Donor Hematopoietic Cell Engraftment Research Articles

Icariin protects against acute graft-versus-host disease while preserving graft-versus-leukemia activity after allogeneic hematopoietic stem cell transplantation

BackgroundAcute graft-versus-host disease (aGVHD), which is mainly mediated by allogeneic T cells, is a decisive factor in the success of allogeneic hematopoietic stem cell transplantation (allo-HCT). Prophylaxis for aGVHD in clinical patients is unsatisfactory, and there is still a huge unmet need for novel approaches. Icariin (ICA) shows potent anti-inflammatory activity and suppresses T cell-mediated immune responses. Thus, ICA is a potential drug for the prevention of aGVHD. However, there is no data assessing the impact of ICA on aGVHD after allo-HCT. PurposeThis study aimed to investigate the protective effect of ICA against aGVHD and its mechanisms. Moreover, the impact of ICA on the graft-versus-leukemia (GVL) effect and engraftment of donor hematopoietic and immune cells were assessed. MethodsDifferent murine models of allo-HCT were developed to study the influence of the ICA on GVHD and GVL effect. Flow cytometry was used to analyze the growth of leukemia cells, alterations in different immune cells, and apoptosis. Cell proliferation was determined using a CCK-8 assay. RNA sequencing and quantitative proteomic analysis were performed to elucidate the underlying mechanisms, which were further verified by polymerase chain reaction or functional experiments. ResultsDifferent concentrations of ICA exhibited opposite effects: low-concentration ICA promoted, while high concentrations suppressed the proliferation and function of T cells. A high dose of ICA administration during days +3 to +5 post-allo-HCT can alleviate murine aGVHD but does not affect the course of chronic GVHD (cGVHD), the GVL effect against both acute myeloid and lymphoblastic leukemia, or the recovery of donor hematological and immune cells. ICA extensively represses the expansion, function, and infiltration of donor alloreactive T cells, while preserving regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Quantitative proteomic analysis showed that downregulation of integrin-linked kinase (ILK) and lymphocyte cytosolic protein 2 (LCP2) expression was possibly associated with ICA-mediated aGVHD protective effects. Furthermore, an inhibitor of ILK, which can alleviate murine aGVHD administered early after allo-HCT. ConclusionThese findings suggest that the bioactivities of ICA are associated with its concentration and that ICA can effectively mitigate aGVHD without losing GVL activity or engraftment of donor hematopoietic and immune cells. Thus, ICA may be a promising drug for preventing aGVHD in clinical settings.

Pharmacokinetics of Briquilimab as a Conditioning Agent for Hematopoietic Stem Cell Transplantation in Patients With Severe Combined Immunodeficiency, Myelodysplastic Syndrome, or Acute Myeloid Leukemia

For successful engraftment of donor hematopoietic stem cells (HSC), conditioning with chemotherapy and/or radiation prior to hematopoietic cell transplantation (HCT) has been required to open marrow niche space and minimize the risk of immune rejection. Briquilimab, a humanized IgG1 monoclonal antibody that blocks the interaction between the c-Kit receptor and stem cell factor on various C-Kit expressing tissues including HSC, is a potential nonmyeloablative conditioning agent in clinical development for patients with severe combined immunodeficiency (SCID), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). This study aimed to characterize pharmacokinetics (PK) and develop a population PK model of briquilimab after single intravenous infusions of 4 different doses in patients with SCID, MDS, or AML receiving HCT. The PK data was collected from 2 different studies: JAS-BMT-CP-001 and JSP-CP-003. JAS-BMT-CP-001 is a phase 1/2 open-label study of briquilimab as a conditioning agent prior to allogenic HCT in SCID patients. The participants received single intravenous infusions of 0.1, 0.3, 0.6, or 1.0 mg/kg. JSP-CP-003 was a phase 1a/b open-label study of briquilimab in combination with a standard conditioning regimen of low dose total body irradiation and fludarabine in MDS or AML subjects undergoing HCT. The participants received a single intravenous dose of 0.6 mg/kg briquilimab. In both studies, briquilimab PK samples were obtained at pre-treatment, 5 minutes post-end of infusion, 4- and 24-hours post-start of infusion, any time between 2 days and 30 days postinfusion, and on the day of HCT prior to donor cell infusion.The population PK model was developed using the PK data from these 2 clinical studies, and the effect of participants’ baseline characteristics on the briquilimab PK was evaluated. PK simulations were performed using the developed PK model to calculate the time to reach target concentrations for HCT. A total of 49 participants (21 SCID adult and pediatric participants with a median age of 12 yr and 28 MDS/AML adult participants with a median age of 70 yr) were included in the PK analysis. A 2-compartment model with combined linear and non-linear elimination best described the PK of briquilimab. Body weight was determined as the sole covariate of the PK parameters among the explored covariates. For a typical subject with a body weight 70 kg, the estimated parameters for clearance, maximum metabolic rate of Michaelis Menten elimination, Michaelis Menten constant, central volume, peripheral volume, and intercompartmental clearance were 17.6 mL/h, 51,434.8 ng/h, 71.5 ng/mL, 3444.0 mL, 1613.3 mL, and 21.2 mL/h, respectively. The median time to reach target concentrations of 500, 1000, and 2000 ng/mL after a single dose of 0.6 mg/kg was calculated as 12.3, 10.4, and 7.7 days, respectively. The PK of intravenous briquilimab was characterized in subjects with SCID, MDS, or AML receiving HCT, and a population PK model was developed to estimate briquilimab clearance to use as a guide to the timing of donor cell infusion post-briquilimab. Body weight was identified as a significant covariate on elimination and volume of distribution of briquilimab.

