Donor Chimerism In Peripheral Blood Research Articles

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

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.

Haploidentical Hematopoietic Cell Transplant with Post-Transplant Cyclophosphamide Expedites Donor-Derived Immune Reconstitution in a Pediatric Severe Aplastic Anemia Cohort

Severe aplastic anemia (SAA) is a life-threatening, idiopathic process involving the immune-mediated destruction of hematopoietic stem cells. Treatment involves immunosuppressive therapy (IST) and/or allogeneic hematopoietic cell transplantation (HCT). The latter has recently been optimized with use of a post-transplant cyclophosphamide (PTCy) based graft-versus-host disease (GvHD) platform, permitting successful HCT with only half-HLA-matched haploidentical donors. In pediatrics, this platform results in >90% overall survival with low rates of GvHD and graft failure. However, reports on donor chimerism and immune reconstitution are limited. We hypothesized that a PTCy platform would expedite CD3 donor engraftment and CD4 T cell recovery. In a phase III clinical trial, we treated three cohorts of pediatric (age </=25 years) patients with SAA requiring alloHCT: MUD, MSD, and HAPLO. The MUD cohort included n=15 recipients of 7-8/8 HLA-matched unrelated donor grafts (n=5 7/8, n=10 8/8) with median age 14 years (range 2-17 years), following cyclophosphamide (Cy) 50 mg/kg, fludarabine (Flu) 200 mg/m 2, rabbit anti-thymocyte globulin (rATG) 9 mg/kg total dose rabbit and low dose total body irradiation (TBI) at 200 cGy. The MSD cohort included n=9 recipients of 8/8 HLA-matched sibling donor grafts with median age 15 years (range 12-24 years), following Cy 200 mg/kg and 9 mg/kg rATG. The HAPLO cohort included n=4 recipients of 4/8 HLA-matched related donor grafts with median age 8.5 years (range 2-21 years) following Cy 29 mg/kg, Flu 150 mg/m 2, 9 mg/kg rATG, and low dose TBI at 200 cGy. GvHD prophylaxis consisted of 30 days of mycophenolate mofetil, and 100 days of a calcineurin inhibitor (followed by an 8-10 week taper), with the HAPLO cohort additionally receiving post-transplant Cy (PTCy) 50 mg/kg/day on days +3 and +4. Peripheral blood lymphocyte counts and peripheral blood and bone marrow donor chimerism were evaluated on days +100, +180 and +365. Recipient characteristics were largely equivalent between cohorts, with 43% receiving IST prior to HCT. Clonality was overrepresented in the HAPLO cohort (2 of 4, 1 with monosomy 7 myelodysplastic syndrome and 1 with a large PNH clone), with only 2 of 15 in the MUD and none in the MSD cohorts. Active controlled infections at the time of HCT were additionally overrepresented in the HAPLO cohort, with disseminated pseudomonas in 1. The MUD cohort had 1 with Rhizopus invasive sinusitis (none in the MSD cohort). Achievement of neutrophil engraftment was statistically significantly delayed in the HAPLO cohort at a median of 17 days compared to 12 for MSD and 14 for MUD, p=0.004), however there were no episodes of primary or secondary graft failure. With only a single late death from intracranial hemorrhage in the MUD cohort, OS was equivalent at 2 years (100% in MSD and HAPLO cohorts, 93.3% in MUD). GvHD rates were low with only 1 (MUD) experiencing grade II acute GvHD, and 1 (MUD) experiencing mild chronic GvHD. Immune reconstitution varied by cohort with statistically significantly greater CD3 peripheral blood donor chimerism (p<0.001) as well as CD4 (p=0.04) and CD8 (p=0.03) T cell counts over time for the HAPLO recipients (CD3 chimerism in Fig 1, CD4 count in Fig 2; 2-way ANOVA fixed effects model). Considering a CD4 >200 cell/uL as clinically meaningful, we assessed the proportion of each cohort achieving this milestone at each timepoint. While each cohort had an increase in proportion achieving CD4>200 over time, the HAPLO cohort reached 50% with CD4 >200 at day +100, compared to 11% MUD and 0% MSD (p=0.09). At day +180, the proportion with CD4 >200 were 36% MUD, 25% MSD and 75% HAPLO (1 yr: 90% MUD, 100% MSD and HAPLO). Donor availability remains a challenge to successful transplant for patients with SAA, and haploidentical matches overcome donor limited accessibility. PTCy as GvHD prophylaxis is associated with low incidences of graft failure and severe GvHD. While PTCy might be anticipated to inhibit donor T cells and immune reconstitution, our data, albeit limited by small cohorts, demonstrates superior and more efficient CD3 donor engraftment and CD4 T cell count recovery following HAPLO HCT with PTCy compared to non-PTCy based MUD and MSD cohorts.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Impact of Ruxolitinib in Myelofibrosis Allogeneic Stem Cell Transplant Outcome—a Pilot Study

