Donor Bone Marrow Cells Research Articles

Unique Roles for Grb2-Associated Binding (Gab) Adaptors in Hematopoietic Stem Cell Homeostasis and Repopulation

Homeostasis of hematopoietic stem cells (HSCs) is essential for the continuous replenishment of the hematopoietic system throughout the entire lifespan of an organism and is a tightly regulated process, controlled by intrinsic and extrinsic signals. Although a variety of molecules controlling HSC homeostasis are known, it remains unclear how signaling is regulated. The growth factor receptor-bound protein 2 (Grb2)-associated binding (Gab) proteins are a family of Pleckstrin homology domain containing adaptor proteins that dock with receptor tyrosine kinases (RTKs) and non-RTKs to control cell activation of ERK and PI3K pathways. The murine Gab family includes Gab1, Gab2, and Gab3. Gab1 and Gab2 are expressed throughout hematopoiesis including HSCs but Gab3 is limited mostly to macrophages, dendritic cells, and T-cells. Previously we reported that Gab2 is intrinsically required for hematopoietic cell responses to early-acting cytokines, with defective hematopoiesis in Gab2-/- mice (obtained from Toshio Hirano, Osaka, Japan; Nishida K et al, Blood, 99, 2002). Little is known about redundancy among Gabs in hematopoiesis and it is possible that roles for Gab1 and Gab3 have been obscured by the prominence of Gab2. Using Gab1flox/flox (Bard-Chapeau EA et al, Nat. Med. 11, 2005) and Gab3-/- mice (obtained from Pam Hankey-Giblin, Penn. State; Seiffert M et al, Mol. Cell. Biol. 23, 2003) we set out to make compound mutant mice. Gab3 knockout mice do not have reported hematopoietic phenotypes. First, we generated Gab2/3 double knockout mice. In the peripheral blood, we observed greater B-lymphocyte and erythroid deficiency compared to wild-type or Gab2-/- mice. In non-competitive bone marrow transplantation, Gab2/3-/- bone marrow was sufficient to reconstitute relatively normal myeloid hematology in irradiated recipients but resulted in significantly reduced donor derived B- and T-lymphocytes relative to wild type or Gab2-/- donor bone marrow cells, which were the same. In competitive bone marrow transplantation against Boy J (CD45.1) we observed sharp reductions in multilineage engraftment of Gab2/3-/- (13-15 fold) compared to Gab2-/- (4-6 fold) mice while Gab3-/- mice were normal. These data combined with the limited expression pattern for Gab3 suggest a novel critical extrinsic role in hematopoiesis. Despite severe multilineage engraftment defects, we surprisingly observed that Gab2/3-/- mice had significantly increased numbers of KLS cells (~2-fold) as well as LT-HSCs (KLSCD150+CD48-, ~3-fold) compared to the wild-type controls. Associated with the increased HSC numbers were cell cycle changes observed by staining with Pyronin Y/Hoechst 33342. During steady-state, Gab2/3-/- KLS cells were less quiescent with a lower percentage of cells in G0 (Gab2/3-/- vs WT: 40.5 ± 6.1 vs 57.4 ± 11.4, N=4, p=0.04) and more cells in G1 (Gab2/3-/- vs WT: 19.5 ± 0.7 vs 13.5 ± 3.5, N=4, p=0.01) phase of the cell cycle. Interestingly, KLS cells from Gab2/3-/- mice were less apoptotic than wild-type control (1.05 ± 0.49% in Gab2/3-/- (N=7) vs 5.97 ± 1.40% in WT KLS (N=10), p<0.001). Together these indicate that the HSCs were expanded but functionally defective. To further explore the mechanisms for Gab2/3-/- defects, we found that Gab1 expression levels were increased 2-fold. Since germline Gab1 deficiency results in embryonic lethality, Gab1 floxed alleles were deleted conditionally using Vav1-Cre to generate viable adult Gab1/2/3-/- mice. Real-time PCR confirmed the average of 94% Gab1 deletion efficiency (N=10) and Western analysis showed no detectable level of Gab1 in splenocytes. Interestingly, Gab1 deletion reversed the KLS expansion observed in the Gab2/3-/- mice indicating that Gab1 has effects that oppose Gab2/3 at the KLS level. However, Gab1/2/3-/- mice had severe defects in multilineage engraftment in competitive repopulation (39-52 fold). Altogether, our data indicate that Gab2/3 play positive roles while Gab1 plays a negative role in HSC homeostasis but all three Gabs are essential for multilineage repopulation. Further studies of Gab1 and Gab3 function in intrinsic and extrinsic regulation of HSC biology and repopulation are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice.

