Donor Cell Chimerism Research Articles

The Effect of Narrow-Band Ultraviolet B Irradiation on the Vascularized Composite Allotransplantation Rat Model.

Vascularized composite allotransplantation (VCA) allows functional and esthetic reconstruction for patients with complex anatomical defects. However, acute and chronic graft rejections are significant obstacles to VCA. Ultraviolet light is an oncogenic environmental hazard. However, ultraviolet B (UVB) has an immunomodulation effect. Therefore, this study aims to elucidate the impact of UVB irradiation on the VCA rat model. The rat vascularized bone marrow allotransplantation model was used. A vascularized bone marrow from a Brown Norway rat (RT1Ac) was transplanted into a Lewis rat (RT1Ab). The allograft and surrounding abdominal skin were exposed to narrow-band ultraviolet B (NB-UVB) (311 nm) radiation with an energy of 1350 mJ/cm2 3 times a week until the end of the study period. There were 5 study groups: syngeneic transplantation (group 1), allogeneic transplantation (group 2), allogenic transplantation-NB-UVB (group 3), allogenic transplantation-antilymphocyte serum (ALS)-tacrolimus (group 4), and allogenic transplantation-antilymphocyte serum-tacrolimus-NB-UVB (group 5). Group 5 had decreased graft survival compared with group 4. In the donor cell chimerism analysis, donor cell chimerism decreased significantly after UVB irradiation and was unresponsive to the administered immunosuppressants. After UVB irradiation, the CD8 T-cell ratio was increased, and the regulatory T-cell ratio was decreased. The preliminary data showed that NB-UVB irradiation of the VCA rat model may decrease graft survival. However, further studies are needed to elucidate the possible mechanisms of this phenomenon.

Exosomes from donor-derived adipose mesenchymal stem cells prolong the survival of vascularized composite allografts.

Donor-derived adipose-derived mesenchymal stem cells (ADMSCs) dampen the alloimmune response and exosomes are reported to have biological activity similar to their parent cells. Here, we investigated the roles of exosomes from donor-derived ADMSCs (ADMSC-exo) in vascularized composite allotransplantation (VCA). Brown Norway-to-Lewis rat hindlimb transplantations were intravenously treated with either exosome from donor-derived ADMSCs or phosphate-buffered saline, combined with a short course of immunosuppression. We established that the treatment with ADMSC-exo prolongs the survival time of VCA grafts. Skin and muscle samples from ADMSC-exo-treated animals showed no histological signs of rejection, but samples from controls showed rejection of degree III. Comparing to the control group, a significant increase of donor cell chimerism, Tr1 and Treg, while a decrease of CD4+ T and Th1 cells were observed in the ADMSC-exo-treated group. Our findings imply that ADMSC-exo may be a valuable and safe treatment for extending VCA graft survival.

Tolerance Protocols in Large Animal VCA Models—Comprehensive Review

The clinical application of vascularized composite allografts (VCA) has continue to expand each year as new transplants are performed. However, chronic rejection and the many side effects from chronic immunosuppression continue to inhibit widespread application of VCA. Immune tolerance to VCA would address these two issues and shift the risk-benefit ratio to allow for the more widespread application of these transplants. Future development of clinical tolerance protocols will be generated from pre-clinical experiments performed in large animal models. This article reviews the current state of large animal tolerance protocols published for VCA. The most successful tolerance protocols thus far include the use of hematopoietic stem cell transplant with non-myeloablative preconditioning and establishment of donor cell chimerism. In addition to this strategy, several groups have examined the inclusion of mesenchymal and adipose-derived stem cells to address the complications related to the use of hematopoietic stem cell transplant (such as graft versus host disease). These large animal models are critical to development of a viable pre-clinical model for tolerance in vascularized composite allografts that can deliver a tolerant state with minimal harm to the recipient of these non-life-saving transplants.

Prediction, management, and prognosis of mixed chimerism after hematopoietic stem cell transplantation in transfusion-dependent pediatric thalassemia patients.

Early-onset mixed chimerism (MC) with a high proportion of residual host cells is considered a signal of graft rejection in patients undergoing allogenic hematopoietic stem cell transplantation for transfusion-dependent thalassemia. In order to prevent graft rejection and minimize the risk of treatment-related graft-versus-host disease (GVHD), we established a hierarchical management system based on chimerism analysis. This retrospective study provides a comprehensive review of the characteristics, interventions, and outcomes of the 38 patients who developed MC after transplantation among the 144 pediatric thalassemia patients between July 2007 and January 2019 at our center. A sibling donor, a blood type-matched donor, conditioning regimens without fludarabine, and transplants containing <10×108 total nucleated cells/kg were identified to be associated with the development of MC. Among the 38 patients developing MC, only four patients rejected the grafts. The response rate to donor lymphocyte infusion (DLI, only for patients receiving sibling donor transplantation) and cytokine immunomodulation without DLI was 70.6% and 42.9%, respectively. Patients that developed GVHD after DLI or cytokine therapy had a more significant increase in donor cell chimerism (16%, range 0%-35%) than those without (8.5%, range -21% to 40%, P=.049). However, even when treatment-related GVHD was included, patients with MC had a lower cumulative incidence of total acute GVHD than patients with complete donor chimerism (29.2% vs 48.0%, P=.030). Interventions based on chimerism analysis were effective in preventing graft rejection and did not increase treatment-related GVHD in thalassemia patients with MC.

