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Extracellular vesicles (EVs) are critical mediators of intercellular communication within the bone marrow niche and have been implicated in numerous features of aging. However, their role in natural hematopoietic stem cell (HSC) aging has not been fully elucidated. The goal of this work was to test the hypothesis that EVs from whole bone marrow (BM-EVs) can modulate the HSC aging phenotype in vivo. With respect to HSC aging, our prior work showed that, in contrast to the well-known reduced functional capacity and prominent myeloid skewing displayed by old immunophenotypically-defined HSCs, old unseparated whole bone marrow (WBM) (24-26-mo-old) had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM in limit dilution competitive bone marrow transplantation, and showed minimal to no myeloid skewing. In order to test the ability of BM-EVs derived from this total WBM population to alter HSC aging phenotype, we first isolated EVs from WBM flushed from old (24-26-month old) and young (6-8-week old) C57/BL6 (CD45.2) mice by differential centrifugation (2000 × g for 30 min, supernatant centrifuged 100,000 × g for 1 hour, BM-EV pellet collected). Utilizing nanoparticle tracking analysis, we found no difference in mean particle size between old and young BM-EVs, but there was an approximately 2-fold increase in the number of EVs from old WBM compared to young WBM. To test the ability of these BM-EVs to alter HSC function in vivo, we injected old CD45.2 mice with 2 x 109 young BM-EVs and young CD45.2 mice with 2 x 109 old BM-EVs mice via tail vein, daily x 3 days. Control mice were injected with age-matched BM-EVs or vehicle alone. At one-month post injection, we isolated total WBM and Lineage negative/c-Kit+/Sca-1+/CD150+ cells (LSK-SLAM) from the EV-exposed or vehicle control mice. We injected either 3 x 105 WBM cells or 400 LSK-SLAM mixed with 3 x 105 healthy WBM competitor cells from young B6.SJL (CD45.1) mice into lethally irradiated young CD45.1 hosts and measured peripheral blood chimerism and lineage contribution by flow cytometry up to 6 months post-transplant. For the young marrow, exposure to old BM-EVs had no appreciable effects on engraftment capacity or lineage distribution. However, old WBM exposed to young BM-EVs exhibited a significant decrease in engraftment (15% ± 5%) when compared to old WBM exposed to age-matched old BM-EVs (61% ± 14%) or vehicle control (47% ± 7%) (% average donor chimerism ± SEM, n=4-5 mice/group, p Disclosures No relevant conflicts of interest to declare.

Patients with severe malaria treated with artesunate sometimes experience a delayed hemolytic episode. Artesunate (AS) induces pitting, a splenic process whereby dead parasites are expelled from their host erythrocytes. These once-infected erythrocytes then return to the circulation. We analyzed hematologic parameters in 123 travelers treated with AS for severe malaria. Among 60 nontransfused patients observed for more than 8 days, 13 (22%) had delayed hemolysis. The peak concentration of circulating once-infected erythrocytes was measured during the first week in 21 patients and was significantly higher in 9 patients with delayed hemolysis than in 12 with other patterns of anemia (0.30 vs 0.07; P = .0001). The threshold of 180 million once-infected erythrocytes per liter discriminated patients with delayed hemolysis with 89% sensitivity and 83% specificity. Once-infected erythrocyte morphology analyzed by using ImageStream in 4 patients showed an 8.9% reduction in their projected area, an alteration likely contributing to their shorter lifespan. Delayed clearance of infected erythrocytes spared by pitting during AS treatment is an original mechanism of hemolytic anemia. Our findings consolidate a disease framework for posttreatment anemia in malaria in which delayed hemolysis is a new entity. The early concentration of once-infected erythrocytes is a solid candidate marker to predict post-AS delayed hemolysis

Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

