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We performed a prospective multicenter study of T-cell receptor αβ (TCR-αβ)/CD19–depleted haploidentical hematopoietic cell transplantation (HCT) in children with acute leukemia and myelodysplastic syndrome (MDS), to determine 1-year disease-free survival (DFS) and compare 2-year outcomes with recipients of other donor cell sources. Fifty-one patients aged 0.7 to 21 years were enrolled; donors were killer immunoglobulin-like receptor (KIR) favorable based on ligand mismatch and/or high B content. The 1-year DFS was 78%. Superior 2-year DFS and overall survival (OS) were noted in patients 80% of recipients had a KIR-favorable donor by our definition, demonstrating that this approach is broadly applicable to groups often unable to find donors. This prospective, multicenter study showed improved outcomes using TCR-αβ/CD19–depleted haploidentical donors using RTC for children with acute leukemia and MDS. Randomized trials comparing this approach with matched unrelated donors are warranted. This trial was registered at https://clinicaltrials.gov as #NCT02646839.

The manufacture of individual patient-derived CAR T-cells is expensive, frequently unsuccessful, too time consuming to benefit acutely ill patients and difficult to scale for large numbers of patients. "Off-the-shelf" T cell products that are banked from healthy donors would improve accessibility, allow rapid treatment, and reduce costs. The major obstacles to the success of allogeneic T cells are graft-versus-host disease (GVHD) and graft rejection, mediated by host and recipient alloreactive T cells, respectively. To address GVHD, we are using allogeneic Epstein-Barr Virus-specific T cells (EBVSTs), which have not produced GVHD in more than 300 recipients. To prevent graft rejection we have expressed a chimeric antigen receptor for CD30 (CD30.CAR) in EBVSTs. CD30 will be upregulated by host alloreactive T cells when they encounter infused CD30.CAR EBVSTs. As a consequence, they will become targets for the CD30.CAR EBVSTs. Previous clinical studies (NCT02917083, NCT01555892, and NCT00062868) have shown that CD30.CAR T cells can destroy CD30+ lymphoma cells through their chimeric receptor, while EBVSTs can kill EBV+ lymphoma cells through their native TCR. Thus, once engrafted, banked CD30.CAR EBVSTs may kill both CD30+ and EBV+ lymphomas through their CAR and TCR respectively without causing GVHD. To assess the safety and activity of banked CD30.CAR EBVSTs, we treated patients with multiply relapsed (median of 4 prior lines of therapy; range 3-5) or refractory CD30-positive lymphomas in a Phase 1 dose escalation study using 4 × 10 7, 1 × 10 8 or 4 × 10 8 CD30.CAR EBVSTs infused after lymphodepletion with cyclophosphamide and fludarabine. Although CD30.CAR killing is not HLA restricted, selection of the CD30.CAR EBVST product for each recipient was based on the best HLA class I and class II match; this should allow endogenous EBV (when present) to boost the in vivo activity of CD30.CAR EBVSTs via their native TCRs, and augment reactivity in patients whose CD30+ malignancies are also EBV+. We have currently treated eight patients, including two at the highest dose level. The infusions have been well tolerated with no dose-limiting toxicities and in particular no cytokine release syndrome (CRS) or GVHD of any grade. We have evaluated seven of the patients. At 6-week evaluation, per Lugano criteria, two patients have had a complete response (one shown in Figure 1) and three have had a partial response (overall response rate of 71%). CD30.CAR EBVSTs were detectable for 1 week in peripheral blood, but there was no evidence of expansion. We are analyzing tumor samples for CD30.CAR EBVSTs, and will continue to assess the safety, efficacy and durability of these responses. Thus banked CD30.CAR EBVSTs can safely be given to allogeneic recipients and may cause significant tumor responses including complete remissions. These cells may be a suitable platform for other "off-the-shelf" CAR-T cell therapies. Figure 1 Figure 1. Disclosures Quach: Tessa Therapeutics: Research Funding. Ramos: Athenex: Research Funding; Novartis: Consultancy; Genentech: Consultancy; Tessa Therapeutics: Patents & Royalties, Research Funding. Grilley: Allovir: Current equity holder in publicly-traded company, Other: Leadership; QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support. Brenner: Athenex: Membership on an entity's Board of Directors or advisory committees; Turnstone Biologics: Membership on an entity's Board of Directors or advisory committees; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; Coya Therapeutics: Membership on an entity's Board of Directors or advisory committees; CellGenix GmbH: Membership on an entity's Board of Directors or advisory committees; Walking Fish