Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

Abstract Introduction Notch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development. Methods: Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis. Results: Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was >90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors. Conclsuion: The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation. Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. Disclosures Stanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.

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CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽

10.1182/blood-2019-132138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1054-1054 ◽

Cited By ~ 1

Author(s):

Hongxing Liu

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Janus Kinase ◽

Gingival Hyperplasia ◽

Hematopoietic Stem ◽

T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

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IL-7 Effects on Thymocyte Progenitors.

Blood ◽

10.1182/blood.v106.11.3318.3318 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3318-3318

Author(s):

Nahed El Kassar ◽

Baishakhi Choudhury ◽

Francis Flomerfelt ◽

Philip J. Lucas ◽

Veena Kapoor ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Notch Signaling ◽

Stromal Cells ◽

T Cell Development ◽

Fold Increase ◽

Cell Development ◽

B Cell Development ◽

Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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The Role of Bone Marrow T Cells in Regulating Hematopoietic Stem Cell Function.

Blood ◽

10.1182/blood.v120.21.2349.2349 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2349-2349

Author(s):

Claudia Brandao ◽

Alexander M. de Bruin ◽

Martijn A. Nolte

Keyword(s):

Bone Marrow ◽

T Cells ◽

Immune System ◽

T Cell ◽

Cd8 T Cells ◽

Feedback Mechanism ◽

T Cell Subsets ◽

Effector Memory ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2349 After immune activation, effector/memory T cells, including virus-specific CD8 T cells, are known to migrate to the bone marrow (BM), where they can be maintained by the production of IL-15 by the stroma; however, it is not yet known whether these T cells also have a function at this site. Since depletion of T cells from allogenic BM grafts compromises HSC engraftment, we hypothesize that T cells can directly influence the balance between differentiation and self-renewal of hematopoietic stem cells (HSCs). To test the ability of T cells to affect hematopoiesis, we performed co-cultures of HSCs and T cells isolated from murine BM. We found that T cells localized in the BM are able to enhance HSC differentiation as well as their self-renewal capacity. This feature is specific for BM central memory (CM) CD8 T cells, since other T cell subsets are not able to affect HSCs to the same extent. Moreover, depletion of CM CD8 T cells from the total BM T cell pool abrogates the impact on HSC differentiation and self-renewal, indicating that this particular T cell population is both sufficient and required for the observed effects. BM CM CD8 T cells do not affect quiescence of HSCs, but do enhance their proliferative capacity, and we found that supernatant from CM CD8 T cells is sufficient for this effect. Interestingly, competitive transplantation assays showed that HSCs cultured with CM CD8 T cells-derived supernatant contribute much better to leukocyte formation than medium-treated HSCs. This effect is seen in both the myeloid and lymphoid compartment, indicating that CM CD8 T cells are able to release soluble factors that support and enhance the multilineage reconstitution capacity of HSCs. Functional studies with blocking antibodies or knock-out mice showed that the supernatant-mediated effect is not caused by the hematopoietic cytokines IL3, IL6, IL21, GM-CSF, RANTES, TNFα or IFNγ. Preliminary data indicate that this feedback mechanism of the immune system on the hematopoietic process in the bone marrow is also present in the human situation, since autologous BM T cells increase the numbers of human HSCs, as well as their differentiation capacity. Overall, these findings demonstrate that T cells have an important function in the BM and that especially CD8 TCM cells can directly influence HSC homeostasis. We postulate that this feedback mechanism of the immune system on the hematopoietic process in the BM is particularly relevant during viral infection, as the efficient migration of virus-specific CD8 T cells to the BM could well benefit the replenishment of the HSC/progenitor cell compartment and restoration of blood cell numbers that got lost upon infection. Disclosures: No relevant conflicts of interest to declare.

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IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v120.21.1554.1554 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1554-1554

Author(s):

Lucy S. Hodge ◽

Steve Ziesmer ◽

Frank J Secreto ◽

Zhi-Zhang Yang ◽

Anne Novak ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Bone Marrow Microenvironment ◽

Waldenström’S Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Prolonged Thrombocytopenia After Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Its Association with an Increase in CD8+ CX3CR1+ Cells in Bone Marrow.

Blood ◽

10.1182/blood.v120.21.3077.3077 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3077-3077

Author(s):

Xiao-hui Zhang ◽

Guo-xiang Wang ◽

Yan-rong Liu ◽

Lan-Ping Xu ◽

Kai-Yan Liu ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Surface Expression ◽

