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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.

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.

Background Hydroxyurea (HU), a chemotherapeutic agent and an approved therapy for sickle cell disease (SCD), increases cGMP levels and thereby increases fetal hemoglobin (HbF) levels in RBC, which generally correlates with decreased hemolysis and reduced disease severity in subjects with SCD. In addition, increased cGMP levels are anti-inflammatory and reduce the adhesion of WBC to the vascular endothelium. However, potential adverse effects of HU such as infertility, susceptibility to infections, or teratogenic effect have been the subject of concerns. Phosphodiesterase-9 inhibitors (PDE9i) may provide an alternative to HU because of their ability to inhibit the degradation of cGMP and increase cellular cGMP levels. Previously described PDE9i were developed to inhibit neuronal PDE9 for neurologic diseases, like Alzheimer's. A PDE9i with low brain penetrance may be preferable for the chronic treatment of SCD adults and children. IMR-687 is a safe, potent, PDE9i being developed specifically for SCD without these liabilities of HU or the high brain penetrance of other PDE9is. Hypothesis We hypothesized that PDE9i's will increase cellular cGMP levels in hematopoietic cells, increase HbF in red blood cells (RBC), inhibit RBC sickling, decrease inflammation (leukocytosis), and inhibit vaso-occlusion in murine models of SCD. Results & Methods IMR-687, is a potent inhibitor of PDE9A (IC50 100-fold higher IC50 with other phosphodiesterases. To determine the ability of IMR-687 to induce cGMP, K562 erythroid cells were exposed to increasing concentrations of IMR-687 or HU and cGMP assessed at 6 hours. IMR-687, at much lower drug concentrations, induced higher levels of cGMP than HU at 6 hours (Fig. Left Panel). We next tested IMR-687 in the Berkeley (Berk) mouse model of SCD. Groups of 7-8 Berk SCD mice were dosed by gavage with either vehicle, 30 mg/kg/day of IMR-687, or 100 mg/kg/day of HU. After 30 days of treatment, both IMR-687 and HU resulted in statistically significant decreases in the percentage of sickled RBCs (Fig. Center Left Panel) and increases in the percentage of HbF positive RBCs relative to controls. In addition, both compounds led to statistically significant decreases in total bilirubin, spleen weight and total leucocyte counts (Fig. Center Right Panel). The ability of IMR-687 to reduce microvascular stasis (% non-flowing venules) was assessed in HbSS-Townes transgenic sickle mice after transient hypoxia and re-oxygenation. Groups of 3 mice/group were dosed orally via drinking water with IMR-687 (30 mg/kg/day), HU (100 mg/kg/day) or vehicle for 10 days. After 10 days of treatment, the mice were exposed to transient hypoxia. Compared with controls, 30 mg/kg/day IMR-687 and 100 mg/kg HU produced statistically significant reductions in microvascular stasis at both 1-hour (Fig. Center Right Panel) and 4-hour time points post-hypoxia. In addition, IMR-687 significantly reduced the proportion of sickled RBCs, increased the number of HbF positive red cells, and reduced the white count. Lastly, low brain penetration of IMR-687 was confirmed after IV dosing in the rat, with brain concentrations >20 times lower than those in the plasma at all time points assessed. Consistent with this, treatment of C57Bl/6J mice with 10 mg/kg/day IMR-687 for 5 days displayed no effect on locomotor activity or classical fear conditioning (an animal model of learning and memory). In contrast, treatment with a brain penetrant PDE9i, developed for the treatment of neurologic diseases, significantly increased conditioned fear responses in these mice. Conclusions IMR-687 increases HbF levels, and reduces red cell sickling, leukocytosis and microvascular stasis, without the observed toxicities of HU. IMR-687 may offer a once a day, oral, safe replacement for HU in the treatment of SCD. Figure Figure. Disclosures McArthur: Imara: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patent; Cydan Development: Employment, Equity Ownership. Maciel:Imara: Research Funding. Chen:Imara: Research Funding. Fricot:Imara: Research Funding. Kobayashi:Imara: Equity Ownership; Cydan Development: Employment. Nguyen:Imara: Research Funding. Parachova:Imara: Patents & Royalties. Abdulla:Imara: Research Funding. Vercellotti:Imara: Research Funding. Svenstrup:Imara: Consultancy, Patents & Royalties. Belcher:Imara: Research Funding; CSL-Behring: Research Funding.

Abstract 247 Background: β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods: Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results: RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary: Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures: Daniel: Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.