Therapeutics: Membership on an entity's Board of Directors or advisory committees; Poseida Therapeutics: Membership on an entity's Board of Directors or advisory committees; Onkimmune: Membership on an entity's Board of Directors or advisory committees; Memgen: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Abintus: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Allovir: Current equity holder in publicly-traded company; Marker Therapeutics: Current equity holder in publicly-traded company. Heslop: Kiadis: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; GSK: Membership on an entity's Board of Directors or advisory committees; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees. Rouce: Tessa Therapeutics: Research Funding; Pfizer: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lapteva: Tessa Therapeutics: Consultancy. Rooney: Tessa: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

While autologous T cell therapies can effectively treat B-cell leukemia and lymphoma, the personalized manufacturing process is difficult to scale, expensive and may fail. Even when autologous products are successfully manufactured, they are not immediately available to acutely ill patients. "Off-the-shelf" T cell products derived from healthy donors that can rapidly be administered, would improve accessibility and reduce the cost of T cell therapy. However, major obstacles to successful allogeneic T cell products include their potential for graft-versus-host disease (GVHD) and graft rejection, mediated by host and recipient alloreactive T cells respectively. To address GVHD, we are using Epstein-Barr Virus-specific T cells (EBVSTs) as our platform since they are virus specific rather than allospecific and have not produced GVHD in more than 300 allogeneic recipients. To prevent graft rejection we have introduced into these EBVSTs, a chimeric antigen receptor for CD30 (CD30.CAR). CD30 is upregulated during the activation of alloreactive T cells, which leads to them becoming targets. The CD30.CAR provides the additional advantage of targeting CD30-positive lymphoma and has proved safe and effective in prior clinical trials (NCT02917083) using autologous CAR-T cells. Hence, we expect off-the-shelf CD30.CAR EBVSTs to eliminate the alloreactive T cells they elicit in allogeneic hosts, and therefore persist for sufficient time to eliminate CD30-positive lymphoma, without causing GVHD. Here we show that CD30.CAR-EBVSTs resist fratricide by masking their own CD30 molecules expressed in cis, but are nonetheless protected from rejection when co-cultured with alloreactive T cells expressing CD30 in trans. Notably, CD30.CAR EBVSTs preserve the function of both their TCR and the CD30.CAR, with retention of EBV specificity and the ability to eliminate CD30-positive tumor cells. We have manufactured a bank of clinical grade CD30.CAR EBVSTs from donors with HLA types designed to provide a partial HLA match for our diverse recipients. Clinical grade CD30.CAR EBVST cultures readily expanded to sufficient numbers for a planned clinical trial and expressed the CD30.CAR on 77% to 99% of cells. All of the lines passed functional release criteria of having greater than 100 IFNɣ spot-forming units (SFU) per 105 cells in response to both latent and lytic EBV antigens, and greater than 20% specific cytolysis against a CD30-positive Hodgkin lymphoma cell line, HDLM2, at an effector to target ratio of 20:1. Although CD30.CAR killing is not HLA restricted, we will select the CD30.CAR EBVST product for each recipient, based on the best HLA class I and class II match. This will allow endogenous EBV to boost the in vivo activity of CD30.CAR EBVSTs, and will provide additional reactivity for patients with CD30-positive and EBV-positive tumors. The IND for the clinical trial (NCT04288726) has been approved and we will recruit patients with CD30-positive lymphomas including Hodgkin lymphoma, diffuse large B cell lymphoma and NK/T cell lymphoma. In summary, we present an approach to making an off-the-shelf T cell therapy that can rapidly translate to the clinic, requires no gene editing, and can serve as a platform for other CAR/TCRs to target a multiplicity of malignancies. Disclosures Quach: Tessa Therapeutics: Research Funding. Brenner:Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Tumstone: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Heslop:Tessa Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ramos:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Kuur Therapeutics: Research Funding. Rouce:Tessa Therapeutics: Other, Research Funding; Novartis: Honoraria. Rooney:Marker Therapeutics: Current equity holder in publicly-traded company, Other: co-founder; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Allovir: Current equity holder in publicly-traded company, Other: co-founder.