Aging is accompanied by loss of tissue homeostasis, deterioration in organ function and an increased incidence of disease. Age-related changes in the adult stem/progenitor compartment likely drive much of this aging phenotype. For the hematopoietic system, it has been shown that the number of hematopoietic stem cells (HSCs) increases with age, but their renewal potential decreases and they exhibit aberrant lineage production with myeloid skewing. These age-related findings have been based primarily on studies of highly purified, immunophenotypically defined HSCs. However, our data indicate that young murine WBM contains a large population of cycling HSCs not represented within the purified HSC population. The goal of this work was to determine the effects of aging on this total population of stem cells in un-separated WBM. We first performed limiting dilution competitive bone marrow transplantation assays to determine the relative frequency of HSCs in old and young murine WBM. We injected serial dilutions of WBM from either old (24 month old) or young (6-8week old) donor C57BL/6 (CD45.2) mice into lethally irradiated young B6.SJL (CD45.1) hosts. Each mouse received 3x105 competitor WBM cells derived from young B6.SJL mice. At 6 months post transplant, we measured peripheral blood chimerism and lineage composition. According to the Poisson distribution, the estimated frequency of stem cells (with positive engraftment defined as ≥1% donor chimerism) was 1 in 9.8 x 103 for old marrow and 1 in 40 x 103 for young marrow, an approximately 4-fold increase in functional stem cells in old marrow, similar to reports in the literature. Interestingly, in contrast to what has been observed with old purified HSCs, the lineage analysis of donor-derived peripheral blood from aged WBM showed minimal to no myeloid skewing (Table, Row A). To test stem cell function in secondary transplantation, lethally irradiated young recipient mice were initially transplanted with old or young donor WBM mixed with equal numbers of young competitor WBM. At 6 months post-transplant, WBM was harvested and serially transplanted into lethally irradiated secondary recipient young mice. Peripheral blood chimerism was analyzed at 6 months. Similar to young WBM, the stem cells within old WBM retained robust stem cell capacity in serial transplantation, and aged WBM continued to show little to no myeloid skewing (Table, Row B, C). Finally, using tritiated thymidine (3H-thymidine) suicide and Hoechst 33342 staining, we determined the cell cycle status of the aged HSCs in WBM. WBM harvested from old or young donor mice was exposed to 3H-thymidine in vitro for 30 minutes, during which time any cell traversing S-phase would be selectively killed. Cells were then mixed with equal numbers of un-manipulated young competitor WBM and transplanted into lethally irradiated young host mice. Controls included donor WBM un-manipulated or incubated with unlabeled thymidine. We found a significant reduction in engraftment capacity in old WBM post 3H- thymidine incubation (Table, Row D), suggesting the presence of cycling HSCs. Utilizing Hoechst 33342, we isolated G0/G1 and S/G2/M cell cycle-specific fractions of old WBM by FACS and injected each population with equal numbers of young competitor WBM into lethally irradiated young recipient mice. At 6 months post transplant, aged WBM cells in S/G2/M contributed to peripheral blood chimerism, albeit to a lesser extent than cells in G0/G1 (Table, Row E). We previously published that, although purified HSCs in young marrow are quiescent, cells in S/G2/M and in G0/G1 from young un-separated WBM had equal engraftment capacity. Thus, it appears that this cycling stem cell population is still present in old marrow but declines with age. In conclusion, in contrast to purified HSCs, functional engraftment capacity in un-separated WBM does not seem to diminish with age, shows minimal to no myeloid skewing, and is due, in part, to cycling stem cells. These data support the existence of a population of HSCs in WBM distinct from conventionally defined purified HSCs, with seemingly differential susceptibility to aging with respect to self-renewal and lineage fate decisions. Thus, we propose that approaches to reverse aging and define mechanisms underlying age-related hematologic diseases may need to be broadened to incorporate a more heterogeneous HSC compartment not fully represented by the purified HSCs. Table Disclosures No relevant conflicts of interest to declare.

