F4/80 Identifies a Subset of Non-Mobilizable Bone Marrow HSCs Involved in Stress-Induced Hematopoiesis

Abstract Hematopoietic stem cell (HSCs) are commonly isolated by using cell surface markers in order to study their hierarchy and functional properties. However, even with the most rigorous methods of isolation, subsets of HSCs likely exhibit functional heterogeneity. We have found that F4/80, an adhesion G-protein coupled receptor well known as a macrophage marker, was expressed on an HSC subpopulation (50-60% of Lin− Sca1− cKit− CD150+ CD34− CD135− cells) in the bone marrow. Interestingly, F4/80 was not expressed on HSCs that have egressed in the blood and on only a small fraction (3%) of splenic HSCs. To evaluate the function of the HSC subset expressing F4/80, we transplanted competitively 200 F4/80+ or F4/80− HSCs using CD45.1/2 congenic system. We found that F4/80+ HSCs exhibited a lower engraftment potential compared to F4/80− HSCs at 16 weeks after transplantation (19.7±4.8% and 37.1±4.5% donor contribution, respectively, P=0.025), although both HSC subsets were able to sustain mixed chimerism for myeloid, B and T cells without significant alteration in lineage bias. Since F4/80+ HSCs were not found in extramedullary tissues (blood or spleen), we tested whether they could be mobilized following G-CSF or CXCR4 antagonist (AMD3100) treatment. Interestingly, F4/80+ HSCs were still retained in the bone marrow after either G-CSF- or AMD3100-induced mobilization whereas F4/80− HSCs were mobilized efficiently. However, the frequency of bone marrow F4/80+ HSCs was reduced in G-CSF-treated animals (3.3-fold, p<0.0001) while the F4/80− HSC frequency was increased (2.2-fold, p<0.0001). F4/80 could act directly to retain HSCs in the bone marrow microenvironment or it could mark a non-mobilizable pool of stem cells. To test the later possibility, we crossed transgenic mice expressing Cre recombinase knocked in the CD169 locus, a marker of bone marrow macrophage, with ROSA26-loxP-stop-loxP-tdTomato (CD169/tomato). We found that CD169/tomato selectively labelled a large fraction of F4/80+ HSCs (31.7±8.4%) by contrast to F4/80− HSCs which were, by and large, not labelled (2.1±1.2%). We next induced HSC mobilization with G-CSF. Strikingly, we found that in a manner similar to F4/80+ HSCs, CD169/tomato+ HSCs were not mobilized in the blood while their numbers were reduced in the bone marrow after G-CSF (5-fold, p=0.014). Since macrophage depletion can induce HSC mobilization (Chow et al., JEM 2011), we tested the effect of macrophage depletion using clodronate liposomes. Interestingly, F4/80+ HSCs in wild-type mice or CD169/tomato+ HSCs were not mobilized following macrophage depletion but were depleted from the bone marrow while F4/80− HSCs underwent a 3-fold expansion. The reduced HSC numbers in marrow upon mobilization by G-CSF suggested a role in survival or proliferation. To evaluate proliferation status at steady state, we performed cell cycle analyses and BrdU incorporation assay which revealed that F4/80+ HSCs significantly more proliferative than F4/80− HSCs either by cell cycle analyses (3.7-fold increase in non-G0 phase, p=0.024) or by BrdU incorporation (2-fold increase, p=0.026). We next assessed the impact of 5-flurouracil (5FU) administration a chemotherapeutic agent that kills cycling cells and induce stress hematopoietic recovery. While both F4/80+ and F4/80− HSCs were depleted 4 days after 5FU treatment, we observed that F4/80+ HSCs expanded dramatically compared to F4/80− HSCs (10-fold and 4.5-fold over F4/80− HSCs at 8 days and 12 days post-5FU, respectively) during recovery phase. Taken together, these results identify F4/80+ HSCs as a strictly resident subset of bone marrow HSCs involved in rapid recovery after hematopoietic stress. Disclosures Frenette: PHD Biosciences: Research Funding; GSK: Research Funding; Pfizer: Consultancy.