Background: Granulocyte colony-stimulating factor (G-CSF) is used after hematopoietic cell transplantation (HCT) to enhance neutrophil recovery in patients rendered neutropenic by the agents used in the preparative regimen (PMID: 9187067). G-CSF is contraindicated in patients with sickle cell disease (SCD) due to reported life-threatening complications ascribed to sickle vasculopathy, presumably secondary to leukocytosis and granulocyte activation (PMID: 11880644). Complications include severe vaso-occlusive pain crises, splenic engorgement, rupture, and death (PMID: 19513902). Individuals with sickle trait tolerate G-CSF without toxicity (PMID: 29743574, PMID: 27167356).The safety/toxicity of using G-CSF to enhance neutrophil recovery following HCT for SCD has not been previously described in the context of vasculopathy that is present in recipients but whose hematologic milieu is altered by transfusions and donor product infusion. Aim/Method: The hypothesis that G-CSF use would be safe in SCD patients following HCT was tested in a multicenter trial (NCT 03128996). Patients underwent HCT from matched or one-antigen mismatched (at HLA-A, -B, -C, -DRB1 if marrow; -A, -B, -DRB1 loci if cord) related or unrelated donor products between 2004 and 2019 following reduced intensity conditioning (RIC) which included hydroxyurea, alemtuzumab, fludarabine, melphalan +/- thiotepa. The regimen results in short-term marrow suppression followed by recovery. GVHD prophylaxis included tacrolimus, short-course methotrexate or mycophenolate or prednisone, and since 2018, abatacept. All patients underwent transfusions prior to commencing conditioning to reduce Hemoglobin S (Hb S) levels to 1.5x103 cells/mL on 3 successive days. The clinical course, outcomes, and toxicities in the first 100 days post-HCT were evaluated as best representing the period of G-CSF influence on SCD recipients. Results: Sixty-four patients with SCD were evaluated post-HCT. The median age at HCT was 10.67 years (range, 1-21). HCT was performed for stroke/increased transcranial doppler velocity (N=32), acute chest syndrome (N=24) and vaso-occlusive episodes (VOE) (N=38). Twenty-nine and 35 patients received related and unrelated donor HCT respectively; 24 were mismatched at one antigen or allele locus. Graft sources included marrow (46), cord (12), peripheral blood (3), marrow + cord (2) and CD34 selected graft with T cell add-back (1). G-CSF was administered for a median of 9.4 days (range 6-33 days) post HCT. Neutrophil engraftment occurred at a median of 14.35 days (range 10-38), platelets at median of 25.2 days (range 12-89). Three patients recovered platelets beyond day 100. Seven patients had primary graft rejection and autologous reconstitution of hematopoiesis. Chimerism analysis in the remaining patients included a median of 93% (range 41-100% in the myeloid lineage; N=30), 89% (range 26-100% in the lymphoid lineage; N=36) and 93% (range 48-100% in whole blood; N=45) on day 100. The incidence of grade I-II and III-IV acute GVHD was 23.4% and 17% respectively during first 100 days. A total of 23 episodes of bloodstream and 7 non-bloodstream sites of bacterial infection were observed in 22 patients. No patient developed SCD related symptoms following post-HCT G-CSF administration. Organ toxicities noted in 17 of the 64 patients were variable [most common were hypertension (4), posterior reversible encephalopathy (3), and renal dysfunction (3)] and expected as part of the transplant process. Conclusion: In this cohort of SCD patients who underwent HCT from different donor sources following RIC and G-CSF to enhance neutrophil recovery, we noted no toxicities attributable to G-CSF. Specifically, no SCD related symptoms or extended hospitalization were encountered in any patient attributable to G-CSF administration. Commencing conditioning at a Hb S level Disclosures No relevant conflicts of interest to declare.

