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Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy, with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin (COO), genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of “unfavorable” molecular signatures.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), and primary human acute myelogenous leukemia (AML) and lymphoblastic leukemia (ALL) cells. Here we show that human leukemia addicts to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG) and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids (BCKAs), whose metabolic processes provide leukemia cells with critical substrates for the TCA cycle and the non-essential amino acids synthesis, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates polycomb repressive complex 2 (PRC2) function, an epigenetic regulator for stem cell signatures, through inhibiting transcription of PRC components, such as zeste homolog 2 (EZH2) and embryonic ectoderm development (EED). Accordingly, BCAA catabolism plays an important role in maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

The developmental pathway for human megakaryocytes remains unclear, and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem- and progenitor-cell populations that specifically expresses genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34+CD38+IL-3RαdimCD45RA- common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte-macrophage potential but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro. The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41+ CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.

Myelodysplastic syndromes (MDS) is a clonal disorder of hematopoietic stem cells (HSCs) characterized by clonal hematopoietic stem cells (HSCs) with cytopenia, morphological abnormalities, genetic alteration, ineffective normal hematopoiesis, and frequent progression to AML. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually come to dominate the bone marrow space. Despite several studies of mesenchymal stem cells (MSCs), one of the principal components of HSC niche supporting normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. In this study, we examined the mechanism by which less-proliferative MDS cells outcompete normal hematopoiesis through the effects on MSCs using serially transplantable Abcg2-induced MDS/AML model we recently generated. The recipient-derived normal BM cells displayed a considerably lower colony output with markedly decreased numbers of the hematopoietic stem progenitor cells (HSPCs) . However, there were no direct effects on the colony-forming ability of the recipient HSPCs co-cultured with MDS/AML cells, indicating that MDS/AML cells inhibited hematopoiesis through alteration of bone marrow microenvironment, such as MSCs, rather than direct interaction between normal and malignant HSCs. We next analyzed histological features of BM specimens. Interestingly, bone sections from the MDS/AML mice showed a reduced trabecular bone and narrowed growth plates. Moreover, micro computed tomography (micro-CT) analysis of the femora showed a significant reduction of the trabecular bone volume in the recipient mice transplanted with the MDS/AML BM cells. We detected decreased bone formation based on the calcein double labeling, but unchanged numbers of the TRAP-positive mononuclear or multinucleated (osteoclastic) cells in the MDS/AML samples, suggesting that the reduced bone volume was caused by suppressed bone formation. The impaired bone formation was also observed in the human MDS patients in terms of lower bone volume and decreased expression of BGLAP, one of osteogenic markers. In line with the above findings, single cell qRT-PCR analyses of mouse MSCs displayed downregulation of a line of osteolineage markers, indicating that MDS/AML cells suppress bone formation through inhibiting osteolineage differentiation of MSCs. Based on the findings, we next examined if re-induction of osteolineage differentiation of the MDS/AML-derived MSCs could rescue the potential of MSCs to support normal hematopoiesis. Importantly, the number of colony-forming cells (CFCs) was significantly restored by inducing differentiation of MDS/AML-derived MSCs toward osteogenic lineage both in vitro and in vivo. These results indicate that the impairment of osteolineage differentiation is the principal cause for an impaired normal hematopoiesis in MDS/AML, and that restoring the supportive niche will be a potential therapeutic option. Since extracellular vesicles (EVs) derived from MDS/AML cells are critical mediators of intercellular communication, we examined the molecular mechanism underlying the dysfunction of MSCs via EVs. As expected, EVs from MDS/AML cells were incorporated into the normal MSCs where osteolineage marker genes were clearly downregulated, and the number of CFCs significantly decreased in the HSPCs co-cultured with MSCs treated by the MDS/AML-derived EVs. Moreover, by comprehensively analyzing microRNAs (miRNAs) enriched in EVs derived from MDS/AML cells, we identified several miRNAs that impaired the differentiation of normal MSCs. These results suggested that miRNAs in EVs derived from MDS/AML cells disrupted the hematopoietic supporting niche through suppressing an osteolineage differentiation of MSCs. Here we uncover a heretofore unrecognized mechanism of bone marrow failure in MDS via the impairment of osteolineage differentiation in MSCs. EVs from MDS cells will be an attractive therapeutic target to restore the supportive niches, such as MSCs, for the remaining normal HSCs. Figure Disclosures No relevant conflicts of interest to declare.

Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and widely performed in clinical practice. Platelet loss is the major complication of PBSC apheresis, and platelet-rich plasma (PRP) return is recommended in case of severe platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss nor the efficacy of PRP return post-apheresis. To address these questions, we assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated efficacy of PRP transfusion on platelet recovery post-apheresis. Platelet counts reduced up to 70% post-apheresis in both allo- and auto-PBSC settings, while severe platelet count decrease (< 50 x 109/L) was only observed in auto-PBSC patients (Figure 1). We next analyzed the relationship between severe platelet (< 50 x 109/L) after apheresis and several clinical factors by using univariate and multivariate analysis for auto-PBSC patients. As shown in Table 1, in univariate analysis, severe platelet counts following auto-PBSC apheresis was found more frequently in patients with lower platelet count, lower percentage of CD34+ cells in PB at pre-apheresis, repeated round of apheresis, and smaller number of collected CD34+ cells. On the other hand, in multivariate analysis, the white blood cell (WBC) counts pre-apheresis was the only significant risk factor of severe platelet count following apheresis (p = 0.038). We finally analyzed the transitions of platelet counts in the setting of apheresis. The median platelet counts at pre-apheresis, post-apheresis, and post-PRP return were 187.0 x 109/L, 132.0 x 109/L, and 154.0 x 109/L for allo-PBSC apheresis, and 147.0 x 109/L, 111.0 x 109/L, and 127.0 x 109/L for auto-PBSC apheresis (p < 0.0001 for all, allo-PBSC donors and auto-PBSC patients, respectively) (Figure 2), indicating that PRP return post-apheresis facilitated a rapid platelet recovery in both allo- and auto-settings. Collectively, our data suggest that WBC counts pre-apheresis is a useful predictor for severe platelet decrease following auto-PBSC apheresis and that PRP return is an effective mean to facilitate platelet recovery post-apheresis. Disclosures No relevant conflicts of interest to declare.

Clearance of apoptotic cells by macrophages is considered important for prevention of inflammatory responses leading to tissue damage. The phosphatidylserine receptor (PSR), which specifically binds to phosphatidylserine (PS) exposed on the surface of apoptotic cells, mediates uptake of apoptotic cells in vitro, yet the physiologic relevance of PSR remains unknown. This issue was addressed by generating PSR-deficient (PSR-/-) mice. PSR-/- mice exhibited severe anemia and died during the perinatal period. In the PSR-/- fetal livers, erythroid differentiation was blocked at an early erythroblast stage. In addition, PSR-/- embryos exhibited thymus atrophy owing to a developmental defect of T-lymphoid cells. Clearance of apoptotic cells by macrophages was impaired in both liver and thymus of PSR-/- embryos. However, this did not induce up-regulation of inflammatory cytokines. These results indicate that during embryonic development, PSR-mediated apoptotic cell uptake is required for definitive erythropoiesis and T lymphopoiesis, independently of the prevention of inflammatory responses. (Blood. 2004;103:3362-3364)

The mechanisms mediating hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF are complex. We have found previously that G-CSF–enforced mobilization is controlled by peripheral sympathetic nerves via norepinephrine (NE) signaling. In the present study, we show that G-CSF likely alters sympathetic tone directly and that methods to increase adrenergic activity in the BM microenvironment enhance progenitor mobilization. Peripheral sympathetic nerve neurons express the G-CSF receptor and ex vivo stimulation of peripheral sympathetic nerve neurons with G-CSF reduced NE reuptake significantly, suggesting that G-CSF potentiates the sympathetic tone by increasing NE availability. Based on these data, we investigated the NE reuptake inhibitor desipramine in HSPC mobilization. Whereas desipramine did not by itself elicit circulating HSPCs, it increased G-CSF–triggered mobilization efficiency significantly and rescued mobilization in a model mimicking “poor mobilizers.” Therefore, these data suggest that blockade of NE reuptake may be a novel therapeutic target to increase stem cell yield in patients.

Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood types in response to various demands through life. HSC functions are tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow (BM). Using Nestin-GFP transgenic mice, we have identified Nestin-GFP+ perivascular stromal cells exhibiting a mesenchymal stem/progenitor cell activity as niche cells. Furthermore, we found two types of Nestin-GFP+ cells expressing different surface markers, Nerve/glial antigen 2 (NG2) and Leptin receptor (Lepr) that are associated with arterioles and sinusoid, respectively, in the BM (Kunisaki et al. Nature, 2013). Both arteriolar and sinusoidal niche cells have been reported to show high gene expression of cytokines essential for HSC maintenance such as CXCL12 and stem cell factor (SCF), however, it remains unknown how the distinct niche cells differentially regulate HSC functions. To investigate the mechanisms, we utilized genetic mouse models, in which CXCL12 or SCF can be deleted in specific cell types. CXCL12 deletion in sinusoidal niche cells by using Lepr-cre/Cxcl12fl/− mice mobilized HSCs and lineage− Sca-1+ c-kit+ (LSK) progenitors into spleen (HSC, CT: Control/DL: Deleted: 760±165 / 2193±557 / spleen, n=6, p Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is a genetic blood disorder characterized by repeated episodes of vaso-occlusion, in which neutrophils play a primary function through their recruitment in inflamed venules and interactions with sickle red blood cells (sRBCs) via activated αMβ2 integrin (Mac-1). Intravital microscopy analyses revealed considerable heterogeneity in Mac-1 activation on adherent neutrophils, suggesting that subsets of neutrophils differ markedly in their pro-inflammatory activity (Nat Med 2009; 15:384). Recently, we found that aged neutrophils, marked by CD62Llo CXCR4hi, represented an overly active subset exhibiting enhanced Mac-1 activation (Blood 2013; 122:324). To further characterize aged neutrophils, we compared their transcriptome with control and in vivo- activated neutrophils. Gene set enrichment analyses revealed that aged neutrophils up-regulated several pathways that were also enhanced during activation, including integrin, leukocyte adhesion, toll-like receptor (TLR), and NFκB signalling pathways (P < 0.05), suggesting a contribution by exogenous inflammatory mediators. Based on the expression profile, we hypothesized that microbiota-derived signals may influence neutrophil aging in the circulation. To test this idea, we analyzed aged neutrophil numbers in antibiotics (ABX)-treated and germ-free (GF) mice, in which the gut microbiota was largely depleted or deficient, respectively. We found significant reductions of aged neutrophil numbers in both models, and the numbers could be significantly restored by either lipopolysaccharide (LPS) gavage or fecal transplantation (Ctrl / ABX / ABX+LPS: 98±14 / 36±11 / 107±25 cells per μl blood; SPF / GF / GF-FT: 56±12 / 4±1 / 15±4 cells per μl blood; P < 0.05). We next investigated whether microbiota-driven neutrophil aging was mediated by TLRs and Myd88 signalling using chimeric mice reconstituted with a mixture of WT and Myd88, TLR4 or TLR2-deficient hematopoietic cells. Interestingly, we observed significant reductions in the aged subset of Myd88, TLR4 or TLR2-deficient neutrophils compared to WT neutrophils in the same mice, and in their capacity to capture fluosphere beads that specifically bound to activated Mac-1 (WT / LysM-cre/Myd88-flox: 10.4±0.3 / 7.0±0.6 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr4-/-: 10.3±0.2 / 6.0±0.2 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr2-/-: 10.0±0.4 / 6.8±0.5 %, 1.3±0.2 / 0.8±0.1 bead per cell; P < 0.01). Analysis of SCD mice (BERK) revealed that the aged neutrophil population was significantly increased (by >10-fold) compared to hemizygous (SA) mice, and the expansion was completely abrogated by microbiota depletion (SA / SCD (SS)-Ctrl / SS-ABX: 71±11 / 981±261 / 124±27 cells per μl blood, P < 0.01). We then challenged SA, untreated and ABX-treated SCD mice with TNF-α to induce acute vaso-occlusion. SCD mice exhibited significant increases in neutrophil adhesion, Mac-1 integrin activation and heterotypic interactions with RBCs compared to SA mice, all of which were markedly reduced by microbiota depletion (SA / SS-Ctrl / SS-ABX: 6.4±0.6 / 18.1±1.0 / 11.2±1.0 adherent neutrophil per vessel; 0.5±0.1 / 1.0±0.2 / 0.3±0.1 bead per cell; 0.1±0.1 / 0.5±0.1 / 0.2±0.1 RBC interaction per vessel per min; P < 0.05), resulting in significantly enhanced blood flow and prolonged survival in ABX-treated SCD mice (P < 0.05). Most interestingly, the splenomegaly of SCD mice was significantly reduced, and liver damage including fibrosis, necrosis and inflammation was dramatically alleviated in ABX-treated SCD mice (P < 0.05). Finally, we evaluated whether the numbers of circulating aged neutrophils were altered in patients with SCD. As Penicillin V antibiotic prophylaxis therapy is recommended for children < 5 years or older patients with immune defects to prevent life-threatening infections, we determined aged neutrophil numbers in this patient population. We found that SCD patients exhibited a marked increase in the numbers of circulating aged neutrophils compared to healthy controls, which were significantly reduced in patients on Penicillin V prophylaxis (Ctrl / SS / SS-PV: 261±40 / 1175±205 / 439±87 cells per μl blood, P < 0.05). Our results raise the possibility that targeting neutrophil aging and the microbiota may have broad implications in our understanding of sickle cell disease pathogenesis and its management that should be further studied in clinical trials. Disclosures No relevant conflicts of interest to declare.

