T-cell Depleted Bone Marrow Research Articles

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Alleviates Chronic Graft- Versus-Host-Disease Via Modulating the CXCR5/CXCL13 Axis

Introduction: Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic hematopoietic cell transplant (allo-HCT) often leading to transplant-mediated mortality. Studies have shown that T follicular helper cells (Tfh) upregulate CXCR5 and migrate into the lymphoid organ follicles in response to CXCL13 promoting germinal center (GC) B cell proliferation and differentiation of plasma cells resulting in pathogenic antibody production and profibrotic pathways associated with cGVHD. In contrast, T follicular regulatory cells (Tfr) modulate the magnitude of the GC response; thus, the balance of Tfr/Tfh could be crucial to mitigating cGVHD.Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers influencing transcription of a multitude of inflammatory genes and our lab has previously reported pharmacological inhibition of BET, through OPN-51107, improves survivability and limits disease progression in murine models of acute GVHD that led to opening the first-in-human clinical trial of OPN-51107 in steroid refractory acute GVHD. We demonstrated BET proteins to be important in modulating acute GVHD pathogenesis, however, mechanisms of BET regulation within cGVHD are largely unknown. Here, using OPN-51107 we aim to further investigate mechanisms of cGVHD pathogenesis and identify successful BET inhibition-based therapeutic approaches. Methods: We used a Bronchiolitis Obliterans model (B6->B10.br) of cGVHD. Mice were administered 120mg/kg cyclophosphamide (D-3 and D-2) and 8Gy irradiation (D-1) as a conditioning regimen followed by B6 T-cell depleted bone marrow cells with and without CD45.1 B6 splenocytes. Mice that did not receive allogeneic splenocytes served as controls and did not develop GVHD. Recipients of allogeneic splenocytes were treated with a BET inhibitor - OPN-51107 at 10mg/kg 3x/weekly, or vehicle, from D28-54(+ 4) via oral gavage. Resting lung elastance and resistance was measured using the Flexivent system. At the time of lung function analysis, lung and liver tissue was harvested and stained via Hesson's Trichrome to identify and quantify collagen deposition. We performed immunofluorescence to quantify alveolar alloantibody deposition. Spleen and lung cells were stimulated with PMA/I and analyzed via spectral flow cytometry. An inflammatory murine cytokine array (#ARY006, R&D Systems) was used to analyze circulating plasma cytokine levels. Results: BET inhibition via OPN-51107 treatment decreased lung resistance (vehicle vs OPN-51107 =1.16Rrs vs 0.83Rrs p<0.05) and elastance (vehicle vs. OPN-51107=62Ers vs. 45Ers p<0.05). OPN-51107 treatment also significantly reduced alveolar antibody (Ig) deposition per area compared to vehicle (vehicle vs. OPN-51107= 4.91% vs. 3.3% p=0.01) and collagen deposition (vehicle vs. OPN-51107=19.0% vs. 12.9% p<0.05) and liver (vehicle vs. OPN-51107=19.3% vs. 2.35% p<0.0001). Congruently, BET inhibition decreased B220-CD19- CD138+mature plasma cells (vehicle vs. OPN-51107=15% vs 9.1% p<0.05) in the lung. Serum cytokine array analysis revealed a significant decrease in circulating CXCL13, relevant to Tfh migration as well as a decrease in other proinflammatory cytokines - CXCL9, MCP-1, IL3, RANTES and G-CSF important for macrophage activation and pathogenicity in OPN-51107 compared to vehicle treated mice. BET inhibition reduced splenic Tfh while increasing Tfr, thereby increasing Tfr:Tfh ratio (vehicle vs. OPN-51107= 5% vs. 9% p<0.05). Furthermore, total splenic CD19+ B-cells (vehicle vs. OPN51107= 70% vs. 33% p<0.001) as well as CXCR5 expression on B-cells were decreased in OPN-51107 treated mice (MFI vehicle vs. OPN-51107= 4744 vs. 571.5 p<0.0001). Conclusion: OPN-51107 improved lung function which correlated with significantly decreased lung fibrosis and antibody deposition in a BO model of cGVHD. BET inhibition reduces cGVHD immune pathology through decreased inflammatory cytokine production, promotion of GC Tfr cell population and modulates B-cell proliferation and/or trafficking. Significance: Using OPN-51107 in a BO model of cGVHD we demonstrate that BET inhibition is a feasible therapeutic strategy to reduce disease severity and improve lung function.

Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1×1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

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

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor peritoneal-derived cells can attenuate graft-versus-host disease after MHC-incompatible bone marrow transplantation in mice

BackgroundGraft-versus-host disease (GVHD) remains one of the most important barriers to bone marrow transplantation (BMT) and quality of life. Peritoneal-derived cells (PCs) can regulate immune function. MethodsThe GVHD mouse model, which was established by injection with T-cell depleted bone marrow cells and spleen cells, was treated with donor PCs. The survival time, weight, GVHD score, and pathological organ damage were used to assess the efficacy of PCs. Various cytokines (e.g., TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10, and TGF-β) and immunocytes (e.g., CD4+, CD8+, Treg, and NK cells) were employed to investigate the preliminary mechanism. The cytokine levels were tested via Luminex liquid chips, real-time PCR, and Western blotting, and the proportion of each T-cell subpopulation was detected via flow cytometry. One-way Analysis of Variance (ANOVA) followed by Tukey's post-test was employed to perform multiple comparisons, and Kaplan–Meier method was used to perform survival analysis. ResultsCompared to the saline group, the PC group harbored a significant reduction in pathological damage to the colon and small intestine, a longer survival time, and a lower GVHD score. The mice in the PC group had significantly higher levels of TGF-β, IL-10, and IL-4 and lower levels of IFN-γ, TNF-α, and IL-15 than those in the saline group. In the PC group, CD8+ and Th1 ratios decreased, while CD4+, Th2, NK, and Treg ratios increased. ConclusionsDonor PCs can attenuate GVHD caused by MHC-incompatible BMT by regulating cytokine levels and immunocyte ratios.