Background: Myelofibrosis (MF) is a clonal stem cell disorder that develops de novo or evolves from a pre-existing myeloproliferative neoplasm such as polycythemia vera or essential thrombocythemia. MF is characterized by dysregulated proliferation of myeloid precursor cells and reactive deposition of connective tissue within the bone marrow, leading to bone marrow fibrosis and eventual failure from ineffective hematopoiesis. Common clinical manifestations include constitutional symptoms such as fatigue, fever, night sweats, weight loss, and hepatosplenomegaly. The clinical course and prognosis of MF is heterogenous, and survival outcomes are largely dependent on risk stratification that incorporates both phenotypic characteristics as well as cytogenetic features. Allogeneic stem cell transplant (alloSCT) is the only available treatment option to cure MF. Ruxolitinib (Rux), a potent JAK1/JAK2 inhibitor, has demonstrated significant effects in improving constitutional symptoms and reducing spleen size in intermediate to high risk patients. Hence, pre-transplant Rux treatment has often been practiced on patients with symptomatic splenomegaly and/or constitutional symptoms. Moreover, there are ongoing studies suggesting improved outcomes in patients treated with Rux before receiving alloSCT. However, the toxicity profile and tolerability profile of Rux treatment in transplant eligible patients remains unclear, especially the impacts of post-transplant Rux maintenance treatment.Method: We retrospectively analyzed the alloSCT outcomes among MF patients who never received Rux, received Rux before alloSCT, and received Rux before and after alloSCT at Penn State Hershey Cancer Institute between 6/2014 and 6/2017.Result: This pilot study analyzed 15 patients with mean age of 58.5 years old (range: 49-70) at the time receiving alloSCT. Ten patients were male and 5 patients were female. Those patients' MF scores were ranging from 2 to 3 (WHO Grade) at diagnosis. Five patients had primary MF and 10 had secondary MF. At diagnosis, all patients had constitutional symptoms, 14 of them had splenomegaly (1 had splenectomy before his MF diagnosis). Seven patients received Rux treatment before alloSCT, 4 received Rux before and after alloSCT, and 4 never received Rux. The mean length of time for Rux treatment before alloSCT was 8.3 months (range:5-20); the mean length of time for receiving Rux post alloSCT was 12.5 months (range:3-23). All patients received reduced intensity conditioning (RIC) regimen (14 patients received Fludarabine/Bu2, 1 patient received Fludarabine/Cyclophosphamide). One patient in the Fludarabine/Bu2 group also received total body irradiation (TBI) as part of conditioning regimen. The mean neutrophils engraft days for patients received Rux before alloSCT was 13.6 (range: 12-19), for patients received Rux before and after alloSCT was 13.3 (range: 12-16), and for patients never received Rux was 16 (range:13-18). All patients were transfusion independent at day 30 (D30) with >95% donor chimerism in peripheral blood. Following alloSCT, 4 patients restarted Rux at a mean time of 3.5 months (range: 3-4.5) post alloSCT. CMV viremia was detected in 5 patients before day 100 of alloSCT; in which 3 patients never received Rux. Fungal pneumonia (1 aspergillus) occurred in 3 patients who received Rux before allSCT. Multiple episodes of GVHD (grade II-III) occurred in 4 patients, however 3 of them never exposed to Rux before. One patient treated with Rux before alloSCT experienced liver GVHD (grade I to II) and responded well to steroids and immunosuppression treatment. The mean spleen size reduction in 6 months after alloSCT among patients received Rux before and after alloSCT was 26% (20 to 32%), among patients received Rux before alloSCT only was 18.2% (6 to 40%), and among patients never received Rux was 17.7% (12 to 24%). All patients are alive and in complete remission.Conclusion:Patients in this pilot study tolerated well to Rux treatment before and after alloSCT with neither prolonged engraft time nor increased toxicity. Before and after alloSCT Rux management may improve overall outcomes. Further study on large patient population is warranted. DisclosuresNo relevant conflicts of interest to declare.

Transplantation Using Bone Marrow from a (very) HLA Mismatched Unrelated Donor in the Setting of Post-Transplant Cyclophosphamide Is Feasible and Expands Access to Underserved Minorities

Background Despite increasing donor options for allogeneic transplantation, including matched/mismatched related donors, matched unrelated donors and cord blood units, a proportion of patients do not find a donor. This is especially relevant in patients from racial/ethnic minorities. Post-transplant cyclophosphamide (PTCY) has successfully overcome barriers related to HLA-mismatching in the related donor setting. We hypothesized that transplantation with a mismatched unrelated donor (MMUD) using PTCY would be feasible and associated with high engraftment and acceptable GVHD incidence. Methods We performed a prospective Phase II study of MMUD bone marrow (BM) transplantation with PTCY for patients with hematologic malignancies. Patients with a suitable HLA matched related or URD were excluded. Patients received a fresh BM graft, followed by PTCY on days +3, +4, Sirolimus/MMF starting on Day+5. Matching for 4-7/8 at HLA-A, -B, -C, and –DRB1 was permitted. We enrolled 80 patients (40 full intensity conditioning [FIC]; 40 reduced intensity conditioning [RIC]) at 11 transplant centers in the U.S. between Dec 2016 and March 2019. Regimen intensity was at the center's discretion. Results Characteristics are shown in Table 1. Importantly, 48% of patients were non-white/Hispanic, 55% had an HCT-CI >2 and 34% had a KPS of 1 HLA allele, 59% were under 30. Overall survival and non-relapse mortality at 100 days were 92% and 5% in the FIC arm, and 90% and 7.5% in the RIC arm (Table 2). Neutrophil recovery was 98% in both arms, with no primary graft failure in the FIC arm, and 7.5% in the RIC arm. Peripheral blood donor chimerism was >75% at all timepoints. Acute GVHD grade III-IV at day 100 was relatively high at 25% in the FIC arm but very low at 2.6% in the RIC arm. Viral reactivations were high (Table 3). Conclusion These early (day 100) results of our prospective study show high rates of engraftment with acceptable rates of GVHD in recipients of MMUD transplantation with PTCY, despite a high degree of HLA mismatch. An important finding is that ethnic minorities represent almost 50% of the study population, showing that this approach expands access to patients in need of potentially curative therapy. BK and HHV-6 virus reactivation/infection were common, the clinical significance of which requires further study. Understanding the risks for and impact of graft failure and acute GVHD is being studied as follow up continues.

