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Acute thrombocytopenia induces activation of long-term hematopoietic stem cells and leads to multipotent progenitor’s exhaustion
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Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 knock-out mice delayed leukemic burden. These results place Gaq as an important target for antileukemic strategies to overcome receptor tyrosine kinase resistance mechanisms.
3057 - MECHANISM OF LONG-TERM ROBUST HUMAN HSC ENGRAFTMENT NSGW41 MICE Susann Rahmig, Nicole Mende, and Claudia Waskow Regeneration in Hematopoiesis, Institute for Immunology, TU Dresden, Dresden, Germany Xenotransplantation models enable the in-depth analysis of human hematopoietic stem cell (HSC) function in vivo.However, human HSC engraftment and self-renewal are limited in xenotransplantation settings restricting the study of mechanisms regulating human HSC function. By introducing a loss-of-function KITreceptor into NOD/SCID Il2rg-/- (NSG) micewe created a novel mouse strain, NOD/SCID Il2rg-/- KitW41/W41 (NSGW41), which combines an impaired endogenous HSC compartment with immunodeficiency. This mouse strain efficiently supports stable long-term engraftment of human HSCs without the need for conditioning and shows highly improved multilineage engraftment, including the myeloid and megakaryocyteerythroid lineages. Thus, we postulate that stable human HSC engraftment is a major prerequisite for the continuous differentiation of all hematopoietic lineages. To investigate whether self-renewal und functionality of engrafted human HSCs is conserved in NSGW41 mice we transplanted titrated numbers of human HSCs in primary and secondary recipients and could show enhanced pick-up, maintenance and expansion of human HSCs in vivo compared to conventional KIT-proficient NSG mice. We hypothesized that endogenous HSCs with a defective KIT receptor have a competitive disadvantage compared to human KIT-proficient HSCs and thus allow for their stable engraftment. In fact, in NSGW41 mice the endogenous HSC pool is significantly decreased after engraftment of human HSPCs, supporting our hypothesis that endogenous HSCs are actively replaced from their niche. Analysis of the murine bone marrow niche revealed a significant expansion of mesenchymal stromal cells (MSCs) after transplantation of human cells. The increase in MSCs strictly correlated with the number of engrafted human HSCs, suggesting that human donor HSCs modulate their niche cells. The transcriptional profile of murine MSCs is also strongly altered after humanization, favoring the expression of genes involved in blood formation and differentiation. We suggest that MSCs provide a niche for human HSCs in mice and that human HSCs actively modify the murine bone marrow niche after xenotransplantation.
3056 - ACUTE THROMBOCYTOPENIA INDUCES ACTIVATION OF LONG-TERM HEMATOPOIETIC STEM CELLS AND LEADS TO MULTIPOTENT PROGENITOR’S EXHAUSTION Be ata Ramasz, Tatyana Grinenko, and Ben Wielockx TU Dresden Medical Faculty, Dresden, Germany Long term hematopoietic stem cells (LT-HSCs) have a limited contribution to blood production during adulthood whereas multipotent progenitors (MPP) drive steadystate hematopoiesis. However, little is known about the role of the different HSC/progenitor populations under mild stress. We used an acute, specific platelet depletion model and found that, LT-HSCs (Lin- Sca-1+c-Kit+ CD48- CD150+) and MPP2 (Lin- Sca-1+c-Kit+ CD48+ CD150+), but not MPP3/4 (Lin-Sca-1+cKit+CD48+CD150-) proliferate in response to thrombocytopenia already after 12h. After transplantation into lethally irradiated mice the MPP2 gave rise to donor platelets significantly faster than LT-HSC or MPP3/4. Acute thrombocytopenia did not influence the repopulation capacity of LT-HSC and MPP3/4, but only led to a dramatic decrease of platelet production by MPP2. To further characterize these populations we found that CD41- LT-HSCs and CD41- MPP2 show faster and stronger activation of proliferation than their respective CD41+ cells. Activation of these particular cells is accompanied with the activation of Stat5 and Erk1,2 signaling pathways. These changes are not dependent on increased concentration of well-known thrombopoietic stimulators, including TPO and IL-6. Together, our data show that acute thrombocytopenia stimulates proliferation of CD41- LT-HSC and MPP2 which leads to the exhaustion of the progenitor pool.