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSCs) for patients that require allogeneic stem cell transplantation. The use of UCB in adults is restricted due to the limited number of HSCs within a single unit. This limitation can be overcome by ex-vivo expansion. Several strategies have successfully expanded the numbers of clinically functional HSCs. Such strategies, including the aryl hydrocarbon receptor antagonist, SR1, pyrimidoindole derivative, UM171, nicotinamide, NAM and more recently, valproic acid (VPA), have entered clinical development. HSC grafts expanded by SR1 and UM171 have been reported to establish rapid and sustained hematological reconstitution in allogeneic transplant recipients [1-3]. Our group has started a clinical trial using VPA-expanded allogeneic UCB grafts (NCT03885947). Initial data have shown multi-lineage donor cell reconstitution with especially rapid T cell and platelet engraftment in patients with treatment refractory acute leukemias. To gain insight into the mechanisms underlying these clinical HSC expansion procedures, we recreated the different approaches to compare the various expanded grafts. Although there is successful clinical data with the NAM approach, we were unable to recreate these conditions in our laboratory and therefore, we could not compare these grafts to those expanded with SR1, UM171 and VPA [4-7]. These comparative studies indicated that each strategy increased to varying degrees both the percentage and absolute numbers of CD34+CD133+CD90+EPCR+ as opposed to cultures treated with their respective cytokine cocktail alone. Although SR1 treatment resulted in the generation of greater numbers of total nucleated cells from purified 1x106 UCB-CD34+ cells (16x107 in SR1 vs 8x107 in UM171 and 7x107 in VPA), the numbers of CD34+CD133+CD90+EPCR+ cells (4x105 in SR1 vs, 7x105 in UM171 and 8.7x105 in VPA) as well as of other CD34+ subpopulations with different degrees of stem cell activity were lower than those observed in VPA and UM171-expanded cultures. In order to understand the underlying mechanism by which these agents act, we isolated CD34+CD90-EPCR- cells and treated with VPA, SR1 or UM171. VPA treatment for 24 hrs resulted in acquisition of CD90 and EPCR expression (80% and 25% of cells acquired CD90 and EPCR, respectively). UM171 treatment led to a modest increase in cells expressing EPCR and CD90. By contrast, SR1 treatment did not induce a phenotypic switch. Comparison of RNA-seq data indicated a strong induction of CD90, EPCR and CD133 in the CD34+ grafts expanded with VPA and UM171, but not in grafts generated with SR1. Twenty five out of 53 and 5 out of 9 genes upregulated more than 2 fold by UM171 and SR1, respectively, were also upregulated in VPA-expanded grafts. Eight out of 39 and 6 out of 77 genes downregulated more than 1.5 fold by UM171 and SR1, respectively, were also downregulated in VPA-expanded grafts. However, grafts expanded with each of these agents shared only three upregulated and three downregulated genes suggesting that different mechanisms underlie the actions of each of these agents. In fact, enhanced histone acetylation induced by VPA was not observed in SR1 and UM171 expanded cells. Both UM171 and VPA did not signal through the AHR pathway, which is required for SR1-mediated expansion. These data suggest that these agents target distinct pathways leading to ex vivo HSC expansion. Since mitochondrial activity is an additional critical regulator of the HSC fate, we examined the mitochondrial profile. VPA-expanded HSCs exhibited a remodeled mitochondrial network associated with low levels of mitochondrial ROS and mass. HSCs expanded with UM171 unlike SR1 displayed low ROS levels. Neither UM171 nor SR1 impacted mitochondrial mass. In vivo transplantation of UM171 and VPA expanded grafts in NSG mice showed similar degrees of human cell chimerism (~25%). Whereas both expanded grafts were capable of establishing multilineage hematopoiesis, we found a greater degree of CD34+ and T cell chimerism in NSG mice engrafted with VPA-expanded grafts. Studies of the engraftment potential of SR1 expanded grafts are ongoing. In conclusion, our findings suggest that although VPA, UM171 and SR1 expand greater numbers of human HSCs that are useful for allogeneic stem cell transplantation, their underlying mechanisms of action and pathways involved are different. Disclosures Schaniel: Summer Road: Research Funding. Hoffman:Merus: Research Funding.

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p Disclosures No relevant conflicts of interest to declare.