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

Isolation of Circulating Plasma Cells in Multiple Myeloma on a Novel Automated System

Abstract Multiple myeloma is a cancer of disordered plasma cells. The disease poses significant challenges for clinical management where cytogenetic analysis, such as FISH (fluorescent in situ hybridization), was commonly used for diagnosis and prognosis. Beads based enrichment of plasma cells are widely used to enhance FISH sensitivity and specificity. However conventional beads-based methods often require time-consuming sample preparation and results in low cell recovery. Here we present an easy and automated method to positively isolated CD138+ plasma cells from bone marrow samples. The method is developed on a novel designed magnetic separation system, MARS®. MARS® is matrix-free magnetic separation platform providing high purity, high recovery, and high capacity for cell isolation. Magnetic bead labeled CD138+ cells are isolated directly from bone marrow and peripheral blood without centrifugation. In the pilot study, U266 cells were spiked into peripheral blood from 0.5% to 2.0% of white blood cells frequency range. In addition, healthy donor bone marrow cell sample with 0.04% plasma cells were processed on MARS®. Plasma cells were isolated at 1mL/min flow rate followed by automated rinsing and releasing on MARS®. 48% to 80% recovery of the plasma cells were obtained. Results show our method has advantage in fast (&amp;lt;30 minutes), easy process with high recovery. Our next objective will be to validate the method on Multiple Myeloma patient specimens and compared to conventional beads-based enrichment methods. Enriched plasma cells will be analyzed by FISH to prove the enhanced sensitivity with better enrichment method.

A Prospective Controlled Trial to Evaluate Safety and Efficacy of in vitro Expanded Recipient Regulatory T Cell Therapy and Tocilizumab Together With Donor Bone Marrow Infusion in HLA-Mismatched Living Donor Kidney Transplant Recipients (Trex001).

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR <35 mL/min/1.73 m2], graft-vs.-host disease or patient death by 12 months) and efficacy (total leukocyte donor chimerism within 28 days post-transplant) are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617.

Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function

Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced "stress" associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2.

CAR2.0 Therapy for the Management of Post-Transplantation Relapse of B-Cell Acute Lymphoblastic Leukemia

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-HCT) is a standard treatment for relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However ~30-40% of patients (pts) still relapse after HCT. We report a cohort of 20 r/rB-ALL pts, who relapsed after HCT, and enrolled in the CAR2.0 study receiving one or two types of CAR-T cells targeting various B-ALL antigens. METHOD: Pts with r/r B-ALL who relapsed after allo-HCT and did not have significant active comorbiditeis, were enrolled in the study. The target antigens were determined based on immunostaining of each pt's leukemia cells, and CAR-T infusions included a single, or a combination of CAR-Ts targeting the following antigens: CD19, CD22, CD123 and CD38. T cells were collected from pts (N=4) or their allogeneic donors (N=16) and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine lymphodepleting therapy before infusion of 0.2-5.8x106 CAR-T/kg per infusion. In addition to disease response, we carefully monitored the quality of apheresis cells, efficiency of gene transfer, T cell proliferation rate, CAR-T infusion dose, and the CAR-T copy number in peripheral blood. RESULTS: Among the 20 enrolled pts, 11 were &amp;lt;18 years of age, and 7 were BCR- ABL (P190) positive. Before CAR-T treatment, 7 pts had ≤grade 2 active graft-versus-host disease (GVHD), and 13 pts received chemotherapy or targeted therapy after their relapse post HCT. Six pts had extramedullary relapse and 2 of them also had bone marrow relapse. The tumor burden in bone marrow ranged from minimal residual disease (MRD) negative to 66% of blasts, based on flow cytometry before CAR-T therapy. Five pts had &amp;gt;10% blasts in bone marrow, 8 pts had &amp;lt;3% blasts, and 7 pts had MRD negative bone marrow (summarized in the Table below). Based on the GVHD history, chimerism state and the available T-cell sources, 16 pts used allogeneic HCT donor T-cells for CAR-T preparation. All pts were full donor chimeras prior to CAR-T infusion, except one pt who had 41% donor cells in bone marrow. Eleven pts received a single CD19 CAR-T infusion, with a mean dose of 1.6x106 CAR-T/kg, and ten achieved an MRD remission and one had progressive disease (PD) within 60 days by flow cytometry. The remaining 9 pts received 2 CAR-Ts (CD19 plus CD22, CD123 or CD38 CAR-Ts) given on the same day, and resulted in 8 CR and 1 PD within 60 days. After CAR-T infusion, no cytokine release syndrome (CRS) was observed in 8 pts, and 12 pts experienced CRS of grade 1, which was consistent with the previously described low toxicity profile of the 4SCAR design. Acute GVHD ≤ grade 2 developed in 5 pts within one month following CAR-T cell infusion but all responded well to supportive care and/or cyclosporine infusion. The 2 pts who developed PD after CAR-T infusion included the one with 41% donor chimerism and had grade 2 GVHD and active infections before CAR-T infusion. The other pt with PD following CAR-T had severe bone marrow suppression, low leukocyte count, infections and was transfusion dependent before enrollment. This emphasizes the need for controlling comorbidities before infusion of CAR-T cells. In summary, total 18 patients (90%) achieved negative MRD remission within 2 months of therapy with acceptable CRS. Four pts relapsed (after being in remission for 3 months) and 14 pts are in continued remission, 6 of which for &amp;gt; 1 year. None of these 20 pts received a second HCT after CAR-T infusion. GVHD developed in 5/16 (31%) pts after donor source CAR-T cell infusion within one month, but all responded well to treatment. CONCLUSION: This study focuses on CAR-T cell therapy following relapse after HCT. While the expanded study is ongoing, we present results of the first 20 pts. Use of donor-derived or recipient-derived CAR-T products in pts who relapsed after allo-HCT is well tolerated and it may prolong life expectancy of these pts while maintaining good quality of life. Table Disclosures No relevant conflicts of interest to declare.