Allogeneic Hematopoietic Cell Transplant For Bone Marrow Failure or Myelodysplastic Syndrome in Dyskeratosis Congenita/Telomere Biology Disorders: Single-Center, Single-Arm, Open-Label Trial of Reduced-Intensity Conditioning Without Radiation

BackgroundDyskeratosis congenita/Telomere biology disorders (DC/TBD) often manifest as bone marrow failure (BMF) or myelodysplastic syndrome (MDS). Allogeneic hematopoietic cell transplant (alloHCT) rescues hematologic complications, but radiation and alkylator-based conditioning regimens cause diffuse whole-body toxicity and may expedite DC/TBD-specific non-hematopoietic complications. Optimization of conditioning intensity in DC/TBD to allow for donor hematopoietic cell engraftment with the least amount of toxicity remains a critical goal of the alloHCT field. Objectives/Study DesignWe report prospectively collected standard alloHCT outcomes from a single-center single-arm open-label clinical trial of bone marrow or peripheral blood stem cell alloHCT for DC/TBD-associated BMF or MDS. Conditioning was reduced intensity (RIC) including alemtuzumab 1mg/kg, fludarabine 200 mg/m2, and cyclophosphamide 50 mg/kg. A previous single-arm open-label phase II clinical trial for the same patient population conducted at the same center, differing only by inclusion of 200 centigray of total body irradiation (TBI), served as a control cohort. ResultsThe Non-TBI cohort included 10 patients (ages 1.7-65.9 years, median follow-up of 3.9 years) compared to the control TBI cohort which included 12 patients (ages 2.2-52.2 years, median follow-up of 10.5 years). Baseline characteristics differed only in total CD34+ cells received, with a median of 5.6 (Non-TBI) compared to 2.6 (TBI) x 106/kg (p=0.02; no difference in total nucleated cells). The cumulative incidence of day +100 grade II-IV acute and 4-year chronic graft-versus-host disease (GvHD) were low at 0 and 10% (Non-TBI) and 8 and 17% (TBI), respectively (acute, p=0.36; chronic, p=0.72). Primary graft failure was absent. Secondary non-neutropenic graft failure occurred in one (Non-TBI cohort). The Non-TBI cohort demonstrated delayed achievement of full donor chimerism but superior lymphocyte recovery. There was no difference in 4-year overall survival at 80% (Non-TBI) and 75% (TBI; p=0.78).MDS as an indication for alloHCT was uncommon, but overall associated with poor outcomes. There were 3 MDS patients in the Non-TBI cohort: 1 relapsed and died at day+387; 1 relapsed at day+500 and is alive 5.5 years later following salvage with a 2nd alloHCT; 1 relapsed at day+1093 and is alive at day +100 after a 2nd alloHCT. There was 1 MDS patient in the TBI cohort who achieved 100% donor myeloid engraftment without relapse but died at day+827 from a bacterial infection in the setting of immune mediated cytopenia. ConclusionElimination of TBI from the RIC regimen for DC/TBD was not associated with significant changes in rates of graft failure, GvHD, and overall survival, but was associated with delayed achievement of full donor chimerism and improved lymphocyte reconstitution. For DC/TBD-associated BMF, TBI appears to be dispensable. Optimal approaches to DC/TBD-associated MDS remain unclear. Larger cohorts are needed to better assess the unique contribution of TBI and donor CD34+ cell dose. Longer follow-up is required to assess differences in DC/TBD complications and late effects.

Monitoring of Red Cell Donor Chimerism in Oncohematological Patients After Allogeneic Hematopoietic Stem Cell Transplantation

Background. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a definitive therapy for patients with different oncological and hematological diseases. The study of red cell chimerism is a crucial process for diagnosing transplant engraftment and functioning during the post-transplantation period.
 Aim. To assess the effect of immunohematological (АВО and HLA donor–recipient matching) and medical (conditioning regimens) parameters on the onset time of post-transplantation donor chimerism which is determined by RBC antigens.
 Materials & Methods. The study enrolled 54 patients at the Kirov Research Institute of Hematology and Transfusiology in Russia (25 female and 29 male patients) aged 3–60 years (median 32 years). All of them received allo-HSCT in 2013–2021. Acute leukemias were identified in 39 patients, 8 patients were reported to have malignant lymphoproliferative diseases, 3 patients had myeloproliferative neoplasms, and 4 patients were diagnosed with aplastic anemia. RBC antigens of donors and recipients were analyzed by gel hemagglutination using Bio-Rad (USA) reagents and equipment.
 Results. The onset time of donor chimerism depends neither on the degree of HLA donor–recipient matching, nor on conditioning regimen. Donor chimerism in recipients with major ABO-incompatibility occurs significantly later than in patients with minor АВО-incompatibility and ABO-identity.
 Conclusion. Monitoring of post-transplantation donor chimerism is an important diagnostic and prognostic tool to assess donor hematopoietic cell engraftment, hematologic recovery, graft rejection, and relapse of the disease. After allo-HSCT, first donor red cells occur in pairs with major АВО-incompatibility later than in pairs with minor АВО-incompatibility or ABO antigen compatibility. Other immunohematological and medical parameters do not affect the development rate of donor chimerism determined by RBC antigens.