Introduction: Systemic sclerosis (SSc) is a chronic autoimmune disease with ongoing high disease related mortality. Three randomized controlled trials proofed efficacy of autologous stem cell transplantation (aHSCT) in patients with severe SSc and superiority over the standard cyclophosphamide pulse therapy. Treatment related mortality is a major point of criticism and different treatment regimen are used in different centers. Methods: This prospective, open, multi-center, non-interventional study of the European Group for Blood and Marrow Transplantation (EBMT) analyzed data for consecutive aHSCT performed in SSc. Every center followed its own local protocol which usually refers to the recent update of the EBMT Guidelines. In order to be eligible for inclusion, patients had to be transplanted for the diagnosis of progressive SSc and aged between 18 and 65 years. Major exclusion criteria were an ejection fraction 50mmHg, creatinine clearance Progression free survival (PFS) was chosen as primary endpoint. Response to treatment, defined as the number of patients who achieve >25% improvement of the skin thickness, measured by the modified Rodnan Skin score (mRSS; range 0-51) and/or ≥10% improvement in forced vital capacity (FVC) or hemoglobin-adjusted diffusion lung capacity for carbon monoxide (DLCO) and relapse rate as well as safety considerations were secondary endpoints. Results: Between 2013 and 2015, 80 patients from 12 centers were followed for a median of 24.1 (5.97 - 59.8) months. Patients had a median age of 43 years at aHSCT and 70% were female. All patients were mobilized with cyclophosphamide (CYC) with a median of 2g/m2 and granulocyte-colony stimulating factor (GCSF) in 98%. In 45% of transplants stem cells were purified using CD34-positive selection. For conditioning, centers used CYC at 200 mg/kg (50 - 240) in 76 patients (95%) or CYC at 100 mg/kg and thiotepa 10 mg/kg in 4 patients. All patients received additional anti-thymocyte globulin (ATG; different brands were used) over 4-5 days. Progression free survival at 1 year was reached by 83.6% of our patients. Response to treatment at 1 year was achieved by 75% (63.7 - 83.2) and 10.1% relapsed or had further progression despite aHSCT. Figure 1 demonstrates the improvement of skin fibrosis over time. Median mRSS decreased from 24 (2 - 49) at baseline to 13 (0 - 40) at month 12, and 10.5 (0 - 32) at month 24. On multivariate analysis, creatine kinase (CK) levels < 250 U/ml, mRSS The 100-day treatment-related mortality for this population was 6.2% (n=5) (CI 2.3 - 13). Three of these 5 patients had a cardiac or cyclophosphamide related event that led to death. During the 24 months of follow up another 3 patients died due to progressive disease, which leads to an overall survival of 91.2% (CI: 85.1 - 97.4) at 2 years. Discussion: Although aHSCT has been proven effective in SSc, we still do not know which regimen is the best. With data from this non-interventional trial we could demonstrate the effectiveness of aHCST in a daily practice setting. Treatment seems to become safer over time with reduction of TRM from 10% in the European phase III Autologous stem cell transplantation international scleroderma trial (ASTIS; JAMA. 2014; 311(24): 2490-8) to 6.2% in our actual study; mainly due to increasing experience of the centers and exclusion of patients with advanced organ damage. Nevertheless indication for aHSCT should also be driven by negative predictive parameters for disease related mortality and it is fairly unlikely to further reduce TRM in these sick patients. We also found a potential value of CD34-selection in these patients. As a retrospective approach demonstrated no benefit to the outcome of SSc patient treated with AHSCT (Bone Marrow Transplant.2016; 51(4): 501-5), these findings have to be confirmed by prospective randomized trials. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Extracellular vesicles (EVs) including exosomes and microvesicles, have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that pretreated irradiated murine bone marrow stem cells with human or murine MSC-EV in vitro, could significantly improve the engraftment capacity of radiation-damaged stem cells up to 9 months post-transplantation. Interestingly, the restoration of engraftment was not significantly observed within the first month of post-transplant, the predominant reversal effect occurred on later period of post-transplant from 3 months up to 9 months. This is indicating a long-term effect of MSC-EVs on reversal of radiation damage of stem cell engraftment capacity. To confirm this hypothesis, in our current study, the effect of human MSC-EVs on reversal of engraftment capacity of bone marrow stem cells post-radiation was investigated by an in vivo study. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-EVs or vehicle were then injected intravenously 24, 48 and 72 hours after irradiation. The whole bone marrow were harvested at 6, 12, 26 and 53 weeks post EV-injection and then transplanted into 950 cGy exposed B6.SJL mice and engraftment evaluated at 1 and 3 months post-transplantation. In those transplanted mice at 6 weeks post-EV injection, there was slight increase in the restoration of engraftment rate (the percent of irradiated mice with EV/Vehicle treatment engraftment rate compared to healthy non-irradiated mice engraftment rate) in EV treated mice (17.58±2.32% compared to untreated mice (13.80±1.41%) after 1 month post-transplantation. However, for those mice transplanted at 12, 26, and 53 weeks post-EV injection, there were the significant higher restorations of engraftment rate in EV treated mice (40.48±6.03%, 33.93±3.76%, and 56.62±3.635) compared to untreated mice (12.39±1.30%, 15.14±2.21%, 36.21±3.63%) after 1 month transplantation respectively. The similar restorations of engraftment were also seen in 3 months post-transplantation. Our study also showed that there was a significant inhibition of stem cell engraftment at 53 weeks post 500cGy whole body radiation mice which was 36.21±3.63% of engraftment rate from healthy mice. Thus our data suggest that there is a long-term effect of MSC-EVs on the restoration of engraftment of stem cells in radiation-exposed mice. Disclosures No relevant conflicts of interest to declare.