Genetic Manipulation Resulting in Decreased Donor Chondroitin sulfate Synthesis Mitigates Gvhd Following Allogeneic Hematopoietic Cell Transplantation in a Murine Model.

Introduction: Severe acute graft versus host disease (GVHD) represents a major risk associated with allogeneic hematopoietic cell transplantation (HSCT). Both acute and progressive GVHD following HSCT are in large part attributable to responses of donor T cells to host allo-antigens. Alloreactive T cell activation can be modulated by signals from the tissue microenvironment (TME). Glycosaminoglycans (GAGs) in the TME or modifying T cell surface proteoglycans are known to regulate T cell function. However, the roles of GAGs in acute GVHD following allogeneic HSCT remain to be elucidated, since a suitable murine model has not been available. We previously established a unique mouse model (T1KO), in which knockout of the chondroitin sulfate (CS) N-acetylgalactosaminyltransferase-1 (T1) gene - encoding the rate-limiting CS-synthesizing enzyme - decreases CS production. We also reported a role for CS in murine hematopoietic stem cells. In the present study, we focus on donor T cell CS levels to assess the role of T cell CS in acute GVHD following allogeneic HSCT. We were able to mitigate the clinical features of GVHD in a murine model using T1KO donors. Methods: Eight- to 12-week-old T1KO mice were generated from a C57BL/6N (WT) strain as donors (H-2b) for allogeneic bone marrow transplantation (BMT). Donor BM cells were then transplanted into eight- to 12-week-old BALB/c recipients (H-2d). Prior to transplantation, recipients were irradiated at a dose of 7 Gy. They then received 5 x 106 BM cells and splenocytes of identical genotype, as well as low (5 x 105), intermediate (1 x 106), or high (4 x 106) doses of CD90.2+ cells from either WT or T1KO donors. For five weeks following BMT, survival was monitored daily and clinical GVHD scores (incorporating body weight, activity, fur condition, alopecia, and tortoiseshell-like mucosae) were calculated three times per week. Results: The peripheral blood CD4+/CD8+ T cell ratio of T1KO mice was equivalent to that of WT mice. Following allogeneic BMT, histopathologic analysis confirmed onset of acute recipient GVHD on day 49 when donors were WT mice. As a result, significant lymphocyte infiltration was observed in target organs (liver, colon, and skin). High-dose transplantation of splenocytes from T1KO rather than WT mice significantly prolonged recipient median survival (27.0 versus 20.5 days, p = 0.02). A similar trend was observed for low- and intermediate-dose transplantation (low: 53.0 versus 29.0 days, p = 0.09; intermediate: 50.5 versus 30.5 days, p = 0.13. Similarly, a significant improvement in day 11 GVHD score was observed following high-dose splenocyte transplantation when donors were T1KO mice (mean scores: 1.00 versus 2.75, p &amp;lt; 0.01). However, no significant differences in GVHD score were observed following low- and intermediate-dose transplantation. Taken together, our results suggest that donor T cells producing lower CS levels alleviate acute GVHD following allogeneic HSCT in a murine model. Conclusion: Alloreactive T cell activation following allogeneic HSCT may be down-regulated by lower CS levels, thereby mitigating GVHD severity. Figure 1 Disclosures No relevant conflicts of interest to declare.

