Cxcl12 Deletion Research Articles

Role of CXCL12-Expressing Bone Marrow Populations in Leukemic Stem Cell Regulation

CXCL12 is the major chemoattractant for hematopoietic stem cells (HSC) in the bone marrow microenvironment (BMM), and plays a major role in HSC localization to their regulatory niches. Studies using genetic drivers/reporters have shown that CXCL12 deletion from perivascular mesenchymal stem cells (MSC) and CAR cells (using Prx1-Cre line) leads to loss of HSC quiescence, frequency and self-renewal potential, while deletion from endothelial cells (using Tek-Cre line) results in modest loss of HSC long-term repopulating activity. In contrast, deletion of CXCL12 from osteoprogenitors (using Osx-Cre line) resulted in HSC mobilization without any effect on HSC function, while deletion from mature osteoblasts (using OCN-Cre line) had no effect on HSC function (Greenbaum et al. Nature. 2013;495(7440):227-30; Ding et al. Nature. 2013;495(7440):231-5.). These studies have been useful in identifying MSC/CAR cells and endothelial cells as important HSC niche components but the BM niches for LSC remain poorly characterized. In the present study, we examined alterations in CXCL12-producing niche cells in the CML BMM, and their role in regulating LSC growth, using the SCL-tTA BCR-ABL mouse model of CML. Our previous studies indicated that CXCL12 levels are reduced in CML compared to normal BM (Zhang et al. Cancer Cell. 2012; 21(4):577). To evaluate the effect of leukemia development on specific CXCL12-expressing cell populations in the BMM, we crossed CXCL12GFP mice (GFP reporter knocked into the CXCL12 locus) with SCL-tTA-BCR-ABL mice to generate CXCL12GFP-SCL-tTA-BCR-ABL mice. CXCL12-expressing cells in the BM were identified by GFP expression. Mice developing CML after BCR-ABL induction by tet-withdrawal demonstrated significantly increased numbers of GFP+ endothelial cells (CD45-Ter119-CD31+) and reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) compared to WT mice. Within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased. To evaluate the contribution of CXCL12-expressing populations towards LSC regulation, we crossed CXCL12f/f mice (loxP sites flanking exon 2) with Tek-Cre, Prx1-Cre, OCN-Cre and OSX-Cre transgenic lines. CML BM cells (CD45.1/2+; 2*106/mouse) were transplanted into lethally irradiated (8Gy) WT (CD45.2) Cre- or Cre+ CXCL12f/f knockout animals, and followed for CML development. When compared to WT mice, CXCL12f/f-Tek-Cre+ animals exhibited significantly reduced engraftment of CML cells (CD45.1/2+) in the BM, with associated reduction in CML myeloid cells (Gr-1+Mac-1+), MEP (CD16/32- CD34-), CMP (CD16/32lowCD34+), MPP (LSK+CD48+) and LTHSC (CD150+CD48-) numbers. No changes in splenic engraftment were seen. To evaluate long-term reconstitution, BM cells from primary transplanted WT or knockout animals were transplanted into lethally irradiated (8Gy) WT secondary recipients. CML engraftment in secondary mice receiving BM from Tek-Cre+ and WT animals was similiar at 12 weeks, indicating that residual LTHSC retained repopulating potential. In contrast, CXCL12f/f-OCN-Cre and CXCL12f/f-Osx-Cre mice did not demonstrate significant differences in total CML cell or CML LTHSC engraftment, but showed increased LMPP engraftment in the BM. Interestingly CXCL12f/f-Prx1-Cre+ animals exhibited significantly increased leukocytosis and BM cellularity, and increased MEP, CMP, LMPP, MPP, STHSC and LTHSC numbers in the BM compared to WT mice. Increased numbers of CML myeloid cells and LSK were seen in the peripheral blood, but no change in splenic engraftment was seen. CML engraftment in secondary mice receiving BM from Prx1-Cre+ animals was significantly increased at 12 weeks compared to WT animals, indicating that the expanded LTHSC population maintained repopulating potential. These results suggest that loss of endothelial cell expressed CXCL12 reduced CML LTHSC in BM, whereas loss of MSC/CAR cell expressed CXCL12 enhances CML LTHSC numbers in BM, in association with increased mobilization to PB. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM endothelial cell and MSC/CAR cells in CML, and indicate significant differences in niche regulation of CML LSC compared with normal HSC. We expect that improved characterization of BM niches in CML will facilitate further dissection of key niche interactions underlying LSC maintenance and expansion. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation intocommon lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.