3058 - RANDOMIZED PHASE II TRIAL OF COMBINATION IDIOTYPE VACCINE AND ANTI-CD3/ANTI-CD28 COSTIMULATED AUTOLOGOUS T CELLS IN PATIENTS WITH MULTIPLE MYELOMA POSTAUTOTRANSPLANTATION Muzaffar Qazilbash1, Edward Stadtmauer2, Veera Baladandayuthapani1, Beryl Tross1, Medhavi Honhar1, Soungchul Cha1, Kunhwa Kim3, Sheetal Rao1, Michael Popescu1, Nina Shah4, Qaiser Bashir1, Krina Patel1, Elizabeth Shpall1, Donna Weber1, Sheeba Thomas1, Jatin Shah1, Robert Orlowski1, Naseem Kerr5, Alfred Garfall2, Adam Cohen2, Karen Dengel5, Carl June2, Richard Champlin1, and Larry Kwak6 1 UT MD Anderson Cancer Center, Houston, United States; 2Perelman School of Medicine University of Pennsylvania, Philadelphia, United States; 3Johns Hopkins School of Medicine, Baltimore, United States; 4Univeristy of California San Francisco, San Francisco, United States; 5University of Pennsylvania Center for Cellular Immunotherapies, Philadelphia, United States; 6City of Hope, Durate, United States
Background: Despite major advances in the treatment of multiple myeloma (MM) only a minority of patients achieve long-term disease control. Immunotherapy combined with autologous hematopoietic stem cell transplantation (auto-HCT) may reduce relapse rates. Immunoglobulin idiotype (Id) conjugated with a carrier protein, Keyhole limpet hemocyanin (KLH), is a tumor-specific antigen in MM. Vaccine-primed, anti-CD3/anti-CD28 costimulated adoptive T-cell transfer can augment humoral and cellular immune responses to vaccination despite cytotoxic therapy. We hypothesized that Id-KLH vaccine + the vaccineprimed costimulated T cells will result in a robust Id-specific humoral and cellular response, compared to a control vaccine (KLH only). Methods: In this randomized, phase II trial, eligible patients were randomized 1:1 to receive either Id-KLH vaccine or KLH-only vaccine, followed by auto-HCT, and then vaccine- primed costimulated T cells followed by two booster doses of the vaccine they were randomized to. Results: A total of 36 patients were enrolled between 1/2013 and 5/2015: 16 (44%) to Id-KLH and 20 (55%) to KLH-only. There was no significant difference between the two groups in patient characteristics. No infusion reactions or dose-limiting toxicity was seen in either arm. Five (31%) and 3 (15%) patients achieved complete remission (CR) by day+180 in the Id-
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 knock-out mice delayed leukemic burden. These results place Gaq as an important target for antileukemic strategies to overcome receptor tyrosine kinase resistance mechanisms.
3057 - MECHANISM OF LONG-TERM ROBUST HUMAN HSC ENGRAFTMENT NSGW41 MICE Susann Rahmig, Nicole Mende, and Claudia Waskow Regeneration in Hematopoiesis, Institute for Immunology, TU Dresden, Dresden, Germany Xenotransplantation models enable the in-depth analysis of human hematopoietic stem cell (HSC) function in vivo.However, human HSC engraftment and self-renewal are limited in xenotransplantation settings restricting the study of mechanisms regulating human HSC function. By introducing a loss-of-function KITreceptor into NOD/SCID Il2rg-/- (NSG) micewe created a novel mouse strain, NOD/SCID Il2rg-/- KitW41/W41 (NSGW41), which combines an impaired endogenous HSC compartment with immunodeficiency. This mouse strain efficiently supports stable long-term engraftment of human HSCs without the need for conditioning and shows highly improved multilineage engraftment, including the myeloid and megakaryocyteerythroid lineages. Thus, we postulate that stable human HSC engraftment is a major prerequisite for the continuous differentiation of all hematopoietic lineages. To investigate whether self-renewal und functionality of engrafted human HSCs is conserved in NSGW41 mice we transplanted titrated numbers of human HSCs in primary and secondary recipients and could show enhanced pick-up, maintenance and expansion of human HSCs in vivo compared to conventional KIT-proficient NSG mice. We hypothesized that endogenous HSCs with a defective KIT receptor have a competitive disadvantage compared to human KIT-proficient HSCs and thus allow for their stable engraftment. In fact, in NSGW41 mice the endogenous HSC pool is significantly decreased after engraftment of human HSPCs, supporting our hypothesis that endogenous HSCs are actively replaced from their niche. Analysis of the murine bone marrow niche revealed a significant expansion of mesenchymal stromal cells (MSCs) after transplantation of human cells. The increase in MSCs strictly correlated with the number of engrafted human HSCs, suggesting that human donor HSCs modulate their niche cells. The transcriptional profile of murine MSCs is also strongly altered after humanization, favoring the expression of genes involved in blood formation and differentiation. We suggest that MSCs provide a niche for human HSCs in mice and that human HSCs actively modify the murine bone marrow niche after xenotransplantation.