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Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

Blood ◽

10.1182/blood-2021-150455 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2596-2596

Author(s):

Manja Wobus ◽

Ekaterina Balaian ◽

Uta Oelschlaegel ◽

Russell Towers ◽

Kristin Möbus ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Immunohistochemical Staining ◽

Research Funding ◽

Fold Increase ◽

Low Risk ◽

Hematopoietic Stem ◽

Healthy Donors ◽

The Impact

Abstract Introduction Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species. Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies. Aim We investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro. Methods Immunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system. MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs. Results Immunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro. Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation. Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%. To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM. Conclusion In summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. Disclosures Balaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

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Megakaryocytes Regulate Hematopoietic Stem Cell Quiescence Via PF4 Secretion

Blood ◽

10.1182/blood.v122.21.3.3 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3-3 ◽

Cited By ~ 1

Author(s):

Ingmar Bruns ◽

Daniel Lucas ◽

Sandra Pinho ◽

Jalal Ahmed ◽

Michele P Lambert ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Research Funding ◽

Fold Increase ◽

Brdu Incorporation ◽

Platelet Factor ◽

Neuraminidase Treatment ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Cell Quiescence

Abstract 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<0.001), followed by a concomitant reduction in platelets (7.0-fold reduction; P<0.0001). Strikingly, Mk depletion led to a 11.5-fold increase (P<0.001) in the number of Lin-c-Kit+Sca-1+CD105+CD150+ HSC due to a 5.5-fold increase in proliferation (determined by BrdU incorporation; P<0.001). Since loss of quiescence has been frequently associated with reduced HSC potential, we tested whether the increase in HSC proliferation after Mk depletion impaired HSC function by competitive transplantations in limiting dilutions. Extreme limiting dilution analysis (ELDA) revealed 625 HSCs with repopulating capacity per femur in DT-treated PF4-cre:iDTR mice which is 4.8-fold more (P<0.01) than in DT-treated PF4-cre transgenic control mice. Importantly, Mk depletion did not result in aberrant hematopoiesis as reflected by WBC and RBC counts in the peripheral blood and progenitor cell (CFU-E, GMP, Pre-GM, PreMegE, Pre CFU-E ) numbers in the BM that were indistinguishable from WT mice. To ascertain whether the effect was mediated by circulating platelets rather BM Mk, we injected mice with neuraminidase, which selectively depletes platelets. Neuraminidase treatment led to a 13.9-fold reduction in blood platelets (P<0.0001), but neither BM Mk nor HSC were significantly altered. Thus, Mk and not platelets regulate BM HSC quiescence. To investigate the mechanisms through which Mk regulate HSC, we screened for factors that have been shown to maintain cell quiescence in several cell types. Of those, PF4 itself, which is expressed exclusively by Mk, had the greatest reduction in the bone marrow extracellular fluid (BMEF) upon Mk depletion as determined by ELISA analyses. We therefore hypothesized that PF4 contributed to Mk-mediated maintenance of HSC quiescence. We first carried out in vitro culture experiments which revealed that recombinant murine (rm) PF4 restrained HSC proliferation in serum-free culture conditions (3.1 fold reduction; P<0.01). This effect was abrogated when PF4 was neutralized by heparin. Following 7 days of rmPF4 injections, mice exhibited a 1.5 fold reduction in HSC numbers (P<0.0002). We confirmed the reduction of functional HSCs by competitive transplantations. Sixteen weeks after transplantation recipient mice transplanted with BM cells from PF4-treated mice showed a significant reduction in the percentage of donor-derived cells (p<0.01). Next, we analyzed HSC numbers in Pf4-/- mice or in huPF4-Tg mice (which overexpress total PF4) in comparison to WT controls. Consistent with its role in HSC quiescence, Pf4-/- showed increased HSC numbers (1.5 fold; P<0.01) and enhanced proliferation (1.6 fold, P<0.05). By contrast, overexpression of PF4 in huPF4-Tg mice led to 1.5-fold (P<0.01) and 2.3-fold (P<0.05) reductions in HSC numbers and proliferation, respectively. These studies demonstrate a feedback loop where an HSC progeny, the megakaryocyte, can directly regulate HSC pool size and proliferation via PF4. Disclosures: Lambert: Amgen: Research Funding; Nestle: Consultancy; GSK: Research Funding.

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Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.861.861 ◽

2006 ◽

Vol 108 (11) ◽

pp. 861-861 ◽

Cited By ~ 1

Author(s):

Viktor Janzen ◽

Heather E. Fleming ◽

Michael T. Waring ◽

Craig D. Milne ◽

David T. Scadden

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Caspase 3 ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

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Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

Blood ◽

10.1182/blood.v112.11.73.73 ◽

2008 ◽

Vol 112 (11) ◽

pp. 73-73

Author(s):

Rebecca L Porter ◽

Benjamin J Frisch ◽

Regis J O’Keefe ◽

Laura M Calvi

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Prostaglandin E2 ◽

Flow Cytometric Analysis ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions ◽

Flow Cytometric ◽

Incorporated Brdu

Abstract HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

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Absence or Blockage of E-Selectin-Mediated Cell Adhesion Delays Hematopoietic Stem Cell (HSC) Turn-Over and Enhances Chemoresistance.