Efficacy of Vecabrutinib Treatment in a Murine Model of Sclerodermatous Graft-Versus-Host-Disease

Introduction. Chronic graft-versus-host disease (cGVHD) can manifest as a complication in patients following allogeneic hematopoietic stem cell transplant resulting in morbidity and mortality. Effective treatment strategies for cGVHD are currently lacking. Ibrutinib, an irreversible BTK inhibitor with activity against several other Tec-family kinases, has been clinically developed for cGVHD treatment due to regulation of pathogenetic B cells and T-cell subsets. Vecabrutinib is a more selective, reversible inhibitor of BTK with a distinct kinase domain interaction, which could result in differentiated safety and activity profiles compared to ibrutinib. Vecabrutinib has the capacity to overcome the C481S mutation that mediates resistance to ibrutinib. Vecabrutinib also also demonstrates activity against ITK, which is expressed in T cells. In this study, we investigated the activity and immune modulation of vecabrutinib treatment in a murine model of sclerodermatous cGVHD. Ibrutinib was utilized for comparison.Methods. A murine model of sclerodermatous cGVHD was initiated by adoptive transfer of T-cell depleted bone marrow plus whole splenocytes from B10.D2 donors into BALB/c recipients that were previously subjected to sub-lethal irradiation. Total bone marrow and irradiation only groups were included as controls. Mice with established cGVHD characterized by weight loss and skin irritation symptoms were treated with vecabrutinib once daily at 50mg/kg by oral gavage or ibrutinib at 30mg/kg in drinking water 5 days per week for approximately 3 weeks beginning on day 27 post-adoptive transfer and ending on day 45 (n=10 mice per group). Body weight and clinical symptoms (appearance, activity, skin symptoms, diarrhea, conjunctivitis) were measured throughout the study. Immunophenotyping for B cells and T cells was performed on spleens collected from euthanized mice by flow cytometry at two timepoints (day 40 during treatment and on day 76 post-treatment). Levels of circulating immunoglobulins were measured by multiplex cytokine assay at both timepoints.Results. Clinical symptoms, including appearance, activity, skin irritation, redness, alopecia and diarrhea were significantly reduced in vecabrutinib- and ibrutinib-treated groups. Furthermore, there was a trend toward a more favorable clinical score overall for the vecabrutinib-treated group, however no statistical difference was observed compared to ibrutinib possibly due to small sample size. Weight loss was slightly elevated during vecabrutinib and ibrutinib treatment compared to vehicle (trend), however mice recovered body weight post-treatment and continued to maintain benefit. On day 40 (during treatment) and day 76 (post-treatment) groups of mice were euthanized for immunophenotyping analysis utilizing a 22-color flow cytometry panel. During treatment, both vecabrutinib and ibrutinib-treated mice retained total B cell numbers but exhibited reduced B-cell activation, proliferation, and number of B220+ CD138+ plasma cells. In addition, B cells secreted less IL-10, and IL-4/5. Expression of antigen-presentation molecules CD80 and CD86 on B cells were unchanged. Total CD3+ T cells, activated and proliferating CD4+ and CD8+ T cells, and cytotoxic granzyme-B+ CD8+ T cells were reduced in treated mice. Interestingly, CD4+ CD25+ FoxP3+ Treg number, expression of PD-1 on Tregs and CD4+ CXCR5+ PD-1+ T follicular helper cells were also reduced in both treatment groups. In addition, there were globally reduced numbers of cytokine-secreting CD4+ cells but no differences in Th1/Th2 or Th17/Treg ratios were observed. Post-treatment, proliferation and cytokine secretion of B cells and T cells were still lowered but less impaired than during treatment. Tregs and PD-1 expression were still reduced post-treatment. Finally, circulating levels of IgA were reduced during and post treatment in vecabrutinib-treated mice compared to vehicle, while IgG1, IgG2b were reduced in both treated groups. No changes in IgM levels were observed in either treatment group. In conclusion, vecabrutinib treatment demonstrated efficacy and beneficially regulated B cell and T cell immune subsets in a preclinical murine model of sclerodermatous cGVHD. Studies to further evaluate differences between vecabrutinib and ibrutinib treatment are ongoing. [Display omitted] DisclosuresFox: Sunesis Pharmaceuticals: Current Employment. Taverna: Sunesis Pharmaceuticals: Current Employment. Pinilla Ibarz: Sellas: Other: ), patents/royalties/other intellectual property; MEI, Sunesis: Research Funding; AbbVie, Janssen, AstraZeneca, Takeda: Speakers Bureau; AbbVie, Janssen, AstraZeneca, Novartis, TG Therapeutics, Takeda: Consultancy, Other: Advisory.

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.