Hematopoietic Mononuclear Cell Transplant with Minimal Gvhd in up to Completely Mismatched Unrelated Recipients

37 patients have been transplanted in a phase 2 protocol to establish chimerism to induce tolerance in up to 0 of 6 matched related and unrelated recipients of living donor renal allografts (KTx). The protocol is based upon tolerogenic CD8+/TCR- facilitating cells (FC) and nonmyeloablative conditioning. Recipients were conditioned with fludarabine (30mg/m2 days -5,-4,-3), cyclophosphamide (50mg/kg day-3 and+3), 200 cGy TBI (day-1) followed by KTx (day0). G-CSF mobilized peripheral blood mononuclear cells were apheresed from the donor >2 weeks before kidney transplantation, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 +cells and FC, and cryopreserved until transplantation on day+1 after kidney transplantation. Immunosuppression consisted of mycophenolate mofetil and tacrolimus. 36 patients have reached at least 1 year of follow up (range 12-105 months) and are the focus of this analysis. Patients ranged in age from 18-65 yrs. Enrollment was agnostic to degree of HLA match; 2 were 6/6 and 3 5/6 matched related using high resolution allele level typing, with the remainder 4/6 to 0/6 matched related (n=15) or unrelated (n=16) Two of the recipients were renal re-transplants. Tacrolimus/MMF immunosuppression (IS) was weaned and discontinued at 1 year if chimerism (>50% whole blood and T cell), normal renal function and normal kidney transplant biopsy were noted. 34 of 36 subjects exhibited peripheral blood donor chimerism at one month post KTx. Durable chimerism allowing for full IS withdrawal developed in 26 (time off IS from 1- 88 months); the majority (23/26) showed >95% donor whole blood/T cell chimerism. Three have exhibited stable-mixed chimerism ranging between 40% - 60%. Three patients failed to develop chimerism. Transient chimerism occurred in 8 patients. Durably chimeric patients retained chimerism after removal of IS, remain rejection-free without donor-specific antibody (DSA) and show immunocompetence to vaccinations. None have had to resume immunosuppression. Transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low-dose IS with stable renal function. There have been two cases of GVHD: one grade 2 lower GI acute GVHD that developed during conversion from tacrolimus to sirolimus that responded to steroids; this patient has developed moderate chronic GVHD of the skin. He is off IS. The second presented late following development of severe gastrointestinal symptoms and manifested treatment-resistant lower GI GVHD with associated tissue-invasive CMV colitis that proved fatal at 11 months post-Tx. There have been two additional kidney graft losses, both related to early infections. A second subject death occurred in a heavy (>100 pack yr) smoker who developed advanced stage lung cancer 4.5 years after Tx. A third subject developed pneumococcal sepsis > 4 years after transplant. He had not undergone the recommended pneumococcal vaccination post HSCT. Overall patient survival is 91.8% and death censored graft survival 94.1%. These rates compare favorably to treatment-related mortality rates after standard of care renal transplant. In summary, high levels of durable chimerism and tolerance with a low (5.5%) incidence of GVHD has been achieved in up to 0 of 6 matched related and unrelated recipients of FCR001 + kidney transplant. Figure Disclosures Ildstad: Talaris Therapeutics Inc: Employment, Equity Ownership, Other: Founder and CSO.

Outcomes of Hematopoietic Cell Transplantation in Patients with Germline SAMD9/SAMD9L Mutations.

Germline mutations in SAMD9 and SAMD9L genes cause MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) (OMIM: *610456) and ataxia-pancytopenia (OMIM: *611170) syndromes, respectively, and are associated with chromosome 7 deletions, myelodysplastic syndrome (MDS), and bone marrow failure. In this retrospective series, we report outcomes of allogeneic hematopoietic cell transplantation (HCT) in patients with hematologic disorders associated with SAMD9/SAMD9L mutations. Twelve patients underwent allogeneic HCT for MDS (n = 10), congenital amegakaryocytic thrombocytopenia (n = 1), and dyskeratosis congenita (n = 1). Exome sequencing revealed heterozygous mutations in SAMD9 (n = 6) or SAMD9L (n = 6) genes. Four SAMD9 patients had features of MIRAGE syndrome. Median age at HCT was 2.8 years (range, 1.2 to 12.8 years). Conditioning was myeloablative in 9 cases and reduced intensity in 3 cases. Syndrome-related comorbidities (diarrhea, infections, adrenal insufficiency, malnutrition, and electrolyte imbalance) were present in MIRAGE syndrome cases. One patient with a familial SAMD9L mutation, MDS, and morbid obesity failed to engraft and died of refractory acute myeloid leukemia. The other 11 patients achieved neutrophil engraftment. Acute post-transplant course was complicated by syndrome-related comorbidities in MIRAGE cases. A patient with SAMD9L-associated MDS died of diffuse alveolar hemorrhage. The other 10 patients had resolution of hematologic disorder and sustained peripheral blood donor chimerism. Ten of 12 patients were alive with a median follow-up of 3.1 years (range, 0.1 to 14.7 years). More data are needed to refine transplant approaches in SAMD9/SAMD9L patients with significant comorbidities and to develop guidelines for their long-term follow-up.