Incidence and Outcome of Late Relapse after Allogeneic Stem Cell Transplantation for Myelofibrosis

In this cross-sectional study, we retrospectively evaluated the files of 227 patients with myelofibrosis who underwent transplantation between 1994 and 2015 for relapse later than 5 years after allogeneic stem cell transplantation (SCT). A total of 94 patients who were alive and in remission at 5 years were identified with follow-up of at least 5 years (median, 9.15 years) after SCT. Thirteen patients (14%) experienced late molecular (n=6) or hematologic (n=7) relapse at a median of 7.1 years while 81 patients did not experience relapse. Relapse patients received either donor lymphocyte infusion (DLI) (n=7) and/or second transplantation (n=4). Of those, 72.7% achieved again full donor cell chimerism and molecular remission, and after a median follow-up of 45 months, the 3-year overall survival rates for patients with or without relapse were 90.9% (95% confidence interval [CI], 77% to 100%) and 98.8% (95% CI, 96% to 100%), respectively (P=.13). We conclude that late relapse occurs in about 14% of the patients and the majority can be successfully salvaged with DLI and/or second allograft. All patients with molecular relapse are alive and support the long-time molecular monitoring in myelofibrosis patients after allogeneic SCT.

Donor's graft ex vivo T-cell depletion with fludarabine reduces graft-versus-host disease signs and improves survival after intestinal transplantation - an experimental study.

Intestinal passenger T leukocytes are responsible for graft-versus-host disease (GvHD) in intestinal transplantation (ITx). We hypothesized that ex vivo fludarabine treatment of the bowel graft would diminish the risk of GvHD and improve overall survival post-transplant. We performed isolated heterotopic small bowel transplantations from Lewis (LEW) to Brown Norway (BN) rat strains, which generated GvHD signs from the fourth day post-transplant. These symptoms included rash, weight loss, piloerection, and diarrhea. The grafts of one of the experimental groups were immersed and sealed in cold Celsior preservation solution with 1000µm fludarabine for 1h, prior to its implantation into recipient animals. No histological signs of intestinal tissue alterations were observed after fludarabine treatment. Fludarabine-treated bowel recipients showed significantly later and milder clinical signs of GvHD and reduced total donor cell chimerism, as determined by flow cytometry using strain-specific anti-HLA antibodies. Additionally, fludarabine treatment prolonged recipients' overall survival (13.5days±0.3days vs. 9.2days±0.5). We conclude that active modification of the intestinal leukocyte composition is advantageous in our ITx animal model. Immunosuppression with fludarabine during the surgical procedure, which could be translated directly to the clinic, protects bowel recipients from GvHD and improves overall post-transplant survival.

Zebrafish as a Model System for the Interrogation of Hematopoietic Stem Cell Engraftment

Hematopoietic stem cell (HSC) transplant is a treatment used to infuse healthy blood stem cells to patients that suffer from a range of blood disorders, such as leukemia. Although transplantations are successful nearly 85% of the time, the associated morbidity and mortality rates are still too high. To improve HSC transplantation outcomes, it is crucial to better understand all facets of HSC engraftment, which includes the seeding, expansion, and differentiation of donor HSC into the recipient. Zebrafish is an ideal animal model to study engraftment due to their small size, high fecundity, and malleable genetics. We have established a novel transplantation model using the zebrafish runx1 W84X bloodless mutant strain. These animals lack endogenous HSC and thus possess empty HSC niche available for donor cell engraftment without the need for pre‐conditioning regimens. Transplantation of donor marrow cells that ubiquitously express green fluorescent protein (ubi:GFP) into runx1 W84X homozygous mutants at two days‐post fertilization (dpf) results in robust GFP+ donor cell engraftment that sustains the blood system of the mutants into adulthood. Sham‐injected and un‐injected embryos served as negative controls. Using this model, we will image transplanted embryos daily to visualize all stages of engraftment. From 4–7 dpf, we observed seeding of GFP+ cells into all hematopoietic tissues including the thymus, caudal hematopoietic tissue, and kidney marrow (equivalent to mammalian bone marrow). We will use confocal microscopy to gain a more in‐depth view of the cell‐to‐cell interactions occurring within these niches to understand engraftment. Additionally, we will use this assay for longitudinal studies to determine what facets predict which animals will sustain high versus low donor cell chimerism. Studying this animal model could advance our understanding of human HSC transplantation outcomes.Support or Funding InformationNIH (Grant # R25‐GM104547); Cancer Research Center (Grant # P30CA013330)