Role of Glucose Transporter 1 in Graft-Versus-Host Disease and Graft-Versus-Tumor Effect

Graft-versus-host disease (GVHD) is a major obstacle to allogeneic hematopoietic cell transplantation. GVHD is induced when donor mature T cells encounter host antigens and get activated. Activated T cells require high metabolic flux through growth-promoting pathways to support clonal expansion. It is generally thought that glycolysis is employed in this process. However, conflicting data have been reported by different groups regarding the role of glycolysis in GVHD. Since glucose transporter 1 (Glut1) is essential for T cell glycolysis, we attempted to define the role of glycolysis in GVHD using donor T cells that are deficient of Glut1.One million T cells from Glut1-knockout (CD4CreGlut1fl/fl) or wild-type (control) C57BL/6 donors were transplanted into lethally irradiated (9.5 Gy) C3H/HeJ mice along with 1×107 donor T-cell-depleted bone marrow (TCDBM) cells. While all recipients of control T cells developed lethal GVHD and died within 20 days after transplantation, eight out of ten CD4CreGlut1fl/fl T cell recipients survived more than 60 days after transplantation (Figure 1, P<0.01). Body weights were slightly lower and clinical scores were slightly higher (P=not significant) in the surviving CD4CreGlut1fl/fl T cell recipients at day +56 when compared to those in the TCDBM alone group, suggesting that CD4CreGlut1fl/fl T cell recipients developed very mild GVHD if any. We further studied the role of Glut1 in CD4 and CD8 T cell mediated GVHD separately using C57BL/6 into BALB/c model. Similar to the results from the total T cell experiment, all CD4CreGlut1fl/fl CD4 and CD8 T cell recipients survived more than 60 days after transplantation. By contrast, 100% control CD4 and 50% CD8 T cell recipients died of lethal GVHD within 60 days after transplantation. These data demonstrate that Glut1 is required for alloresponses and glycolysis is indispensasble in the development of lethal GVHD.To understand the mechanism by which CD4CreGlut1fl/fl T cells mediate less GVHD, we first studied their ability to respond to alloantigens in mixed lymphocyte reaction. The results indicated that CD4CreGlut1fl/fl T cells have dramatically decreased ability to respond to BALB/c antigens when compared with the control T cells. Consistent with the in vitro data, T cell expansion was 59.4% less in CD4CreGlut1fl/fl T cell recipients than that in control T cell recipients when measured at day +7 after GVHD induction. Apoptosis was increased in alloantigen-activated (CD69+) CD4 but not in CD8 T cells from CD4CreGlut1fl/fl mice when compared with that from control mice when measured 16 hours after the initiation of culture. However, more cell death was observed in both activated CD4 and CD8 T cells from CD4CreGlut1fl/fl mice than that from control mice. In addition, alloreactive CD4CreGlut1fl/fl T cells failed to sustain activated mTORC1 signaling, as assessed by phosphorylation of the downstream p70 S6 kinase target, small ribosomal subunit S6. These results indicate that, upon response to alloantigens, T cells are prone to apoptosis and cell death and lead to decreased clonal expansion in the absence of Glut1, suggesting a mechanism by which CD4CreGlut1fl/fl T cells have decreased ability to induce GVHD.To determine whether absence of Glut1 in T cells affects graft-versus-tumor (GVT) effects, we transplanted 1×106 T cells from Glut1-knockout (CD4CreGlutfl/fl) C57BL/6 donors into lethally irradiated (8.5 Gy) BALB/c mice along with 1×107 donor TCDBM cells. At the time of transplantation, the recipients were challenged with 1×105 BCL1 cells, a BALB/c-derived leukemia/lymphoma cell line. While all TCDBM alone recipients died of leukemia/lymphoma and all control T cell recipients died of lethal GVHD, all CD4CreGlut1fl/fl T cell recipients survived more than 100 days after transplantation without leukemia/lymphoma (Figure 2), indicating that T cells deficient of Glut1 preserve GVT effect.The results from this study definitely demonstrate that glycolysis is essential for the development of GVHD. Inhibition of glycolysis in T cells increases apoptosis and cell death and suppresses clonal expansion of allospecific T cells. Interestingly, GVT effect is preserved despite the inhibition of glycolysis. These data suggest that glycolysis could potentially be targeted for prevention and treatment of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

Syngeneic Fecal Microbiota Transplant Effectively Attenuated Graft-Versus-Host Disease after Bone Marrow Transplantation in Mice