Abstract 1172: JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms

Abstract The myeloproliferative neoplasms (MPNs) are stem cell disorders characterized by hematopoietic stem cell (HSC) expansion and an increased propensity to develop hematologic malignancies. The acquired kinase mutation JAK2V617F plays a central role in MPNs. In this work we tested the hypothesis that the JAK2V617F-bearing megakaryocytes (MKs), an important component of the hematopoietic niche, promotes HSC dysfunction in MPNs. Methods JAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice (Radek Skoda, Switzerland) were crossed to generate MK lineage-specific human JAK2V617F knock-in mouse line (Pf4/FF1). Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline. Results We previously reported that, at 28 wks of age, the Pf4/FF1 mice developed a murine myeloproliferative syndrome with moderate thrombocytosis, splenomegaly, increased marrow MKs, and a 3-fold increase of CD45+CD201+CD150+CD48- HSCs with increased donor engraftment in a competitive repopulation assay compared to Pf4-cre control mice. (Zhan Leukemia 2016; Zhang Stem Cells 2018) Rigorous, extremely sensitive RT-PCR assays eliminated the possibility that HSCs in Pf4/FF1 mice express the mutant kinase. To study the effect of JAK2V617F-bearing MKs on HSC function during aging, we followed the Pf4/FF1 mice up to 2 years of age. At 2 yr, there were significant neutrophilia in Pf4/FF1 mice but no change was observed in hemoglobin, platelet count, or marrow HSC cell numbers compared to age-matched control mice. Indeed, while HSC numbers significantly increased in old (2-yr) control mice compared to young (6-mo) control mice (3.3-fold, p=0.0002, n=4-8), there was no significant increase of HSCs between old and young Pf4/FF1 mice (1.6-fold, p=0.158, n=6-8). A competitive marrow transplantation experiment was performed in which CD45.2 donor marrow cells from 2-yr old Pf4/FF1 or control mice were injected intravenously together with competitor CD45.1 wild-type marrow cells intro lethally irradiated wild-type recipient (CD45.1) (n=8-9 in each group). During a 4-month follow up, old HSCs from the Pf4/FF1 mice displayed hallmarks of murine hematopoietic aging including a lower peripheral blood donor chimerism, increased myeloid blood cell production, and decreased lymphoid cells compared to old HSCs from control mice. To begin to understand how JAK2V617F-bearing MKs promote the aging phenotype of HSCs in old Pf4/FF1 mice, qRT-PCR of purified HSCs identified up-regulation of p21 (2-fold, p = 0.019) in Pf4/FF1 mice compared to controls. Conclusion The Pf4/FF1 mice, in which the JAK2V617F expression is restricted to MKs, developed a myeloproliferative phenotype at young age and accelerated stem cell aging at old age. The precise mechanism by which JAK2V617F-bearing MKs affect the MPN marrow niche and stem cell dysfunction during disease progression deserve further investigation. Citation Format: Helen Wong, Melissa Castiglione, Molly Quan, Huichun Zhan. JAK2V617F-mutant megakaryocytes contribute to stem cell aging in myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1172.

577 Successful allogeneic epidermal grafting of chronic recessive dystrophic epidermolysis bullosa wounds from hematopoietic cell donors in the outpatient setting

Chronic wounds, or non-healing epidermal erosions present >6 weeks, are common, contribute to morbidity, and lack definitive therapy in recessive dystrophic epidermolysis bullosa (RDEB). The immune tolerance established by allogeneic hematopoietic cell transplantation (HCT) permits adoptive transfer of donor epidermal grafts and establishment of induced mosaicism. In a prospective, open label clinical trial for post-HCT RDEB patients with stable peripheral blood donor chimerism of ≥5%, up to 3 chronic wounds were grafted. Epidermal skin grafts measuring 5 cm2 were obtained from related HCT donors in the outpatient setting using the CelluTomeTM Epidermal Harvesting System. Wound size was measured at baseline, 6, 12, and 52 weeks. Nine patients received a total of 23 epidermal grafts at a median of 1207 days (range 267-3111) post-HCT. The mean (±SD) percent reduction in wound surface area achieved was 61.8% (±31.3), 85.1% (±20.3), and 90.9% (±16.8), at 6, 12, and 52 weeks, respectively, each significantly reduced compared to baseline (p < 0.0001). Donor harvest sites healed quickly with no scarring, though mild hypopigmentation was noted in 5 of 9. Recipient reported benefits include absence of blistering, touch sensation equivalent to native skin, and normal temperature regulation at donor sites. Evaluation of one graft following completion of the study at 1 year revealed wild-type type VII collagen staining by immunofluorescence, normal ultrastructure including intact anchoring fibrils at the dermal-epidermal junction by electron microscopy, and full thickness skin whole DNA donor chimerism of 42% (compared to 16% in concurrently biopsied native skin). Persistence of the donor graft beyond 1 year and observed migration of donor-grafted cells into adjacent wound suggest the epidermal grafts include non-terminally differentiated cells.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.