MRD detected by flow cytometry (FC) or PCR has been associated with key outcomes after HCT for ALL. In a prospective multicenter trial (NCT02646839; Pediatric Blood and Marrow Transplant Consortium [PBMTC] ONC1401), we performed a planned sub-study of NGS-MRD to predict outcomes pre- and post-HCT for ALL patients (n=57, median follow-up 523 [range 58-1198] days post-HCT). In addition, we performed analyses of relevant clinical factors to assess their relationship to EFS. The larger trial studying the role of KIR favorable haploidentical vs. other transplantation approaches for children diagnosed with ALL, acute myeloid leukemia and myelodysplastic syndrome will be reported separately. We evaluated baseline blast samples from 74 patients for dominant BCR/TCR rearrangements and to follow MRD by NGS. Dominant rearrangements were identified in 100% of B-ALL patients, 96.8% (61/63) in BCR and 3.2% in TCR gamma. For T-ALL patients, rearrangements were identified in 62.7% (7/11), with the remaining 37.3% being polyclonal. Patients proceeded to HCT only if they were in morphological remission. Pre-HCT NGS-MRD from bone marrow (BM) was highly predictive of EFS (n=29 P=0.027, Figure 1) and although NGS-MRD from peripheral blood (PB) did not reach statistical significance due to decreased sample size (n=27, P=0.17, Figure 2), it trended similarly. In BM NGS-MRD negative patients, relapse was exceptionally low with all events due to transplant related mortality (TRM). There did not appear to be a benefit of acute graft-vs-host-disease (aGVHD) in NGS-MRD- patients (Figure 3) or chronic (c)GVHD, but sample size was a limitation. Pre-HCT, 10% of the BM samples were MRD+ by FC, but 35% were MRD+ by NGS. Direct comparison of NGS-MRD in BM and PB with FC MRD pre- and post-HCT showed improvements positive and negative predictive power (data not shown). Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources (Figure 4) with decreased incidence of GVHD [aGVHD > grade 2: n=1 (3.3%) and extensive cGVHD: n=1(3.3%)]. TBI (total body irradiation) based myeloablative conditioning (TBI/TT [Thiotepa]/CY [Cyclophosphamide], TBI/CY, or TBI/VP16; ± anti-thymocyte globulin [ATG]) and non-TBI reduced toxicity (Flu [Fludarabine]/Mel [Melphalan]/TT; ± ATG) had identical EFS (P= 0.31). TRM was very low 8.7%(n=5) in this population (n=57)) and rescue of relapse was high for the duration of follow up to date, resulting in similar OS for MRD- vs. MRD+ patients (P= 0.15), likely due to rescue with cell/immunotherapy. We next examined the interaction of obesity, using body mass index (BMI) based on height/weight pre-HCT, in the context of NGS-MRD on EFS. The BMI score was converted to a percentile through population norms for age and gender and defined thresholds published by the Centers for Disease Control and prevention (CDC). Lean patients (< 85th percentile [%]) overall had better survival than the overweight (OW)/obese (85-94%/≥95%) (Figure 5: P=0.016). But among the lean patients, NGS-MRD still had an effect, with NGS-MRD+ patients had poorer EFS. Same was observed with the OW/obese group, where being NGS MRD+ adds further reduction in survival. Overweight/Obese patients who were pre-HCT MRD- had survival closer to the lean/MRD+ patients. We observed decreased EFS by weight category and NGS-MRD (Figure 6: P= 0.02). In conclusion, NGS-MRD was highly predictive for EFS regardless of HCT preparative approach or graft Alpha/Beta Depletion. NGS improved predictive power for MRD positivity compared to FC. NGS was able identify rearrangements suitable for MRD tracking in all B-ALL and in most of T-ALL samples. In BM NGS-MRD negative patients, relapse was exceptionally low. There did not appear to be a benefit of aGVHD or cGVHD in NGS-MRD- patients. Alpha/beta depleted haploidentical grafts had similar outcomes to other stem cell sources with decreased incidence of severe GVHD. We observed decreased EFS by weight category and NGS-MRD. Lean patients had better survival, after HCT, than OW/Obese patients. But among the lean patients, NGS-MRD+ patients had poorer