Blood ◽

10.1182/blood.v114.22.564.564 ◽

2009 ◽

Vol 114 (22) ◽

pp. 564-564

Author(s):

Ingrid G Winkler ◽

Valerie Barbier ◽

Bianca Nowlan ◽

Theodore Smith ◽

John T Patton ◽

...

Keyword(s):

Stem Cell ◽

Cell Adhesion ◽

Hematopoietic Stem Cell ◽

Adhesion Molecule ◽

Research Funding ◽

Fold Increase ◽

Brdu Incorporation ◽

Adhesive Interaction ◽

Hematopoietic Stem

Abstract Abstract 564 The behaviour of a hematopoietic stem cell (HSC) is regulated by its immediate micro-environment or niche. We have identified a novel function for the adhesion molecule E-selectin which is constitutively expressed on bone marrow (BM) vasculature. Using mice knocked-out for E- (E-/-) or P-selectin (P-/-) genes, we investigated whether selectin absence alters HSC behaviour in vivo. We found HSC cycling in the absence of E-selectin to be significantly delayed 2.5-fold in BrdU incorporation assays compared to either P-/- or WT (mice were administered BrdU for 3d then BrdU incorporation in BM Lineage-KIT+Sca1+(LKS+)CD34- or LKS+CD48-CD150+cells measured). To confirm these findings, LKS+ cells were stained with rhodamine123, a vital dye retained by metabolically active cells but not quiescent HSC. More LKS+ cells from E-/- mice were rhodamine dull (34±2%) than WT (23±1%; p=0.037) confirming that a greater proportion of HSC from E-/- mice are quiescent. We then determined whether administration of E-selectin antagonists alone could similarly delay HSC turnover. Mice were administered the glycomimetic E-selectin antagonist GMI-1070, for set periods of time before harvest. We found HSC turnover to be significantly delayed following GMI-1070 administration (1.4 fold less BrdU incorporation, p=0.011) with a concomitant 1.4-fold increase in the number of Rho123 dull LSK+ quiescent HSC per femur (p=0.020). Non-cycling, quiescent HSC are known to be more resistant to chemotherapy and irradiation. Indeed 7 days following 5-FU administration, we found that E-/- mice had faster BM HSC recovery / less HSC damage compared to WT mice, both by phenotype analysis and in a competitive long-term reconstituting assay. Following 5-FU administration the number of reconstituting units/femur in WT mice decreased 5.1-fold but only decreased 2.3-fold in similarly treated E-/- mice. Interestingly, when mice were pre-treated with GMI-1070 before 5-FU, there was significantly enhanced blood neutrophil recovery compared to mice administered 5-FU alone (blood neutrophils were 710±205 ×103/mL with GMI-1070, compared to 234±141 ×103/mL without, at day 9 post-5-FU, p=0.0001). Similarly when mice were severely irradiated and test bleeds performed weekly, a more rapid haematopoietic recovery was observed in E-/- compared to WT mice. In summary, we have identified a novel function for the adhesion molecule E-selectin. HSC turnover is dramatically reduced in E-/- mice an effect that can be replicated by transient administration of E-selectin antagonist mimetics. Furthermore blood leukocyte and HSC numbers recover faster following cytotoxic or irradiation injury in the absence or blockage of E-selectin-mediated cell adhesion. Thus E-selectin may well be a crucial component of the proliferative HSC niche regulating HSC turnover. Blockage of E-selectin adhesive interaction by GMI-1070, a novel E-selectin antagonist that has completed phase I clinical trails, may represent a promising treatment for the protection of HSC during chemotherapy. Disclosures: Winkler: Glycomimetics Inc: Research Funding. Smith:GlycoMimetics, Inc: Employment. Patton:GlycoMimetics, Inc: Employment. Magnani:GlycoMimetics, Inc.: Employment. Levesque:Glycomimetics Inc.: Research Funding.

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Candidemia-Induced Emergency Granulopoiesis Consists of Successive Dual Waves Triggered by the Shift From C/EBPalpha to C/EBPbeta Dependency.