CXCR4 is required for progression of T cell acute lymphoblastic leukemia

Abstract Of the most common childhood cancer, acute lymphoblastic leukemia (ALL), 15% of cases are caused by transformation of T cell progenitors (T-ALL). While survival rates have vastly improved for newly diagnosed T-ALL, substantial challenges remain in the treatment of relapsed disease and in the minimization of side-effects from central nervous system prophylaxis. Hence, the search for more effective, less toxic treatments continues. Homing receptors play multiple roles in cancer progression, yet little is known about the trafficking of T-ALL. Upon profiling homing receptor expression, we found high levels of the chemokine receptor CXCR4 on the surface of mouse and primary human T-ALL cells. Furthermore, T-ALL cells localized in close proximity to CXCL12-producing stromal cells in mouse femoral bone marrow. Deletion of Cxcl12 in vascular endothelial cells limited leukemia progression, suggesting a vascular niche for T-ALL. In parallel, we found CXCR4 expression to be essential for T-ALL maintenance and progression. Deletion of Cxcr4 in mouse T-ALL after disease onset led to disease remission and prolonged survival, and CXCR4 antagonism using the small molecule inhibitor AMD3465 suppressed human disease in a patient-derived xenograft model. Loss of CXCR4 signaling in T-ALL cells reduced Myc abundance, which was previously reported to regulate leukemia-initiating cell activity. In line with this, CXCR4-deficient T-ALL cells failed to establish disease when adoptively transferred into secondary hosts. Together, our findings highlight an important contribution of microenvironment in regulating T-ALL pathogenesis, and suggest targeting CXCL12:CXCR4 signaling as a powerful new strategy for treating this aggressive disease.

Prolyl-4 Hydroxylase 2 (PHD2) Deficiency in Endothelial Cells and Hematopoietic Cells Induces Obliterative Vascular Remodeling and Severe Pulmonary Arterial Hypertension in Mice and Humans Through Hypoxia-Inducible Factor-2α.

Vascular occlusion and complex plexiform lesions are hallmarks of the pathology of severe pulmonary arterial hypertension (PAH) in patients. However, the mechanisms of obliterative vascular remodeling remain elusive; hence, current therapies have not targeted the fundamental disease-modifying mechanisms and result in only modest improvement in morbidity and mortality. Mice with Tie2Cre-mediated disruption of Egln1 (encoding prolyl-4 hydroxylase 2 [PHD2]; Egln1(Tie2)) in endothelial cells and hematopoietic cells exhibited spontaneous severe PAH with extensive pulmonary vascular remodeling, including vascular occlusion and plexiform-like lesions, resembling the hallmarks of the pathology of clinical PAH. As seen in patients with idiopathic PAH, Egln1(Tie2) mice exhibited unprecedented right ventricular hypertrophy and failure and progressive mortality. Consistently, PHD2 expression was diminished in lung endothelial cells of obliterated pulmonary vessels in patients with idiopathic PAH. Genetic deletions of both Egln1 and Hif1a or Egln1 and Hif2a identified hypoxia-inducible factor-2α as the critical mediator of the severe PAH seen in Egln1(Tie2) mice. We also observed altered expression of many pulmonary hypertension-causing genes in Egln1(Tie2) lungs, which was normalized in Egln1(Tie2)/Hif2a(Tie2) lungs. PHD2-deficient endothelial cells promoted smooth muscle cell proliferation in part through hypoxia-inducible factor-2α-activated CXCL12 expression. Genetic deletion of Cxcl12 attenuated PAH in Egln1(Tie2) mice. These studies defined an unexpected role of PHD2 deficiency in the mechanisms of severe PAH and identified the first genetically modified mouse model with obliterative vascular remodeling and pathophysiology recapitulating clinical PAH. Thus, targeting PHD2/hypoxia-inducible factor-2α signaling is a promising strategy to reverse vascular remodeling for treatment of severe PAH.