Immune Mediated Cytopenias Following Allogeneic Hematopoietic Cell Transplantation for Non-Malignant Disease: Risk Factors and Treatment Response in the Pediatric Population

Background: Immune mediated cytopenias (IMC) - isolated or combined hemolytic anemia, thrombocytopenia, and neutropenia - are increasingly recognized as potentially serious complications following allogeneic hematopoietic cell transplantation (HCT) for non-malignant disorders (NMD). However, IMC incidence, severity, response to therapy, and associated risk factors are not well defined.Methods: Pediatric patients undergoing HCT for NMD between 2010 and 2017 at a single institution were identified, excluding those experiencing graft failure or death prior to day +100. Demographics and HCT characteristics were obtained from a prospectively-maintained, institutional database. A retrospective chart review identified cases of IMC with positive direct antiglobulin test, anti-platelet antibody, and/or anti-granulocyte antibody; and documented treatment(s) and response. Composite IMC severity, incorporating peak acuity and response to therapy, was defined as mild (outpatient management with supportive care or a single course of therapy), moderate (outpatient management with 2 or more courses of therapy), or severe (inpatient management with 2 or more courses of therapy). Factors hypothesized to be associated with IMC included younger age, reduced intensity conditioning, mixed or declining donor chimerism, and inherited metabolic disorder as indication for HCT. For the first year after transplant, peripheral blood donor chimerism, B-cell reconstitution, and IgG and IgM levels were obtained.Results: A total of 271 NMD HCT recipients were included, with 53 (20%) exhibiting IMC, 20 (38% of cases) with multiple cell lines affected, diagnosed at a median of 131 days post-HCT (range, 30-264 days). IMC disease was mild, moderate, or severe in 56%, 32%, and 11%, respectively. For those with moderate or severe IMC, all received steroid therapy, with a response rate of 13%. Eighty-seven percent received additional therapy including IVIG, rituximab, bortezomib, and/or vincristine. Resolution of moderate IMC required an average of 2 individualized treatment courses, while severe IMC responded after an average of 6. Notably, no patients died of IMC. Univariate analysis revealed statistically significant association between IMC and younger age at HCT (median 3.1 vs. 7.1 years, p<0.01) and inherited metabolic disorder as the indication for HCT (p<0.01). Patient sex, recipient-donor HLA-match, stem cell graft source, intensity of conditioning, and presence of acute graft-versus-host disease were not significantly different between those with and those without IMC. Following HCT, a decline in peripheral blood donor CD3+ chimerism by at least 10% from day +60 to +100 was more common in those with IMC (p<0.01), although 50% of patients with IMC did not demonstrate a decrease in donor T-cell engraftment. The percent donor CD3+ chimerism at day +100 trended lower in the IMC group compared with the non-IMC group (71% vs 88.5%, p=0.10). The kinetics of patient B cell, IgG, or IgM recovery was not associated with IMC.Conclusion: Immune mediated cytopenias following HCT for NMD in a large pediatric cohort was common at 20%, with nearly half of those affected exhibiting clinically significant courses before disease resolution. Elucidation of risk factors may inform modifications to conditioning regimens or other approaches to prevent IMC. We found younger age at HCT, inherited metabolic disorder as HCT indication, and declining donor T-lymphoid chimerism as risk factors for IMC. However, the pathogenesis of IMC post-HCT for NMD remains elusive. Further insights may arise from investigation into the role of infections/molecular mimicry and complement activation. DisclosuresMiller:Sangamo Therapeutics: Employment.

CD3+/CD19+ Depleted Matched and Mismatched Unrelated Donor Hematopoietic Stem Cell Transplant with Targeted T Cell Addback Is Associated with Excellent Outcomes in Pediatric Patients with Nonmalignant Hematologic Disorders

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell–replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3+ T cell and CD19+ B cell depletion using CliniMACS, followed by a targeted addback of 1 × 105 CD3+ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell–replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.

Long-term Follow-up of a Phase 2 Clinical Trial to Induce Tolerance in Living Donor Renal Transplant Recipients

37 pts have been transplanted in a phase 2 protocol to induce tolerance in recipients of living donor renal allografts (KTx). The protocol is based upon tolerogenic CD8+/TCR-facilitating cells (FCRx) and nonmyeloablative conditioning. Recipients were conditioned with fludarabine (30mg/m2 days -5,-4,-3), cyclophosphamide (50mg/kg day-3 and+3), 200 cGy TBI (day-1) followed by KTx(day0). A G-CSF mobilized peripheral blood mononuclear cell product was apheresed from the donor >2 weeks pre-KTx, processed to remove graft-versus-host disease (GVHD)-producing cells yet retain CD34 +cells and FC, and cryopreserved until use day+1 post-KTx. 36 pts have reached at least 1 year of FU (range 12-105 months) and are the focus of this analysis. Pts ranged in age from 18-65 yrs. Enrollment was agnostic to degree of HLA match; 2 pts were 6/6 and 3 5/6 related, with the remainder 4/6 to 0/6 matched related (n=15) or unrelated (n=16) Two were re-transplants. Tacrolimus/MMF immunosuppression (IS) was weaned and discontinued at 1 year if chimerism, normal renal fcn and normal KTx biopsy were noted. 34 of 36 exhibited peripheral blood donor chimerism at one month post KTx. Durable chimerism allowing for full IS withdrawal developed in 26(time off IS from 1- 88 months); the majority (23/26)showed full (>95%) donor whole blood/T cell chimerism. Transient chimerism was seen in 8 pts. Durably chimeric pts retained chimerism after removal of IS, remain rejection-free w.o DSA, and show immune competence. Transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low-dose IS with stable renal fcn. There have been two cases of GVHD: one grade 1-2 acute GI GVHD that responded to steroids; this pt has developed chronic GVHD. The second presented late following development of symptoms and manifested treatment resistant GI GVHD with associated CMV that proved fatal at 11 months post-Tx. There have been two additional graft losses, both previously reported and related to infections. A second subject death occurred in a heavy (>100 pack yr) smoker who developed advanced stage lung cancer 4.5 years after Tx. Overall patient survival is 94.4% and death censored graft survival 94.1%. In summary, high levels of durable chimerism and tolerance with a low (5.5%) incidence of GVHD has been achieved in recipients of FCRx + LDKTx.