Blood ◽

10.1182/blood.v116.21.3778.3778 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3778-3778

Author(s):

Sakiko Satake ◽

Hideyo Hirai ◽

Nobuaki Shime ◽

Rina Nagao ◽

Ruriko Tanaka ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Steady State ◽

Early Phase ◽

Cathepsin G ◽

Brdu Incorporation ◽

Mouse Bone Marrow ◽

Granulocytic Differentiation ◽

Hematopoietic Stem ◽

Factor C

Abstract Abstract 3778 Introduction: Granulocyte is a major cellular component in the front line of host defense. The number of granulocytes must be tightly tuned to meet the demand, because both the shortage and the excess of granulocytes can be harmful to the host. During emergency situations such as infections, granulocytes are replenished from peripheral pools and bone marrow production. As the half-life of granulocytes is quite short, granulopoiesis, de novo production of granulocytes in bone marrow, plays an important role during emergency. We have previously shown that granulopoiesis at steady state is largely dependent on a transcription factor, C/EBPalpha, whereas emergency granulopoiesis is dependent on C/EBPbeta (Hirai H, et al. Nature Immunol., 2006). However, the precise developmental stage where the shift from C/EBPalpha dependency to C/EBPbeta dependency takes place is almost unknown. The aim of this study is to dissect the process of granulopoiesis by a novel flow cytometric method and to elucidate the molecular mechanisms involved in the regulation of emergency granulopoiesis. Methods: 4 ≂ 106 cfu Candida albicans were intravenously injected to induce emergency granulopoiesis. Mouse bone marrow cells were harvested and stained with a combination of fluorescent-conjugated antibodies including anti-c-kit, anti-CD34, anti-Ly6G antibodies and markers for other lineages. Then the stained cells were analyzed or sorted by flow cytometry. After eliminating the cells which lost potential to give rise to granulocytes, the remaining cells were dissected into five subpopulations (#1≂ #5) according to the expression levels of c-kit and Ly6G. #1 is c-kithigh Ly6Glow cells, @ #2: c-kitint Ly6Glow, #5: c-kitlow Ly6Ghigh, and the cells residing between #2 and #5 are divided into #3 and #4. Cell number, gene expressions and cell cycle status of each population were analyzed before and after inducing emergency granulopoiesis. @ Results and Discussions: Wright-Giemsa staining and qRT-PCR for granule proteins (cathepsin G, myeloperoxidase, neutrophil elastase2, lactoferrin and MMP9) in each population indicated that lower c-kit expression and higher Ly6G expression correlated well with granulocytic differentiation and that the granulopoiesis progresses from # 1 to #5 in this order both at steady state and during emergencies (Figure 1). Then we applied this method to candidemia-induced emergency granulopoiesis. In vivo BrdU incorporation analysis showed immediate acceleration of the cell cycle in the most immature population (#1) and in one of the intermediate populations (#2). Chronological monitoring of each population after inducing candidemia revealed that rapid increase in mature granulocytes (#5) preceded the replenishment from the most immature population (#1). These results suggested that there are two distinct gwavesh in granulopoiesis at the early phase of infection, a rapid supply (first gwaveh) of granulocytes from relatively mature population (#2≂ #4), and a further and sustained supply (second gwaveh) originated from more immature populations (#1) including hematopoietic stem/progenitor cells (Figure 1). Transcripts of C/EBPalpha were significantly downregulated in #1≂ #4 at the early phase of infection, while those of C/EBPbeta were maintained in all the subpopulation (Figure 2), suggesting that shift from C/EBPalpha dependency to C/EBPbeta dependency took place at multiple developmental steps in granulopoiesis. C/EBPbeta has less inhibitory effects on cell cycle than C/EBPalpha while their abilities to induce granulocytic differentiation are similar (Hirai H, et al. Nature Immunol., 2006). The shift toward C/EBPbeta dependency may trigger the dual waves in emergency granulopoiesis, which demands both differentiation and proliferation of granulocytic precursors. Disclosures: No relevant conflicts of interest to declare.

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CD166 Engagement Augments Mouse and Human Hematopoietic Stem and Progenitor Function Via Activation of Stem Cell-Associated and Cell Cycle Pathways

Blood ◽

10.1182/blood-2018-99-119251 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1282-1282

Author(s):

Joydeep Ghosh ◽

Jing Zhang ◽

Chi Zhang ◽

Xinxin Huang ◽

Safa F. Mohamad ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Signaling Pathways ◽