Abstract IA23: Leukemia development and the microenvironment

Abstract Myeloproliferative neoplasms (MPNs) are hematological malignancies originated by hematopoietic stem cells (HSCs) carrying mutations, most frequently in JAK2, CALR or MPL genes. These diseases, which cannot be cured in most cases, are characterized by overproduction of myeloid and erythroid-megakaryocytic lineages. However, it remains unclear how some mutations, that are frequently common in disparate MPNs, can cause different clinical outcomes. Moreover, current treatments are mostly symptomatic and are only capable of slowing down the disease, but generally they cannot eradicate it. This suggests that other factors critically contribute to MPN progression. Our research group has demonstrated specific alterations of the microenvironment of HSCs in MPN. We have shown that mutated HSCs can only expand and trigger disease after damaging bone marrow (BM) nerve terminals and mesenchymal stem cells (BMSCs), overcoming the control that this environment can exert over mutated HSC proliferation and migration. Therefore, preserving the HSC niche might be a complementary strategy to treat these diseases. This approach is being currently tested in a multicenter phase-II clinical trial (clinicaltrials.gov identifier NCT02311569). MPNs can be considered a pre-leukemic state since MPN patients have a higher risk of developing acute myeloid leukemia (AML), which is highly aggressive and exhibits poor response to chemotherapy. Work from other groups and our preliminary data suggest that acute leukemic cells can transform the BM microenvironment to promote leukemogenesis. We have studied the contribution of nestin+ BMSCs to the development of acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene. For this purpose we have used a doxycycline-inducible rtTA;MLL-AF9 mouse strain, which closely mimics the human disease. Development of AML in rtTA;MLL-AF9 mice caused a significant reduction of stromal cells in the BM but, contrasting our previous findings in MPN, it did not seem to affect the number of BM nestin+ cells. To study the possible contribution of nestin+ cells to leukemia development, we partially depleted nestin+ cells by inducing diphtheria toxin expression in these cells using Nestin-creERT2;iDTR mice previously transplanted in a competitive fashion with preleukemic and normal BM cells. We have shown previously that nestin+ cell depletion using this strategy can accelerate MPN. In contrast, nestin+ cell depletion had the opposite effect in AML, since it selectively diminished leukemia burden in BM, spleen and blood, and reduced the number of primitive leukemic cells, without affecting normal residual hematopoiesis. Combined nestin+ cell depletion and standard chemotherapy further exaggerated the elimination of BM leukemic cells. Among primitive leukemic cells, MLL-AF9+ lin- ckitlow sca1- cells were most significantly expanded in leukemia and were most significantly decreased upon nestin+ cell ablation, an effect that seemed to be further enhanced by simultaneous chemotherapy. This was not associated with detectable changes in the cell cycle profile of leukemic progenitors. Moreover, non-proliferative (ki67-) lin- cells were not found enriched in the vicinity of nestin+ cells, and Cxcl12 deletion in nestin+ cells, which increased thrombocytosis in our previous MPN studies, did not affect AML progression. In order to identify changes in leukemic progenitors following Nes+ cell depletion, we compared the gene expression profiles of lin- ckitlow sca1- cells isolated from control and Nes+ cell-depleted, MLL-AF9 expressing mice. Next-generation sequencing assays identified downregulation of gene sets related to HSC self-renewal, protein synthesis and degradation, and mitochondrial respiration in leukemic progenitors obtained from Nes+ cell-depleted bone marrow. The data suggests that AML-AF9+ leukemic progenitors impose changes in Nes+ cells that promote leukemogenesis. Using a novel co-culture system, we are currently testing possible mediators of the pro-survival effect of BMSCs over leukemic progenitors. Citation Format: Abel Sánchez-Aguilera, Vaia Stavropoulou, Daniel Martín-Pérez, Alexandar Tzankov, Jürg Schwaller, Simón Méndez-Ferrer. Leukemia development and the microenvironment. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA23.