ICOSL+ plasmacytoid dendritic cells as inducer of graft-versus-host disease, responsive to a dual ICOS/CD28 antagonist.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). CD146 and CCR5 are proteins that mark activated T helper 17 (Th17) cells. The Th17 cell phenotype is promoted by the interaction of the receptor ICOS on T cells with ICOS ligand (ICOSL) on dendritic cells (DCs). We performed multiparametric flow cytometry in a cohort of 156 HCT recipients and conducted experiments with aGVHD murine models to understand the role of ICOSL+ DCs. We observed an increased frequency of ICOSL+ plasmacytoid DCs, correlating with CD146+CCR5+ T cell frequencies, in the 64 HCT recipients with gastrointestinal aGVHD. In murine models, donor bone marrow cells from ICOSL-deficient mice compared to those from wild-type mice reduced aGVHD-related mortality. Reduced aGVHD resulted from lower intestinal infiltration of pDCs and pathogenic Th17 cells. We transplanted activated human ICOSL+ pDCs along with human peripheral blood mononuclear cells into immunocompromised mice and observed infiltration of intestinal CD146+CCR5+ T cells. We found that prophylactic administration of a dual human ICOS/CD28 antagonist (ALPN-101) prevented aGVHD in this model better than did the clinically approved belatacept (CTLA-4-Fc), which binds CD80 (B7-1) and CD86 (B7-2) and interferes with the CD28 T cell costimulatory pathway. When started at onset of aGVHD signs, ALPN-101 treatment alleviated symptoms of ongoing aGVHD and improved survival while preserving antitumoral cytotoxicity. Our data identified ICOSL+-pDCs as an aGVHD biomarker and suggest that coinhibition of the ICOSL/ICOS and B7/CD28 axes with one biologic drug may represent a therapeutic opportunity to prevent or treat aGVHD.

Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1−/− donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.

Desensitization using imlifidase and EndoS enables chimerism induction in allosensitized recipient mice.

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients who have been sensitized to donor antigens is challenging. Donor‐specific antibodies (DSA) are a dominant barrier toward successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allosensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor‐specific IgG using imlifidase and endoglycosidase of Streptococcus pyogenes (EndoS), which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody‐mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion, and nonlethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS, can be used for inducing donor hematopoietic chimerism in allosensitized recipient mice in combination with other desensitization strategies.

Inducing Donor MHC Chimerism with Bone Marrow Derived Exosomes in Non-Human Primates

Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on expression of donor MHC molecules in the host's primary and secondary lymphoid organs. Here, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells. Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). Two H38- cynomolgus macaque recipients received conditioning consisting of ATGAM and thymic irradiation, with or without total body irradiation (TBI) before a heart and kidney co-transplantation. Both recipients underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38+ donor bone marrow derived exosomes was infused into H38- recipients on post-operative day 2. Image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, spleen and lymph nodes. Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in both animals. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Tissues collected from the total body irradiated NHP showed that H38+ cross-dressed cells were also present in the lymph nodes, thymus, and spleen. In a separate study, a NHP recipient treated with an exosome protocol without TBI is currently 54 days post-transplantation with a well-functioning cardiac allograft. Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow to induce allograft tolerance. The use of exosomes would allow for the reduction of recipient conditioning and would eliminate the risk of GVHD making this approach clinically safer than traditional BMT.

Characterization of a Novel CD8+ B Cell Subset in Monkeys Tolerant of Heart Allografts

We have previously reported that transient mixed hematopoietic chimerism induced via donor bone marrow transplantation can achieve tolerance to cardiac allografts in non-human primates (NHPs). The purpose of this study was to measure the frequency and properties of a unique subset of CD8+ B cells, recently identified in our laboratory, in tolerant monkeys. Cynomolgus Macaques underwent organ transplantation followed by injection of donor bone marrow cells with mixed chimerism conditioning consisting of 3Gy TBI, 7Gy thymic irradiation, anti-thymocyte globulin, and a short course of anti-CD154 mAb and CyA therapy. The frequencies of different B cell subsets were measured in the peripheral blood of ten monkeys pre- and post-transplantation. In addition, PBMCs were isolated from peripheral blood of two tolerant animals (recipients of heart plus kidney or heart plus thymus cotransplants) 847 and 274 days post-bone marrow and FACS-sorted into five groups: T cells (CD3+), whole B cells (CD20+), mature B cells (CD20+CD8-CD27+), naïve B cells (CD20+CD8-CD27-), and newly identified CD8+ B cells (CD20+CD8+). mRNA was extracted from lysates of each cell population and sequenced using an Illumina NextSeq 500. During the reconstitution phase of the conditioning protocol, CD8+ B cells exhibited a phenotype reminiscent of transitional B cells (CD20+CD27-IgMhiCD38hi) and the percentage of these cells among peripheral B cells was much higher than that observed in naïve animals (23.4% vs 2.5% respectively; p<0.001). In the two tolerant animals, the CD8+ B cells gene expression suggested an immature profile which included the expression of CD21, CD38, and CD24. Interestingly, however, CD8+ B cells showed an increased expression of the gene encoding for the regulatory protein CD72. In addition, CD8+ B cells had greater expression of CD69 which is involved with TGFbeta production. The prevalence of CD8+ B cells expressing genes involved in regulatory immunity after bone marrow transplantation suggests that these cells may play a role in tolerance induction to heart allografts in NHPs. Work is in progress to further characterize these novel cells.