Fold Increase ◽

Marker Genes ◽

Pathway Enrichment Analysis ◽

Hematopoietic Stem ◽

Upregulated Genes ◽

The Impact ◽

Cells Cultured

Abstract Functions of hematopoietic stem and progenitor cells are regulated by cellular signaling networks and by the cellular and non-cellular elements of the hematopoietic niche. CD166 is highly expressed on both human and murine hematopoietic stem cells (HSC) and on murine osteoblasts and has been identified as a critical regulator of their functions. CD166 engages in trans-homophilic interactions with other CD166 molecules. However how homophilic or heterophilic engagement of CD166 (with CD6) improves HSC function is unknown. Since we described that CD166 is expressed on murine and human HSC and murine osteoblasts, we hypothesized that CD166-CD166 homophilic engagement is critical for the hematopoiesis enhancing activity (HEA) that is mediated by osteoblasts. CD166+LSK cells cultured for 7d on either WT osteoblasts or recombinant murine CD166 (rmCD166) showed increased colony forming units (CFU) compared to CD166-LSK cells cultured identically (p<0.05). Moreover, after 7d, the frequency of Lin-Sca1+ cells was higher in co-cultures containing rmCD166 and CD166+LSK than cultures initiated with CD166-LSK (p<0.05). Next, we determined the effect of loss of CD166 expression on progenitors and osteoblasts on hematopoietic functions using CD166 knockout mice (CD166-/-). CD166-/-LSK cells co-cultured with rmCD166 showed decreased CFU production compared to WT LSK cultured similarly (p<0.05). In addition, WT HSC cultured on CD166-/-osteoblasts for 7d showed decreased fold change in CFU relative to WT HSC cultured over WT osteoblasts (p<0.05). In vivo transplantation studies to corroborate our in vitro results are ongoing. To determine if CD166-CD166 interactions also enhance hematopoietic functions of human HSC, we cultured CD34+CD166+ and CD34+CD166- HSC isolated from cord blood with recombinant human CD166 (rhCD166). On d7, CD34+CD166+ HSC cultured with rhCD166 showed 30±4-fold increase in CFU production compared to d0, whereas CD34+CD166- HSC had only 9±0.4-fold increase. Taken together, these data suggest that CD166-CD166 homophilic interactions enhance hematopoietic functions and loss of this homophilic interaction negatively impacts HSC. To elucidate the underlying signaling mechanism of CD166-CD166 mediated HEA, we cultured WT or CD166-/-SLAM LSK cells with rmCD166 for 20hr and performed single-cell (sc) RNA seq. We analyzed for differential gene expression (DEG) using LGMT model and identified 518 upregulated and 174 downregulated genes in CD166-/-HSC compared to WT. Following pathway enrichment analysis of DEGs, we identified 148 canonical pathways enriched by the upregulated genes in CD166-/-HSC, including cell cycle, translational regulation, and mitochondria-related signaling pathways. 268 pathways were impacted by the downregulated genes in CD166-/-HSC, including oxidative stress response, and metabolism. Moreover, CXCR4 signaling, PDGF signaling and glucocorticoid receptor signaling pathways were also downregulated in CD166-/-HSC. A cell trajectory reflecting associations among cells revealed a single cluster of CD166-/-HSC. CD166-/-HSC are linked with low expression of stemness marker genes, and high expression of genes regulating cell cycle, oxidative phosphorylation, and glucose metabolism. In addition, ER-stress and oxidative stress responsive genes are overexpressed in CD166-/-HSC. In CD166-/-HSC, genes of all enriched pathways were highly connected in the co-expressed networks, which indicates that in HSC, the impact of loss of CD166 on cell cycle, metabolism, growth factors and stemness pathways is highly associated. Sixteen hub genes including Suclg1, Eif4a1, Cox4i2, Jak3, Runx3, and Cdk6 were identified in the co-expression network. We next applied bi-clustering algorithm QUBIC to identify modules of co-upregulated genes to analyze the transcriptomic variations of DEG in CD166-/-HSC and identified 39-gene co-upregulation modules forming one big block in CD166-/-HSC and 35-gene modules forming three blocks in WT HSC indicating that modules closely corresponded to CD166's impact over different cell states rather than on individual pathways. Overall, our studies suggest that homophilic CD166 interactions involving HSC are required for maintenance of essential pathways that sustain HSC function and progenitor cell production including stemness, mitochondrial function, metabolism, cell cycle and growth factor signaling. Disclosures No relevant conflicts of interest to declare.

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Loss of Abelson Interactor-1 Is Linked to Inflammatory Hematopoiesis and Accelerated Aging of Hematopoietic System

Blood ◽

10.1182/blood-2018-99-118393 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1285-1285

Author(s):

Anna Dorota Chorzalska ◽

John Morgan ◽

Max Petersen ◽

Diana O Treaba ◽

Adam J Olszewski ◽

...