Immunodeficient mice are used to reconstitute human hematopoiesis and analyze human hematopoietic function in vivo by xenotransplantation of hematopoietic stem cells (HSCs). It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD is critical for efficient human cell engraftment in xenotransplantation models. We have reported that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows the mouse SIRPA to bind human CD47, preventing activation of recipient macrophages to engulf human hematopoietic cells (Takenaka et al., Nature Immunol., 2006). We then developed a C57BL/6.Rag2nullIl2rgnull mouse line harboring the NOD-type Sirpa, named the BRGS mouse. The engraftment efficiency of human HSCs in BRGS mice was equal to that of NOD-Rag2nullIl2rgnull mice. This BRGS strain should be useful for further genetic modification because its background is simplified (Yamauchi et al., Blood, 2013). One of the persistent problems with the current xenograft models is that reconstituted human hematopoiesis is B-lymphoid dominant, whereas the engraftment levels of human myeloid cells, including megakaryocyte/erythrocyte lineage cells are quite low. In mouse syngeneic and allogeneic transplantation, recipients bearing mutation in the receptor tyrosine kinase Kit can accept donor HSCs without irradiation preconditioning. These data suggest that the Kit mutation impairs endogenous murine HSC self-renewal and allows donor HSCs efficiently to access to niche space. To develop a more efficient xenograft model, we further introduced a loss-of-function KitWv into the BRGS strain to deteriorate self-renewal and reconstitution capability of host HSCs. Here we show that this new mouse strain, termed the BRGSK mouse, showed very efficient human cell engraftment including myeloid, erythroid and megakaryocyte lineage cells. CD34 + cells obtained from human umbilical cord blood were transplanted into sublethally-irradiated BRGSK and BRGS mice at the age of 6-8 weeks by intrafemoral injection. At 8-12 weeks after transplantation, the average human hematopoietic chimerism defined by human CD45+ cells was more than 90% in the BM and this chimerism was maintained over 24 weeks in homozygous BRGSK recipients. Furthermore, the chimerism of human CD33+ myeloid cells in the BM was significantly improved (~5.7% in BRGS vs. ~32.5% in BRGSK). Surprisingly, CD71- CD235a+ mature erythrocytes and CD41+ platelets were detected in the BM. All human erythroid series including mature erythrocytes, CD71+ CD235a- early erythroblasts and CD71+ CD235a+ late erythroblasts were clearly seen (~0.1% in BRGS vs. ~42.2% in BRGSK). Immunohistochemical staining showed that these human erythroid cells were positive for human hemoglobin subunit alpha. Confocal immunofluorescence imaging revealed that human erythroid cells formed large erythroid islands, independently from mouse erythroid islands in the sternal marrow of recipient BRGSK mice. A number of CD41+ megakaryocytes were scattered in the BM. The BRGSK mice also well supported terminal differentiation of myeloid cells such as neutrophils, eosinophils, basophils, mast cells, monocytes, and dendritic cells. Thus, reconstitution and maintenance of human myelo-erythropoiesis is significantly improved in the new BRGSK strain. The new BRGSK xenograft model should be a strong tool to investigate normal and malignant human hematopoiesis. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Vaso-occlusion is one of the most common complications in sickle cell disease (SCD). Our previous studies have shown that neutrophils play an important role in promoting sickle cell vaso-occlusion by capturing sickle red blood cells (sRBC) through activated αMβ2 integrin (Mac-1). However, high Mac-1 activity was only observed in a small subset of adherent neutrophils (Nat. Med. 2009;15:384). Recent studies have shown that a CD62Llow CXCR4high subset of neutrophils represents the cells that have truly aged in vivo (Cell. 2013;153:1025). Since aged neutrophils may have experienced activation signals when they survey the whole body, we hypothesized that they are over-active cells that promote sickle cell vaso-occlusion. To test this possibility, we first analyzed the correlation between CD62L expression and Mac-1 activity of adherent neutrophils using multichannel fluorescence intravital microscopy and albumin-coated fluosphere beads that specifically bind activated Mac-1. We found a strong inverse correlation where neutrophils with lower CD62L expression showed greater bead binding capacity (P < 0.001). Mice deficient in P-selectin (Selp-/-), an adhesion molecule required for neutrophil clearance from the circulation, showed a dramatic increase in the percentages of CD62Llow CXCR4high aged neutrophils (wild-type / Selp-/-: 9.0 ± 1.0% / 74.7 ± 2.1%, P < 0.01). When purified neutrophils from Selp-/- mice were transferred into wild-type recipients, these aged neutrophils exhibited significantly higher Mac-1 activity compared to those purified from wild-type animals (wild-type / Selp-/-: 0.26 ± 0.06 / 1.52 ± 0.35 beads per adherent neutrophil, P < 0.05). Since microbial products derived from the microbiota could translocate into the system and modulate the innate immunity (Nat. Rev. Microbiol. 