Distinct Contributions By Perivascular Niche Cells in Hematopoietic Stem Cell Maintenance

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<0.05) and blood (LSK, CT/DL: 177±36 / 668±156 / mL blood, n=5, p<0.05), but had no effect on HSC numbers in the BM (CT/DL: 1435±101 / 1194±75 / femur, n=6, p=0.085), which is consistent with a previous report (Ding and Morrison, Nature, 2013). Furthermore, assessments of endogenous HSC localization using whole-mount 3D imaging technology revealed that the deletion of CXCL12 in Lepr+ niche cells had no impact on HSC location (KS-test: Two-sample Kolmogorov-Smirnov test, p=0.9981). By contrast, deletion of CXCL12 in NG2-cre derived cells, which recombines efficiently in the entire Nestin-GFP+ non-endothelial stromal fraction including both peri-arteriolar and peri-sinusoidal cells (96.9±1.3%), and overlapping with Lepr+ cells (88.5±1.6%) and CXCL12-abundant reticular cells (90.7±1.4%), led to a robust reduction of HSC numbers in the BM (CT/DL: 1487±87 / 179±40 / femur, n=10, p<0.0001) with HSC and progenitor mobilization into spleen (HSC, CT/DL: 705±262 / 3550±540 / spleen, n=6-8, p<0.01) and blood (LSK, CT/DL: 494±178 / 5357±896 / mL blood, n=5-7, p<0.01). In addition, deletion of CXCL12 in NG2-cre targeted cells led to HSC displacement away from arterioles (KS-test: Two-sample Kolmogorov-Smirnov test; p=0.001). To examine further a role of CXCL12 produced by NG2+ arteriolar niches on HSC maintenance, we generated tamoxifen-inducible NG2-creERTM/Cxcl12fl/− mice. Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduced the number of HSCs in the BM (CT/DL: 1617±160 / 960±95 / femur, n=10-13, p=0.0013), which was confirmed functionally by a competitive repopulation assay. Moreover, 3D imaging revealed that HSCs were located further away from arterioles in NG2-creERTM/Cxcl12fl/− marrow (KS-test: p<0.0001), suggesting a role for arteriolar niches in CXCL12-mediated HSC maintenance. As niche cells synthesize several factors, we evaluated the contribution of arteriolar niches in SCF synthesis, a cytokine shown to be critical for HSC maintenance. As expected, deletion of SCF in NG2-cre targeted cells led to a significant reduction of HSC numbers in the BM (CT/DL: 606±85 / 96±23 / femur, n=5-7, p<0.0001). To further evaluate functions of SCF produced by distinct vascular niches, we also compared these mice with deletions using Lepr-cre or tamoxifen-inducible NG2-creERTM mice. We found that deletion of SCF in Lepr-cre targeted cells showed a significant reduction of HSC numbers in the BM (CT/DL: 690±84 / 220±83 / femur, n=3-4, p<0.0118), consistent with previous studies (Ding et al., Nature, 2012), whereas there was no significant change observed in NG2-creERTM/SCFfl/− mice, suggesting that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important source of SCF in the BM. These results highlight distinct contributions of perivascular cells primarily located in separate vascular niches, arteriolar and sinusoidal, in HSC maintenance and mobilization. DisclosuresNo relevant conflicts of interest to declare.

CXCL12-Producing Vascular Endothelial Niches Control Acute T Cell Leukemia Maintenance

The role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of Cxcr4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.