EFS. Disclosures Abdel-Azim: Adaptive: Research Funding. Dvorak:Jazz Pharmaceuticals: Consultancy; Alexion Inc: Consultancy. Bunin:PRA Health Sciences: Other: Immediate family member employed. Walters:Editas Medicine: Consultancy; TruCode: Consultancy; AllCells, Inc: Consultancy. Cairo:Jazz Pharmaceuticals: Other: Advisory Board, Research Funding, Speakers Bureau; Miltenyi: Other: MTA; Osuka: Research Funding. Kitko:Novartis: Consultancy, Honoraria; Mallinckrodt: Honoraria. Jacob:Adaptive Biotechnologies: Employment, Other: shareholder. Wing:Miltenyi Biotec: Employment. Pulsipher:Medac: Honoraria; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Other: Education for employees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Evidence from clinical investigations and animal models indicate that chronic graft versus host disease (cGVHD) results from defective thymic generation of functional and self-tolerant T cell populations following hematopoietic stem cell transplantation (HSCT). We have previously demonstrated that the rare subset of T cells that naturally express 2 T cell receptors (TCRs) on the cell surface as a result of incomplete allelic exclusion are predisposed to respond to auto- and alloantigens. Dual TCR T cells disproportionately participate in pathologic alloreactivity in HSCT patients and mouse models of acute GVHD. These findings, combined with observations demonstrating that dual TCR T cells represent a physiologic reservoir of unique TCRs that evade negative selection, prompted us to examine the role of thymic selection and dual TCR T cells in cGVHD. To study the role of post-transplant thymopoiesis in generation of potentially pathogenic dual TCR T cells, we used a mouse model of syngeneic bone marrow transplantation into lethally-irradiated recipients. Radiation-induced damage to the thymic stroma was characterized by disruption of thymic architecture and loss of cortical and medullary thymic epithelial cells (TECs). This damage resulted in significantly increased generation of dual TCR T cells following transplantation of congenically-marked syngeneic T cell-depleted bone marrow. Two-fold increased production of dual TCR T cells persisted for at least 20 weeks after transplantation. These data demonstrate the hazard for production of T cells predisposed to pathogenic reactivity in the post-transplant environment, and suggest that dual TCR T cells could be a source of T cells causing cGVHD. To examine involvement of dual TCR T cells in cGVHD, we analyzed peripheral blood samples from patients after allogeneic HSCT (> 12 months post-transplant) using our previously utilized pair-wise TCRVa labeling flow cytometry approach. Flow cytometry analysis revealed that dual TCR T cells were present at increased frequencies in patients with cGVHD (n = 10, 8.3% + 1.1%, P = 0.028) compared to patients without cGVHD (n = 3, 2.5 + 1.1%) or healthy age-matched controls (n = 5, 1.9 + 0.4%). Dual TCR T cells from patients with cGVHD had an activated CD69+ phenotype as compared to T cells expressing only a single TCR from the same patient. Single-cell TCRa/TCRb sequencing confirmed the increased frequencies of dual TCR T cells specific to activated T cells in patients with cGVHD. Repertoire analysis of TCRs sequenced from single cells indicated that the increase in dual TCR T cells was polyclonal. The single-cell sequencing approach enabled multiplexed examination of T cell lineage-associated transcription factors and cytokines. Single-cell transcriptional profiling demonstrated that dual TCR T cells demonstrated predominantly pro-inflammatory and cytotoxic phenotypes with expression of Tbet and perforin. This is in contrast to T cells expressing only a single TCR from the same patient, or dual TCR T cells from healthy control patients, which had a quiescent phenotype. These data indicate a role for dual TCR T cells in mediating cGVHD. Together, these results suggest that dual TCR T cells may be an important link between post-transplant T cell development and cGVHD. Disclosures No relevant conflicts of interest to declare.