In B lymphocytes, induction of apoptosis or programmed cell death (PCD) by Fas (CD95/APO-1) is suppressed by the triggering of CD40. This suppression controls various aspects of the humoral immune response, including antibody affinity maturation. The opposing effects of these receptors are also crucial to B-cell homeostasis, autoimmune disease, and cancer. Cytoprotection by CD40 involves activation of protective genes mediated by NF-κB transcription factors; however, its basis remains poorly understood. Here, we report that, in B cells, Gadd45β is induced by CD40 through a mechanism that requires NF-κB and that this induction suppresses Fas-mediated killing. Importantly, up-regulation of Gadd45β by CD40 precedes Fas-induced caspase activation, as well as up-regulation of other NF-κB–controlled inhibitors of apoptosis such as Bcl-xL and c-FLIPL. In the presence of Gadd45β, the Fas-induced apoptotic cascade is halted at mitochondria. However, in contrast to Bcl-xL, Gadd45β is unable to hamper the “intrinsic” pathway for apoptosis and in fact appears to block Fas cytotoxicity herein by suppressing a mitochondria-targeting mechanism activated by this receptor. These findings identify Gadd45β as a critical mediator of the prosurvival response to CD40 stimulation and provide important new insights into the apoptotic mechanism that is triggered by Fas in B cells.

Adult hematopoietic stem cells (HSCs) provide an attractive target for gene modification strategies for the correction of genetic blood disorders. A significant barrier to the translation of these strategies to the clinic has been the decline in numbers of fully functional HSCs associated with ex vivo culture. In order to create optimal conditions for gene modification, HSCs are typically cultured with cytokines to trigger their proliferation. Cytokine stimulation, however, induces stem cell differentiation, resulting in loss of long-term marrow repopulating potential. To overcome this barrier, we treated two sources of adult HSCs, G-CSF mobilized peripheral blood (mPB) and bone marrow (BM) CD34+ cells with valproic acid (VPA), a histone deacetylase inhibitor. This approach has previously been shown to promote the expansion of fully functional cord blood HSCs (Chaurasia et al. JCI, 2014). Cryopreserved mPB or BM CD34+ cells were thawed and cultured in serum-free media supplemented with stem cell factor, FLT-3 ligand, thrombopoietin and interleukin 3. The cells were primed with cytokines for 16 hours followed by addition of VPA (1mM) and incubation in the presence of VPA and cytokines for 7 days. The viability, number of total nucleated cells (TNC), CD34+ cells and frequency of various HSC subpopulations were determined shortly after thawing the primary cells (PCs) and at daily time points, in both VPA-treated cultures and cultures containing cytokines alone (control). The viability of mPB and BM cells treated with VPA for 7 days, each exceeded 90%. While not significantly affecting the expansion of TNC, VPA treatment maintained a higher percentage of CD34+ cells throughout the culture period, and led to a higher expansion of CD34+ cells compared to that observed in control cultures (29 vs 5.5-fold in mPB and 21 vs 5-fold in BM, p Since the phenotype of cells expanded ex vivo does not always correlate with function, we used aldehyde dehydrogenase (ALDH) activity as a functional marker of HSCs. VPA-expanded mPB CD34+ cells showed significantly higher ALDH activity compared to PCs and controls, after both 3 and 7 days of incubation (Table 1). We have previously shown that epigenetic reprogramming induced by VPA is linked to mitochondrial remodeling and suppression of ROS (Papa et al. ASH 2016). We used the mitochondrial membrane potential dye TMRM to assess mitochondrial activity, and found a significant decrease in TMRM in VPA-treated cultures from mPB, as compared to control cultures, at 3 and 7 days (Table 1). This reduction in mitochondrial membrane potential is associated with primitive HSCs. To test their functional capacity, we transplanted the progeny of 2X105 mPB and BM CD34+ cells cultured with or without VPA, into sub lethally irradiated NSG mice. The degree of human cell chimerism in mouse bone marrows was determined 16 weeks later by human CD45 expression. While mice transplanted with grafts from the control cultures had limited or no human chimerism (range 0-1.4%), those transplanted with VPA-treated mPB grafts had up to 50% human chimerism (range 2-50%) (Table 1). The engrafted cells belonged to both myeloid and lymphoid lineages. Comparable results were observed using BM. Several groups have demonstrated the application of expansion methods adapted from cord blood to adult HSCs. These efforts met with successful albeit modest results, perhaps owing to a limited effect of the evaluated agents on adultHSCs. To our knowledge, the above studies represent the most robust demonstration of expansion of HSCs from adult sources, evident as early as after 3 days of culture. These studies strongly support the evaluation of VPA treatment in HSC gene modification strategies. Disclosures No relevant conflicts of interest to declare.