Naked Antibody-Based Conditioning Targeting CD47 and CD117 Plus Janus Kinase Inhibition Enables Toxin-Free, Radiation-Free, and Chemotherapy-Free Fully Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for numerous malignant and non-malignant hematologic diseases. To enable engraftment of donor hematopoietic stem cells (HSCs), recipients undergo conditioning with chemotherapy and irradiation to make marrow space for incoming donor HSCs and suppress host immune responses that may lead to graft rejection. These cytotoxic conditioning regimens carry risks of severe toxicities which prevents many patients from accessing the lifesaving benefit of allo-HSCT. Previously, we developed fully ablative CD45- and cKit-targeted antibody-drug conjugates (ADC) which, when combined with Janus kinase 1/2 (JAK1/2) inhibitors, enabled fully mismatched allo-HSCT with full donor chimerism and minimal toxicity while providing durable antileukemia activity (Persaud et al (2022), ASH Abstract). However, even ADCs carry potential risks of acute and chronic toxicities that may preclude their common use as HSCT conditioning agents. Moreover, unlike malignant diseases, allo-HSCT for non-malignant diseases does not require the conditioning regimen to provide antitumor benefit and partial donor chimerism may be sufficient for cure, strongly favoring the use of less intense conditioning strategies with the fewest possible toxicities. We hypothesized that naked antibody-based conditioning strategies can achieve the therapeutic benefits of allo-HSCT for non-malignant hematologic diseases, obviating exposure to toxic ADC payloads. METHODS: We combined unconjugated antibodies targeting CD47 and cKit with JAK1/2 inhibition to perform minor histocompatibility antigen (miHA)-mismatched (129/Sv→B6-Ly5.1) and fully mismatched (BALB/c→B6) murine allo-HSCT. In some full mismatch HSCT experiments, recipients included B6-gp91phox -/- mice, which serve as a model of chronic granulomatous disease (CGD) due to their deficiency in a catalytic subunit of NADPH oxidase. Mice received 100 µg anti-CD47 (clone mIAP410; BioXcell) 8 days before HSCT (d-8) as a priming dose to reduce the severity of CD47 blockade-induced anemia. Next, mice received 500 µg anti-cKit (clone ACK2; BioLegend) on d-6 and daily 500 µg doses of anti-CD47 from d-6 through d-2. For JAK1/2 inhibition, mice received oral ruxolitinib (Incyte; 2 mg drug/g chow) starting d-3 or d-1 and continuing until d+28. On d0, recipients were transplanted with 20 x 10 6 whole bone marrow cells from 129/Sv (miHA) or BALB/c (fully mismatched) mice. Peripheral blood donor chimerism was followed monthly by flow cytometry for CD45.2 + (miHA) or H-2D d+ (fully mismatched) cells. Oxidative burst among granulocytes in B6-gp91phox -/- recipients was assessed by dihydrorhodamine 123 (DHR123) staining after phorbol 12-myristate 13-acetate (PMA) stimulation. RESULTS: CD47 and cKit antibodies plus ruxolitinib enabled multilineage engraftment in both miHA and fully mismatched allo-HSCT, including myeloid lineage donor chimerism of 95-99% (Figure 1A). As expected, CD47 blockade induced significant but transient morbidity from anemia during the peritransplant period; notably, a shorter duration of ruxolitinib treatment before HSCT (starting d-1 instead of d-3) improved the tolerability of the regimen without impacting engraftment success. Allo-HSCT of B6-gp91phox -/- mice led to near complete replacement of host granulocytes in peripheral blood with donor granulocytes, whose DHR123 positivity upon PMA stimulation indicated an intact oxidative burst capacity (Figure 1B). CONCLUSIONS: Combination of antibodies targeting CD47 and cKit with JAK1/2 inhibition provides a novel conditioning strategy for allo-HSCT in mouse models, achieving high-level, stable multilineage engraftment without radiation, chemotherapy, or exposure to toxic ADC payloads. The near complete donor chimerism among granulocytes made it particularly well suited to correcting phagocyte oxidase function in the gp91phox -/- model of CGD. Future studies seek to 1) mitigate the peritransplant anemia caused by CD47 blockade using Fc-silenced anti-CD47 reagents, 2) determine the proportions of donor- versus recipient-derived myeloid cells in non-hematopoietic tissues of allo-HSCT recipients, and 3) use murine infection models to evaluate the impact of allo-HSCT on host defense in transplanted B6-gp91phox -/- recipients.

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N= 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished invitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid invitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

Curative islet and hematopoietic cell transplantation in diabetic mice without toxic bone marrow conditioning.