Keyword(s):

Bone Marrow ◽

Animal Models ◽

Research Funding ◽

Molecular Mechanisms ◽

Bone Marrow Failure ◽

Accelerated Aging ◽

Fold Increase ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Oncology Research

Abstract Background: Hematopoietic stem cells (HSC) ensure homeostasis and lifelong maintenance of hematopoietic system, but with age, they gradually lose quiescence, self-renewal potential, and system restoration capacity. HSC aging results in a differentiation shift towards myeloid lineage, anemia, thrombocytosis, decrease in T and B cells, imbalance in macrophage function, and increased osteoclast activity. Mechanisms involved in HSC aging include increased mTOR activity and ROS production, impaired autophagy, epigenetic reprograming, and cumulative DNA damage. Intriguingly, cellular and molecular similarities between aging and inflammation have led to a novel concept of "inflammation-associated aging of hematopoiesis". Understanding the molecular mechanisms responsible for this process may impact strategies targeting age-related diseases, including neoplasms. However, to date only few primary animal models of inflammation have shown bone marrow failure, so new animal models need to be established to provide mechanistic insight into the long-term implications of chronic inflammation on the hematopoietic system. We have previously shown that bone marrow-specific deletion of an adapter protein Abelson interactor-1 (Abi1) leads to a myeloproliferative neoplasm (MPN)-like disease in 35-56-week-old mice, mechanistically associated with increased activity of Src Family Kinases (SFKs), STAT3 and NF-κB. At both transcript and protein levels, Abi-1 is also significantly reduced in HSCs and granulocytes from patients with primary myelofibrosis (PMF), and Abi-1-deficient HSC in human PMF show increased SFK-STAT3-NF-κB signaling (Chorzalska, ASH 2017). Methods: Myeloid/lymphoid, stem/progenitor populations profiling by FACS, bone marrow transplantation assays, transcriptomics and proteomics analyses as well pro-inflammatory cytokine profiling and histopathology analyses were performed on the transgenic Abi-1KO mice carrying bone marrow-selective knockout at 4 weeks post-recombination, upon confirming both inducible inactivation of the Abi1flox allele and loss of Abi-1 protein in the marrow (Fig.1A, B). Results: To better understand initial systemic events that lead to the development of MPN-like disease in aged Abi-1KO mice we have now characterized early changes within the hematopoietic system associated with loss of Abi-1. Blood count analysis indicated leukocytosis, anemia and thrombocytosis, and an increase in the fraction of myeloid (CD11b+/Gr-1+) as well as macrophage/monocyte (F4/80+) cells at the expense of lymphoid (B220+) cells in Abi-1KO relative to Abi-1WT mice (Fig.1C). Previously reported 2.6-fold increase in Abi-1KO LT-HSCs (Chorzalska, ASH 2017) was now shown to be associated with 30% increase in number of LT-HSCs is in the S/G2/M phases of the cell cycle relative to Abi-1WT LT-HSCs (Fig. 1D). Lethally irradiated recipient C57BL/6 wild-type mice transplanted with bone marrow cells from Abi-1KO relative to Abi-1WT mice (in the absence of competitor cells) showed progressive loss of chimerism in primary and secondary recipients (Fig. E). Genome-wide gene expression analysis of Abi-1WT vs. Abi-1KO LSK cells showed significant overexpression of genes regulated by or involved in regulation of the NF-κB pathway (Fig. 1F). Plasma cytokine levels showed 2-fold increase in IL-1B, IL-12, IL-17, IL-23, IL-27, and MCP-1 and nearly 10-fold increase in INFγ (Fig. 1G). Label-free, intensity-based quantitative proteomic analysis of bone marrow from 20-week-old Abi-1KO and Abi-1WT mice showed abundance of peptides derived from Mac-1, myeloperoxidase, STAT1, STAT3, and SFKs - Hck and Fgr, confirming not only activation of SFKs and STAT3 signaling, but also increase in proteins associated with myeloid lineages (Fig. 1H). Loss of bone density (Fig. 1I) and a significant decrease in thymus size (Fig. 1J) were observed in Abi-1KO mice relative to Abi-1WT mice. Conclusions: In sum, phenotypic analyses performed 4-10-weeks post Abi1 gene inactivation indicate changes consistent with accelerated aging of hematopoietic system that are mechanistically linked to inflammatory SFK-STAT3-NF-κB signaling. To our knowledge this is the first animal model linking accelerated inflammation-driven aging of hematopoietic system to development of an MPN in aged mice. Disclosures Olszewski: Genentech: Research Funding; TG Therapeutics: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Reagan:Pfizer: Research Funding; Alexion: Honoraria; Takeda Oncology: Research Funding.