Background: Myeloablative hematopoietic cell transplantation (HCT) provides a cure for children with hemoglobinopathies, but transplant related early and late morbidity remains a challenge. Toxicities associated with myeloablative chemotherapy include early complications such as mucositis, hemorrhagic cystitis, seizures, and hepatic injury. Late effects such as infertility and compromised linear growth impair long-term quality of life, often decreasing enthusiasm for the procedure. Reduced intensity preparative regimens are especially attractive in children with non-malignant disorders to mitigate these toxicities but have been associated with increased risk of graft rejection. The primary objective of this study was to determine the toxicity and efficacy of a non-myeloablative preparative regimen using alemtuzumab, fludarabine, and melphalan followed by HCT from HLA matched related donors (MRD) in children with hemoglobinopathies. Methods: Following institutional review board approval, and parent and/or patient consent, participants were enrolled at 18 centers. Children < 21 years of age with severe sickle cell disease (SCD) manifestations, or transfusion dependent (> 8 red blood cell transfusions per year) thalassemia with a MRD and a performance status > 40 were eligible for inclusion. The preparative regimen included alemtuzumab (total dose 48 mg) IV (between days –22 and –19), fludarabine (30 mg/m2/day) (days –8 to –4) and melphalan (140 mg/m2) on day -3. Graft versus host disease (GVHD) prophylaxis included a calcineurin inhibitor (tapered after day 100, and methotrexate (7.5 mg/m2 on days 1, 3 and 6) or mycophenolate mofetil. Five patients also received methylprednisone (1 mg/kg/day) between days 1 and 28; this practice was discontinued in 2007. Results: A total of 52 children (43 with SCD and 9 with thalassemia), median age 11 years (range, 10m - 20y) underwent HCT between March 2003 and July 2014. Of these, 46 received bone marrow, 5 received marrow and cord blood (CB), and 1 received CB alone. Median follow up was 35.5 months (range, 3 – 136). Forty-nine children were alive at last follow up (Figure 1); 48 were symptom-free; one CB recipient had disease recurrence following graft rejection and successfully underwent a 2nd HCT. No hepatic veno-occlusive disease was noted. Three deaths 6, 11 and 21 months post HCT were from GVHD related causes [bronchiolitis obliterans (n=1); infection with GVHD (n=2)]. The cumulative incidence of graft failure and transplant related mortality was 1.9% and 5.7% respectively. The mean time to engraftment of neutrophils (ANC >500/cu mm) and platelets (>50,000/cu mm) was 13.0 (range 5-21) and 25.9 (range 8-120) days respectively. Three patients had neurologic toxicity (seizures/PRES) post HCT. At the time of last follow up, 35 patients (67%) were complete donor chimera (>90% donor) and 16 (31%) were mixed chimera (28-89% donor). Acute GVHD (grade 1-3) was noted in 30.8%. Four had grade I, 6 had grade II, and 6 had grade III GVHD. No patient developed grade IV aGVHD. Chronic GVHD was noted in 13%. Of 43 patients that were alive without disease and >6 months post HSCT, 38 had successfully discontinued all immune suppression and maintained donor chimerism post withdrawal. Immune reconstitution was robust by the end of the first year post transplant; infectious complications (23 of 30 CMV+ recipients had reactivation) were noted primarily in the first 3 months post HSCT. Conclusions: Children with hemoglobinopathies undergoing MRD HCT tolerated this non-myeloablative preparative regimen well, with minimal early toxicities. The event-free survival rate was comparable to those achieved with myeloablative regimens. Withdrawing immunosuppression was not associated with graft loss or recurrent GVHD. GVHD complications resulted in a low mortality rate. Alternative GVHD prophylaxis may further improve event-free survival. No gonadal toxicity (determined by hormone levels) has been identified to date in 15 HCT recipients of pubertal age (>13 years). Follow up however is early and in progress to determine long-term toxicities. These results support consideration of reduced intensity conditioning regimens for HCT in children with hemoglobinopathies as an alternative to standard myeloablative regimens. Figure 1: Overall/Event-free survival following non-myeloablative conditioning andMRD transplants for hemoglobinopathy Figure 1:. Overall/Event-free survival following non-myeloablative conditioning andMRD transplants for hemoglobinopathy Disclosures No relevant conflicts of interest to declare.