T Cell Subsets ◽

Control Group ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Abstract 3077 Background: Since prolonged thrombocytopenia (PT) is an independent risk factor for poor clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the underlying mechanisms need to be understood in order to develop selective treatments. Previous studies1–4 have suggested that abnormalities in B cells may play a role in the pathogenesis of PT. However, abnormalities in B cells alone do not fully explain the complete pathogenic mechanisms of PT. Our previous studies5 showed that the frequency of megakaryocytes with a ploidy value ≤ 8N was significantly increased in patients who developed PT after allo-HSCT compared to the control group. Mechanisms concerning the megakaryocyte hypoplasia in PT after allo-HSCT are not well understood. Design and Methods: PT was defined as a platelet count ≤80 × 109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia, such as aGVHD, disease recurrence, CMV infection, or antiviral drug treatment at three months post-HSCT when all other blood cell counts had return to normal.5 We analyzed T cell subsets in bone marrow (BM) and peripheral blood (PB) from allo-HSCT recipients with and without PT (n = 23 and 17, respectively) and investigated the expression characteristics of homing receptors CX3CR1, CXCR4 and VLA-4 by flow cytometry. Futhermore, Mononuclear cells (MNCs) from PT patients and controls were cultured with and without autologous CD8+ T cells in vitro, and clarify the effect of activated CD8+ T cells on the ploidy and apoptosis of megakaryocytes in the bone marrow. Results: The results demonstrated that the percentage of CD3+ T cells in the BM was significantly higher in PT patients than the experimental controls (76.00 ± 13.04% and 57.49 ± 9.11%, respectively, P < 0.001), whereas this difference was not significant for the PB (71.01 ± 11.49% and 70.49 ± 12.89%, respectively, P = 0.911). While, some T cell subsets in the BM and PB from allo-HSCT recipients with PT were not significantly different from that of the experimental control group, such as CD8+ T cells, CD4+ T cells, CD4+ CD25bright T cells (regulatory T cells), CD44hi CD62Llo CD8+ T cells and naive T cells (CD11a+ CD45RA+). Furthermore, the surface expression of homing receptor CX3CR1 on BM T cells (64.16 ± 14.07% and 37.45 ± 19.66%, respectively, P < 0.001) and CD8+ T cells (56.25 ± 14.54% and 35.16 ± 20.81%, respectively, P = 0.036), but not in blood, were significantly increased in PT patients compared to controls. For these two groups of patients, the surface expression of CXCR4 and VLA-4 on T cells and CD8+ T cells from both BM and PB did not show significant differences. Through the study in vitro, we found that the activated CD8+ T cells in bone marrow of patients with PT might suppress apoptosis (MNC group and Co-culture group: 18.02 ± 3.60% and 13.39 ± 4.22%, P < 0.05, respectively) and Fas expression (MNC group and Co-culture group: 21.10 ± 3.93 and 15.10 ± 2.33, P <0.05, respectively) of megakaryocyte. In addition, megakaryocyte with a ploidy value ≤ 8N (MNC group: 40.03 ± 6.42% and 24.54 ± 4.31%, respectively, P < 0.05) was significantly increased in patients with PT compared to the control group. Conclusions: In conclusion, an increased surface expression of CX3CR1 on T cells may mediate the recruitment of CD8+ T cells into the bone marrow in patients with PT who received an allo-HSCT. Moreover, CD8+CX3CR1+ T cells, which can have significantly increased numbers in bone marrow of patients with PT, likely caused a reduction in the megakaryocyte ploidy, and suppressed megakaryocyte apoptosis via CD8+ T cell-mediated cytotoxic effect, possibly leading to impaired platelet production. Therefore, treatment targeting CX3CR1 should be considered as a reasonable therapeutic strategy for PT following allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

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Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v120.21.3465.3465 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3465-3465

Author(s):

Edyta Pawelczyk ◽

Heba A Degheidy ◽

Allison L Branchaw ◽

Kenn Holmbeck ◽

Steven R Bauer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Methyl Cellulose ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Mineral Density ◽

Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

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Ammonium Ions Derived from Spontaneous Decomposition of L-Glutamine Inhibit Lymphoid Differentiation from Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v110.11.2244.2244 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2244-2244

Author(s):

Gerald J. Spangrude ◽

Birgitta Johnson ◽

Scott Cho ◽

Xiaosong Huang ◽

L. Jeanne Pierce

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Progenitor Cells ◽

Stromal Cell ◽

T Cell Development ◽

Cell Development ◽

Ammonium Ions ◽

Hematopoietic Stem ◽

Lymphocyte Differentiation

Abstract The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

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CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Blood ◽

10.1182/blood.v116.21.1452.1452 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1452-1452

Author(s):

Tiffany Simms-Waldrip ◽

Michelle Yoonha Cho ◽

Kenneth Dorshkind ◽

Kathleen M Sakamoto

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Proliferation ◽

Research Funding ◽

Myeloid Cells ◽

Myeloid Cell ◽

Wild Type ◽

In Vivo Models ◽

Hematopoietic Stem

Abstract Abstract 1452 The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment. The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations. At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

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Notch target Hes5 ensures appropriate Notch induced T- versus B-cell choices in the thymus

Blood ◽

10.1182/blood-2007-03-079855 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2615-2620 ◽

Cited By ~ 17

Author(s):

Barbara Varnum-Finney ◽

Mari H. Dallas ◽

Keizo Kato ◽

Irwin D. Bernstein

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Notch Signaling ◽

Lower Threshold ◽

Wild Type ◽

Notch Ligand ◽

Cell Commitment ◽

B Cell Precursors

Notch signaling establishes boundaries in the thymus by inducing T-cell commitment and inhibiting a B-cell choice. Here, we show a significant 1.6-fold increased generation of B-cell precursors in thymuses from mice deficient for Notch target Hes5 compared with wild-type littermates. We further show that culture of bone marrow–derived progenitors with increasing densities of purified immobilized Notch ligand (Delta1ext-IgG) induced increased expression of Notch targets Hes1 and Hes5, and that although Hes5-deficient progenitors responded appropriately to high densities of ligand, they misread intermediate and low densities. Together, our results suggest that to ensure an appropriate outcome in the thymus in response to a lower threshold of induced Notch signaling, induction of the additional target Hes5 is required.

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