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

After lung transplantation (LuTx), a transient chimerism of donor cells exists in the blood of recipients due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor CD8+ and CD4+ T and NK cells and to investigate whether they might represent tissue-resident memory (TRM) cells. Lymphocyte dynamics in recipient blood were determined in 97 lung transplant patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry with lineage-, tissue-, and donor HLA class I allele-specific mAb. The same makers were used to determine the phenotype of lymphocytes present in organ storage solution (perfusate, n=102), recipient explanted lung parenchyma (n=28) and donor trachea (n=17). In peripheral blood of all recipients, donor-derived CD4+ and CD8+ T and CD56dim NK cells were detected at T0, T24 and 3 wks after LuTx and had higher CD69 expression compared to recipient cells (p<0.05) and were mostly CD25- CCR7- memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In lung parenchyma and trachea, most CD69+ T and NK cells showed coexpression of other tissue residency markers such as CD103, CD49a and PD-1 with significant enrichment in trachea (p<0.05). These markers were not found in circulating donor lymphocytes and perfusates, indicating that they represent distinct memory T and NK subsets. Donor T and NK cells showed higher IFN-γ production with PMA/ionomycin stimulation compared to recipient cells (p<0.05). The presence of these particular TRM cells did not have an impact on the development of PGD 24h after LuTx. However, patients with high frequencies of donor T cells showed a trend towards a CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). Our results demonstrate that donor T and NK cells found in the blood of LuTx recipients are a distinct subset from circulating lymphocytes and TRM cells present in lung tissue, since they express CD69 but lack expression of other classical TRM markers. They display a higher functional capacity despite the onset of immunosuppression. Donor T cells might be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

Regulatory T cells promote alloengraftment in a model of late-gestation in utero hematopoietic cell transplantation

AbstractIn utero hematopoietic cell transplantation (IUHCT) has the potential to cure congenital hematologic disorders including sickle cell disease. However, the window of opportunity for IUHCT closes with the acquisition of T-cell immunity, beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Here we report that regulatory T cells can promote alloengraftment and preserve allograft tolerance after the acquisition of T-cell immunity in a mouse model of late-gestation IUHCT. We show that allografts enriched with regulatory T cells harvested from either IUHCT-tolerant or naive mice engraft at 20 days post coitum (DPC) with equal frequency to unenriched allografts transplanted at 14 DPC. Long-term, multilineage donor cell chimerism was achieved in the absence of graft-versus-host disease or mortality. Decreased alloreactivity among recipient T cells was observed consistent with donor-specific tolerance. These findings suggest that donor graft enrichment with regulatory T cells could be used to successfully perform IUHCT later in gestation.

Monitoring minimal residual/relapsing disease after allogeneic haematopoietic stem cell transplantation in adult patients with acute lymphoblastic leukaemia.

Relapse after allogeneic haematopoietic stem cell transplantation (SCT) is a major cause of death in patients with acute lymphoblastic leukaemia (ALL). Here, we retrospectively analysed the contributions of lineage-sorted donor cell chimerism (sDCC) and quantitative PCR (qPCR) targeting disease-specific genetic rearrangements to detect minimal residual/relapsing disease (MRD) and predict impending relapse in 94 adult ALL patients after SCT. With a median follow-up of surviving patients (n = 61) of 3.3 years, qPCR and/or sDCC measurements turned positive in 38 patients (40%). Of these, 22 patients relapsed and 16 remained in complete remission. At 3 years, qPCR and/or sDCC positive patients showed an increased incidence of relapse (50% vs. 4%, p < 0.0001), decreased relapse-free survival (RFS, 40% vs. 85%, p < 0.0001), and decreased overall survival (OS, 47% vs. 87%, p 0.004). Both, qPCR and sDCC pre-detected 11 of 21 relapses occurring within the first two years after SCT and, overall, complemented for each other method in four of the relapsing and four of the non-relapsing cases. Patients receiving pre-emptive MRD-driven interventions (n = 11) or not (n = 10) showed comparable median times until relapse, RFS, and OS. In our single centre cohort, qPCR and sDCC were similarly effective and complementary helpful to indicate haematological relapse of ALL after SCT.

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

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

Chromatin Remodeling Subunit Brm Regulates Hematopoietic Stem Cell Self-Renewal By Limiting Intracellular Valine Levels

