated primed C57BL/6 cells at a median of 25 days after HSCT. In contrast, > 90% of mice that received the same number of PD - treated cells survived > 60 days without evidence of GVHD (see figure). Additionally, third-party C3H/Hej mice that received the same number of PD-treated cells died of GVHD, confirming the selective depletion of the alloimmune response for BalB/c by 2-Se-Cl. The high selectivity of this novel photosensitizer may have broad applications, including targeting the bioenergetics in alloimmune, autoimmune, and malignant T cells to provide alternative treatment options and improve clinical outcomes for patients with diseases such as GVHD, systemic lupus erythematosus, or peripheral T cell lymphoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.