2011;9:233), we hypothesized that microbiota-derived signals could regulate the neutrophil aging process. To test this idea, we depleted the microbiota by treating mice with ampicillin, neomycin, metronidazole and vancomycin for 4 weeks. In these mice, the numbers of CD62Llow CXCR4high aged neutrophils were significantly decreased, and this reduction was reversible by intragastric gavage of Lipopolysaccharide (LPS; Control / Antibiotics-treated (ABX) / ABX + LPS: 87.2 ± 20.3 / 12.9 ± 2.0 / 83.9 ± 60.0 cells / μL blood, P < 0.05 between first two groups). To analyze the kinetics of neutrophil aging in vivo, we transferred blood from CD45.1+ mice into CD45.2+ control or antibiotics-treated mice, and monitored the percentages of CD62Llow CXCR4high population in CD45.1+ donor neutrophils. Strikingly, the aging process of donor neutrophils was significantly slower in antibiotics-treated recipients (Control / ABX: 78.2 ± 3.4% / 45.7 ± 7.7%, 5h after transfer, P < 0.01; 95.5 ± 0.7 / 67.1 ± 7.8%, 9h after transfer, P < 0.05). Since MyD88 mediates the signaling of most toll-like receptors (TLRs), we analyzed the neutrophil aging phenotypes in LysM-Cre/MyD88-flox mice, in which MyD88 is specifically deleted in the myeloid lineage. Similarly, we observed a dramatic decrease in the numbers of aged neutrophils in these mice (wild-type / LysM-Cre/MyD88-flox: 70.1 ± 22.2 / 23.8 ± 3.7 cells / μL blood, P = 0.08), and also a significantly slower aging process when we transferred blood from LysM-Cre/MyD88-flox mice into wild-type recipients (wild-type / LysM-Cre/MyD88-flox: 15.0 ± 3.1% / 5.0 ± 1.0%, 10min after transfer, P=0.09; 49.7 ± 5.2% / 12.0 ± 1.5%, 5h after transfer, P < 0.05; 59.6 ± 2.0% / 19.5 ± 3.2%, 9h after transfer, P < 0.01). To test whether neutrophil aging was relevant in SCD, we depleted the microbiota in a humanized SCD mouse model (“Berkeley” mice). Five weeks after depletion, we observed a dramatic decrease in the numbers of aged neutrophils (Control SCD / ABX SCD: 4392 ± 574 / 819 ± 358 cells/ μL blood, P < 0.01), and a significant improvement of splenomegaly (Control SCD / ABX SCD: 691.7 ± 46.4 / 453.5 ± 28.5 mg, P Disclosures: No relevant conflicts of interest to declare.

Although most hematopoietic stem cells (HSCs) are quiescent under homeostasis in the adult bone marrow, they are actively proliferating during development. Definitive HSCs, marked by the ability to repopulate a lethally irradiated adult mouse, are first detectable in the aorta-gonad-mesonephros region around E10.5, and then colonize the fetal liver (FL) to expand in this organ until E15 when hematopoietic activity shifts to the fetal bone marrow. The role of the microenvironment, or niche, in the regulation of HSCs in the FL, a site of physiological expansion, is unclear. Fetal liver sinusoidal cells as well as hepatic progenitors have been proposed as sources of supportive signals that drive the exponential expansion of HSCs, however, exact cell type(s) supporting HSC expansion have not been defined. Since stromal cells marked by Nestin-GFP form niches for HSCs in the BM, we have hypothesized that similar cells exist in the FL. Indeed, a rare population of Nestin+ cells (CD31-CD45-Ter119-) comprising 0.02 ± 0.01% of nucleated FL cells was isolated. FL Nestin+ cells express surface markers similar to their BM counterparts, including PDGFRα, CD51, and endoglin. Since BM Nestin+ cells are enriched in mesenchymal stem and progenitor cell (MSPC) activity, we next tested this activity in FL Nestin+ cells. We found that the entire CFU-F forming capacity of the FL was contained within FL Nestin+ cells, further suggesting similarities of these cells between these two hematopoietic organs. In addition, FL Nestin+ cells were enriched for the HSC maintenance genes SCF and CXCL12, suggesting that they may also serve as the HSC niche in the FL. FL Nestin+ cells were localized on the abluminal side of large-bore arteries and expressed the pericyte markers, α-smooth muscle actin and NG2. To investigate the function of the FL Nestin+NG2+ cells in vivo, we generated a genetic depletion mouse model of NG2+ cells (NG2-cre / inducible diphtheria toxin-A (iDTA) transgenic mice). We found that E14.5 NG2-Cre;iDTA mouse embryos developed normally compared to fl-DTA control littermates. The frequency and absolute numbers of HSCs per FL, however, were reduced by about 40% in NG2-Cre;iDTA embryos (control / depleted; 0.01146 ± 0.0008% / 0.00660 ± 0.0008% of nucleated cells, p=0.0015; 606 ± 76 / 377 ± 48 HSCs/FL, p=0.03, , N=6-7 per group) suggesting that Nestin+ cells are required to maintain HSCs/progenitors in vivo. To interrogate further the relationship between FL Nestin+ cells and HSCs, we adopted the re-aggregate organ culture assay (Sheridan, Genesis 2009). Sorted lineage- FL hematopoietic progenitor cells and FL parenchymal cells either with or without sorted Nestin+ cells were re-aggregated and cultured for 7 days in serum-free, cytokine-free, media. After 7 days of culture, we found that CD150+ CD48− CD41− Lineage− HSCs were maintained in re-aggregates containing Nestin+ cells, but not when Nestin+ cells were absent. To confirm that FL Nestin+ cells were essential to maintain functional HSCs, re-aggregated cells cultured for 7 days were transplanted together with competitor bone marrow into lethally irradiated mice. We found that the contribution to the peripheral blood at 8 weeks post-transplant was only observed in the re-aggregated group containing Nestin+ cells. These preliminary data indicate that factors derived from FL Nestin+ cells are required to maintain HSCs in re-aggregate cultures. These findings suggest that HSCs inhabit similar microenvironments in temporally and spatially distinct hematopoietic organs. Further studies on differences between Nestin+ cells in these tissues may shed light on the mechanisms that determine the finely tuned quiescence, self-renewal and differentiation of HSCs. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) adapt to the varying needs for the replenishment of terminally differentiated blood cells by switching between quiescence, self-renewal and differentiation. A tight regulation of this balance is crucial to ensure hematopoietic homeostasis over a lifetime. In the bone marrow (BM), HSCs reside in specialized microenvironments, or niches, that regulate HSC behavior. Although all niche cells identified thus far have a non-hematopoietic origin (e.g., mesenchymal stem cells, osteoprogenitors, endothelial cells and sympathetic nerves), less is known about HSC regulation by its progeny. During confocal imaging studies of whole-mount BM, we have noted an association between HSCs and megakaryocytes (Mk); indeed up to 50% of Lin-CD41-CD48-CD150+ HSC were in direct contact with Mk. HSC were located at an average of 15.4 µm-distance to Mk. This distribution was not random (p=6x10-8) as demonstrated by two-sample Kolmogorov-Smirnov test. To assess the functional role of Mk in HSC regulation in vivo, we bred PF4-cre mice (in which the Cre recombinase gene is under the control of the Mk-specific platelet factor 4 (PF4) promoter) with iDTR mice (where cre-mediated recombination induces expression of the diphtheria toxin (DT) receptor). After 7 days of DT treatment PF4-cre:iDTR mice showed specific BM Mk depletion (5.3-fold reduction; P Disclosures: Lambert: Amgen: Research Funding; Nestle: Consultancy; GSK: Research Funding.

Abstract 638 A prevailing idea suggests that quiescent hematopoietic stem cells (HSCs) reside near osteoblasts in the bone marrow (BM) whereas actively cycling HSCs are found near sinusoids. However, this idea is not been proven experimentally. Our recent studies have identified Nestin+ perivascular cells containing all BM mesenchymal stem cell activity as a candidate HSC niche cell (Nature 2010;466:829). To gain more insight in the 3-dimensional (3D) structure of the HSC niche, we have established a novel whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between vascular structures and endogenous CD150+CD48−CD41−Lineage− HSCs in the sternal or femoral BM can be precisely determined. Our imaging analyses in both bone types revealed a prominent sinusoidal network interrupted by rare small caliber (∼10μm) arterioles whose identity was confirmed by staining with artery-specific Alexa Fluor 633 (Nat Methods 2012;9:273) or Sca-1 (Cell Stem Cell 2009;4:263). 3D analyses using Nes-GFP transgenic mice showed two distinct types of Nestin+ cells associated with distinct vascular structures: Nes-GFPbright cells exhibiting a pericyte-like morphology were tightly associated with arterioles (referred to as periarteriolar Nestin (Nesperi) cells) whereas more abundant Nes-GFPdim cells exhibiting a reticular shape (Nesretic cells) were largely associated with sinusoids. We sorted Nesperi and Nesretic cells based on GFP expression and cell size for further characterization. Nesperi cells were very rare (∼0.002% of BM) and expressed ∼3-fold and 10–50-fold higher levels of genes associated with HSC maintenance and retention (Cxcl12, Kitl, Angpt1, Vcam1) compared to Nesretic cells and Nes-GFPneg CD45−CD31−Ter119− stromal cells, respectively. These factors were down-regulated after G-CSF stimulation in Nesperi cells but not in Nesretic cells, which led us to hypothesize that Nesperi cells played an important role in HSC maintenance. We examined the spatial correlation between endogenous HSCs and Nesperi cells by whole-mount immunostaining and found that 38.6 ± 2.9% and 54.9 ± 6.8% of HSCs were localized within 20μm and 40μm distance from Nesperi cells, respectively. After G-CSF treatment, HSCs entered cell cycle (control/G-CSF; 86.8 ± 2.6%/52.2 ± 3.8% in G0, as defined by Ki67 and Hoechst 33342 staining, p=0.002) and expanded around Nesperi cells (control/G-CSF; 34.3 ± 1.0%/48.2 ± 1.6% in 0–20μm proximity, p 80μm distance, p80μm distance, p Disclosures: No relevant conflicts of interest to declare.

Abstract 505 Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (> 10-fold, p 7-fold, p Disclosures: No relevant conflicts of interest to declare.

Abstract 1099 Leukocyte recruitment is a crucial component of immune homeostasis. The sequential multistep process leading to leukocyte migration is thought to be regulated on demand at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves regulate rhythmic recruitment of leukocytes in tissues. While oscillations in the numbers of circulating leukocytes ranged from 4.1 to 7.8 × 106 cells/ml blood (P Disclosures: No relevant conflicts of interest to declare.