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Novel Improvement of Haploidentical Hematopoietic Stem Cell Transplantation for Advanced Refractory/Relapsed Acute Leukemia

Introduction The advanced refractory/relapsed acute leukemia has a very dismal prognosis even with the salvage allogeneic hematopoietic stem cell transplantation (allo-HSCT). To develop a novel transplant protocol to achieve a rapid engraftment and quick graft-versus leukemia (GVL) effect is crucial. Based on previous observation, the G-CSF primed bone marrow (BM) plus peripheral blood mononuclear cells (PBMNCs) could not only increase mononuclear cells and CD34 cells, but also change T-cell biology by down-regulating the expression of adhesion and CD28/B7 molecules and increase the absolute number of D2APCS, which promotes a T-cell shift from T1 to T2 subset to secrete a higher anti-inflammatory cytokines of IL-4 and IL-10. After going through a rigorous conditioning, the recipients' T-cells are incapable to attack host. We propose that reinfusion of G-CSF primed donor PBMNCs to recipient on transplant day + 6 may cross mismatched MHC barrier thus accelerating engraftment. Patients and Methods From April 2018 to June 2019, 30 advanced-stage patients with refractory/relapsed acute leukemia were enrolled and underwent a salvage haploidentical (haplo)-HSCT. The median age was 12 (2-63) years with M/F ratio of 17/13. There were 21 patients with acute myelocytic leukemia (AML, 70%), 4 -cell acute lymphoblastic leukemia (B-ALL, 13%), 2 biphenotypic acute leukemia (BAL, 7%), 1 juvenile myelomonocytic leukemia (JMML, 3%), and 2 T-lymphoblast leukemia (T-LBL, 7%). The median BM blasts were 40% (5%-90%). Twenty-eight patients received a conditioning regimen including fludarabine 30mg/m2/d×4 day, cytarabine 2.0g/m2/d×4 days, busulfan 3.2mg/kg/d Q6h×4days, cyclophosphamide 1.5g/m2/d×2 days and melphalan 110mg/m2/d×1 day. Two patients with T-LBL received BCNU+Etoposide+Cytarabine+Melphalan (BEAM) regimen. Graft-versus-host disease (GVHD) prophylaxis contained methotrexate (MTX), cyclosporine A (CsA) and mycophenolate mofetil (MMF), plus ATG (Rabbit anti-human thymocyte immunoglobulin, Sangstat) 1.5mg/kg/d iv daily from day -4 to -1 and additional 1.5mg/kg on day +7 (total dose of 7.5 mg/kg). In addition, anti-CD25 monoclonal antibody (basiliximab) 20 mg on day+1 and +4 was given iv. Donors were primed with G-CSF at 3-4 ug/kg/d sq d1-5. On day 4 of G-CSF, donor BM cells were harvested and were infused unmanipulated on the same day to patient on transplant day 01. On day 5 of G-CSF injection, donor primed PBMNCs were harvested, part of which were immediately infused unmanipulated to recipient on transplant day 02. Part of the cells were cryopreserved and stored in the liquid nitrogen, which were then thawed and reinfused unmanipulated to recipient on day +6 (Table 1). Results All patients achieved a trilineage engraftment with a median time of 13.5 (5-16) days. The median neutrophil and platelet engraftment were 13 (11-19) days and 10 (5-22) days respectively (Fig.1). Among 29 evaluable patients, acute GVHD occurred in 10 (34.5%, Fig. 2) with a median time of 32 (14-60) days, including 5 cases (17%) of grade Ⅱ, 3 (10%) of grade Ⅲ and 2 (7%) of grade Ⅳ. Among the 28 evaluable patients for chronic GVHD, only 6 (21.4%, Fig.3) developed limited chronic GVHD with a median time of 103 (90-180) days. Twenty seven out of thirty (90%) patients achieved disease-free survival (DFS) with a median follow-up time of 6 (5-14) months. Three patients died from transplant related complication, including infection for 2 and GVHD for 1 respectively. Conclusion In this study, with an intensive myeloablative condition and salvage haplo-HSCT, by reinfusing additional unmanipulated donor PBMNCs to recipients on day +6 during their transplantation, a short engraftment time without engraftment failure, low lethal GVHD incidence, and a lower infection rate were able to be achieved. More importantly, most patients remain DFS at a median follow-up time of 6 months. Despite short-term follow-up. the outcomes are particularly encouraging. Disclosures No relevant conflicts of interest to declare.

Integrative Study of EZH2 Mutational Status, Copy Number, Protein Expression and H3K27 Trimethylation in AML/MDS Patients

Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p&amp;lt;0.05 (indicated by asterisks). Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.