Kinetics of Langerhans cell chimerism in the skin of dogs following 2Gy TBI allogeneic hematopoietic stem cell transplantation.

BackgroundLangerhans cells (LC) are bone marrow-derived cells in the skin. The LC donor/recipient chimerism is assumed to influence the incidence and severity of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In nonmyeloablative (NM) HSCT the appearance of acute GVHD is delayed when compared with myeloablative conditioning. Therefore, we examined the development of LC chimerism in a NM canine HSCT model.Methods2 Gy conditioned dogs received bone marrow from dog leukocyte antigen identical littermates. Skin biopsies were obtained pre- and post-transplant. LC isolation was performed by immunomagnetic separation and chimerism analysis by PCR analyzing variable-number-of-tandem-repeat markers with subsequent capillary electrophoresis.ResultsAll dogs engrafted. Compared to peripheral blood chimerism the development of LC chimerism was delayed (earliest at day +56). None of the dogs achieved complete donor LC chimerism, although two dogs manifested a 100 % donor chimerism in peripheral blood at days +91 and +77. Of interest, one dog remained LC chimeric despite loss of donor chimerism in the peripheral blood cells.ConclusionOur study indicates that LC donor chimerism correlates with chimerism development in the peripheral blood but occurs delayed following NM-HSCT.

In Utero Hematopoietic Cell Transplantation Induces Peripheral Tolerance By Upregulating Two Types of Nontraditional Regulatory T Cells

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism across immune barriers without myeloablation/immunosuppression. It has the potential to induce donor specific tolerance (DST) for postnatal nonmyeloablative cellular transplants to treat target diseases such as Sickle cell disease. Partial deletion of donor reactive host T cells and host reactive donor T cells is responsible for tolerance and the absence of GVHD following IUHCT. The remaining nondeleted donor and host specific T cells are thought to undergo anergy. The mechanisms by which anergy and peripheral tolerance are achieved following IUHCT are not completely understood. Studies in both mice and humans have demonstrated the importance of regulatory T cells in maintaining tolerance following postnatal allogeneic transplantation. Subsets of regulatory T cells include traditional regulatory T cells (Tregs; CD4+ CD25+ FoxP3+), Type 1 regulatory T cells (Tr1 cells; CD4+ CD49b+ CD223+), and LAP+ regulatory T cells (LAP+ cells; CD4+ CD25- LAP+). These T regulatory populations inhibit T cell activation and proliferation through, among other mechanisms, the secretion of immunosuppressive cytokines including IL-10. During pregnancy, there is upregulation of circulating IL-10 levels in maternal and fetal blood, and maternal antigen specific fetal Tregs are induced in peripheral lymphoid tissue. We propose that IUHCT takes advantage of these tolerogenic features of the fetus to promote the peripheral induction of regulatory T cells capable of suppressing reactive T cell clones that escape thymic deletion.Methods: 10x106 bone marrow cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP (H2Kb) mice were injected intravenously into allogeneic gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd). Uninjected B6 FoxP3GFP and Balb/c FoxP3GFP mice served as controls. Mice were sacrificed at 2 and 4 weeks of age. Peripheral blood donor chimerism was determined by flow cytometry at the time of sacrifice. Splenocytes were analyzed for expression of CD4, H2Kb, H2Kd, CD49b, CD223, CD25, and LAP to determine the percentage of donor and host Tregs, Tr1 cells, and LAP+ cells within the CD4+ population. CD4+ splenocytes were also assessed for intracellular IL-10 expression. Statistically significant differences between chimeric and uninjected control animals were determined using a two-tailed student's t-test.Results: Chimeric animals demonstrated a 4- and 5-fold increase in donor Tr1 cells compared to uninjected B6 FoxP3GFP controls at 2 (2.4% vs. 0.6%, p=0.03) and 4 weeks of age (2.4% vs. 0.5%, p=0.007). There was no significant difference in host Tr1 cell levels of chimeric mice compared to uninjected Balb/c FoxP3GFP controls at either 2 (0.73% vs. 0.61%, p=0.4) or 4 weeks of age (1.0% vs. 0.53%, p=0.09). Similar to Tr1 cells, LAP+ cells of donor origin were increased compared to B6 FoxP3GFP controls at 2 (2.2% vs. 0.8%, p=0.008) and 4 weeks of age (3.2% vs. 0.6%, p=0.03). Additionally, host LAP+ cells were increased compared to Balb/c FoxP3GFP controls at 2 (2.6% vs. 1.5%, p=0.0004) and 4 weeks of age (3.0% vs. 1.1%, p=0.0005). The increase in Tr1 and LAP+ cells was associated with a significant decrease in traditional Tregs in both the host and donor population at 2 and 4 weeks of age (Figure 1). Elevated levels of Tr1 and LAP+ cells did not correlate with the absolute levels of donor cell engraftment but was dependent on achieving a threshold level of chimerism known to be associated with DST. Furthermore, the MFI of intracellular IL-10 among donor CD4+ splenocytes was significantly increased compared to those from B6 FoxP3 controls at 2 weeks of age (Figure 2), demonstrating immunosuppressive activity consistent with elevated Tr1 and LAP+ levels.Conclusion: The induction of donor and host tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. DST also allows for postnatal nonmyeloablative cellular transplants to increase engraftment to therapeutically relevant levels. We demonstrate a potentially important role that nontraditional Tr1 and LAP+ regulatory T cells play in maintaining donor cell engraftment and peripheral tolerance in the setting of IUHCT. Future investigations into the ability of these cells to augment tolerance induction after inefficient IUHCT have the potential to expand the clinical promise of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Use of Alefacept for Preconditioning in Multiply Transfused Pediatric Patients with Nonmalignant Diseases