Chromatin remodeling complexes facilitate gene expression and control cell fate decisions. The ATPase subunit of chromatin remodeling complex BRG1 is essential for stem cell function, but the role of its paralog Brm remains essentially unknown. To assess a role(s) for Brm in hematopoietic cell regulation in vivo, we studied hematopoietic stem (HSCs) and progenitor cells (HPCs) in bone marrow (BM) of Brm -/- vs. wildtype (WT) control mice. While BM from Brm -/- mice contain increased numbers of rigorously-defined phenotypic populations of long- and short-term repopulating HSCs and granulocyte macrophage progenitors (GMPs) and increased numbers and cycling status of functional HPC (assessed by CFU-GM, BFU-E, and CFU-GEMM colony assays), they were defective in self-renewal capacity upon in vivo serial transplantation using congenic mice (CD45.2+ donor cells, CD45.1+ competitor cells, and F1 (CD45.2+/CD45.1+) recipient mice). Increased numbers of HSCs from Brm-/- BM failed to show competitive advantage over wild type (WT) control BM cells in primary (1°) transplantation in lethally irradiated mice (based on month 4 donor cell chimerism and phenotypically defined HSC numbers). Moreover, 2° and 3° engraftment at 4 months post transplantation each, a measure of HSC self-renewal capacity, revealed much reduced engraftment of donor Brm -/- BM cell chimerism and HSC numbers compared to the extensive 2° and 3° engraftment of control WT BM. No significant differences in myeloid/lymphoid ratios were noted in 1° or 2° engrafted mice, suggesting no differentiation lineage bias of donor Brm -/- BM cells. This demonstrates a critical role for Brm in controlling in vivo self-renewal of mouse BM HSCs. Valine [(2S)-2 amino-3 methylbutanoic acid (C5H11N02)] is implicated in hematopoietic regulation, since depleting dietary valine permitted non-myeloablative mouse HSC transplantation (Taya et. al. Science 354:1152-1155, 2016). Metabolic analysis of lineage negative (lin-) cells demonstrated that valine, but not leucine, levels were very highly elevated in Brm -/- BM cells, thus linking intracellular valine levels with Brm expression. Exogenously added valine significantly increased basal oxygen consumption rates of both total WT BM and WT lin- cells, but not of total or lin-Brm -/- BM cells in vitro (via Seahorse machine analysis). To study effects of valine on HPCs, we assessed the addition of exogenously added valine on mouse BM and human cord blood (CB) cells cultured in the presence of cytokines with either non-dialyzed or dialyzed fetal bovine serum (FBS). Valine, but not leucine, dose-dependently enhanced HPC (CFU-GM, BFU-E, and CFU-GEMM) colony formation and secondary replating capacity of cytokine stimulated CFU-GM and CFU-GEMM derived colonies of normal mouse BM cells in vitro in presence of non-dialyzed FBS; additional enhanced valine effects were noted when dialyzed FBS (lacking valine and other amino acids) was used. Valine also enhanced mouse BM HPC survival in vitro in context of delayed addition of growth factors, and cytokine stimulated (SCF, FL, TPO) ex-vivo expansion of normal mouse BM HSCs and HPCs. Valine enhancement of the above noted functional mouse BM HPC assays in the presence of dialyzed FBS was also apparent with low density and CD34+ purified CB cells, demonstrating that valine effects are not species specific. Our results suggest that valine is an enhancing factor for HSC maintenance of self-renewal capacity and HPC proliferation, and that Brm gene expression limits intracellular valine levels, thereby controlling HSC self-renewal and HPC proliferation. This information is of potential use for future translation to modulate self-renewal of HSCs and survival and proliferation of HPCs for clinical advantage. Disclosures No relevant conflicts of interest to declare.

Obstructive sleep apnea and craniofacial appearance in MPS type I-Hurler children after hematopoietic stem cell transplantation

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal storage disorder characterized by severe multi-systemic organ manifestations including obstructive sleep apnea syndrome (OSAS). Hematopoietic stem cell transplantation (HSCT) is the treatment of choice in severe MPS I (MPS IH, Hurler syndrome). However, the effect of HSCT on OSAS in MPS IH still remains unclear. The purpose of this study was to analyze respiratory patterns during sleep following HSCT in MPS IH children and to relate these findings to craniofacial abnormalities. Overnight polysomnographies of nine MPS IH children (mean age: 8.2years) previously treated with HSCT were retrospectively analyzed. Magnetic resonance images of the head were assessed with regard to soft and hard tissue abnormalities of the upper respiratory tract. The mean apnea hypopnea index (AHI) was 5.3 events/h (range, 0.3-12.2), and the majority of apnea/hypopneas were obstructive. Whereas two patients had severe OSAS (AHI > 10) and two moderate OSAS (5 > AHI < 10), five patients had no evidence of OSAS (AHI < 2.0). Donor cell chimerism was significantly lower in MPS IH patients with OSAS as compared to patients without OSAS (p < 0.001). The upper airway space and the maxilla were significantly smaller and the adenoids larger in MPS IH patients with OSAS as compared to those of non-OSAS patients. OSAS was only observed in MPS IH patients with graft failure or low donor cell chimerism. Conversely, successful HSCT seems to ameliorate adenoid hyperplasia and maxillary constriction in MPS IH patients and thereby minimizes the risk of OSAS at least at younger ages.

PS1555 STUDY OF IMMUNE HEMOLYTIC DISEASE AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background:Immune hemolytic disease (IHD) is a main and severe complication after hematopoietic stem cell transplantation (HSCT). Allogeneic immune‐hemolysis is that immune cells and antibodies from the recipient attack red blood cells from donor, which is defined as host versus graft (HVG). We analyzed retrospectively our data and given a summary as below.Aims:To try to illustrate and proven the causes of Post‐HSCT IHD, which is GVG or HVG, by analysis based on serum titer of ABO antibody, chimerism rate of donor cell, and responds of therapy.Methods:707 HSCTs from our center since January 2010 to March 2017, were retrospectively analyzed. The item of analysis included age, gender, ABO blood‐type, diseases and transplant type, etc. Standard of diagnosis of Post‐HSCT IHD included: 1: after HSCT, 2:hemoglobin ≤80 × 109/L for three time in two consecutive weeks, 3: needed regular transfusion, and 4 positive Coomb's test. Standard of partial remission: without gaining CR standard but ≥ 50% decline of transfusion. Statistic methods included stata13 chi‐square test.35 cases were divided into three groups by host blood‐type antibody titer and donor cell chimerism. Group 1(n = 16), all cases were donor‐recipient ABO blood‐type mismatched and 100% donor cells chimerism but recipient ABO blood‐type antibodies occurrence: antibody titer high (more than pre‐HSCT value) or delayed decline (more than 3 months after HSCT). Group 2 (n = 13), mixed donor cell chimerism (75–98.8%). Group 3 (n = 6) full donor cell engraftment, 100% donor cell chimerism without ABO blood‐type antibody from recipient. In group 3, four with donor‐recipient ABO mismatch, 3 cases with acute GVHD and 5 cases with positive CMV test. In G1, ABO blood‐type antibodies from recipient can indirectly prove host immune cell existence. In G2, chimerism &lt;100% may directly prove host immune cell existence. In G3, although we had no direct evidence to prove recipient cells existence and some cases were along with GVHD, by responds of therapy host cells were still thought to be existence according to hypothesis of immune cells escapes and immune cells harbor in lymph nodes.Results:Post‐HSCT IHD incidence was 4.95%, 336 patients with ABO blood‐type mismatch, 27 suffered from post‐HSCT IHD, In contrast, oppositely only 8 patients had post‐HSCT IHD among 339 patients with ABO blood‐type match (7.14% versus 2.36%, p = 0.001). Post‐HSCT IHD occurred in 10 cases in 40 HSCT pairs of A blood‐type donor and O blood‐type recipient (A‐O pair), 2 in 54 B‐O pairs and 2 in 10 AB‐O pairs. statistic significant difference was found when comparing three groups (p = 0.029). According to Chi‐square test, Mixed chimerism compared with 100% implantation, in adverse implantation the probability of immuno‐hemolysis was greater, it was statistically significant(P = 0.000, P &lt; 0.05). The percentage of hemolysis after transplantation for patients with matched sibling donors was 3.44%, for patients with unrelated donors was 7.38%(P = 0.019&lt;0.05), which appeared that the probability of post‐transplantation hemolysis by unrelated donors was greater.Summary/Conclusion:The cells and antibody leading post‐HSCT IHD from host cells were proved by all 35 patients with post‐HSCT IHD, who was cured with tapering immunosuppress drugs, plus or not Rituximab, and giving or not DLI. High titer recipient blood‐type antibody was a direct evidence of existence of host immune cells. Tapering immunosuppress drugs, plus or not Rituximab and giving or not DLI were a correct therapy for post‐HSCT IHD.