Mixed hematopoietic chimerism can promote immune tolerance of donor-matched transplanted tissues, like pancreatic islets. However, adoption of this strategy is limited by the toxicity of standard treatments that enable donor hematopoietic cell engraftment. Here, we address these concerns with a non-myeloablative conditioning regimen that enables hematopoietic chimerism and allograft tolerance across fully mismatched major histocompatibility complex (MHC) barriers. Treatment with an αCD117 antibody, targeting c-Kit, administered with Tcell-depleting antibodies and low-dose radiation permits durable multi-lineage chimerism in immunocompetent mice following hematopoietic cell transplant. In diabetic mice, co-transplantation of donor-matched islets and hematopoietic cells durably corrects diabetes without chronic immunosuppression and no appreciable evidence of graft-versus-host disease (GVHD). Donor-derived thymic antigen-presenting cells and host-derived peripheral regulatory Tcells are likely mediators of allotolerance. These findings provide the foundation for safer bone marrow conditioning and cell transplantation regimens to establish hematopoietic chimerism and isletallograft tolerance.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

Outcomes of Patients with Baseline Marrow Fibrosis at Allogeneic Hematopoietic Cell Transplantation

Introduction:Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative option for several relapsed and refractory hematologic malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Success of allo-HCT lies in complete engraftment of donor hematopoietic cells and reconstitution of donor hematopoiesis. Among the well-defined factors of poor engraftment include HLA disparity between recipient and donor, T-cell-depleted grafts, reduced-intensity conditioning, and low stem cell dose. However, bone marrow reticulin fibrosis (MF) was not clearly defined as a risk factor.Marrow fibrosis has not been observed to be associated with impaired engraftment in primary myelofibrosis patients undergoing allo-HCT. However, in patients with MDS a pre-existent MF showed a poor engraftment and secondary graft failure; Particularly, in patients with higher WHO grade fibrosis tend to have poor donor chimerism and transfusion dependency. But, this was not associated with any adverse effect on survival. Further studies have showed the time to neutrophil engraftment is prolonged in those with higher degree of MF but not in the lower grades. Studies evaluating these findings in AML with marrow fibrosis are lacking.The current study is aimed at estimating the incidence of poor engraftment in AML/MDS patients with MF who underwent allo-HCT at our institution and to compare the incidence between those with and without MF.Methods:We retrospectively reviewed all AML/MDS patients that underwent allo-HCT between 2006 and 2015 at Washington University, St Louis. We included patients aged >18, and that underwent first allo-HCT. Patients that underwent second or later allo-HCT were excluded. Pre-transplantation bone marrows biopsies were evaluated for evidence of reticulin fibrosis and WHO grade (low grade/0-1, high grade/2-3) prior to allo-HCT. Primary end point was complete donor engraftment status measured using PCR based single-tandem repeats at day 30 post-allo-HCT. Secondary end point was to evaluate neutrophil engraftment, absolute platelet engraftment, day 100 engraftment status and survival outcomes. Neutrophil and platelet engraftment were defined per standard CIBMTR criteria.The association between primary outcome and clinical risk factors was examine via univariate and multivariate logistic regression. The associations between neutrophil/platelet engraftment and clinical risk factors were examined via univariate and multivariate general linear modeling. The relationship between marrow fibrosis grade and overall survival, as well as competing risk survival of relapse and non-relapse mortality, was investigated by Cox proportional hazard regression.Results:Seven hundred and twenty-nine patients were included in the final analysis (grade 0/1 n=662; grade2/3 n=67) (Table 1). Patients in higher-grade fibrosis are relatively older (61 vs 56 years) are more likely to have MDS, likely to have active disease at transplantation (55% vs. 31%), and have had reduced intensity conditioning. Compared to patients with completed engraftment, patients in no-engraftment group are older (61.3 vs. 55.5; p= <0.0001) and a greater number of patients in that group have a higher grade of fibrosis (16% vs. 8%; p= 0.0064). Univariate analysis shows higher grade of MF is associated with engraftment failure at day 30 (odds ratio 2.247 95%CI 1.24-4.07; p= 0.008). However, in multivariate analysis, this significance was lost (odds ratio 1.77; CI 0.90-3.50; p= 0.10). MF grade was not associated with neutrophil and platelet engraftment, engraftment status at 100 days, overall survival, relapse free survival and non-relapse mortality. Factors associated with completed engraftment at day 30 post-allo-HCT are summarized in Table 2.Conclusions:Marrow fibrosis preceding allo-HCT was not associated with engraftment failure at day 30 or after transplantation with the adjustment for other risk factors.MF was not associated with other engraftment parameters or survival. Further analyses evaluating the effect of resolution of MF on survival outcomes is underway. It is imperative that several disease and transplantation characteristics play significant role than MF itself in engraftment failure supporting the theory that engraftment is an immunologic function. [Display omitted] DisclosuresVij:Amgen: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria, Research Funding; Konypharma: Honoraria; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Jazz: Honoraria; Celgene: Honoraria.

Alemtuzumab/Low Dose TBI Conditioning Facilitates Stable Long-Term Donor Hematopoietic Cell Engraftment from Sibling Donors in Children with Sickle Cell Disease