Haematopoietic stem cell niches: new insights inspire new questions

Haematopoietic stem cell (HSC) niches provide an environment essential for life-long HSC function. Intense investigation of HSC niches both feed off and drive technology development to increase our capability to assay functionally defined cells with high resolution. A major driving force behind the desire to understand the basic biology of HSC niches is the clear implications for clinical therapies. Here, with particular emphasis on cell type-specific deletion of SCL and CXCL12, we focus on unresolved issues on HSC niches, framed around some very recent advances and novel discoveries on the extrinsic regulation of HSC maintenance. We also provide ideas for possible paths forward, some of which are clearly within reach while others will require both novel tools and vision.

This Niche Is a Maze; An Amazing Niche

The cellular identity of niche cells that regulate hematopoietic stem cell (HSC) self-renewal and differentiation has been debated for several years. Two recent studies in Nature (Ding and Morrison, 2013; Greenbaum etal., 2013) have shed light into the bone marrow stromal subsets making CXCL12, a chemokine critical for HSC maintenance.

Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches.

While haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche1, most published experimental manipulations of the HSC niche have also impacted the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs1 and restricted progenitors2,3 reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine, CXCL12, for HSC and restricted progenitor maintenance. Cxcl12DsRed knock-in mice showed that Cxcl12 was primarily expressed by perivascular stromal cells and at lower levels by endothelial cells, osteoblasts, and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or Nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors, but not HSCs or myeloerythroid progenitors and did not mobilize these cells into circulation. Different stem/progenitor cells thus occupy distinct cellular niches in bone marrow: HSCs in a perivascular niche and early lymphoid progenitors in an endosteal niche.

CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance

Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts and perivascular stromal cells including endothelial cells, CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin-green fluorescent protein (GFP)-positive mesenchymal progenitors have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C-X-C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations. Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1-cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

CXCL12 Production by Early Mesenchymal Progenitors Is Required for Hematopoietic Stem Cell Maintenance

AbstractAbstract 510Hematopoietic stem cells (HSCs) reside in a specialized microenvironment in the bone marrow that provides key signals required for HSC maintenance. Neither the stromal cell population(s) that comprise this stem cell niche nor their HSC-regulatory signals are well defined. In particular, there is controversy about the relative importance of the endosteal niche, which is comprised of osteoblasts and their progenitors, and the perivascular niche, which is comprised of endothelial cells, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem and progenitor cells (including Nestin-GFP+ cells). CXCL12 is a key component of the stem cell niche that regulates HSC trafficking and function. CXCL12 is constitutively expressed by several stromal cell populations, including endothelial cells, osteoblasts, and perivascular stromal cells. Here we generated a floxed allele of Cxcl12 to conditionally delete Cxcl12 from candidate niche cells in the bone marrow and assess the effect on HSCs. Deletion of Cxcl12 in endothelial cells and mature osteoblasts was mediated by the Tie2-Cre recombinase (Cre) and osteocalcin (Oc)-Cre transgenes, respectively. To target Cxcl12 deletion in CAR cells and osteoprogenitors, we used the osterix (Osx)-Cre transgene. Finally, to target multipotent mesenchymal progenitors, we used the Prx1-Cre transgene. Prx1 is a transcription factor expressed early during limb bud mesoderm development, and Prx1-Cre targets all cells derived from limb bud mesoderm.We first performed lineage mapping studies using transgenic mice carrying a knock-in of the green fluorescent protein (GFP) gene into the Cxcl12 locus. As reported previously, the highest CXCL12 expression was observed in CAR cells, which is a heterogeneous perivascular stromal cell population that contains osteoprogenitors. Lineage mapping with Osx- and Prx1-Cre show that both transgenes mediate efficient and equivalent recombination in nearly all mature osteoblasts, osteoblast progenitors, and CAR cells. Prx1-Cre, but not Osx-Cre, also targets a novel subset of PDGFRα+ Sca+ mesenchymal progenitors. We show that deletion of Cxcl12 from mature osteoblasts has no effect on HSC number or function. Deletion of Cxcl12 from Osx-Cre-targeted stromal cells, which includes osteoprogenitors and CAR cells, results in constitutive mobilization of hematopoietic progenitors; however, HSC number and function are normal. Cxcl12 deletion in endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 in mesenchymal progenitors is associated with a marked loss of HSCs and HSC quiescence. In addition to endothelial cells, we thus identify a novel Prx1-Cre targeted subset of mesenchymal progenitors that appears to be necessary to support HSCs. These data show that expression of CXCL12 from stromal cells in the perivascular region is required for HSC maintenance. Disclosures:No relevant conflicts of interest to declare.