Transfusion-related alloimmunization is a potent barrier to the engraftment of allogeneic hematopoietic stem cells in patients with nonmalignant diseases (NMDs). Memory T cells, which drive alloimmunization, are relatively resistant to commonly used conditioning agents. Alefacept, a recombinant leukocyte function antigen-3/IgG1 fusion protein, targets CD2 and selectively depletes memory versus naive T cells. Three multiply transfused pediatric patients with NMD received a short course of high-dose i.v. alefacept (.25 mg/kg/dose on days −40 and −9 and .5 mg/kg/dose on days −33, −26, −19, and −12) before undergoing unrelated allogeneic transplant in the setting of reduced-intensity pretransplant conditioning and calcineurin inhibitor–based post-transplant graft-versus-host disease (GVHD) prophylaxis. Alefacept infusions were well tolerated in all patients. Peripheral blood flow cytometry was performed at baseline and during and after alefacept treatment. As expected, after the 5 weekly alefacept doses, each patient demonstrated selective loss of CD2hi/CCR7−/CD45RA− effector memory (Tem) and CD2hi/CCR7+/CD45RA− central memory (Tcm) CD4+ and CD8+ T cells with relative preservation of the CD2lo Tem and Tcm subpopulations. In addition, depletion of CD2+ natural killer (NK) cells also occurred. Neutrophil recovery was rapid, and all 3 patients had 100% sorted (CD3/CD33) peripheral blood donor chimerism by day +100. Immune reconstitution (by absolute neutrophil, monocyte, and lymphocyte counts) was comparable with a cohort of historical control patients. All 3 patients developed GVHD but are all now off immune suppression and >2 years post-transplant with stable full-donor engraftment. These results suggest that alefacept at higher dosing can deplete both memory T cells and NK cells and that incorporating CD2-targeted depletion into a reduced-intensity transplant regimen is feasible and safe in heavily transfused patients.

Graft-Versus-Host Disease After Simultaneous Pancreas–Kidney Transplantation: A Case Report and Review of the Literature

Graft-versus-host disease (GVHD) after solid organ transplantation is rare and usually fatal. We present, to our knowledge, the second successfully treated case in a simultaneous pancreas-kidney (SPK) transplant recipient. A 29-year-old female with end-stage renal disease from type 1 diabetes mellitus received an SPK transplant from a male donor, with rabbit-antithymocyte globulin induction. Twelve days posttransplant, she was readmitted with abdominal pain, nausea and vomiting. She developed leukopenia, abnormal liver enzymes, fever and a skin rash. Skin biopsy showed interface dermatitis consistent with allergic reaction versus GVHD. Fluorescence in situ hybridization of the skin biopsy showed 28% of cells had a Y chromosome confirming GVHD. Short tandem repeats (STR) enriched for CD3+ cells from peripheral blood showed a mixed chimerism. She was successfully treated with a single plasmapheresis to remove antithymocyte globulin, high-dose steroids, photopheresis and high tacrolimus levels (12-15 ng/mL). Five months after transplantation, she has normal renal function and white blood cell count, normal hemoglobin A1C and no evidence of peripheral blood donor chimerism. In conclusion, early diagnosis of GVHD after SPK transplantation may allow successful treatment. STR enriched for CD3+ may be useful to evaluate the response to therapy.