Introduction: The gut microbiota plays an important role in maintaining intestinal homeostasis and outcomes after allogeneic hematopoietic stem cell transplantation (allo-HCT). Post-transplant loss of microbiota diversity in patients has been associated with increased infections, graft-versus-host disease (GVHD), disease relapse, and treatment-related mortality. Case studies of fecal microbiota transplantation (FMT) from healthy donors into patients with steroid-resistant gastrointestinal GVHD suggest that FMT may restore the normal microbiota and improve GVHD. However, it remains unclear what constitutes the “optimal” target microbiota and how different changes in the microbiota may impact GVHD. A particularly interesting question is whether the goal of FMT should be to replicate the original recipient microbiota (recipient FMT) or to transfer the donor microbiota (donor FMT), given that the donor microbiota may be more compatible with the transplanted donor immune system.Methods: To test the hypothesis that donor FMT improves GVHD, we evaluated FMT in the context of our well-established allo-HCT acute GVHD model. T-cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into BALB/c mice without causing GVHD. T cells (1 x 106) from C57BL/6 donors were then be added back to induce GVHD. The BALB/c recipient microbiota was modified by FMT and/or antibiotics. FMT was performed by gavaging freshly collected stool from healthy mice of stem cell donor strain (B6, donor FMT) or recipient strain (BALB/c, recipient FMT). To avoid the potential mechanical injury caused by oral gavage that may affect results, we also evaluated another method of FMT in which allo-HCT recipients were housed with healthy mice, resulting in FMT via coprophagy. To test the effect of antibiotics, some recipient mice were supplied with imipenem and vancomycin in drinking water for three days before FMT. Proliferation of luciferase-labeled T cells was monitored using bioluminescent imaging.Results:As expected, BALB/c mice receiving TCD BM from C57BL/6 had no GVHD, while BALB/c mice receiving TCD BM with 1 x 106 T cells had significant GVHD-related mortality. Contrary to our original hypothesis, donor FMT significantly worsened mortality, while recipient FMT significantly improved mortality (Fig. 1a). The addition of antibiotics particularly exacerbated early morality in the donor FMT group (Fig. 1b) while antibiotics had minimal effect in the recipient FMT setting (Fig. 1c). Interestingly, even in the context of TCD BM alone, donor FMT worsened survival, and this effect was enhanced with the addition of antibiotics (Fig. 1d). Deaths were early and consistent with infection on necropsy.To better understand the mechanisms by which FMT affect survival, we observed T-cell proliferation using bioluminescent imaging of luciferase-labeled T cells. Mice that received recipient FMT had significantly lower luminescence in GVHD major target organs compared to the mice that received donor FMT, indicating less T-cell proliferation (Fig. 2), which is consistent with less T-cell activation and less GVHD.Similar results were seen in co-housing experiments, in which co-housing with healthy recipient animals improved body weight and GVHD scores (Fig. 3). Preliminary 16s ribosomal RNA sequencing results support our hypothesis that FMT changes the microbiome, and these changes in the microbiome are linked to clinical outcomes. We will also explore the pathological changes in the gut microenvironment and the potential mechanism.Conclusion: Recipient FMT in the context of allogeneic HCT helped restore the gut microbiota, suppress T-cell responses in GVHD-associated organs, and ameliorated GVHD. In contrast, donor FMT resulted in a high risk of bacterial infections as well as more severe GVHD. This study may provide crucial clinical guidance for using FMT as a potential treatment for GI GVHD. [Display omitted] DisclosuresSung:Novartis: Research Funding; Merck: Research Funding; Cellective: Research Funding.

Targeting T Cell Bioenergetics By Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells to Prevent Graft-Versus-Host Disease While Maintaining Antitumor Immunity

Immune effector cell therapy is a potent form of cancer treatment with a high curative potential for certain human malignancies. Yet, the success of current approaches is offset by associated toxicities, including acute graft-versus-host-disease (GVHD). In the allogeneic setting, GVHD is caused by an attack on the recipient's tissues by donor 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 photosensitizer 2-Se-Cl with the ability to accumulate in alloreactive T cells in proportion to degree of oxidative phosphorylation. 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. We have previous demonstrated the ex vivo photodepletion of alloreactive T cell using 2-Se-Cl successfully prevented GVHD in a lethal model of the disease with full retention of antipathogen immunity and 3rd party responses. To evaluate for the retention of antitumor immunity after photodepletion with 2-Se-Cl, a well-established complete MHC antigen-mismatched murine model of hematopoietic stem cell transplant (HSCT) was employed. To prepare the cells for photodepletion, donor C57BL/6 splenocytes were cocultured for 4 days with irradiated (20 Gy) Balb/c splenocytes, and then photodepleted (PD) with 2-Se-Cl. On the day of HSCT, recipient Balb/c mice were irradiated with 8.5Gy, and received 7.5 x106 T cell-depleted (TCD) C57BL/6-derived bone marrow cells accompanied by 2.5 x106 PD - treated cells, or 105 recipient-derived (A20, Balb/c-derived) leukemia cells (engineered to express firefly luciferase), or both. To evaluate for the retention of 3rd party responses, C3H/HeJ were irradiated (9.5 Gy) and received a combination of 7.5 x106 TCD C57BL/6-derived bone marrow cells accompanied by 2.5 x106 PD - treated cells. All mice were monitored for signs of GVHD according to a well-established mouse GVHD grading system. Three animals underwent HSCT in each group in 3 independent experiments. Recipient Balb/c mice that received PD-treated splenocytes survived > 45 days without evidence of GVHD or leukemia, equivalent to controls (recipients of TCD bone marrow cells only). In contrast, mice that were inoculated with leukemia at time of HSCT without the addition of PD-treated splenocytes died of leukemia progression (see figure). Additionally, all third-party C3H/HeJ mice that received the same number of PD-treated cells died of lethal GVHD, consistent with the selective depletion of the alloimmune response towards BalB/c by 2-Se-Cl. These results demonstrate that ex vivo application of 2-Se-Cl protects against lethal GVHD while maintaining antitumor immunity. The high selectivity of this novel photosensitizer may have broad applications, including the rapid production of immune effector cell products with potent antitumor activity and a low potential for toxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Immune Reconstitution and Outcome After HLA-Nonidentical T-Cell–Depleted Bone Marrow Transplantation for Severe Combined Immunodeficiency: A European Retrospective Study of 116 Patients