BackgroundHematopoietic stem cell transplant (HCT) is the only established curative approach for sickle cell disease (SCD). Due to the high toxicity profile of myeloablative regimens, some adult patients with severe sickle cell anemia are not eligible for such therapy. However, a new adult nonmyeloablative protocol (National Institutes of Health, NIH protocol) described by Hsieh et al. (2014) was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. The NIH protocol includes alemtuzumab, low dose total-body irradiation (TBI) of 300 cGy, and prolonged sirolimus for graft-versus-host disease (GVHD) prophylaxis for those recipients with a matched-sibling donor (MSD).The Alberta Children's Hospital has adopted this published regimen for MSD HCT in adolescents and younger children with SCD since the NIH data was presented in 2013.To our knowledge, there is no published literature describing the application of the NIH protocol in a pediatric population; the youngest subject in the referenced adult study was 16 years of age.MethodsThis retrospective cohort describes outcomes of nonmyeloablative allogeneic MSD HCT in children and adolescents with SCD who underwent HCT between 2013-2017. Fourteen patients with SCD under 18 years of age at the time of HCT received the NIH conditioning regimen. HLA-matched siblings had either normal hemoglobin or sickle cell trait and of the appropriate weight/height to donate peripheral blood stem cells (PBSCs) without blood priming of the apheresis circuit.The conditioning regimen consisted of alemtuzumab 1.0 mg/kg administered subcutaneously daily divided over five days (Days -7 to Day -3). Patients received a single TBI dose of 300 cGy on day -2, with testicular shielding for male recipients. GVHD prophylaxis consisted of a sirolimus loading dose of 3mg/m2/dose (PO) on day -1, followed by 1mg/m2/dose once daily starting on Day 0. PBSCs were typically collected on Day -3 to allow for cancellation of the HCT before TBI in the event of failed mobilization. Unmanipulated PBSCs were infused fresh on Day 0. Patients were eligible for early discharge post-transplant even prior to neutrophil engraftment. Weaning of sirolimus was initiated no earlier than 1-year post-HCT and if donor T-cell chimerisms were greater than 50%. Treatment failure was defined as clinical manifestation of SCD, failure to start weaning sirolimus by 2-years post-HSCT, or a failure to complete a sirolimus wean by 2.5 years post-HCT.ResultsFourteen patients (3 M,11 F) are included on this retrospective study. Median age at HCT was 11 years. Post-HCT follow-up ranges from 1 to 48 months (median 17). There were no failed donor stem cell mobilizations. Patients received a median of 11.4x106 CD34 cells per kilogram of body weight.All patients were discharged home prior to neutrophil engraftment with a median hospital stay of 7 days (range 5-19). All patients had donor neutrophil engraftment at a median of 21 days (mean= 22, SD= 4) and platelet engraftment at median of 14 days (mean= 12, SD= 14). Six patients never required platelet transfusion. Readmission after initial discharge was required in 9/14 patients with an average of 1 readmission/patient (range 1-4), typically for fever or CMV pre-emptive therapy. One patient developed septic shock with Acinetobacter ursingii related to poor central line care at home.All patients are currently alive. Post-HCT, there have been no cases of graft failure, sickling crises, veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, PRES, or PTLD. Although 6/14 patients had CMV reactivation, only 3 patients required pre-emptive therapy. None had CMV/EBV disease.There were no treatment failures as defined by protocol. Currently, 8 patients are off sirolimus, 1 is currently weaning, with no secondary graft failure events. Only 2 patients over 1-year post-HCT have not initiated sirolimus weaning. There have been no cases of acute or chronic graft-versus-host disease.ConclusionsNonmyeloablative conditioning regimen is safe and effective as curative therapy for children with SCD. Outpatient management pre-engraftment was feasible in this population. In contrast to adult data, sirolimus weaning was possible in most patients by one-year post-HCT. Graft integrity was maintained after discontinuation of sirolimus in all eligible patients. [Display omitted] DisclosuresGuilcher:Patient Access Solutions: Consultancy; Canada Market Research: Consultancy; Jazz pharmaceuticals: Consultancy. Bruce:Bristol-Meyers-Squibb - consultant 2016-2017: Consultancy; ovartis consult and ad board 2015-2016: Consultancy; Apopharma 2015: Consultancy.

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance.

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Engineered IL-2 Cytokine-Cytokine Receptor Complex Enables Selective Expansion of Regulatory T Cells and Facilitates Establishment of Organ Transplantation Tolerance

Mixed hematopoietic cell chimerism induction is a promising approach to establish transplantation tolerance, though achievement of donor cell engraftment in patients given non-myeloablative conditioning remains challenging. Adoptive transfer of regulatory T cells (Tregs) has been shown to improve alloengraftment in animal models. However, ex vivo Treg expansion for the purpose of infusing large numbers is technically challenging. An innovative approach is to engineer an orthogonal IL-2 (oIL-2) cytokine-receptor pair that selectively interacts with each other and transmits native IL-2 signals, but does not interact with the natural cytokine or receptor counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this ortho cytokine-receptor combination into Treg with the goal of improving donor hematopoietic cell engraftment upon adoptive transfer in a murine mixed chimerism model to induce tolerance to a transplanted organ allograft. CD4⁺CD25^hiFoxp3^GFP+cells were isolated from Foxp3^GFP-BALB/c mice. Purified Tregs were preactivated and transduced with the construct for murine oIL-2 receptor β chain (oIL-2Rβ) through a retroviral vector. Transduction efficiency was approximately 40%. Transduced oIL-2Rβ⁺Tregs (oTreg) were injected at 1 × 10⁶ /mouse together with 15 × 10⁶ bone marrow cells (BMCs) that were obtained from luciferase transgenic C57BL/6 donor mice into WT BALB/c recipient mice that received 3.3-Gy total body irradiation and anti-CD40L mAb (0.3 mg, ip on d0) as preconditioning. Recipients were treated with PBS, WT IL-2, or oIL-2 (both 25K IU/day) for 14 days. Without oTreg transfer, donor cells were rejected by d10 as measured by bioluminescence. Improvement of engraftment by oTreg transfer was not statistically significant without IL-2 injection (P = 0.13 compared to BMC + PBS group on d30). WT IL-2 injection increased CD8⁺T cells and resulted in rapid graft rejection. On the other hand, oIL-2 injection significantly increased %Foxp3^GFP+ in CD4⁺T cells (PBS; 4.3±1.6%, oIL-2; 9.3±4.7%, each n=10, P=0.007) without increasing CD8⁺T cells. Consequently, donor cell engraftment was significantly improved in this group (P<0.0001 compared to BMC + PBS group on d30). Those recipients accepted heart allografts that were transplanted from C57BL/6 donor mice after chimerism induction, indicating the establishment of donor specific tolerance. Because ortho IL-2 did not have an impact when Tregs were transferred without oIL-2Rβ transduction, we concluded that specific interactions between oIL-2 and oIL-2Rβ resulted in improved acceptance of the heart allografts. Our data indicates that selective IL-2 stimulation improves Treg potential for facilitation of engraftment and for the induction of transplantation tolerance. Cytokine receptor engineering could be a powerful option to manipulate target cells such as Treg in vivo.