Manipulation of Induced Foxp3+ Regulatory T Cells for Prevention of Mouse Acute Graft-Versus-Host Disease

Background Foxp3+ regulatory T cells (Tregs) comprise of natural (n) and induced (i) Treg subsets play an important role in immune system. Currently, isolation of nTregs and in vitro-expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, iTregs can be generated in vitro from naïve T cells and to a large number of iTregs in short time. As we known, regulatory T cells would decay after a period of time, in vivo or in vitro. Keeping a certain number of iTregs during the GVHD treatment is necessary, it should be the best to provide iTregs to the patient more than single usage. Aim Manipulated supplements of TGF-β1-induced Foxp3+ regulatory T cells should be a good way for prevention from acute graft-versus-host disease within a short time. Investigation was performed via animal model. Methods Splenocytes from C57BL/6 mice were used as a source of naïve T cells by a CD4+ naïve T cell isolation Kit. To induce Foxp3+ regulatory T cells (iTregs), the CD4+ naïve T cells were incubated with anti-CD3/CD28 coated 24-well plate in the presence of IL-2 (20U/ml) and TGF-b1 (50ng/ml) for 3 days. Foxp3+iTregs were harvested and identified as the expressions of CD4+/CD25+/FoxP3+/CD127- via flow cytometry (Fig.1). In this experiment, recipients (BALB/c) were irradiated with 800cGy and then infused with donor (C57BL/6) bone marrow cells with (TCD-BM+CD4T) or without donor T cells (TCD-BM) by intravenous injection. TCD-BM+CD4T cells mice would appear aGVHD phenotype. 8x106 Foxp3+ iTregs were injected into the TCD-BM with donor T cell mouse one or twice (TCD-BM+CD4 T +iTreg) for immunosuppression assay as shown in Fig.2. Mouse GVHD phenotype, body weights and survival rates were investigated lasting for over 90 days. Tissue sections were stained with haematoxylin-eosin. Results According to our preliminary data, it indicated the injection of iTregs in the prevention of aGVHD should be feasible (Fig.3). Consequently, we have tried to investigate preventative efficiency of repeated iTregs supplements in TCD-BM mice. First of all, we compared the single-dose of iTregs with the repetition-dose of iTregs in aGVHD prevention. The data showed in Fig.4. The data showed that the survival rate was 73.3% in repeated treatment in mice, however, the survival rate was only 45.8% in single-dose of iTregs mice within 24 days. As the TCD-BM survival rate was 76.1%. It indicated that the repetition-dose of iTregs would prevent the occurrence of aGVHD, and the survival rate was similar as the bone marrow transplantation mice. The BM-CD4T mice with aGVHD phenotype could survive no more than 10 days. Furthermore, we investigated the survival time of the continual iTreg supplements mice. The data showed in Fig.5. After 90 days later, the body weight of iTregs treated mice could maintain the recovery efficiency to 83.8±2.1% and the survival rate to 78%, comparing with the TCD-BM mice was 88.8±0.6% and 73%. All of these mice could keep alive more than 90 days. Using histographic staining, we confirmed the aGVHD prevention with repeated supplement of Foxp3+iTregs to the CD4T mice (Fig.6). The mice, administration of CD4T cells with bone marrow cells, failed to survival for the serious damage of intestine villi (Fig.6A) and Peyer's patches (Fig.6B). In contrast, CD4T mice with Foxp3+-iTregs (iTregs) could survival more than 90 days and intestine villi were recovered after 90 days (Fig.6A). Peyer's patches are an important gut associated lymphoid tissue in small intestine and play a crucial role in immune response. Therefore, we have investigated the changes of Peyer's patches (Fig.6B). As the recovery of mice with iTregs for twice, the Peyer's patches reappeared after 90 days later. It indicated that keeping more iTregs in vivo could more efficient on prevention of aGVHD. It indicated that more alive iTregs to prevent GVHD occurrence more efficient and may provide the information pre-clinically. Conclusion We showed that repetition supplement of iTreg cells to TCD-BM+CD4T-treated mice, could maintain the mice in high survival rate. Therefore, we may provide more of the functional iTregs to GVHD patients, continuously. It's a good way to prevent the occurrence of GVHD. The result should develop a novel-cell based approach for potentially reducing the risk of acute GVHD clinically. Disclosures No relevant conflicts of interest to declare.