AbstractWe have performed a retrospective analysis of the development of T- and B-cell functions after HLA-nonidentical T-cell–depleted bone marrow transplantation (BMT) performed in 193 patients with severe combined immunodeficiency (SCID) at 18 European centers between December 1982 and December 31, 1993. One hundred sixteen of 193 patients were alive with evidence of engraftment 6 months after BMT. Development of T-cell function occurred earlier than B-cell function and was achieved more frequently up to the time of last follow-up. The median time to achieve normal T-cell function was 8.7 months, whereas the median time to achieve normal B-cell function was 14.9 months. Twenty-four patients died later than 6 months post-BMT, mainly due to chronic graft-versus-host disease (cGVHD) and/or viral infection. Absence of T-cell reconstitution 6 months after BMT, unlike absence of B-cell reconstitution, was associated with a poor outcome. Two additional factors were associated with a poor outcome: presence of cGVHD 6 months after BMT and B− SCID versus B+ SCID. However, two of these three factors remained as significant prognostic factors in a multivariate analysis: the absence of T-cell function and the presence of cGVHD 6 months after BMT. Analysis of the factors influencing the development of immune reconstitution showed that T- and B-cell functions occurred earlier and more frequently in B+ SCID versus B− SCID patients. Acute GVHD was associated with a slower development of T-cell function at 6 months, and cGVHD had a negative influence on the development of T-cell function afterwards, but neither acute nor chronic GVHD was found to influence the development of B-cell function. Once engraftment occurred, whether patients had or had not received Busulfan in the conditioning regimen did not influence the kinetics and quality of T-cell function development. In a multivariate study, two factors were found to influence the T-cell function 6 months after BMT: type of SCID and acute GVHD. The results of this retrospective analysis should lead to new protocols adapted to SCID disease, considering that disease-related as well as BMT-related parameters influence the development of immune function and thereby long-term outcome after HLA-nonidentical T-cell–depleted BMT.

Glucagon-like Peptide-2 (GLP-2) Treatment Reduces Gvhd-Related Mortality in a Pre-Clinical Transplant Model Via Expansion of Macrophages with Tolerogenic Potential

The gastrointestinal (GI) tract is a major target in GVHD. Conditioning-induced damage and mucosal barrier disruption are important factors in GVHD, however therapies targeting these processes have not been identified. Glucagon-like-peptide 2 (GLP-2) is an enterocyte-specific growth factor produced by L cells that has regenerative potential in models of GI damage. Its impact on the mucosal immune system has not been elucidated. We sought to examine the therapeutic and immunologic effect of GLP-2 in murine GVHD. We employed a major MHC-mismatched GVHD model (C57BL/6J → BALB/cJ). Mice were treated with 800nmol/kg/day of Elsiglutide (a GLP-2 analogue, provided by Helsinn) or vehicle beginning on D+1 for 30 days. Treatment with GLP-2 significantly improved survival and GVHD scores (Fig. 1A), while increasing small intestine mass and villi length (Fig 1B). GLP-2 also reduced T-cell infiltration into the jejunum (Fig. 1C). Analysis of intestinal immune cells by 28-color flow cytometry revealed dramatic differences between treatment groups in both myeloid- and T-cells. On D+14, GLP-2 led to an increased proportion of donor CSF-1R+ macrophages in the lamina propria (LP) (Fig. 2A) - cells that support the maintenance of the intestinal stem cell niche (Sehgal, Nat Commun, 2018). On D+21 the LP donor myeloid compartment was further altered, especially in MHC IIlow F4/80+ CD64+ macrophages (Fig. 2B, C). Here GLP-2 treatment expanded macrophages with lower expression of the co-stimulatory molecules CD80 and CD86 as well as the phagocytic marker CD206, whilst increasing the inhibitory molecule SIRPα, consistent with a tolerogenic phenotype. GLP-2 treatment also increased CX3CR1 expression on MHC IIlow macrophages with reduced Ly6C - a phenotype associated with physiologic macrophage maturation and linked to the resolution of colitis (Zigmond, Immunity, 2012). Vehicle-treated mice, conversely, had predominance of Ly6Chigh MHC IIlow LP macrophages reminiscent of an early infiltrating phenotype and near absence of mature macrophages, suggesting an impaired monocyte-macrophage transition that was restored by GLP-2. In addition, GLP-2 treatment led to significant changes in donor intraepithelial lymphocytes on D+21 (Fig. 2D), where CD8 T cells exhibited decreased CD27, CD103 and CXCR3 expression but higher PD-1, suggesting less activation. To assess potential mechanisms for the differences in macrophage and T-cell phenotype, we examined the impact of GLP-2 on the intestinal microbiota. A syngeneic BALB/cJ model was used to explore the effects of GLP-2 independent of GVHD. Stool samples from D+0, D+14, and D+28 were subjected to 16S rRNA sequencing. Vehicle-treated mice had distinct β-diversity clusters at all time-points, showing a transplant effect on the microbiota (Fig. 3A). GLP-2-treated mice had near-complete cluster overlap between D+0 and D+14, suggesting attenuation of the impact of conditioning. GLP-2 treated mice were significantly enriched for Akkermansia muciniphila and Bacteroidales S24-7 family at D+14 and D+28 (Fig. 3B). These taxa have been associated with anti-inflammatory properties and A. muciniphila abundance is linked to epithelial mucin production, which is increased by GLP-2. We then assessed the role of microbial communities in the protective effect of GLP-2 by conducting an allogeneic transplant with 3 caging conditions; 1) vehicle and GLP-2 treated mice caged together, 2) caged separately, or 3) caged separately plus oral antibiotics. We observed a clear cage effect where co-housing the treatment groups improved the survival of vehicle treated mice (Fig. 3C), suggesting transferal of the therapeutic effect via the microbiome. Antibiotic administration also dampened the beneficial effect of GLP-2. Finally, we conducted a GvL experiment by co-transplanting Luc-A20 and monitoring tumor progression via bioluminescence imaging. Both GLP-2 and vehicle-treated mice eliminated the tumor, whereas mice receiving T-cell depleted bone marrow showed tumor progression (Fig. 3D). In summary, our results demonstrate high therapeutic potential of GLP-2 in GVHD. GLP-2 administration led to reduced mortality, modified the microbiome and altered the intestinal immune response to a more tolerogenic state. This novel mechanism sheds light on the role of the enteroendocrine system in maintaining gut homeostasis and sets the stage for therapeutic clinical trials. Figure 1 Disclosures Uhlemann: Allergan: Research Funding; GSK: Research Funding; Merck: Research Funding. Reshef:Gilead: Consultancy; Magenta: Consultancy; Novartis: Consultancy; Monsato: Consultancy; Atara: Consultancy; BMS: Consultancy.