Abstract 139 Chemotherapy regimens commonly produce long-term hematopoietic injury, possibly through a lesion to the hematopoietic stem cell (HSC) and/or its niche but the mechanisms have yet to be elucidated. Nestin-expressing mesenchymal stem cells, under regulation of the sympathetic nervous system (SNS), are a critical component of the HSC niche (Nature. 2010; 466:829). Since peripheral neuropathy is a common complication of chemotherapy, we hypothesized that chemotherapy-induced sympathetic bone marrow (BM) neuropathy could impair BM regeneration. To test this issue, we sympathectomized mice using 6-hydroxydopamine (6OHDA) or diphtheria toxin (DT) injection into TH-Cre-iDTR mice, which express the DT receptor exclusively on catecholaminergic neurons. In both models, SNS ablation led to reduced BM regeneration following 5-fluorouracil (5FU) challenge, as determined by the delayed recovery in BM cellularity (1.7-fold↓*), clonogenic progenitors (CFU-C; 2.5-fold↓*) and Lin−Sca-1+c-Kit+Flt3− cells (LSKF cells 2.5-fold↓*). Further, inhibition of β3 adrenergic signaling with the antagonist SR5920A impaired BM recovery to the same extent as 6OHDA sympathectomy: BM cells (1.6-fold↓*), CFU-C (1.6-fold↓*), LSKF (1.6-fold↓*). We then treated mice with the chemotherapy drug cisplatin, which induces neuropathies in mice (2.8-fold↓* in SNS fibers in BM). Cisplatin-treated mice exhibited reduced survival after BM transplantation (66% vs 100%* for cisplatin-treated and control, respectively) due to delayed regeneration: BM cells (4.6-fold↓*), CFU-C (8.8-fold↓*), LSKF (6.4-fold↓*). To evaluate the potential benefit of neuroprotection, we treated mice with 4-methylcatechol (4-MC), a powerful inducer of Nerve Growth Factor (NGF), together with cisplatin or 6OHDA, which led to the protection of SNS fibers (1.8-fold↑* and 11-fold↑* respectively). This prevented transplantation-associated death and increased BM recovery following transplantation or 5FU administration: BM cells (1.9- and 1.6-fold↑, respectively *), CFU-C (3.6- and 1.4-fold↑*), LSKF (4.5- and 2.4-fold↑*). To ascertain whether 4-MC/NGF acted specifically on the nervous system, rather than on hematopoietic or stromal cells, we used TrkAneo/neo mice, which are genetically deficient in the NGF receptor in non-neuronal tissues (Development. 2004;131:5185). Administration of 4-MC to sympathectomized TrkAneo/neo mice enhanced BM regeneration after 5FU: BM cells (2.0-fold↑*), CFU-C (2.0-fold↑*), LSKF (2.2-fold↑*), suggesting that 4-MC acts directly on neurons to rescue hematopoiesis. Since deletion of p53 promotes neuron survival after genotoxic damage (Neurodegeneration. 1996;5:233), we intercrossed mice expressing TH-Cre mice with p53flox/flox animals to delete p53 specifically in catecholaminergic neurons. SNS fibers of TH-Cre;p53flox/flox mice were protected after cisplatin treatment compared to TH-Cre;p53+/flox controls, and this was associated with improved BM recovery after transplantation: BM cells (2.4-fold↑*), CFU-C (2.9-fold↑*), LSKF (6.5-fold↑*) when compared to TH-Cre;p53+/flox animals. Thus, these various pharmacologic and genetic models strongly suggest that chemotherapy-induced sympathetic neuropathy prevents BM regeneration. To evaluate further the mechanisms, we investigated whether BM neuropathy might lead to dysfunctional HSC niche. SNS injury using 6OHDA or cisplatin treatment led to Nestin+ cell expansion and accumulation (1.7- and 3.8-fold↑*, respectively). Treatment with 4-MC reverted this phenotype (1.8-fold↓* for 6OHDA and 1.5-fold↓* for cisplatin), indicating that Nestin+ cell expansion is due to reduced SNS signaling. SNS injury also led to increased Nestin+ cell sensitivity to genotoxic insult as determined by increased Nestin+ cell death and apoptosis (1.4-fold↑*) 24h after 5FU injection, which reduced Nestin+ cell numbers during regeneration (2.1-fold↓* 12 days after 5FU). By contrast, the number of osteoblasts and endothelial cells were unaffected. These results indicate that adrenergic signals promote the quiescence of HSC niche cells and that sympathetic neuropathy renders the niche vulnerable to genotoxic insult, impairing hematopoietic regeneration. Neural protection may represent a novel strategy to maintain hematopoietic reserve and enhance transplantation efficiency in chemotherapy-treated patients. *P Disclosures: No relevant conflicts of interest to declare.