To the editor: We read the manuscript by Parmar with interest. In an exploratory analysis, these investigators found that mismatching of major histocompatibility complex class I–related chain A (MICA) between donor and recipient was associated with gastrointestinal (GI) acute graft-versus-host

Xenografting immunodeficient mice has been employed as a surrogate human hematopoietic stem cell (HSC) assay, however, erythroid output has not been reliably reported, limiting the usefulness of this model for erythroid disorders. We have previously demonstrated that busulfan preconditioning is sufficient to produce stable, high level engraftment of human cells in NOD/SCID/IL2Rγ null mice. Importantly, this high level engraftment can be achieved with low mortality, substantially reducing the number of animals required for experiments requiring long-term follow-up. Supplementation with human holo-transferrin (Tf) allows the detection human erythrocytes in this chimeric mouse assay at low levels, providing a potential model for the study of disorders affecting human red blood cells (2007 ASH meeting #3594). In the current work, we extend our observations and establish practical in vivo erythroid assay system of human HSCs in the humanized mouse model by the addition of an in vitro culture. Bone marrow (BM) from humanized mice containing 24.8±8.7% human cells (n=6) was first exposed to recombinant human (rHu) SCF+IL3 for 3 days in order to specifically enrich for human cells in the mixed chimera. After 3 days, human cells comprised 68.1±8.5% of the culture. The enriched cells were then cultured with rHu EPO+SCF+TGF-β for 7 additional days and then in rHu EPO+SCF for 7 days. After culture, and 98.9±1.47% of cells were of human origin. After centrifugation, the pellets were visibly red. Cells were assayed with both human CD71+ and GPA+ by flow cytometry: 64.0±7.44% were CD71+ and 69.2±7.02% were GPA+, and human α, β, and γ globin were confirmed by hemoglobin electrophoresis and mass spectrometry. In order to determine the utility of this approach, we tested 3 possible applications of this methodology: gene marking erythroid progeny, modeling of human hemoglobinopathies, and modeling of hemoglobin switching. We first transplanted human cord blood (CB) CD34+ cells after lentiviral transduction with a vector encoding GFP following busulfan conditioning. Three months post-transplant, bone marrow was harvested and placed in the in vitro culture. After in vivo culture, 98.9% of cells were of human origin and 60.7% were CD71+ and 72.3% were GPA+. The majority of CD71+ or GPA+ cells were GFP+ (82.3% and 87.7%, respectively). We subsequently transplanted human PB CD34+ cells derived from individuals with sickle cell trait (SCT) as we have previously demonstrated that these cells, unlike those from individuals with sickle cell disease, can be safely mobilized and processed. Further, the percentage of HbS expressed can be reliably measured. Three months post transplant, HbS was easily detectable by hemoglobin electrophoresis. Finally, we sought to address whether this model accurately reflects human erythropoiesis by examining hemoglobin switching after transplanting either CB expressing HbF or PB HSCs expressing HbA and monitoring the output of HbF and HbA over time. Early post-transplant, bone marrow derived from CB recipients expressed predominantly HbF after culture whereas that derived from PB HSC recipients expressed predominantly HbA. HbF declined during follow up and was replaced by HbA over 6 months of follow up from CB recipients, whereas HbA expression remained stable from PB HSC recipients. The time course of hemoglobin switching is similar to human ontogeny. In summary, our practical approach to model human erythropoiesis in the xenograft mouse should prove useful in the both the study of human erythroid disorders as well as therapeutic interventions.