S912 LOCAL INTERFERON‐ALPHA DELIVERY INTO THE MYELOMA MICROENVIRONMENT BY GENE‐ENGINEERED AUTOLOGOUS STEM CELL TRANSPLANTATION

Background:Multiple Myeloma (MM) is an incurable malignancy heralded by an immunosuppressive Tumor Microenvironment (TME). Therapeutic attempts aiming at reprogramming the TME and unleashing innate and adaptive immunity may synergize with current anti‐MM drugs, deepen responses and ultimately prolong long‐term survival.Aims:We developed an ex vivo Gene Therapy (GT) approach that locally delivers Interferon‐alpha (IFNa) into the TME through the Tie2+ myeloid progeny of transplanted gene‐modified Hematopoietic Stem and Progenitor Cells (HSPCs).Methods:Murine Lin‐ or human Cord Blood (huCB) HSPCs were transduced with lentiviral vectors (LV) expressing, respectively, murine or human IFNa under transcriptional control of the Tie2 promoter and post‐transcriptional control by miR‐126–3p thereby targeting IFNa expression to a subset of tumor‐infiltrating monocytes. IFNa‐LV‐transduced or mock‐transduced HSPC were transplanted into Vk∗Myc transgenic mice after they have spontaneously developed a serum M‐spike (immunocompetent mouse model) or into NSGW41 mice that were successively challenged with a luciferase+ MM.1S human myeloma cell line (human xenograft model). Myeloma disease progression was monitored by serum protein electrophoresis (Vk∗Myc), whole body bioluminescence imaging (MM.1S) and bone marrow plasma cell burden at the endpoint. In the MM1.S model, IFNa‐GT was combined with systemic Daratumumab (i.p.), Lenalidomide (i.p.) and Carfilzomib (i.v.) treatment.Results:In the spontaneous Vk∗Myc mouse model of myeloma, Ifna GT halted myeloma progression (4 months PFS of 58.3% vs. 15.8% in control) and reduced mortality by &gt;50%. Treatment response was associated with a higher proportion of CD8+ T cells in the BM and seen at donor cell chimerism as low as 4%. Mechanistic studies on the tumor microenvironment are ongoing. The NSGW41 xenograft model transplanted with huCB HSPCs allowed the development of a larger and more diverse human myeloid compartment compared to the classical NSG model. Even in the absence of adaptive immunity, IFNa‐GT resulted in a significant reduction in BM myeloma burden (13.4+/‐2.2% vs. 26.3+/‐3.7% in mice transplanted with control huCB cells; n = 19 vs. 18), independently confirmed by BLI. Interestingly, the IFNa‐GT group showed an increased content of human CD33+ HLA‐DR+ CD11c+ dendritic cells (including a pro‐inflammatory SLAN+ subset) and increased levels of CD16 and HLA‐DR expression on human myeloid cells within the myeloma‐bearing BM, suggestive of myeloid cell reprogramming towards a more immune‐stimulatory state. Moreover, IFNa‐GT induced an upregulation of CD38 expression (on average 1.4 fold on myeloma cells). This provides a rationale for combining IFNa‐GT with other anti‐MM drugs. Indeed, the anti‐MM effect of Daratumumab, Lenalidomide or Carfilzomib was enhanced by IFNa‐GT, without excessive toxicity on the HSPC graft.Summary/Conclusion:Our studies conducted both in the immunocompetent Vk∗Myc murine model as well as in the hematochimeric xenotransplantation setting demonstrate that IFNa‐GT has robust anti‐MM effect and may subvert the immunosuppressive MM TME, acting both on myeloma cells as well as on the immune infiltrate. A phase I/II dose‐escalation study of IFNa‐GT in patients with early‐relapsed multiple myeloma has recently been approved and is starting accrual in March 2019.