Endothelial Protective Effects of Defibrotide Reduce Acute Graft Versus Host Disease after Experimental Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathophysiology of acute GVHD involves donor T cell- and inflammatory cytokine-mediated injury to the patient's tissues and causes significant morbidity and mortality. Defibrotide is a mixture of single stranded nucleotides that has a number of pharmacological effects that contribute to its endothelial protective properties. We hypothesized that preventive treatment with defibrotide will reduce the incidence and severity of GVHD due to its endothelial protective and anti-inflammatory properties and potentially does not alter graft versus leukemia (GVL) responses. Methods Recipient B10.BR mice received Cyclophosphamide 120 mg/kg per day on day-3 and -2, and received lethal total body irradiation (750cGy) on day 0, followed by the infusion of donor C57BL/6 T-cell depleted bone marrow cells. To induce GVHD, respective groups were co-infused with splenocytes, while non-GVHD controls received BM cells only. Mice were treated with defibrotide at the dose of 800mg/kg per body weight or vehicle control for the first week daily and then three times/week thereafter. For GVL experiments, C57BL/6 recipients were lethally irradiated (12Gy) with TBI and transplanted with C3H.SW donor TCD bone marrow alone or in combination with 3 million T-cells along with C1498-luc and treated with defibrotide or vehicle controls. Results Allogeneic defibrotide treated recipients demonstrated significantly better survival when compared to control group (83.34% vs 54.55% on day +28). Clinical GVHD scores were significantly worse after day +14 in control group when compared to defibrotide treated recipients. Both at day +7 and on day +28 allogeneic defibrotide treated recipients showed significantly less organ GVHD in gut, lung and liver (p Conclusion Defibrotide treatment through its anti-inflammatory and endothelium-protective effects reduces the incidence and severity of acute GVHD, while not impairing GVL activity.

β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P&lt; 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P&lt;0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No 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.

Discovery of DWP213388, a Potent ITK and BTK Selective Dual Inhibitor with Excellent Efficacy and Safety, for Treating Gvhd

Introduction: Graft-versus-host-disease (GVHD) is a severe complication in patients undergoing hematopoietic stem cell transplantation (HSCT) and one of the major causes of death in patients receiving HSCT despite immunosuppressant drug and intensive therapy. GVHD is a rare disease characterized by excessive activation of T and B cell pathways and drugs such as Ibrutinib and steroids are used to improve the survival rate of GVHD patients by controlling immune cell signaling, yet the use of these drugs is limited due to toxicity. It is necessary to develop new treatment agents for GVHD, because of the limitations of these existing treatment agents. Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are critical for T and B cells activation, and dysregulation of this process has been known to increase proinflammatory cytokines and cause various inflammatory diseases. Since ITK and BTK signaling pathway play a major role in T and B cell regulation in GVHD, we thought that ITK and BTK selective dual inhibition may act as a promising agent that have beneficial effects on GVHD. So, we developed a novel ITK and BTK selective dual inhibitor, DWP213388, to modulate both T and B cell pathways without any significant off-target effects, thus to ultimately improve survival rate in GVHD without any serious side effect. Methods: Inhibition of ITK and BTK enzyme activity and selectivity against Cys-family kinase group were evaluated by biochemical activity assay. Target occupancy in human T and B cells was measured by quantifying unbound ITK or BTK in ELISA-based assay using biotinylated-DWP213388. To measure occupancy, mouse spleen and thymus were extracted after oral administration of DWP213388. Cellular assays for BCR or TCR stimulation-dependent activation and cytokine secretion were determined in human PBMC. In vitro mixed lymphocyte reaction (MLR) assay, CD4+ T cells were purified from spleens of B6 mice and were co-cultured with irradiated Balb/c splenic APCs as stimulators that were incubated in the presence or absence of DWP213388. In vivo MLR test, recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of splenocytes with B6. The efficacy of DWP213388 was investigated in mouse GVHD model; Recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of T cell depleted bone marrow (TCD-BM) and splenocytes with B6. In CMV study, DWP213388 was administered orally and an inoculum of 1 x 104 PFU/head MCMV in PBS was injected intraperitoneal. into each mouse on day 0. Five and ten days after the MCMV infection, the liver was removed. The NK cell and CD8 T cell was measured by FACs and virus titration was measured by RT-PCR. Results: We developed a novel, potent dual target inhibitor, DWP213388, with ITK and BTK IC50 values of 1.0 nM and 0.4 nM, respectively. More importantly, this compound is highly selective against ITK and BTK, yet has low affinity toward EGFR. DWP213388 potently occupied both of its targets at low concentration, which shows successful inhibition of TCR/BCR-dependent CD69 expression. DWP213388 suppressed T and B cell subsets such as Th1, Th2, Th17 and plasma cells and inhibited T cell proliferation in both in-vitro and in-vivo MLR assays. In mouse GVHD model, DWP213388 at 10 mg/kg improved clinical symptoms and maintains 100% survival rate (vs Ibrutinib 85%). It showed 73% and 62% occupancy of BTK and ITK. To address any potential compromising immune response due to dual inhibition of T, B cell by DWP213388, we conducted a CMV study to evaluate the effects on infection. DWP213388 does not reduce the number of NK cells and CD8+ T cells, and it does not affect virus killing. Therefore, it has a low risk of infection. These results suggest that DWP213388 may serve as a next generation therapeutic agent for GVHD. Conclusion: We developed a novel, highly potent, and selective dual inhibitor of ITK and BTK, DWP213388. We demonstrated that DWP213388 has potent pharmacological activities compared to Ibrutinib in various cellular models and a mouse GVHD model. DWP213388 intends to improve efficacy and safety by specifically inhibiting ITK and BTK without inhibiting EGFR, which is known to cause severe side effects. Therefore, DWP213388 may serve as a next generation therapeutic agent for GVHD. Disclosures No relevant conflicts of interest to declare.