G-CSF-Induced Alterations in the Bone Marrow Microenvironment Suppress B Lymphopoiesis

Abstract 1246Infectious stress is associated with a shift in the bone marrow from lymphopoiesis to granulopoiesis. Expression of granulocyte colony-stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced during infection. We previously reported that G-CSF treatment is associated with marked suppression of B lymphopoiesis in murine bone marrow. After 5 days of G-CSF treatment (250 μg/kg), total B cells in the bone marrow were reduced 8.1 ± 0.9-fold. Pre-pro-B cells were reduced 1.6 ± 0.3-fold, pro-B cells 12.4 ± 1.9-fold, pre-B cells 5.6 ± 0.8-fold, immature B cells 7.5 ± 1.2-fold, and mature naïve B cells 83 ± 7.6-fold. B-committed lymphoid progenitors (BLP) were modestly but significantly decreased (1.4 ± 0.2-fold), while common lymphoid progenitors (CLP) were not affected by G-CSF treatment. Increased apoptosis of mature naïve B cells in the bone marrow was observed. Studies of G-CSF receptor deficient (Csf3r−/−) bone marrow chimeras show that G-CSF acts in a non-cell intrinsic fashion to suppress B lymphopoiesis. Consistent with this observation, we show that G-CSF treatment results in decreased expression in the bone marrow microenvironment of multiple B-supportive factors including CXCL12, interleukin-6, interleukin-7, and B cell activating factor (BAFF). Prior studies have established that CXCL12-abundant reticular (CAR) cells in the bone marrow play a key role in B cell development. CAR cells are perivascular stromal cells that express very high levels of CXCL12 and are in direct contact with pre-pro-B cells. G-CSF treatment did not affect CAR cell number. However, RNA expression profiling of sorted CAR cells showed that expression of several genes associated with B cell development are significantly decreased by G-CSF, including CXCL12 (4.2 ± 1.5-fold). In addition to CAR cells, other stromal cells in the bone marrow express CXCL12, including osteoblasts and endothelial cells. To assess the role of CXCL12 production by each of these cell types to B lymphopoiesis, we generated Cxcl12flox mice and crossed them with mice expressing the following tissue-specific Cre-recombinase transgenes: Osteocalcin-Cre (Oc-Cre) targeting mature mineralizing osteoblasts; Osterix-Cre (Osx-Cre) targeting CAR cells and all osteolineage cells; or Prx1-Cre targeting mesenchymal progenitors and their progeny. Deletion of Cxcl12 using Oc-Cre or Osx-Cre had a similar effect on B cell development, with an isolated loss of mature naïve B cells in the bone marrow (2.7 ± 0.5 and 4.1 ± 1.7-fold, respectively). In contrast, deletion of Cxcl12 using Prx1-Cre resulted in severe suppression of B lymphopoiesis that included a loss of CLP (3.3 ± 2.0-fold), BLP (5.6 ± 4.3-fold), and pre-pro-B cells (12.4 ± 5.1-fold). Interestingly, treatment of Prx1-Cre Cxcl12flox/- mice with G-CSF resulted in additional B cell loss, indicating that deletion of Cxcl12 in mesenchymal stromal cells is not sufficient to fully recapitulate G-CSF-induced B cell suppression. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. While G-CSF-induced inhibition of CXCL12 expression from stromal cells contributes to B cell suppression, additional alterations in the microenvironment also contribute to this phenotype. Disclosures:No relevant conflicts of interest to declare.