Fetoscopic versus Ultrasound-Guided Intravascular Delivery of Maternal Bone Marrow Cells in Fetal Macaques: A Technical Model for Intrauterine Haemopoietic Cell Transplantation

Introduction: Significant limitations with existing treatments for major haemoglobinopathies motivate the development of effective intrauterine therapy. We assessed the feasibility of fetoscopic and ultrasound-guided intrauterine haemopoietic cell transplantation (IUHCT) in macaque fetuses in early gestation when haemopoietic and immunological ontogeny is anticipated to enable long-term donor cell engraftment. Material and Methods: Fluorescent-labelled bone marrow-derived mononuclear cells from 10 pregnant Macaca fascicularis were injected into their fetuses at E71–114 (18.9–170.0E+6 cells/fetus) by fetoscopic intravenous (n = 7) or ultrasound (US)-guided intracardiac injections, with sacrifice at 24 h to examine donor-cell distribution. Results: Operating times ranged from 35 to 118 min. Chorionic membrane tenting and intrachorionic haemorrhage were observed only with fetoscopy (n = 2). Labelled cells were stereoscopically visualised in lung, spleen, liver, and placenta. Donor-cell chimerism was highest in liver, spleen, and heart by flow cytometry, placenta by unique polymorphism qPCR, and was undetected in blood. Chimerism was 2–3 log-fold lower in individual organs by qPCR than by flow cytometry. Discussion: Both fetoscopic and US-guided IUHCT were technically feasible, but fetoscopy caused more intraoperative complications in our pilot series. The discrepancy in chimerism detection predicts the challenges in long-term surveillance of donor-cell chimerism. Further studies of long-term outcomes in the non-human primate are valuable for the development of clinical protocols for IUHCT.

Specific Bone Marrow Mesenchymal Subsets in Patients with Myelodysplastic Syndromes Harbor Molecular Perturbations That Alter the Dynamics of Competition between Pre-Leukemic Clones and Normal Cells

Myelodysplastic syndromes (MDS) are clonal disorders of aberrant hematopoietic stem cells. The role of the hematopoietic microenvironment (HMEV) in the initiation and maintenance of MDS remains unclear, though model systems have provided strong evidence for the potential of specific mesenchymal subsets, mostly osteoprogenitor cells, within the HMEV as a driver of the disease. Distinct behavior of various models emphasizes the importance of analyzing specific and highly defined populations rather than bulk mesenchymal cells when investigating patient-derived bone marrow (BM) samples.We have developed a platform that allows us to prospectively isolate specific human BM mesenchymal subsets with a comparable transcriptional profile to mouse osteoprogenitor cells that have been shown to drive hematological malignancies when perturbed (Raaijmakers et al., 2010; Dong et al, 2016). Using a combination of three cell surface markers (CD271, CD146, CD106) that were previously shown to prospectively enrich for mesenchymal cells in human BM, we isolated and performed transcriptional and functional analysis of four distinct subsets within the non-hematopoietic (CD45, CD235), non-endothelial (CD31) compartment of the human BM. The four populations are labeled 1A (CD271+CD106-), 1B (CD271+CD106+), 2A (CD271+CD146+CD106-), and 2B (CD271+CD146+CD106+) (Figure 1A). Comparative analysis of differentially expressed gene sets based on RNA-seq within the human and mouse mesenchymal populations identified population 2B to be the most similar to osterix (OSX)-labeled mouse osteoprogenitor cells, whereas population 1A was more similar to osteocalcin (OCN)-labeled mouse mature osteoblasts. In animal models, genetic perturbations specifically in OSX but not OCN labeled cells resulted in hematological malignancy. Based on that observation, we hypothesized that population 2B is the cell subset most likely to harbor molecular perturbations in the BM of MDS patients. We analyzed populations 2A, 2B, 1(1A+1B) and another population 3 which is devoid of colony forming cells and encompasses all CD271-CD146- non hematopoietic stromal cells in 16 human MDS patients. BM from 11 patients undergoing hip replacement surgeries was used as a source of age- and gender-matched normal controls.Despite the heterogeneity of the disease, principal component analysis revealed that the transcriptional profile of normal samples clearly separated from MDS patients mostly in populations 2B and 1 (Figure 1B). Functional annotation of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) demonstrated minimal overlap of enriched terms between the four populations. Thus our data demonstrate transcriptionally distinct subsets of BM mesenchymal cells and identify signatures of transcriptional deregulation which differentiate these cell subsets in MDS patients from normal individuals.Osteopontin, an extracellular matrix protein expressed by osteoblasts among other cells, was the most significantly differentially expressed gene in population 2B of MDS patients.To study the effect of the gene expression perturbations on MDS evolution in vivo, we created a chimeric model where BM cells expressing the Nup98/HoxD13 (NHD13) translocation were transplanted in competition with wild type cells in wild type (WT) or osteopontin knock out (Opn-KO) recipients. Animals were conditioned using immunotoxin to avoid the damaging effect of irradiation on the microenvironment. Following chimerism of donor cells in peripheral blood over 24 weeks demonstrated a competitive advantage of NHD13 cells in the Opn-KO recipients leading to a significant difference in survival when compared to NHD13 cells transplanted in wild type mice (Figure 1C, D).Altogether, our data provide evidence that specific mesenchymal progenitor subsets in the BM of MDS patients harbor molecular perturbations that contribute to the competitive advantage of MDS over normal hematopoietic cells. DisclosuresSykes:Clear Creek Bio: Equity Ownership, Other: Co-founder.

