Browsing by Author "Doan, Phuong L"

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    Alphavirus Replicon Particle Vaccine Breaks B Cell Tolerance and Rapidly Induces IgG to Murine Hematolymphoid Tumor Associated Antigens.
    (Frontiers in immunology, 2022-01) Su, Hsuan; Imai, Kazuhiro; Jia, Wei; Li, Zhiguo; DiCioccio, Rachel A; Serody, Jonathan S; Poe, Jonathan C; Chen, Benny J; Doan, Phuong L; Sarantopoulos, Stefanie
    De novo immune responses to myeloid and other blood-borne tumors are notably limited and ineffective, making our ability to promote immune responses with vaccines a major challenge. While focus has been largely on cytotoxic cell-mediated tumor eradication, B-cells and the antibodies they produce also have roles in anti-tumor responses. Indeed, therapeutic antibody-mediated tumor cell killing is routinely employed in patients with hematolymphoid cancers, but whether endogenous antibody responses can be incited to blood-born tumors remains poorly studied. A major limitation of immunoglobulin therapies is that cell surface expression of tumor-associated antigen (TAA) targets is dynamic and varied, making promotion of polyclonal, endogenous B cell responses appealing. Since many TAAs are self-antigens, developing tumor vaccines that enable production of antibodies to non-polymorphic antigen targets remains a challenge. As B cell responses to RNA vaccines are known to occur, we employed the Viral Replicon Particles (VRP) which was constructed to encode mouse FLT3. The VRP-FLT3 vaccine provoked a rapid IgG B-cell response to this self-antigen in leukemia and lymphoma mouse models. In addition, IgGs to other TAAs were also produced. Our data suggest that vaccination with RNA viral particle vectors incites a loss of B-cell tolerance that enables production of anti-tumor antibodies. This proof of principle work provides impetus to employ such strategies that lead to a break in B-cell tolerance and enable production of broadly reactive anti-TAA antibodies as potential future therapeutic agents for patients with hematolymphoid cancers.
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    Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration.
    (Cell Rep, 2016-11-01) Yan, Xiao; Himburg, Heather A; Pohl, Katherine; Quarmyne, Mamle; Tran, Evelyn; Zhang, Yurun; Fang, Tiancheng; Kan, Jenny; Chao, Nelson J; Zhao, Liman; Doan, Phuong L; Chute, John P
    Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10(m/+) mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10(+/+) mice. After total body irradiation (TBI), Grb10(m/+) mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10(+/+) mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo.
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    Epidermal growth factor regulates hematopoietic regeneration after radiation injury.
    (Nat Med, 2013-03) Doan, Phuong L; Himburg, Heather A; Helms, Katherine; Russell, J Lauren; Fixsen, Emma; Quarmyne, Mamle; Harris, Jeffrey R; Deoliviera, Divino; Sullivan, Julie M; Chao, Nelson J; Kirsch, David G; Chute, John P
    The mechanisms that regulate hematopoietic stem cell (HSC) regeneration after myelosuppressive injury are not well understood. We identified epidermal growth factor (EGF) to be highly enriched in the bone marrow serum of mice bearing deletion of Bak and Bax in TIE2-expressing cells in Tie2Cre; Bak1(-/-); Bax(flox/-) mice. These mice showed radioprotection of the HSC pool and 100% survival after a lethal dose of total-body irradiation (TBI). Bone marrow HSCs from wild-type mice expressed functional EGF receptor (EGFR), and systemic administration of EGF promoted the recovery of the HSC pool in vivo and improved the survival of mice after TBI. Conversely, administration of erlotinib, an EGFR antagonist, decreased both HSC regeneration and the survival of mice after TBI. Mice with EGFR deficiency in VAV-expressing hematopoietic cells also had delayed recovery of bone marrow stem and progenitor cells after TBI. Mechanistically, EGF reduced radiation-induced apoptosis of HSCs and mediated this effect through repression of the proapoptotic protein PUMA. Our findings show that EGFR signaling regulates HSC regeneration after myelosuppressive injury.
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    Ex vivo expansion of murine and human hematopoietic stem cells.
    (Methods Mol Biol, 2014) Doan, Phuong L; Chute, John P
    Hematopoietic stem cells have the capacity to self-renew and give rise to the entirety of the mature blood and immune system throughout the lifespan of an organism. Here, we describe methods to isolate and culture murine bone marrow (BM) CD34(-)ckit(+)Sca1(+)Lineage(-) (CD34(-)KSL) hematopoietic stem cells (HSCs). We also describe a method to measure functional HSC content via the competitive repopulation assay. Furthermore, we summarize methods to isolate and culture human CD34(+)CD38(-)Lineage(-) cells which are enriched for human hematopoietic stem and progenitor cells.
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    Protein tyrosine phosphatase-σ regulates hematopoietic stem cell-repopulating capacity.
    (J Clin Invest, 2015-01) Quarmyne, Mamle; Doan, Phuong L; Himburg, Heather A; Yan, Xiao; Nakamura, Mai; Zhao, Liman; Chao, Nelson J; Chute, John P
    Hematopoietic stem cell (HSC) function is regulated by activation of receptor tyrosine kinases (RTKs). Receptor protein tyrosine phosphatases (PTPs) counterbalance RTK signaling; however, the functions of receptor PTPs in HSCs remain incompletely understood. We found that a receptor PTP, PTPσ, was substantially overexpressed in mouse and human HSCs compared with more mature hematopoietic cells. Competitive transplantation of bone marrow cells from PTPσ-deficient mice revealed that the loss of PTPσ substantially increased long-term HSC-repopulating capacity compared with BM cells from control mice. While HSCs from PTPσ-deficient mice had no apparent alterations in cell-cycle status, apoptosis, or homing capacity, these HSCs exhibited increased levels of activated RAC1, a RhoGTPase that regulates HSC engraftment capacity. shRNA-mediated silencing of PTPσ also increased activated RAC1 levels in wild-type HSCs. Functionally, PTPσ-deficient BM cells displayed increased cobblestone area-forming cell (CAFC) capacity and augmented transendothelial migration capacity, which was abrogated by RAC inhibition. Specific selection of human cord blood CD34⁺CD38⁻CD45RA⁻lin⁻ PTPσ⁻ cells substantially increased the repopulating capacity of human HSCs compared with CD34⁺CD38⁻CD45RA⁻lin⁻ cells and CD34⁺CD38⁻CD45RA⁻lin⁻PTPσ⁺ cells. Our results demonstrate that PTPσ regulates HSC functional capacity via RAC1 inhibition and suggest that selecting for PTPσ-negative human HSCs may be an effective strategy for enriching human HSCs for transplantation.