Reduced Intensity Conditioning for Haploidentical Bone Marrow Transplantation in Patients with Symptomatic Sickle Cell Disease: BMT CTN Protocol 1507

Background: Hematopoietic cell transplantation (HCT) has curative potential in sickle cell disease (SCD). A reduced intensity haploidentical bone marrow transplant (haplo-BMT) platform piloted by investigators at Johns Hopkins, addressed key barriers in HCT for SCD, donor availability and toxicity, but had a graft rejection rate of 43%. (Blood. 2012;120(22):4285-4291). Subsequently, a Vanderbilt University Medical Center supported multi-institution learning collaborative with London, France and United States was developed in 2013 with the objective of reducing this graft rejection rate. Evolution of this haplo-BMT platform with the addition of only thiotepa, resulted in a reduction in the graft rejection rate to 7% (1 of 15), an event free survival (EFS) of 93% (14 of 15), &amp;gt; 95% stable donor engraftment at 6 months and 100% overall survival (Biol Blood Marrow Transplant. 2019 Jun;25(6):1197-1209). Based on the preliminary data from the learning collaborative, we initiated an NIH sponsored BMT CTN, dual-strata, phase II, multi-center prospective clinical trial, (clinicaltrials.gov # NCT03263559) to estimate event free survival (EFS) at 2 years in children with neurological morbidity and adults with severe SCD. Study Design and Methods: Eligibility: Patients are stratified into 2 groups: (1) children with SCD (Hb SS or Sβ+ Thalassemia) between 5.00 - 14.99 years of age with central nervous system (CNS) disease (stroke, elevated transcranial Doppler (TCD) with evidence of intracranial vasculopathy; silent cerebral infarction) and (2) adults with severe SCD (Hb SS, SC, Sβ° thalassemia, Sβ+ Thalassemia, SD, S-OArab), between 15.00 - 45.99 years of age who have had a stroke, recurrent ACS, recurrent vaso-occlusive pain episodes, chronic transfusions to prevent sickle complications and tricuspid valve regurgitant jet velocity (TRJV) ≥2.7 m/sec. Participants must have an HLA haploidentical first degree relative donor with 2, 3, or 4 (out of 8) HLA-mismatches who is willing and able to donate bone marrow. Those with an HLA-matched sibling are excluded. Primary objective is to estimate EFS at 2 years after haplo-BMT in patients with SCD enrolled in each stratum. Secondary objectives include determining the effect of haplo-BMT on clinical and laboratory manifestations of SCD by 2 years post-transplant and determining the incidence of other transplant-related outcomes. A key secondary analysis will be central adjudication of CNS progression in children and adults with evidence of previous CNS injury. Treatment Description: Preconditioning with hydroxyurea 30mg/kg daily (Day -70 to Day -10). The conditioning regimen includes: Thymoglobulin (rATG) (0.5mg/kg on Day -9, 2mg/kg on Day -8, Day -7), Thiotepa 10mg/kg on Day -7, Fludarabine (30mg/m2 on Day -6 to Day -2), Cyclophosphamide 14.5mg/kg on Day -6 and Day -5, and TBI 200cGy on Day -1. Day 0 is the day of infusion with non T-cell depleted bone marrow. Cyclophosphamide 50 mg/kg and Mesna 40mg/kg is given on Days +3 and +4, and GVHD prophylaxis (sirolimus and mycophenolate mofetil) begin on Day +5 (Figure). Study Accrual and Endpoints: The target sample size is 40 patients enrolled and treated in each stratum; if the stopping rule for graft failure is triggered within the first 12 evaluable participants in either stratum using the 200cGy dose of TBI and the DSMB and sponsor approve the pre-defined switch in conditioning regimen, then accrual for that strata will restart under the modified conditioning regimen with 400 cGy TBI. The estimated accrual period is 4 years. The rate of overall mortality by Day 180 after starting hydroxyurea therapy pre-transplant, acute grade III-IV GVHD at 100 days, and severe chronic GVHD at 18 months post-transplant will be monitored by a sequential probability ratio test (SPRT) for censored exponential data for each of these. Graft failure at 100 days post-transplant will be monitored by using a SPRT for binary data. Conclusion: This NIH sponsored BMTCTN phase II trial of HLA-haploidentical HCT is active at 30 BMT CTN sites. This study will determine if a modulated conditioning regimen has adequate donor engraftment, with acceptable risks of transplant-related morbidity and mortality to support a future comparative clinical trial in SCD. Figure Disclosures Walters: AllCells, Inc: Consultancy; TruCode: Consultancy; Editas Medicine: Consultancy. Brodsky:Achillion: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding.