3056 - ACUTE THROMBOCYTOPENIA INDUCES ACTIVATION OF LONG-TERM HEMATOPOIETIC STEM CELLS AND LEADS TO MULTIPOTENT PROGENITOR’S EXHAUSTION Be ata Ramasz, Tatyana Grinenko, and Ben Wielockx TU Dresden Medical Faculty, Dresden, Germany Long term hematopoietic stem cells (LT-HSCs) have a limited contribution to blood production during adulthood whereas multipotent progenitors (MPP) drive steadystate hematopoiesis. However, little is known about the role of the different HSC/progenitor populations under mild stress. We used an acute, specific platelet depletion model and found that, LT-HSCs (Lin- Sca-1+c-Kit+ CD48- CD150+) and MPP2 (Lin- Sca-1+c-Kit+ CD48+ CD150+), but not MPP3/4 (Lin-Sca-1+cKit+CD48+CD150-) proliferate in response to thrombocytopenia already after 12h. After transplantation into lethally irradiated mice the MPP2 gave rise to donor platelets significantly faster than LT-HSC or MPP3/4. Acute thrombocytopenia did not influence the repopulation capacity of LT-HSC and MPP3/4, but only led to a dramatic decrease of platelet production by MPP2. To further characterize these populations we found that CD41- LT-HSCs and CD41- MPP2 show faster and stronger activation of proliferation than their respective CD41+ cells. Activation of these particular cells is accompanied with the activation of Stat5 and Erk1,2 signaling pathways. These changes are not dependent on increased concentration of well-known thrombopoietic stimulators, including TPO and IL-6. Together, our data show that acute thrombocytopenia stimulates proliferation of CD41- LT-HSC and MPP2 which leads to the exhaustion of the progenitor pool.
3058 - RANDOMIZED PHASE II TRIAL OF COMBINATION IDIOTYPE VACCINE AND ANTI-CD3/ANTI-CD28 COSTIMULATED AUTOLOGOUS T CELLS IN PATIENTS WITH MULTIPLE MYELOMA POSTAUTOTRANSPLANTATION Muzaffar Qazilbash1, Edward Stadtmauer2, Veera Baladandayuthapani1, Beryl Tross1, Medhavi Honhar1, Soungchul Cha1, Kunhwa Kim3, Sheetal Rao1, Michael Popescu1, Nina Shah4, Qaiser Bashir1, Krina Patel1, Elizabeth Shpall1, Donna Weber1, Sheeba Thomas1, Jatin Shah1, Robert Orlowski1, Naseem Kerr5, Alfred Garfall2, Adam Cohen2, Karen Dengel5, Carl June2, Richard Champlin1, and Larry Kwak6 1 UT MD Anderson Cancer Center, Houston, United States; 2Perelman School of Medicine University of Pennsylvania, Philadelphia, United States; 3Johns Hopkins School of Medicine, Baltimore, United States; 4Univeristy of California San Francisco, San Francisco, United States; 5University of Pennsylvania Center for Cellular Immunotherapies, Philadelphia, United States; 6City of Hope, Durate, United States
Background: Despite major advances in the treatment of multiple myeloma (MM) only a minority of patients achieve long-term disease control. Immunotherapy combined with autologous hematopoietic stem cell transplantation (auto-HCT) may reduce relapse rates. Immunoglobulin idiotype (Id) conjugated with a carrier protein, Keyhole limpet hemocyanin (KLH), is a tumor-specific antigen in MM. Vaccine-primed, anti-CD3/anti-CD28 costimulated adoptive T-cell transfer can augment humoral and cellular immune responses to vaccination despite cytotoxic therapy. We hypothesized that Id-KLH vaccine + the vaccineprimed costimulated T cells will result in a robust Id-specific humoral and cellular response, compared to a control vaccine (KLH only). Methods: In this randomized, phase II trial, eligible patients were randomized 1:1 to receive either Id-KLH vaccine or KLH-only vaccine, followed by auto-HCT, and then vaccine- primed costimulated T cells followed by two booster doses of the vaccine they were randomized to. Results: A total of 36 patients were enrolled between 1/2013 and 5/2015: 16 (44%) to Id-KLH and 20 (55%) to KLH-only. There was no significant difference between the two groups in patient characteristics. No infusion reactions or dose-limiting toxicity was seen in either arm. Five (31%) and 3 (15%) patients achieved complete remission (CR) by day+180 in the Id-