Previous studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

The safety and efficacy of filgrastim as an adjunct to acute myeloid leukemia (AML) induction and consolidation therapy was assessed in this prospective double-blind, randomized, placebo-controlled, multicenter trial. A total of 521 consecutive de novo AML patients aged 16 or more years were randomized to receive filgrastim (5 μg/kg/d subcutaneously) or placebo after standard induction as well as consolidation chemotherapy. Blinded study drug was given from 24 hours after chemotherapy until the absolute neutrophil count was ≥1.0 × 109/L for 3 consecutive days. The overall complete remission rate was 68%. After a median follow-up of 24 months (range 5 to 40) the median disease-free survival was 10 months (95% confidence interval [CI], 8.7 to 10.8) and the median overall survival was 13 months (95%CI, 12.2 to 14.6). These did not differ between treatment groups. Patients receiving filgrastim experienced neutrophil recovery 5 days earlier after induction 1 than those receiving placebo (P < .0001). This was accompanied by reductions in the duration of fever (7 v 8.5 days; P = .009), parenteral antibiotic use (15 v 18.5 days; P = .0001), and hospitalization (20 v 25 days; P = .0001). Similar reductions were seen after induction 2 and the consolidation courses. There was a significant reduction in the number of patients requiring systemic antifungal therapy in the filgrastim group during induction treatment (34% v 43%; P = .04). In conclusion, filgrastim is safe in that it had no negative impact on the prognosis of the AML patients. In addition, it effectively reduced the duration of neutropenia, leading to significant clinical benefits by reducing the duration of fever; requirement for parenteral anti-infectives, specifically amphotericin B; and the duration of hospitalization.

Introduction: malaria is one of the most frequent hematological diseases worldwide. Because the malaria parasite Plasmodium falciparum develops mainly in red blood cells (RBC), splenic retention of infected and uninfected RBC is likely a key player in the variable susceptibility of humans to malaria. Age and ethnicity are important determinants of the manifestations of malaria in Africa (Reyburn JAMA 2005, Dolo Am J Trop Med Hyg 2005; Greenwood Ann Trop Med Parasitol 1987; Torcia PNAS 2008), Asia (Price Am J Trop Med Hyg 2001), and in travelers (Seringe Emerg Infect Dis 2011). We had speculated that variations in the splenic sensing of RBC contribute to the innate protection/susceptibility of infants against distinct forms of severe malaria and to the pathogenesis of chronic malaria (Buffet Curr Opin Hematol 2009; Buffet Blood 2011). Here, we explore the deformability and morphology of circulating RBC in populations living in a malaria-endemic area. Materials and methods: experiments were embedded in an integrated study driven by Institut de Recherche pour le Développement, which aims at the identification of genetic, epidemiologic and anthropologic determinants of susceptibility to malaria. IRB approval was obtained from Institut des Sciences Biomédicales Appliquées, Benin. Clinical and biological data were collected at the beginning of the rainy season from 627 individuals, belonging to 4 different ethnic groups living in sympatry in Atakora, North Benin and included age, gender, ethnicity, body temperature, presence and grade of splenomegaly, rapid diagnostic test for malaria (RDT), thick film and rapid hemoglobin determination with HemoCue©. Venous blood was collected for determination of RBC morphology and deformability. Using microsphiltration, a RBC filtering method that uses microsphere layers to mimic the mechanical retention of RBC in the splenic red pulp (Deplaine Blood 2011) we quantified the ability of a mix of labeled and non-labeled RBCs to squeeze between calibrated slits, results being expressed as retention or enrichment rates (RER) of subject's RBC compared to normal RBC (from a single French O-positive donor) stored in blood bank conditions. Microsphiltration has been adapted to high-throughput experimentation using microplates (Duez AAC 2015). Experiments were performed in a field laboratory established on site; microplates were prepared in Paris and brought to North Benin in luggage with constant care to avoid shocks during transportation. All RBC samples were filtered in triplicate less than 8 hours after blood collection. Up- and downstream samples were brought back to France at 4°C in sealed micro-well plates and analyzed for individual RER calculation