Case Report: Haploidentical Stem Cell Transplantation In a 78 Year-Old Patient

An older chronologic age has been a consistent predictor of poor outcomes in hematopoietic stem cell transplantation (HSCT), mainly due to non-relapse mortality (NRM). Therefore, non-curative treatment strategies are commonly adopted for these patients. However, mortality and treatment toxicity has decreased as a result of improved supportive measures, such as reduced intensity conditioning regimens and optimized infection management. T-cell replete haploidentical HSCT emerged as a feasible alternative for leukemia patients without substantial differences in outcomes when compared to fully matched related donor. We report an old adult woman treated with haploidentical HSCT.A 78 year-old female patient presented with anemia, leukocytosis, thrombocytopenia and blasts in the peripheral blood. Diagnosis of acute myelogenous leukemia was established. Conventional cytogenetic demonstrated chromosome eight trisomy, and FISH was negative for other common MDS/AML cytogenetic abnormalities. FLT3-ITD and NPM1 mutations were negative. Her medical history was negative except for heavy smoking. Considering the patients advanced age, the first attending physician chose not to administer intensive treatment and started on decitabine 20 mg/m2 for 5 days. She was refractory to the first-line treatment with persistent cytopenias and blasts in the peripheral blood four weeks after treatment was started. Comprehensive geriatric assessment was performed. She was considered independent for Basic Activities of Daily Living (ADL score 6) and Instrumental Activities of Daily Living (IADL score 27), without cognitive impairment in mini-mental state examination (MMSE score 30), at risk of malnutrition in mini nutritional assessment (MNA 9). As she was considered fit, we decided to perform high-dose chemotherapy with idarubicin and cytarabine, but, once more, the disease was refractory. A rescue regimen was attempted with high-dose cytarabine and mitoxantrone, again, with no response. After discussing pros and cons with the patient and the family, we decided to start Another regimen consisting of topotecan and high dose cytarabine immediately followed by allogeneic hematopoietic stem cell transplantation (HSCT). At day 14, she had 3% blasts in the BM aspirate a T-cell replete haploidentical HSCT using her 52 year-old son as donor and mobilized peripheral blood as stem cell source was performed. Conditioning regimen consisted of fludarabine, cyclophosphamide, TBI 2Gy and post-transplant cyclophosphamide. Graft versus host disease (GVHD) prophylaxis consisted of mycophenolate mofetil and cyclosporine. She had neutrophil engraftment with complete donor chimerism at day+15 and platelet engraftment at day+17. At day+48, she had mild (stage II) skin acute GVHD resolved with topical steroids. Cyclosporine was withdrawn at day+ 93. Due to high relapse risk, the patient was started on monthly post-transplant azacitidine 36 mg/m2. At day+100 the patient remained in complete remission, complete donor chimerism in peripheral blood and bone marrow. Functionality was preserved (ADL score 6 and IADL score 24), presented discrete cognitive impairment (MMSE 28) and malnoutrition (MNA 5). She is now at day+182, doing well and performing again all usual daily activities.To the best of our knowledge, this is the oldest patient treated with haploidentical HSCT. Post transplant cyclophosphamide as T cell depletion strategy in haploidentical HSCT is well tolerated and widely available, being therefore an excellent alternative for patients without conventional donors who require immediate transplant. Older adults with hematologic malignancies are a heterogeneous group and decisions based on chronological age alone are clearly inappropriate. Recently, geriatric assessment proved to be an important prognostic tool in acute leukemia and may be useful in HSCT. In experienced centers, haploidentical HSCT in older adults may be a safe procedure and more accurate pre-transplantation risk stratification tools should be developed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Close To Clinics: Acute Gvhd In a Novel Chemotherapy Based Minor Mismatch Transplantation Model

GVHD (graft-vs-host disease) is one of the major complications after allogeneic hematopoietic stem cell transplantation leading to substantial morbidity and mortality. Several murine models are used to investigate this systemic inflammatory disease in vivo and to identify possible targets for clinical treatment. However, current well-established murine GVHD models have their clinical limitations as they are based on major histocompatibility mismatches and only lethal irradiation as conditioning. Development of new treatment options would be forwarded by the availability of a more clinically relevant murine model.To reflect the clinical situation of transplantation and GVHD, we established a new murine GVHD model which is based on Chemotherapy conditioning and MHC-matched bone marrow transplantation (BMT). In LP/J -> C57BL/6 mouse model, our chemotherapy transplantation protocol resulted in stable full donor chimerism and development of acute GVHD.C57BL/6 recipients were conditioned with a 7-day busulfan-cyclophosphamide Chemotherapy protocol. Mice received for 5 days 20 mg/kg/day busulfan intraperitoneally (i.p.) and the last three days additionally 100 mg/kg/day cyclophosphamide. After a two-day resting phase, recipients were transplanted with 1.5 x 107 bone marrow and 2 x 106 splenic T cells from LP/J donors. Recipients showed good engraftment with already 80 % engraftment in peripheral blood at day 15 after BMT. 90 % donor chimerism in peripheral blood and bone marrow could be shown already at day 30 after BMT.LP/J and C75BL/6 mice share the same haplotype (H2b) with only mismatches at different minor histocompatibility loci. As in clinical BMT, GVHD still arises after MHC-matched transplantation because of multiple minor mismatches. We could show in our LP -> C57BL/6 model typical features of acute GVHD. Allogeneic transplanted animals showed significant decreased survival compared to syngeneic transplanted animals (Figure 1A). GVHD animals exhibited GVHD-typical weight loss (Figure 1B) and a significant increased clinical GVHD index consisting of five clinical parameters: weight loss, posture, activity, fur and skin (Figure 1C). Our model showed a strong skin phenotype, especially between day 15 and 25 after BMT extensive scurf spread over the whole body combined with fur loss. Pronounced weight loss and highest clinical GVHD index were monitored between day 15 and 25, implicating this period as acute phase of GVHD. Histopathologic analysis of GVHD organs (large bowel, liver and skin) confirmed acute GVHD symptoms in our model as histopathological scores after Lerner (1974,Transplant Proc 6:367-371) were significantly increased in GVHD animals compared to BMT recipients without GVHD (Figure 1D). [Display omitted] We next characterized systemic inflammation in our model by determining the number of circulating inflammatory cells in peripheral blood at different time points by flow cytometry. CD8+ T cells were significantly increased in GVHD animals in acute phase (day 15 after BMT), whereas CD4+ T cells were elevated in late phase (day 50-60 after BMT). These data suggests, that in our model two inflammatory phases occur, an early acute CD8-driven and a late CD4-driven phase.Finally, we could demonstrate GVHD-typical T cell infiltration in GHVD target organs in acute phase (Figure 2). In large and small bowel, liver and skin CD4 and CD8 infiltration was significantly elevated in GVHD animals, however, CD8 infiltration was more prominent. We conclude that CD8 T cells play a prominent role in acute phase of GVHD in our model. [Display omitted] In summary, we could establish a novel chemotherapy based minor mismatch GVHD model which shows good engraftment, typical features of acute GVHD and systemic and target organ specific inflammation. Our murine GVHD model closely resembles the clinical situation of MHC-matched stem cell transplantation and may help to better understand pathogenic mechanisms in GVHD and develop new treatment options. Disclosures:No relevant conflicts of interest to declare.