ALBERT

Description: Abstract 1361 Poster Board I-385 The expression of Nur77/NR4A1, one member of the Nur Orphan Nuclear Receptor family, is tightly regulated in many cell types, including myeloid and lymphoid lineages, and can change rapidly in response to a variety of external stimuli. Best known for its role in regulating apoptosis by either transcribing lethal genes or by initiating mitochondrial production of cytochrome c, Nur77 may also play other non-apoptotic roles. In an effort to understand the fate and function of cells that upregulate Nur77, we developed a transgenic Nur77-eGFP reporter mouse. Our analysis of cells in the peripheral blood of these mice reveals two populations of circulating cells that express GFP (Nur77) ‘constitutively’: T lymphocytes and a subset of monocytes. Our preliminary data suggest that the GFP+ T cells, which, interestingly are enriched for regulatory T cells, are recent thymic emigrants that have upregulated Nur77 as a consequence of TCR stimulation during maturation. Our observations also indicate that the GFP+ monocytes define a subpopulation of ‘patrolling’ cells that may ultimately differentiate into splenic dendritic cells. Understanding the identity of these circulating GFP+ cells should provide us with insights into the regulation and function of Nur77 itself. The analysis may also clarify the significance of phenotypic differences among blood cell subtypes by providing additional information about cell origin and activity. We thank the Arnold and Mabel Beckman Foundation (HH), HHMI (TB), and NSF (#MCB-0744570 (JP and NC)) for their support for this work. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4723 ‘Resident’ or ‘patrolling’ monocytes have recently been described as a unique subset of myeloid cells that differ in phenotype and function from classic inflammatory monocytes. Resident monocytes are distinguished by their ‘crawling’ migration on endothelial surfaces, their immunosuppressive activity, and their unique pattern of surface protein expression: Ly6Clo, CX3CR1hi, CCR2-, LFA-1+, and PDL-1+. Using a Nur77-eGFP reporter mouse, we show that all murine patrolling monocytes, but not conventional monocytes express Nur77+ (also known as NR4A1), a member of the Nur orphan nuclear receptor family that regulates cell fate decisions several cell types and inhibits leukemogenesis. Nur77 is an immediate early response gene, and we have recently determined that it is a sensitive indicator of antigen receptor signaling among T cells. We therefore propose that its expression among patrolling monocytes is an indication that these cells are actively receiving signals, perhaps via interactions with the endothelium. We are currently working to identify these signals and our preliminary data suggest that adhesion molecules play an influential role. We thank the Arnold and Mabel Beckman Foundation (HH), and NSF (MCB-0744570 (JP and NC)) for their support for this work. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2339 Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4782 The molecular basis for the balance between self-renewal and differentiation that regulates hematopoietic stem cell (HSC) development is poorly understood. Recent studies suggest that levels of expression of the orphan nuclear hormone receptor Nur77 (nr4a1), previously implicated in thymic selection, may play a pivotal role in HSC fate decisions. Previously published quantitative PCR data suggest that murine HSCs express relatively high levels of Nur77 mRNA. Genetic deletion of Nur77 and its close family member Nor-1 (nr4a3) in mice resulted in the development of acute myeloid leukemia (Mullican et al. Nat Med, 13:730;2007), consistent with Nur77's role as an anti-leukemogenic transcription factor, but also suggestive of a role in HSC homeostasis. To more closely investigate the role of Nur77 in normal hematopoiesis, we constructed a transgenic reporter mouse in which GFP is driven by the Nur77 promoter (eGFPNur77). Unexpectedly, we found that fewer than 3% of freshly isolated bone marrow HSCs (Lin-c-Kit+Sca-1+, IL-7R-) and common lymphoid progenitors (CLP = Lin-c-Kit+Sca-1+, IL-7R+), and no common myeloid progenitors (CMP) expressed Nur77 in our novel reporter system. Interestingly, culture of Lin- bone marrow cells with serum-containing medium resulted in a marked increase in Nur77 (GFP) expression among the majority of HSC's and CLP's, but not CMP's. Further studies indicate that a heat-insensitive component of serum, also present in plasma, is responsible for this induction. Taken together with published data, our studies suggest that induction and modulation of Nur77 may play a previously unappreciated role in self-renewal and lymphoid versus myeloid lineage commitment decisions of HSCs. We are currently working to understand the developmental difference in HSC/CLP/CMP responsiveness and to identify the serum component(s) responsible for upregulating Nur77. Disclosures: No relevant conflicts of interest to declare.