Rchimerism: An R Package for Automated Chimerism Data Analysis

A quantitative chimerism test monitors engraftment of donor hematopoietic stem cells or relapse of leukemias or lymphomas in hematopoietic stem cell transplantation patients. The most common method used for chimerism testing is PCR amplification of short tandem repeat loci, followed by capillary gel electrophoresis. Manual data analysis is tedious and time consuming, as it involves the selection of informative loci and the repetition of quantifying chimerism percentage for multiple loci from multiple cell types. It is also susceptible to human errors. Currently, there is no free software to fully automate chimerism data analysis. Rchimerism, an R shiny package, was developed to automatically pick informative loci, calculate chimerism percentage, and display the results through a user-friendly interface. The accuracy of the program was compared with manual calculation on 60 patient samples with 100% concordance. Compared with manual calculation, Rchimerism drastically reduces analysis time from 20 to 40 minutes for single donor transplantation samples and from 40 to 80 minutes for double donor transplantation samples to >1 minute. Rchimerism can be downloaded and used freely by noncommercial laboratories.

Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.

Allogeneic bone marrow transplant in the absence of cytoreductive conditioning rescues mice with β-thalassemia major.

β-thalassemia is a group of inherited blood disorders that result in defects in β-globin chain production. Cooley anemia (CA), or β-thalassemia major, is the most severe form of the disease and occurs when an individual has mutations in both copies of the adult β-globin gene. Patients with CA fail to make adult hemoglobin, exhibit ineffective erythropoiesis, experience severe anemia, and are transfusion dependent for life. Currently, allogeneic bone marrow transplantation (BMT) is the only cure; however, few patients have suitable donors for this procedure, which has significant morbidity and mortality. In this study, a novel humanized murine model of CA is rescued from lethal anemia by allogeneic BMT in the absence of cytoreductive conditioning. A single intravenous postnatal injection of allogeneic bone marrow results in stable, mixed hematopoietic chimerism. Five months after transplantation, donor cells accounted for approximately 90% of circulating erythrocytes and up to 15% of hematopoietic stem and progenitor cells. Transplanted mice are transfusion independent, have marked improvement of hematological indices, exhibit no growth retardation or signs of graft-versus-host disease, and are fertile. This study describes a method for the consistent engraftment of allogeneic donor hematopoietic cells that rescues a humanized mouse model of CA from lethal anemia, all in the absence of toxic cytoreductive conditioning.

Hematopoeitic Stem Cells and Their Progenitors Critically Require Autophagy to Promote Early Engraftment Following Allogeneic Stem Cell Transplantation

Stem cell transplantation (SCT) is the only curative therapy for the majority of hematological malignancies and relies on successful engraftment of donor hematopoietic stem cells (HSC) to reconstitute the patient's hematopoiesis and immunity. Graft-versus-host disease (GVHD) and graft failure are complications of SCT that contribute to significant morbidity and mortality. Recent studies have established a role for autophagy in the long-term survival and function of HSCs and as a regulator of hematopoiesis. We investigated the contribution of autophagy to HSC in the setting of allogeneic transplantation. Using LC3-GFP bone marrow as a graft source in the well-established B6 into F1 model of GVHD, together with imaging flow cytometry, we demonstrated increased autophagy (LC3-GFP punctae) within donor HSC and progenitor cells in GVHD compared to non-GVHD recipients. To assess the contribution of autophagy to HSC development and function we used mice deficient in Atg5, an essential protein for the autophagy pathway. In primary transplants, ATG5 KO foetal liver (FL) had reduced ability to engraft lethally irradiated congenic recipient mice compared to WT FL. Furthermore, competitive transplantation of 50% ATG5 KO FL cells together with 50% WT FL cells demonstrated that ATG5 KO cells had a reduced capacity to reconstitute compared to congenic WT FL cells. Finally, to address the role of autophagy in HSC in the setting of GVHD, we transplanted WT or ATG5 KO FL ± T cells into lethally irradiated B6D2F1 recipients. At D10 post-transplant ATG5 KO recipients failed to establish tri-lineage engraftment in peripheral blood and lacked expansion of myeloid precursor cells in the BM, leading to engraftment failure and significantly reduced survival compared to WT FL recipients (50% WT vs 10% Atg5 KO p<0.0001). This early engraftment failure was confirmed to be autophagy specific using VAVcre x Atg7fl/fl donor mice, in which Atg7 deficiency is restricted to hematopoietic cells. Furthermore, the essential requirement for autophagy in early progenitors and HSC was confirmed using LysMcre Atg7fl/fl mice, which ablate Atg7 from the granulocyte-macrophage progenitor population onwards. Survival, or engraftment, in mice receiving the LysMcre Atg7fl/fl donor graft did not differ significantly from those receiving a WT graft. We demonstrate that autophagy is increased in the GVHD setting and that without autophagy early myeloid precursors fail to provide short term reconstitution leading to primary graft failure and mortality. Thus, intervention to increase autophagy in these cells post-transplant may improve engraftment in the clinic. DisclosuresNo relevant conflicts of interest to declare.