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Impact of Genomic Alterations on Outcomes in Myelofibrosis Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.2301.2301 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2301-2301 ◽

Cited By ~ 1

Author(s):

Raajit K. Rampal ◽

Roni Tamari ◽

Nan Zhang ◽

Caroline Jane McNamara ◽

Franck Rapaport ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Triple Negative ◽

Unrelated Donor ◽

Reduced Intensity Conditioning ◽

Hematopoietic Stem ◽

Disease Characteristics ◽

Donor Type ◽

The Impact ◽

Reduced Intensity

Abstract Introduction: The impact of genomic alterations, such as mutations in ASXL1, on the risk of disease progression and leukemic transformation in patients with myelofibrosis (MF) is well established. Further, emerging data suggests that the number and type of mutations may impact response to therapies such as ruxolitinib or imetelstat. Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the only potentially curative treatment for MF patients. However, the impact of somatic mutations on overall survival (OS) and relapse-free survival (RFS) is poorly understood. Using next-generation sequencing of pre-transplant blood and bone marrow samples from a well clinically-annotated cohort of MF patients who underwent allo-HSCT, we sought to determine the impact of mutational burden on outcomes. Methods: A multicenter retrospective analysis of a cohort of 84 patients was carried out. This included 52 patients treated on the MPD-RC 101 prospective study (NCT00572897), 18 patients treated at Prince Margaret Hospital, and 14 patients treated at Memorial Sloan Kettering Cancer Center. Patient and transplant characteristics are displayed in Table 1. DNA was extracted from pre-transplant bone marrow aspirate samples or peripheral blood samples. High-throughput sequencing of a panel of genes was performed. Average coverage of 829x (standard deviation of ±130) was obtained. Mutect was utilized to call single point variants (comparing our samples to a pool of normal samples) and PINDEL was used to call short insertions and deletions. We excluded all mutations present in at least one database of known non-somatic variants (DBSNP and 1000 genomes) and absent from COSMIC. Univariate Cox regression and Kaplan-Meier graphics were used to investigate the association of patient, transplant, and disease characteristics with OS and RFS. Results: JAK2V617F was the most frequent mutation detected in 41(48.8%) patients (Table 2). Eighteen patients (21.4%) had triple negative disease (negative for JAK2, MPL, and CALR mutations). Univariate analysis included the following: patient characteristics (age, gender), transplant characteristics (related vs. unrelated donor, matched vs. mismatched donor and myeloablative vs. reduced intensity conditioning) and disease characteristics (DIPSS and presence of mutations). Decreased OS was associated with unrelated donor status (HR 2.09, 95% CI: 1.03-4.23, p=0.04), reduced intensity conditioning (HR 4.21, 95% CI: 1.01-17.59, p=0.049), triple negative disease (HR 2.09, 95% CI: 1.02-4.30, p=0.04), and presence of U2AF1 (HR 2.53, 95% CI: 1.10-5.81, p=0.03) or SUZ12 mutations (HR 3.92, 95% CI: 1.19-12.21, p=0.02). Decreased RFS was associated with unrelated donor status (HR 2.27, 95% CI: 1.16-4.45, p=0.02), and the presence of SUZ12 mutation (HR 6.97, 95% CI: 2.37-20.49, p<0.001). A descriptive decrease in RFS in patients with U2AF1 (HR 2.15, 95% CI: 0.94-4.88, p=0.07) was observed but did not reach statistical significance. Importantly, mutations previously reported to be associated with reduced OS and RFS in the non-transplant setting, such as ASXL1, EZH2, IDH1/2, and SRSF2, were not associated with poorer outcomes in this analysis in transplanted patients. In an exploratory multivariate analysis including donor type (related vs. unrelated) and presence of U2AF1 and SUZ12 mutations, there was a significantly reduced OS and RFS in patients who harbor these mutations regardless of donor type (OS: HR 5.30, 95% CI: 2.08-13.47, p<0.001; RFS: HR 5.49, 95% CI: 2.27-13.30, p<0.001). In patients without the above mutations, having an unrelated donor was associated with worse OS (HR 2.55, 95% CI: 1.09-5.96, p=0.03) and RFS (HR 2.61, 95% CI: 1.17-5.83, p=0.02, Figure 1). Conclusions: Our analysis demonstrates that mutations previously associated with poor prognosis in MF, such as ASXL1, do not appear to confer a worsened prognosis in patients undergoing allo-HSCT, suggesting transplant may be able to overcome the impact of these mutations. However, mutations in SUZ12 and U2AF1 are associated with reduced OS in univariate and multivariate analysis (together with donor type). Further studies with larger cohorts of patients are indicated to validate these findings, and to elucidate the impact of these mutations on disease biology. Disclosures Rampal: Incye and CTI: Consultancy. Mascarenhas:Janssen: Research Funding; CTi Biopharma: Research Funding; Promedior: Research Funding; Merk: Research Funding; Incyte: Research Funding. Mesa:Galena: Consultancy; Gilead: Research Funding; Promedior: Research Funding; Incyte: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Ariad: Consultancy; Novartis: Consultancy. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

