CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.

Bone marrow (BM) transfer from flagellin-treated mice has been reported to improve the survival of lethally-irradiated mice. Although the mechanism for flagellin’s antiviral and antibacterial effects have been elucidated, there remains a gap in knowledge regarding its radioprotective effects. Here, we report that flagellin treatment results in a 5-fold increase in the proliferation of Lin-Sca-1+C-Kit+(LSK) cells, a heterogeneous stem and multipotent cell population in BM, with the most striking increase within the ST-HSC, MPP2 and MPP3 subpopulations. Furthermore, the presence of TLR5 but not NLRC4 on radiosensitive, non-LSK cells in BM was both sufficient and necessary for the observed phenomenon. Finally, adoptive transfer of MPP3 cells along with an insufficient amount of whole bone marrow cells (WBM) to lethally-irradiated mice significantly improved their survival, recapitulating the effects of Whole bone marrow from flagellin-treated mice.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences. / 2024-11-01

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13159号 / 論医博第2146号 / 新制||医||1029(附属図書館) / (主査)教授 河本 宏, 教授 前川 平, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis (Ph. D.)--Indiana University, 2008. / Title from screen (viewed June 24, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Maureen Harrington. Includes vita. Non-Latin script record. Includes bibliographical references (leaves 126-132).

Embryonic stem (ES) cells possess the remarkable ability to form cells and tissues from all three germ layers, a characteristic known as pluripotency. In particular, the generation of ES cell-derived hematopoietic cells could serve as an alternate source of hematopoietic stem cells for transplantation in place of bone marrow cells, which are limited by donor availability and high immunogenicity. The advantages of ES cell-derived hematopoietic cells over bone marrow cells include a greater proliferative capacity, which alleviates the problems of donor shortage, and low level expression of MHC antigens, which suggests immune privilege. However, it is unclear whether the immune system is capable of recognizing and rejecting ES cell-derived hematopoietic cells following transplantation. The observation that ES cell-derivatives express low levels of MHC class I, the predominant inhibitory ligand for NK cells, led us to hypothesize that ES cell-derived hematopoietic progenitor cells (HPC) are susceptible to NK cell-mediated killing. To test this hypothesis, we first generated HPCs from murine ES cells ectopically expressing HOXB4, a homeobox transcription factor that confers hematopoietic self-renewal, and confirmed that HPCs expressed low levels of MHC class I antigens. To specifically investigate the role of NK cells in regulating the in vivo engraftment of HPCs, we transplanted NK-replete Rag2-/- or NK-deficient Rag2-/-γc-/- mice with HPCs. We observed permanent HPC engraftment in Rag2-/-γc-/- mice; however, HPC engraftment was significantly reduced in Rag2-/- mice and was eventually eliminated over time. Bone marrow harvested from these animals showed that HPC-derived Lin-c-kit+ and Lin-Sca-1+ progenitor cells, critical progenitor cells for long-term hematopoietic engraftment, were deleted in Rag2-/- but not in Rag2-/-γc-/- mice. Next, we focused on the mechanism of NK cell activation by HPCs. Increased expression of the cytotoxic proteins Granzyme B and Perforin in the NK cells of HPC-transplanted Rag2-/- mice confirmed in vivo NK cell activation. Phenotypic analysis of HPCs revealed high level expression of H60, a ligand of the NK activating receptor NKG2D, and neutralization of H60 rescued HPCs from NK cell-mediated killing. Altogether, our results demonstrate that NK cells are a major barrier to the successful engraftment of ES cell-derived hematopoietic cells, underlining an important role of the innate immune system in regulating the long-term engraftment of ES cell derivatives.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

<p>Immunodeficient-causing diseases such as HIV and leukemia have no cures, often require meticulous treatments and result in high morbidity or mortality. Although bone marrow transplants are an option for a subset of leukemia patients, the shortage of donors and the requirement for donor matching restricts the efficacy of this treatment option. Therefore there is a prominent clinical need for alternative sources of hematopoietic stem/progentior cells with lymphopoietic potential. Recently we described the direct conversion of human dermal fibroblasts to multilineage hematopoietic progenitors by ectopic expression of OCT4. This direct conversion method was used to assess whether OCT4-transduced fibroblasts had the capacity to derive cells of the lymphoid lineage. This work shows the transient co-expression of CD34 and CD45 of fibroblasts within 7 days of OCT4 transduction followed by stable expression of CD45 on fibroblasts by day 15. The acquisition of hematopoietic markers, however, did not coincide with colony formation as previously described. Furthermore, CD45+ cells that were enriched and cultured in hematopoietic conducive conditions did not acquire co-expression of CD34 as previously shown. Interestingly, CD34 expression was shown to be inversely correlated with OCT4 expression. Therefore the constitutive expression of OCT4 may have (1) inhibited the acquisition of CD34 expression on CD45+ cells (2) downregulated the expression of CD34 on the day 7 CD34+CD45+ fibroblasts, thereby resulting in the transient expression of these markers. Furthermore, this work shows that expression of CD45 on OCT4-transduced fibroblasts is required for survival on the MS5 stromal cell line used to support hematopoietic progenitors with lymphopoietic potential, while supplementation of CD45+ fibroblasts with hematopoietic progenitor supportive conditions resulting in co-expression of CD34 and CD45 is required for acquisition of CD19, a pan-B cell marker on CD45+ fibroblasts. These findings suggest OCT4-transduced fibroblasts have lymphopoietic potential.</p> / Master of Science (MSc)

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.