Deletional and regulatory mechanisms coalesce to drive transplantation tolerance through mixed chimerism.

Establishing donor-specific immunological tolerance could improve long-term outcome by obviating the need for immunosuppressive drug therapy, which is currently required to control alloreactivity after organ transplantation. Mixed chimerism is defined as the engraftment of donor hematopoietic stem cells in the recipient, leading to viable coexistence of both donor and recipient leukocytes. In numerous experimental models, cotransplantation of donor bone marrow (BM) into preconditioned (e.g., through irradiation or cytotoxic drugs) recipients leads to transplantation tolerance through (mixed) chimerism. Mixed chimerism offers immunological advantages for clinical translation; pilot trials have established proof of concept by deliberately inducing tolerance in humans. Widespread clinical application is prevented, however, by the harsh preconditioning currently necessary for permitting BM engraftment. Recently, the immunological mechanisms inducing and maintaining tolerance in experimental mixed chimerism have been defined, revealing a more prominent role for regulation than historically assumed. The evidence from murine models suggests that both deletional and regulatory mechanisms are critical in promoting complete tolerance, encompassing also the minor histocompatibility antigens. Here, we review the current understanding of tolerance through mixed chimerism and provide an outlook on how to realize widespread clinical translation based on mechanistic insights gained from chimerism protocols, including cell therapy with polyclonal regulatory T cells.

211At-Anti-CD45 Radioimmunotherapy Can Replace TBI Prior to Haploidentical Bone Marrow Transplantation and Yield Long-Term Hematopoietic Engraftment

Haploidentical bone marrow transplantation (haplo-BMT) is both clinically effective and widely available because related-donors can be identified for nearly all recipients. Despite the curative promise of this approach, many patients with hematologic malignancies will relapse after haplo-BMT and more effective preparative regimens are necessary. We have shown that anti-CD45 radioimmunotherapy (RIT) delivers high-doses of radiation to hematolymphoid organs while minimizing the radiation exposure to non-targeted tissues. The efficacy of beta-emitting radionuclides may be limited by their relatively low decay energies (0.66 – 2.3 MeV). We have thus investigated the higher energy alpha-emitter astatine-211 (211At) (average decay energy of 6.8 MeV), for targeted anti-CD45 radioimmunotherapy (RIT) in lieu of total body irradiation (TBI) prior to haploidentical BMT in a murine leukemia model to decrease relapse rates.Groups of five B6SJLF1/J mice (allotype H2-Db) received escalated activities (20, 30 or 40 μCi) of 211At-anti-CD45 antibody [100 μg (0.67 nmol) of B10-30F11] given by tail vein injections on day -2 in place of TBI prior to BMT. Animals received cyclophosphamide (CY; 200 mg/kg/day) on days –3, –2, or –1, and +2 for graft-versus-host disease prophylaxis, either alone, or with fludarabine (FLU; 100 mg/kg/day) for 4 days starting day -6. Transplanted mice received 1.5 × 107 haploidentical bone marrow cells from CB6F1/J mice (allotype H2-Dd) on day 0. Peripheral blood from recipient mice was then assayed monthly by flow cytometry to measure chimerism as the percentage of donor (H2-Dd) circulating CD8+ cells.The highest activity delivered of 40 µCi 211At-anti-CD45 RIT was uniformly lethal without BMT rescue, whereas 60% of transplanted mice at this dose survived to assessment at 1 month. Mice treated with 30 µCi of 211At-anti-CD45 RIT with pre- and post-transplant CY and without TBI or FLU, had high levels of engraftment with an average of 83.7 ± 5.8% donor CD8+ cells 1 month after haploidentical BMT (Table 1). The addition of FLU to 211At-anti-CD45 RIT with CY did not significantly improve chimerism levels, with mean donor CD8+ cells in mice treated with 40 µCi 211At-anti-CD45 RIT of 64.5 ± 41.6% compared to 60.0 ± 13.9% in the absence of FLU (p=0.8668). In addition, mice that received 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on either day –3, –2, or –1 showed mean donor CD8+ cells of 83.7 ± 5.8%, 49.9 ± 29.6% and 55.0 ± 46.2% 1 month after haploidentical-BMT, respectively. Importantly, chimerism levels remained stable 2 months after haploidentical BMT with mean donor CD8+ cells of 80.4 ± 16.6%, 47.0 ± 37.7% and 63.2 ± 10.7% in mice treated with 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on day –3, –2, and –1, respectively. Engraftment using 40 or 30 µCi 211At-anti-CD45 RIT was comparable to using 850 or 1000 cGy TBI (mean donor CD8+ cells of 70.2 ± 18.8% and 60.0 ± 4.6%, respectively) prior to haploidentical BMT. RIT alone without any chemotherapy was insufficient to facilitate clinically relevant rates of donor engraftment, as mice treated with 30 µCi 211At-anti-CD45 RIT and no FLU, CY or TBI had 15.9 ± 7.1% mean donor CD8+ cells 1 month after haploidentical BMT.These results suggest that 211At-anti-CD45 RIT prior to haploidentical BMT with pre– and post–transplant CY can result in high levels of donor hematopoietic cell engraftment in the absence of TBI and FLU. This conditioning regimen may be less toxic and more effective at preventing relapse than TBI-based approaches due to the high linear energy transfer of the alpha emissions, or the high decay energy of targeted 211At deposited over its short effective path-length. On-going studies are assessing the efficacy and toxicity associated with 211At-anti-CD45 RIT compared to a TBI-based haploidentical BMT using a syngeneic murine leukemia model.Abstract 2417. Table 1Preparative Therapy and CD8+ Donor Chimerism at 1 monthGroupFLU(100mg/kg/d)pre-BMTCY(200mg/kg)TBI211 At-anti-CD45RITpost-BMTCY(200mg/kg)Donor CD8+ %1–day –3–30 µCiday +283.7 ± 5.82–day –2–30 µCiday +249.9 ± 29.63–day –1–30 µCiday +255.0 ± 46.24–day –3–40 µCiday +264.5 ± 41.65d –6 to –3day –3–40 µCiday +260.0 ± 13.96–––30 µCiday +215.9 ± 7.17–day –3––day +24.4 ± 0.68––1000 cGy–day +260.0 ± 4.6 DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic chimerisms: friends or foes?