Artesunate Inhibits Graft-Versus-Host Disease in Mice Via a Mechanism of Inducing Mitochondrial Calcium Overloading in Activated T Cells

Despite pharmacological prophylaxis using calcineurin (CN) inhibitors (i.e., cyclosporin A and Tacrolimus), graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Upon antigen stimulation, activated T cell receptor (TCR) signaling triggers rapid Ca2+ influx. This induces both CN-mediated NFAT activation, and increase of mitochondrial Ca2+ content, a major driver of metabolic activity. However, mitochondrial Ca2+ overload triggers opening of the mitochondrial permeability transition pore and cell death. We hypothesize that pharmacologically increasing mitochondrial Ca2+ load may decrease T cell survival capability, thereby reducing the GVH reaction. If this can be accomplished, it may lead to new strategies for inhibition of GVHD, which is conceptually different from the use of CN inhibitors. To test this hypothesis, we employed a high throughput drug screening system with the proliferation and cytotoxicity of TCR-activated human T cells as the readout, and screened the NPL-800 library (https://www.timtec.net) composed of 800 pure natural compounds. With a stringent criterion in consideration of dose-dependent effect, 26 compounds stood out for reducing the count of activated human T cells with a reduction rate of at least 30% at both 1.0uM and 10.0uM. Positive hits included inhibitors of DNA synthesis, the Na-K-ATPase and mitochondrial metabolism. We were particularly interested in artesunate (ART), which is a derivative of artemisinin that has been used for treating malaria in patients. While artemisinin acts by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in P. falciparum malaria, which causes passive endoplasmic reticulum (ER) Ca2+ depletion and the subsequent cytosolic Ca2+ influx, ART did not inhibit SERCA in T cells. Ex vivo culture assays showed that ART dose-dependently reduced the survival of TCR-activated murine T cells. This inhibitory effect of ART was abrogated by inhibiting Ca2+ influx using BTP2, a potent inhibitor of store-operated Ca2+ channels. Furthermore, treatment of murine CD8 T cells with ART induced significant increases in mitochondrial Ca2+ loading upon TCR activation. These data suggest that inhibition of T cell survival by ART was dependent on TCR activation-induced Ca2+ influx and associated with enhanced mitochondrial Ca2+ uptake. We examined the impact of ART on GVHD in Balb/c mice receiving C57BL/6 (B6) mouse T cell-depleted bone marrow (TCD-BM) and CD4+ T cells. Intraperitoneal injection of ART (10 mg/kg, every other day) from day 1 to day 28 after transplantation reduced clinical signs of GVHD in these recipients and significantly improved their overall survival. Similar inhibition effects of ART on GVHD were observed in miHA-mismatched B6 anti-Balb/b and haplo-identical B6 anti-BDF1 mouse models of GVHD. Further investigations showed that in vivo administration of ART caused significant decreases in the number of host-reactive donor T cells in the spleen and liver of Balb/c mice 7 days after transfer of B6 TCD-BM plus CD4+ T cells. ART treatment did not affect the capacity of donor T cells to produce effector cytokines (e.g., IFN-g and TNF-α) in individual cells. Importantly, in vivo administration of ART preserved anti-leukemia activity of donor T cells and did not impair the reconstitution of hematopoiesis and lymphocytes. Collectively, our findings indicate that pharmacologically increasing mitochondrial Ca2+ loading may have significant implications in the development of novel strategies to prevent GVHD and other T cell-mediated inflammatory disorders in a broad context. Since ART therapy has been clinically approved, this work could be immediately translated into patients. Disclosures No relevant conflicts of interest to declare.

Vasoactive intestinal polypeptide synthesis by somatic tissues in transplant recipients limits GVHD in allo-BMT

Abstract Both donor and recipient hematopoietic cells regulate GvHD in allo-BMT, but the role of somatic host tissues in the induction and maintenance of GvHD is less clear. Vasoactive Intestinal peptide (VIP) is a neuropeptide with potent immune-regulatory activity. Our goal was to examine the role of VIP synthesis by recipient tissues on donor T cell activation and GvHD in allo-BMT. We used MHC mis-matched allo-BMT models, B10.BR--&amp;gt;B6 and Balb/c--&amp;gt;B6, irradiating WT and VIP-KO B6 mice with 11Gy on day −1 and transplanting with 5 × 10E6 T cell depleted (TCD) bone marrow (BM) plus 0, 1 × 10E6, or 3 × 10E6 T cells from MHC mis-matched donors. B6--&amp;gt;B6 radiation chimeras lacking VIP in either hematopoietic or non-hematopoietic cells were created by performing B6 WT--&amp;gt;VIP-KO and VIP-KO--&amp;gt;WT syngeneic transplants. Production of VIP in transplant recipients was visualized with immune-fluorescent staining of tissues using an anti-VIP antibody. Transplanting TCD BM plus 3 × 10E6 donor T cells from B10.BR or Balb/c donors in VIP KIO mice led significantly increased GVHD-mortality compared with WT recipients. Donor-derived T cells in VIP-KO recipients had significantly higher Th1 and Th17 polarization. B10.BR--&amp;gt;B6 second allogeneic transplantation of radiation chimeras caused lethal GVHD mortality in VIP-KO-- &amp;gt;VIP-KO and WT--&amp;gt;VIP-KO mice, but not in WT--&amp;gt;WT or VIP-KO--&amp;gt;WT B6 mice, demonstrating the protective effect of VIP was due to synthesis by somatic recipient cells. Immunofluorescent imaging of allo-BMT recipients showed up-regulation of VIP in lungs post-transplant and high VIP production within neurons innervating the lungs. Short-term administration of VIP from D0 to D10 prevented GvHD-mortality in VIP-KO recipients transplanted BM &amp; T cells.