Hoxblinc Is Aberrantly Expressed in Acute Myeloid Leukemia and Functions As a Potent Oncogenic Long Non-Coding RNA in Leukemogenesis

Aberrant expression of long non-coding RNAs (lncRNAs) might contribute to the development and progression of leukemia. However, functional studies on the actual role of lncRNAs during the development of leukemia remain scarce, and very few lncRNAs have been shown to be involved in leukemogenesis. HoxBlinc is an anterior HoxB gene-associated intergenic lncRNA. It is a cis-acting lncRNA and functions as an epigenetic regulator to coordinate anterior HoxB gene expression. Giving the dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation, HoxBlinc might be an oncogenic lncRNA of leukemia.To determine whether HOXBLINC lncRNA is aberrantly expressed in human AML samples, we performed RT-qPCR on bone marrow mononuclear cells (BMMNCs) from a cohort of 73 AML patients. A dramatic up-regulation of HOXBLINC was observed in over 60% of the patients. When TCGA-AML datasets of a cohort of 179 AML patients were analyzed for their HOXBLINC expression, a significant portion of these AML patients had high levels of HOXBLINC expression. Interestingly, AML patients with high HOXBLINC expression (the top thirty percentile of patients) had a significantly shortened survival as compared to patients with low HOXBLINC expression (the bottom thirty percentile).To investigate the impact of HoxBlinc overexpression on normal hematopoiesis and the pathogenesis of hematological malignancies in vivo, we generated a HoxBlinc transgenic(Tg) mouse model. Within 1 year of age, 67% of the HoxBlincTg mice (10 of 15) died or were sacrificed because of a moribund condition due to AML.We then assessed whether overexpression of HoxBlinc affects the pools of HSC/HPCs by flow cytometric analysis on the BM cells of young WT and HoxBlincTg mice (8-10 weeks of age). HoxBlincTg BM had a dramatically greater number of LT-HSC, ST-HSC, MPP cells, and a significantly higher percentage of GMP, but a lower percentage of MEP/CMP cell populations as compared to WT group. To determine the effect of HoxBlinc overexpression on the function of HSC/HPCs, we performed paired-daughter cell assay, replating assay and liquid culture on sorted LT-HSC, LSK or LK cells from young WT and HoxBlincTg mice, the results indicate that transgenic expression of HoxBlinc enhances HSC self-renewal and impairs HSC/HPC differentiation.To assess whether HoxBlinc overexpression-mediated changes in HSC/HPC function are cell-autonomous, we performed competitive transplantation assays to examine the repopulating capacity of HoxBlincTg BM cells. When the donor cell chimerism was analyzed kinetically in the PB of recipient mice, the CD45.2 cell population remained ~50% in mice receiving WT BM cells, whereas the CD45.2 chimerism in the recipients transplanted with HoxBlincTg BM cells steadily increased. Interestingly, mice receiving HoxBlincTg BM cells developed AML at 2-6 months after transplantation.Previous data reported that HoxBlinc can recruit the Setd1a/Mll1 histone H3K4 methyltransferase complex to mediate formation of the active topologically associated domain (TAD) in the anterior HoxB locus for transcription of the anterior HoxB genes. In this study, LSK or LK cells sorted from young WT and HoxBlincTg mice were analyzed by RNA-seq, ATAC-seq, H3K4me3 CHIP-seq and 4C analysis. Mechanistically, HoxBlinc overexpression alters HoxB locus chromatin three-dimensional organization to enhance enhancer/promoter chromatin accessibility and coordinate the expression of not only HoxB1-5 but also HoxA9, Runx1, Meis1 and so on, which are critical genes for HSC regulation and/or leukemogenesis.Our study provides novel insights into the HSC regulation by lncRNAs and identifies HOXBLINC, which coordinates to maintain an oncogenic transcription program for leukemic transformation, as a potent oncogenic lncRNA in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Rituximab Combined with Donor Lymphocyte Infusion Cured Immunohemolysis after Refractory Hematopoietic Stem Cell Transplantation

BackgroundImmuno-hemolytic anemia is a severe complication after allogeneic hematopoietic stem cell transplantation (HSCT). Primary or secondary reaction between recipient lymphocytes and donor RBC antigens was considered as one of cause of post-transplantation immune-hemolysis (PTI). Different treatments can be administered according to the donor cell chimeric status and antibody titer of blood types of the donor in recipient.Objective:To find the ways of treatment of PTI after HSCT.Methods:A retrospective analysis about the complications of 707 patients underwent HSCT in our center from January 2010 to July 2017 was made. Of them, PI occurred in 28 patients with positive direct Coomb's test. By donor cell chimerism status, the 28 patients with PTI were divided into two groups. In group 1 (n=11), the patients presented full donor chimerism and received rituximab or/and steroid treatment only; In group 2 (n=17), the patients presented mixed chimerism and received rituximab and gradually decreasing immunosuppressive agents with/without, thereafter, donor lymphocyte infusion (DLI).Results: A total of 28 patients was cured after treatment. Of 11 cases in group 1, five patients developed grade 1-2 acute GVHD and four of which developed pancytopenia. Of 17 patients with mixed chimerism (75-99.5%) in group 2, four had post-HSCT pure red blood cell aplasia and the major blood type incompatibility between the donor and recipients. In group 2, six patients with PTI were cured by rituximab and gradually decreasing immunosuppressive therapy only; 11 patients had no respond to the treatment of these treatments, and were cured after combined with DLI.Conclusion:When PTI occurred, chimerism status may help us to determine how to treat PTI. For patients with full donor cell chimerism, it may be fit to give rituximab and to reduce immunosuppressive agent gradually. DLI may be given to the patients with mixed chimerism and no responding to the combined therapy of rituximab and immunosuppressive agent reduction. DisclosuresNo relevant conflicts of interest to declare.