In humans, chimerisms (Chs) can occur naturally or be induced through artificial means. Feto-maternal Chs are natural and result from the spontaneous trafficking of hematopoietic or other types of cells across the placenta during pregnancy. These Chs can be transient or persist for many years and even decades. Mixed hematopoietic Chs (MHChs) can also be artificially induced, and they might have profound effects on the modulation of the immune system, which can be used for inducing donor-specific tolerance in recipients of allogeneic organ transplantation. Nonetheless, the main obstacle for the establishment of such Chs is that they require the engraftment of donor hematopoietic cells, which at present can only be accomplished using relatively toxic regimens that preclude its widespread use and currently restricts its application to some special patients, in which both a solid organ (e.g. a renal allograft) and a marrow transplantation are necessary. However, it is likely that less toxic strategies are developed that can be clinically applicable in the next decade to induce tolerance in organ transplantation. A variant of Chs is the molecular Chs, in which a proportion of the hematopoietic cells would be transduced to express a transgene (e.g. encoding a therapeutic protein, an auto-antigen, or an allergen), so that specific tolerance to these molecules is induced. This might have therapeutic applications in fields such as replacement and genetic therapies, autoimmune diseases, or allergy. Keywords: Mixed hematopoietic chimerism; feto-maternal chimerism; immune tolerance; hematopoietic transplantation; organ transplantation; molecular chimerism (Published: 25 September 2014) Citation: Advances in Regenerative Biology 2014, 1 : 24429 - http://dx.doi.org/10.3402/arb.v1.24429 Abstract in popular science Chimeras are mythological creatures composed of a lion, a snake, and a goat. In biology, chimeras refer to individuals composed of two or more types of genetically distinct cells that derive from different fertilized eggs. Some chimerisms (Chs) occur spontaneously, such as the feto-maternal Chs, which are very common and are due to the bidirectional pass of blood cells through the placenta. These Chs can be transient or they may persist for decades and contribute to the generation of different tissues. Although these Chs have been reported to be associated with a higher risk of certain diseases (e.g. fetal Ch and scleroderma in the mother), the biological role and the clinical significance of feto-maternal Chs are uncertain. When the Chs are caused only by blood cells (e.g. hematopoietic stem cells), they are called mixed hematopoietic Chs (MHChs). These MHChs can be created artificially as occurs in patients with leukemia or non-malignant diseases undergoing hematopoietic transplantation. Indeed, these patients usually become full quimeras (which means that 100% of their blood cells will be donor-derived for the rest of their lives). However, for the donor stem cells to engraft, the recipients must receive a therapy that eliminates (ablates) their own hematopoietic cells. A main limitation of these preparatory therapies (conditioning) is their high toxicity. However, since the creation of MHChs only requires a partial ablation, it can be done using less toxic conditioning regimens. The main clinical interest of MHChs is that they result in donor-specific immune tolerance, as both types of immune cells (donor and recipient) will be educated in a peaceful coexistence. So, although the conditioning regimens still need to be refined, such MHChs can be used to induce tolerance in recipients of organ transplantation, which no longer will require immunosuppressive drugs. In an analogous manner, Chs can be created using autologous hematopoietic cells that have been genetically modified with gene therapy vectors to produce a therapeutic protein, an autoantigen, or an allergen, so that a very robust tolerance to these molecules can be induced. This has potential applications in fields such as replacement and genetic therapies, autoimmune diseases, or allergy.