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Short-Term Fasting Induces Cell Cycle Arrest of Immature Hematopoietic Cells and Increases the Number of Naive T Cells in the Bone Marrow of Mice

Blood ◽

10.1182/blood.v128.22.5047.5047 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5047-5047

Author(s):

Teruhito Takakuwa ◽

Yasuhiro Nakashima ◽

Hideo Koh ◽

Takahiko Nakane ◽

Hirohisa Nakamae ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Research Funding ◽

Bone Marrow Cells ◽

Short Term ◽

Hematopoietic Stem ◽

And Control

Abstract Calorie restriction has long been studied not only as a way to prolong longevity but also as an approach to improve cancer prevention and treatment. Dietary restriction may prevent senescence of the immune and hematopoietic systems. In addition, short-term fasting before chemotherapy can reduce hematological toxicity in cancer patients. We studied the influence of fasting on immune cells, hematopoietic stem cells, and progenitor cells in the bone marrow and spleen of mice. Six-to-twelve-week old C57BL/6 mice were starved for 48 hours before analysis. We collected bone marrow and splenic cells from starved and control mice. After 48 hours of fasting, the body weight significantly decreased by an average of 24.1% compared to that of normal control mice. Calorie restriction caused a significant decrease in peripheral white blood cell count by an average of 48.3%, but hemoglobin level and platelet count were less affected. The averaged total number of bone marrow cells in the fasting group was significantly lower than that in the normal control group (2.45×107 versus 3.14×107, P < 0.01). In fasted mice there was a significant reduction in the hematopoietic stem cell count, using detection based on the lineage- c-kit+ Sca-1+, compared to control mice (0.83×105 versus 1.24×105, P < 0.05). In contrast, there was no significant difference for progenitor cells detected based on the lineage- c-kit+ Sca-1- (6.81×105 versus 7.75×105, P = 0.11). We performed colony assays with bone marrow cells from fasted and control mice. There was no difference between the two groups for not only the primary colony assay but also for the secondary and tertiary assays. Annexin V and 7-AAD analysis by flow cytometry showed that the rates of early and late apoptosis were almost identical in hematopoietic stem cells and progenitor cells, on comparing fasted and control mice. Furthermore, DNA cell cycle analysis of progenitor cells showed that short-term fasting caused a significant increase in the percentage in G0/G1 phase (83.1% versus 70.7%, P < 0.05) and decreases in the S and G2/M phases. These results imply that immature bone marrow cells retained their proliferative capacity by maintaining cell cycle arrest after short-term fasting, a finding that may account for the protective effect of starvation against chemotherapy in cancer patients. Calorie restriction caused a significant decrease in B cells in bone marrow (5.38×106 versus 8.1×106, P < 0.05) and especially in the spleen (6.65×106 versus 33.0×106, P < 0.001), and also significantly decreased T cells in the spleen (3.91×106 versus 14.5×106, P < 0.01). To our surprise, we detected a remarkable increase in the number of T cells in the bone marrow of fasted mice (1.25×106 versus 0.91×106, P < 0.01). Of greatest significance CD44- naive CD8+ T cells were dramatically increased in fasted bone marrow (1.74×106 versus 0.47×106, P < 0.01), and CD44- naive CD4+ T cells were also increased (0.23×106 versus 0.07×106, P < 0.05). In contrast, the numbers of CD62L- CD44+ effector memory and CD62L+ CD44+central memory T cells were not substantially changed after starvation. The increased naive T cells had no activated markers and appear to have migrated into bone marrow in a resting state without proliferating there. Short-term fasting decreased the number of hematopoietic stem cells but progenitor cells remained in a relatively quiescent state, with a prolonged DNA cell cycle and retention of colony-forming capabilities. The number of T cells in the bone marrow of fasted mice also increased dramatically, especially naive CD8+ T cells which probably migrated in from other lymphoid tissues. These findings imply that immature hematopoietic cells and some lymphoid cells can survive starvation while maintaining their function. The mechanisms by which T lymphoid cells promptly accumulate in bone marrow during starvation are under investigation. Disclosures Koh: Pfizer: Consultancy, Honoraria. Nakane:Mundipharma KK: Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Hino:Nippon Shinyaku: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Alexion: Honoraria, Research Funding.

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