in the next 2 weeks by flow cytometry. Results: over 10 days, 262 adults and 249 children were included, 31% Bariba, 17% Gando (genetically related to Bariba), 24% Otamari, 27% Peulhs. Prevalences of splenomegaly, positive RDT, and fever were 13%, 27%, and 2%, respectively. Of 629 blood samples collected, 511 could be analyzed. RER of controls remained stable with time and across 17 microfiltering plates, with a median (IQR) retention rate of 12% (5% - 21%). Ethnicity and age were the only two factors associated with statistically significant differences in RER (figures 1 and 2). Infants (less than 2 year-old) had a more important enrichment than older children and adults (median in 2 years old or less 287%; 3 to 5 years 103%; 6 to 10 years: 64%; more than ten years: 91%; p=0.0161). Peulhs and Otamari also had higher median enrichment rates than Bariba and Gando (RER: 122% and 118%, 75%, 64%, respectively; p=0.0246). Conversely, splenomegaly, gender, positivity of RDT or anemia at the time of sampling were not associated with RER. Discussion: higher averaged enrichment rates in specific ethnic subgroups, namely Peulhs and Otamari, likely result from a more stringent splenic retention, leaving more deformable RBC in circulation. An innate spleen-RBC interaction process was also observed in infants, which is consistent with the higher incidence of severe malarial anemia and splenomegaly observed in this population (Reyburn JAMA 2005; Price Am J Trop Med Hyg 2001). Our results show that innate factors (e.g. ethnicity and age) tend to influence the deformability of RBC, and therefore the phenotypic expression of malaria in Africa. Ongoing experiments aim at deciphering the mechanisms responsible for these differences. Disclosures No relevant conflicts of interest to declare.

Umbical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs) for allogeneic transplantation of patients suffering from hematological malignancies and genetic disorders. Its use, however, as an HSC graft for adults has been restricted due to the limited numbers of HSCs within a single UCB unit. We have previously reported that UCB CD34+ cell numbers can be expanded over a seven-day period of incubation by ex vivo treatment with a cytokine combination and the histone deacetylase inhibitor, valproic acid (VPA) (Chaurasia et al. J Clin Invest. 2014:124(6): 2378-2395). In this study, we addressed the mechanism underling this ex-vivo expansion of UCB-derived CD34+cells. We found that VPA triggered two distinct phases of HSC behavior. The first phase (days 1-4) was characterized by a prompt elevation of the expression of stem cell phenotypic markers (CD90 and CD49f), which was associated with acetylation of histone H3, and expression of pluripotency genes including OCT4, NANOG, and SOX2. During this period, the cells underwent a limited number of cell divisions. The second phase (days 4-7) was characterized by a greater number of cell divisions that resulted in a drastic increase in the absolute number of phenotypically defined HSCs. Both acetylation of H3 and expression of the pluripotency genes were transient events since they were no longer evident during the second phase. During the entire incubation period, VPA-induced HSC cells maintained a distinct mitochondrial metabolic profile characterized by low mitochondrial potential, ROS levels and mass. Remarkably, the p38 activity, a downstream target of ROS, was also suppressed in VPA-expanded HSCs. Given that both ROS and p38 activity lead to exhaustion and limit the lifespan of the pool of HSCs, our findings suggest that the VPA-expanded HSCs are equipped with mechanisms that monitor and ensure their fitness.In addition, removal of VPA from the culture after 24, 48 and 72hrs of incubation reversed the acetylation of H3 and the expression of the pluripotency genes. These events were further accompanied by the rapid loss of stem cell marker expression (24hrs after removal) leading to decreased HSC numbers. Importantly, upon VPA removal, the expanded cells exhibited an increase in mitochondrial ROS, mass and potential suggesting that the epigenetic reprograming and mitochondrial remodeling triggered by VPA were linked and were both reversible events. Both transcript and protein levels of p53 were also increased during the first phase but not during the second phase of the ex-vivo expansion of CD34+cell. Importantly, removal of VPA diminished the up-regulation of p53 during the first phase. We further assessed the role of p53 in VPA stem cell expansion with pifithrin-α, a small molecule inhibitor