Abstract 398 Previous studies have revealed that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner in a process controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature 2008;452:442). Here, we have evaluated the constitutive recruitment of hematopoietic cells back to the BM. We have observed using high-speed multichannel fluorescence intravital microscopy (MFIM) significant circadian oscillations in the number of adherent BM cells in sinusoids with a nadir in the morning (Zeitgeber time, ZT5: 0.97 ± 0.17 adherent cells/ 100 μm2 vessel area) and a peak at night (ZT13: 2.54 ± 0.53 adherent cells/100 μm2, p = 0.007) after adoptive transfer on a 12 hour light-12 hour darkness cycle. Flow cytometric analyses revealed that the majority (∼70 %) of homed BM cells were Gr-1+ Mac-1+ myeloid cells. To investigate the underlying mechanisms, we have examined the expression levels of P- and E-selectins and VCAM-1, essential homing receptors for progenitor cells in the BM, and found that their mRNA and protein expression on BM endothelium oscillated over the course of a day with the peak expression overlapping the time of the highest cell adhesion numbers (ZT13). To examine the role of the SNS in this process, we surgically sympathectomized mice by unilateral section of the superior cervical ganglion (SCGx) whose neurons project into the calvarial vasculature, while performing sham surgery on the contralateral side. Sympathectomy abolished circadian fluctuations in the number of adoptively transferred adherent cells to the denervated calvarial BM compared to the control side in the same animals (nerve-intact side: ZT5 / ZT13: 1.66 ± 0.10 / 2.41 ± 0.08 cells / 100 μm2, p Disclosures: No relevant conflicts of interest to declare.

We have recently shown that hematopoietic stem cells (HSCs) are released into peripheral blood in a circadian manner, controlled by the sympathetic nervous system (SNS) through the regulation of CXCL12 levels in the bone marrow (BM) (Mendez-Ferrer et. al. Nature2008;452:442). In addition to the oscillations in circulating HSC numbers, we have also observed circadian fluctuations in the numbers of blood leukocytes in mice maintained on a standard 12h light - 12h dark cycle. Systemic leukocyte counts (4129 ± 521/μl (12 pm) vs 2000 ± 233/μl (8 pm); p=0.003) and their subsets, neutrophils (2762 ± 118 vs 732 ± 45/μl; p=0.02) and lymphocytes (5997 ± 151 vs 1867 ± 661/μl; p=0.03), were significantly reduced at night compared to the morning. We hypothesized that peripheral leukocyte counts are regulated by their interactions with endothelial cells in the BM microcirculation. To test this hypothesis we analyzed the constitutive interactions of leukocytes with BM microvessels using intravital microscopy to evaluate whether the trafficking of leukocytes in the BM microvasculature is also subjected to circadian regulation. We found that the absolute number of rolling leukocytes (18.0 ± 0.2 (12 pm) vs 31.1 ± 3.5 (8 pm) x103/mm2;p=0.004) and the rolling flux fraction (RFF, i.e. rolling/systemic leukocyte counts)(6.5 ± 0.6 vs 20.7 ± 3.1 %; p=0.0001) were increased three-fold at night, inversely correlating with the number of circulating leukocytes. Adoptive transfer experiments of fluorescently labeled BM leukocytes also revealed a two-fold increase in the number of recruited adherent leukocytes at night compared to when cells were injected in the morning (0.97 ± 0.17 (12 pm) vs 2.54 ± 0.53 (8 pm)/vessel area (μm2);p=0.007). Flow cytometry analyses revealed that the majority of these recruited cells were Mac-1+/Gr-1+ myeloid cells. This suggests that the capacity of the BM to recruit leukocytes fluctuates over the course of a day. Moreover, adoptively transferred cells formed clusters at specific sites in the BM at night. To investigate the mechanisms, we first subjected mice lacking both endothelial selectins (P-and E-selectins) to the same experimental protocols. Double deficient mice did not exhibit circadian variations in PB (9725 ± 1185/μl (12 pm) vs 8271 ± 1394/μl (8 pm); p=0.50) or BM (RFF 0.28 ± 0.05 vs 0.34 ± 0.05 %; p=0.48), and did not show clusters of adoptively transferred cells in the BM. To investigate the role of the SNS, we sympathectomized mice with 6-hydroxydopamine. Chemical sympathectomy significantly reduced the circadian differences of leukocyte rolling on BM endothelium (RFF 7.8 ± 1.1% (12 pm) vs 14.9 ± 2.8% (8 pm); p=0.01) and leukocyte numbers in PB (2750 ± 322 vs 2225 ± 363/μl; p=0.32). In addition, the number of adherent adoptively transferred cells in the BM did not show significant fluctuation (1.66 ± 0.26 vs 1.54 ± 0.20/vessel area(μm2);p=0.72). These results suggest that the process of homing/cluster formation is dependent on constitutive, oscillatory expression of P- and/or E-selectins and regulated by the SNS.