Ruxolitinib, a JAK1/JAK2 Selective Inhibitor, Ameliorates Gastrointestinal and Cutaneous Pathogenesis in MHC-Mismatched Preclinical Models of Acute and Chronic Graft-Versus-Host Disease

Background: Allogeneic hematopoietic cell transplantation (alloHCT) is potentially curative for a variety of hematologic malignancies. However, rather than targeting tumor cells, alloreactive donor T cells may attack healthy recipient tissue triggering graft-versus-host disease (GVHD) resulting in morbidity and mortality. Acute GVHD (aGVHD) clinically presents with gastrointestinal (GI) pathology and is primarily T cell mediated. In contrast, chronic GVHD commonly demonstrates skin inflammation and fibrosis with an autoimmune-like phenotype. Pharmacological inhibition of JAK/STAT signaling has the potential to regulate aberrant immune cell functions involved in the pathogenesis of acute and chronic GVHD. Aim: Characterize the effectiveness of ruxolitinib to modulate in vitro and in vivo immunological mechanisms that are described to be critical in acute and chronic GVHD pathogenesis. Materials and Methods: MHC-mismatch aGVHD was induced in recipient BALB/c mice receiving C57BL/6 donor transplant. Sclerodermatous cGVHD was induced in C57BL/6 mice using LP/J strain donor splenocytes and bone marrow cells. Ruxolitinib or vehicle was orally administered twice daily. Efficacy was assessed by GVHD symptom scores, cachexia, and histological analysis of end organ damage. Mixed lymphocyte reactions (MLR) were cultured for five days in the presence of ruxolitinib for changes in activation markers and cytokine release. Results: Ruxolitinib treatment ameliorated daily aGVHD severity scores with concomitant reduction of GI tract infiltration and tissue damage. Overall aGVHD mice survival was significantly improved with JAK1/2 inhibition. Therapeutic ruxolitinib treatment initiated after cGVHD symptom onset significantly inhibited the chronic-progressive symptom phenotype and reduced tissue markers of inflammation and fibrosis. Ruxolitinib demonstrated concentration-dependent inhibition of activated T cells and reduced JAK-STAT signaling cytokines considered relevant in GVHD inflammation. Conclusion: Ruxolitinib, a potent and selective JAK1/2 inhibitor, significantly ameliorated pro-inflammatory pathways in vitro and in vivo which are thought to be highly relevant for acute and chronic GVHD pathogenesis. On May 24, 2019, the FDA approved ruxolitinib for steroid-refractory acute graft-versus-host disease (aGVHD) in adult and pediatric patients 12 years and older. Ruxolitinib is currently being evaluated in clinical trial (REACH3: NCT03112603) for patients with steroid-refractory cGVHD. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Juvekar:Incyte corporation: Employment, Equity Ownership. Oliver:Incyte corporation: Employment, Equity Ownership. Yao:Incyte corporation: Employment, Equity Ownership. Smith:Incyte corporation: Employment, Equity Ownership.

VCAM1 Confers Innate Immune Tolerance on Hematopoietic and Leukemic Stem Cells

Hematopoietic stem cells (HSCs) home to the bone marrow (BM) via, in part, the interactions with Vascular Cell Adhesion Molecule-1 (VCAM1). Upon migrating into the BM, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here, we show that VCAM1 is also expressed on healthy Lineage− c-Kit+ Sca1+ CD48− CD150+ HSCs and upregulated on acute myeloid leukemia (AML) cells where it serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping in the context of major histocompatibility complex (MHC) class-I presentation. Using a line in which HSCs are hemizygous for MHC (H-2b/H-2q) and Vcam1 can be conditionally deleted by breeding Vcam-1 floxed (Vcam1fl/fl) mice with a Csf1r-iCre transgenic line (referred to as Vcam1cKO), we find that Vcam1 deletion in HSCs leads to striking defects in hematopoietic recovery when donor and recipients are mismatched (donor H-2b/q; recipient H-2b). Remarkably, wild-type donor BM cells with haplotype-mismatched genotype exhibit engraftment and survival similar to their syngeneic (H-2b) Vcam1fl/fl (control) and Vcam1cKO counterparts. In vivo phagocytic assays show that Vcam1cKO;H-2b/q HSCs are phagocytosed by host H-2b immune phagocytes whereas Vcam1-sufficient H-2b/q HSCs are not targeted. Clearance is not dependent on irradiation-induced damage since the same phenotype is observed in parabiotic mice where Vcam1cKO;H-2b/q HSCs are not able to engraft their wild-type (H-2b) parabiont partner. Mechanistically, we show that MHC-mismatched HSCs are recognized at least in part by paired Ig-like receptor-B (PIR-B), a negative regulator of immune cells upon recognition of MHC-I molecules, expressed on α4β1+ murine phagocytic myeloid cells. Transplantation assays revealed that in absence of PIR-B inhibitory signals, Vcam1 deletion leads to significant reductions (~60%) in the early engraftment of syngeneic Vcam1cKO;H-2b/b cells compared to Vcam1fl/fl;H-2b/b cells, suggesting that the absence of VCAM1 also promotes cell clearance by syngeneic Pirb-/- phagocytes. That VCAM1 expression can provide innate immune tolerance indicates that this pathway may be of use for cancer cells. Indeed, alterations in VCAM1 expression has been reported in various cancer cell types, including AML. To evaluate the effect of genetic Vcam1 deletion and MHC-mismatch on AML progression, we have transduced Vcam1cKO;H-2b/q and Vcam1fl/fl;H-2b/q cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, analysis of primary AML recipient BM reveals a marked reduction (&amp;gt;99%) of phenotypic Vcam1cKO leukemic stem cells compared to Vcam1fl/fl control. Accordingly, the survival of secondary AML recipient mice is significantly prolonged in mice harboring Vcam1cKO AML cells relative to Vcam1fl/fl AML. Leukemia progression is markedly enhanced by phagocyte depletion (using clodronate liposomes), and the engraftment defect of Vcam1cKO AML cells is completely rescued, indicating an important role for phagocytes in the establishment of Vcam1-deficient AML. Analysis of The Cancer Genome Atlas (TCGA) database reveals that high VCAM1 expression is associated with the poor prognosis of patients with AML (P=0.03). In addition, VCAM1 is significantly overexpressed in Lineage− CD34+ CD38− CD90− short-term repopulating HSCs from sorted human AML BM samples. VCAM1 overexpression in human AML MOLM13 cancer cells promotes disease progression and significantly (P=0.005) shorter survival. Furthermore, VCAM1 blockade using novel monoclonal antibodies after implantation of primary human AML into immunodeficient mice blunts disease progression, and extends survival of mice. Altogether, these results suggest that VCAM1 engagement serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping, allowing HSCs to migrate and engraft, and offers a novel strategy to eliminate cancer cells via modulation of the innate immune tolerance. Disclosures Wei: Albert Einstein College of Medicine, Inc: Patents &amp; Royalties. Verma:Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding. Frenette:Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents &amp; Royalties; Pfizer: Consultancy.