of p53 function. P53 inhibition led to a significant elevation of the mitochondrial ROS without affecting cellular viability. Moreover, treatment with pifithrin-α reduced both the percentage and the absolute number of HSCs induced by VPA. Conversely, co-treatment with VPA and a low concentration of nutlin (1μM), an inhibitor of p53 degradation decreased further mitochondrial ROS levels and augmented the percentage of HSCs induced by VPA. By contrast, treatment with higher concentrations of nutlin (10μM) led to HSC cell death. These findings raise the possibility that VPA induces a moderate up-regulation of p53 as a stress-response mechanism to cope with oxidative stress. It seems plausible that p53 along with alterations in mitochondrial mass and potential are among the mechanisms required to repress oxidative stress and thus, are implicated in the HSC expansion by VPA. Collectively, our data establishes that VPA expands ex-vivo the pool of HSCs from CD34+ CB cells by triggering epigenetic modifications that are tightly linked to mitochondrial remodeling. Notably, these events are reversible and do not result in the malignant transformation of the CD34+ cells. Moreover, these data indicate that VPA modulates and maintains a critical threshold of p53 that is required for HSCs expansion but is not sufficient to trigger the exhaustion and death of HSCs. In this context, VPA also represses p38 activity that if up-regulated can limit the lifespan of HSCs. These studies demonstrate that effective ex vivo HSC expansion requires the coordination and retention of a number of critical biological events that define an HSC Disclosures No relevant conflicts of interest to declare.

The storage lesion encompasses a series of biochemical and molecular modifications that alter erythrocytes during hypothermic storage, reducing transfusion yield. Indeed, in humans, up to 25% of transfused erythrocytes are cleared from the circulation in a few hours (Luten et al., 2008). The mechanisms underlying this rapid clearance are not fully elucidated but it is reasonable to assume that these erythrocyte alterations are sensed by the spleen, resulting in retention. Among those, membrane shedding may have a major impact on post-transfusion clearance of erythrocytes since it causes a progressive decrease in the surface-volume ratio of the cell, leading to the loss of its flexible biconcave shape. The proportion of "damaged" erythrocytes cleared in the hours following transfusion increases with the duration of storage while the deformability measured by ektacytometry (Frank et al., 2013) progressively decreases during this period. To characterize the morphological alteration of stored erythrocytes, we used imaging flow cytometry (Imagestream X Mark II, AMNIS°). This technology allows a simultaneous high-speed multispectral imaging of cells in brightfield, darkfield, and 9 channels of fluorescence. It combines the ability of conventional flow cytometry to analyze a very high number of events with a powerful exploration of cell morphology. We analyzed the morphological, biochemical, metabolic, and bio-mechanical characteristics of erythrocytes stored in optimal blood bank conditions for 6 donors, at Day 3, 21, 28, 35 and 42 of the storage period. This longitudinal study of parameters such as mean corpuscular volume, intracellular ATP level, hemolysis, osmotic fragility, deformability and the plasma levels of ions and metabolites has highlighted a great inter-donor variability in the storage lesion process. Moreover, Imagestream analysis of front views of sharp, single cells revealed a subpopulation of small erythrocytes. The "projected surface area" distribution on normalized frequency plots was bimodal in 5 of 6 concentrates, showing a well-demarcated subpopulation of less than 62 µm2. The proportion of this sub-population increased with storage from 0.5-3.4% at D3 to 4-23.5% at D42 (p These morphological alterations have been considered irreversible (Berezina et al., 2002) and are reminiscent of those associated with mechanical clearance of erythrocytes in the spleen of patients with hereditary spherocytosis (Mohandas et al., 2008). We hypothesize that these storage-induced small erythrocytes correspond to the subpopulation of "damaged" erythrocytes that are rapidly cleared after transfusion. Confirmation of these findings using ex-vivo perfusion of human spleens and observational studies in transfused patients would support the use of imaging flow cytometry to predict transfusion yield. Disclosures No relevant conflicts of interest to declare.