Safety Analysis of Intra-Patient Dose- Study of Crenolanib Maintenance Therapy in Patients with FLT3 Mutant AML Following Allogeneic Hematopoietic Stem Cell Transplant

Background: FLT3 inhibitors (like sorafenib and midostaurin) have been administered as maintenance therapy post allogeneic stem cell transplantation (SCT) to reduce persistent relapse risks in FLT3-mutant AML patients. Reduced doses of both sorafenib and midostaurin have been found to be tolerable in the post-HSCT setting. Crenolanib is a highly potent and selective FLT3-targeted TKI that has activity as a single-agent and combined with chemotherapy in patients with FLT3-ITD and/or FLT3-TKD mutations. We here report the outcomes of safety and tolerability of crenolanib maintenance in FLT3 mutant AML patients after allo-HSCT (NCT02400255).Methods: To assess the tolerability of crenolanib maintenance in post-SCT AML patients and evaluate the appropriate dose for such patient population, a clinical trial of crenolanib maintenance therapy was performed in patients (age ≥ 18) with FLT3 mutant AML who had undergone SCT. Enrollment criteria included patients with FLT3-ITD, or FLT3-TKD positive disease at any point prior to SCT, having first SCT, with ≥ 50% T cell donor chimerism, adequate engraftment with complete remission (CR) at post-SCT evaluations. Patients needed to enroll between 42 and 90 days post-transplant without uncontrolled infection and graft versus host disease (GvHD).Initially, the study was designed for patients to be treated with crenolanib 80 mg TID (240 mg daily. Due to initial tolerability in the first patients (n=4), the design was changed to an intra-patient dose-escalation, in which patients received crenolanib starting at a dose of 60 mg BID for a month and then escalated to 80 mg BID and finally 80 mg TID as tolerated.As of July 2018, 24 patients, median age 53.5 years (range 31-74) have been enrolled and received crenolanib maintenance therapy. Disease status at SCT was CR (n=10, 42%), CR without count recovery (CRi, n=12, 50%), and active disease (n=2, 8%). The minimal residual disease (MRD) by multicolor flow cytometry was evaluable in 22 CR/CRi patients at SCT and was deemed to positive in 5 (23%). Conditioning regimen was myeloablative (20, 83%) or reduced intensity (4, 17%). Donors were matched related (n=11, 46%), matched unrelated (n=11, 46%) or haploidentical (n=2, 8%).After 4 patients enrolled, the trial design was altered to allow for intra-patient dose escalation. Ten patients were never able to escalate above 60mg BID, one patient stayed at 60 mg TID, 12 patients escalated to 80 mg BID, of those 12, 7 were able to escalate to 80 mg TID.The median days on crenolanib was 474 days (4-728 days) and median number of cycles was 17.5 cycles (1-26 cycles). Of the 21 patients no longer on study, 6 were due to relapse with median time to progression of 17 days (7-76 days) after first dose of crenolanib. Of the 6 relapses, four patients were positive for MRD prior to transplant and two had active disease. Two patients came off study due to noncompliance with study procedures, two were due to withdrawal of consent, 7 were patient decision due to side effects, one was due to suicidal ideation, and one was for insurance non-payment. Only one patient completed the planned 24 cycles of treatment with crenolanib 60 mg BID. Currently, four patients remain on study.Observed side effects were predominantly grade 1 and 2 with the most common (regardless of attribution) being nausea (62%), vomiting (38%), and diarrhea (33%), 13 adverse events that were grade 3 were reported likely attributable to crenolanib, no grade 4 side effects reported. There were two grade 2 GVHD-AEs, one grade 1 and one ungraded GVHD-AEs reported. One patient had a grade 3 rash that was confirmed as GVHD.Conclusion: These interim results suggest that crenolanib can be safely given at a dose of 160 mg to 240 mg total daily in the post-SCT setting. Two randomized phase III trials have been initiated to investigate the efficacy of crenolanib with chemotherapy vs chemotherapy alone in R/R FLT3 mutated AML as well as crenolanib vs midostaurin following chemotherapy in newly diagnosed FLT3 mutated AML (NCT03250338, EudraCT 2017-001600-29; NCT03258931). Post HSCT crenolanib maintenance will be offered at 100 mg BID (200 mg daily) in both trials. DisclosuresOran:AROG pharmaceuticals: Research Funding; ASTEX: Research Funding; Celgene: Consultancy, Research Funding. Shpall:Affirmed GmbH: Research Funding. Agrawal:Arog: Employment. Champlin:Sanofi: Research Funding; Otsuka: Research Funding.