Third Party Type 2 Innate Lymphoid Cells Treat and Prevent GI Tract Acute GvHD

Donor T cells present in the stem cell or bone marrow inoculum are critical to the induction of acute graft-versus-host disease (aGvHD). AGvHD of the gastrointestinal (GI) tract that is non-responsive to corticosteroid therapy is significant problem for patients undergoing allogeneic hematopoietic stem cell (allo-HSCT) transplantation with less than 15% of these patients alive one year after diagnosis. Previously, we have shown that the infusion of donor innate lymphoid type II (ILC2) cells could prevent and treat acute GvHD of the lower GI tract. This approach for clinical translation is cumbersome as it would require the generation of donor derived ILC2 cells for each recipient. Thus, the ability to use third party ILC2 cells would provide an "off the shelf" reagent that could be used to prevent aGvHD. Here, we show that third party ILC2 cells can effectively prevent and treat lower GI tract aGvHD. Co-transplantation of a single dose of third party ILC2s with MHC completely mismatched donor T cells significantly improved median survival of recipients by 11 days and reduced the clinical scores in mice. However we did not observe an improvement in overall survival (OS). When we evaluated a mechanism for this, we found that third party ILC2 cells did not persist in GvHD target organs. To overcome issues with persistence, we infused multiple doses of third party ILC2s on days 0, 7, 14, and 21 in recipient mice receiving complete MHC mismatched donor T cells and BM. Multiple infusions during early aGvHD significantly increased survival rates (Fig. 1A) and significantly reduced clinical scores (Fig. 1B) of recipients post-BMT, with 60% of the recipients surviving more than 60 days. Prevention of aGvHD with ILC2s can be clinically beneficial. However, it would be more advantageous if third party ILC2s could be used to treat active aGvHD. To evaluate their therapeutic benefit, ILC2s were injected 7 days after transplant, a time when donor T cells are activated in GvHD target organs, with additional injections at days 14 and 21. Interestingly, infusion of ILC2s as a treatment increased overall survival, increasing median survival by 10 days and reducing clinical scores from day 20 to day 30 compared to untreated recipients (Fig. 1C and 1D). These findings demonstrate multiple infusion of third party ILC2 cells can prevent and treat acute GvHD. To evaluate the mechanism of activity of ILC2 cells, we utilized donor BM cells lacking expression of the IL-13 receptor. Prevention of aGvHD was significantly diminished with the donor BM cells lacked the ability to respond to IL-13. Additionally, we found using FoxP3-DTR mice that the absence of Tregs modestly but significantly diminished the function of ILC2 cells. Thus, our data are consistent with IL-13 generation by ILC2 cells acting on donor myeloid-derived suppressor cells and Tregs, which greatly extends our initial findings. Finally, we show that ILC2 cells in vitro are not killed by corticosteroids paving the way for clinical trials for steroid non-responsive patients. Disclosures Stefanski: Novartis: Consultancy, Speakers Bureau. Blazar:RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding. Serody:Merck: Research Funding; GlaxoSmithKline: Research Funding.