Regulatory T Cell Amelioration of Graft-versus-Host Disease following Allogeneic/Xenogeneic Hematopoietic Stem Cell Transplantation Using Mobilized Mouse and Human Peripheral Blood Donors.

The present studies examined experimental transplant outcomes using mobilized peripheral blood from mice and humans together with FoxP3+Treg cells. Donor mice were treated with filgrastim and / or plerixafor and their peripheral blood (PB) displayed significant elevations in hematopoietic stem and progenitor populations. Some of these PB donors were concurrently administered a Treg expansion strategy consisting of a TL1A-Ig fusion protein low dose rIL-2. A significant increase (4-5x) in the frequency Tregs occurred during mobilization. C3H.SW PB was collected from mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into MHC-matched B6 (H2b) recipients. Recipients of TrEM, exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients against leukemia levels. Next, huTregs (CD4+CD25+CD127lo) from a healthy human PB mobilized donor were expanded ex-vivo prior to transplant into NSG/ NOD-scid IL2Rgammanull mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of lethality and clinical xGVHD scores. Notably, post-transplant, PB huTregs levels remained elevated and the frequency of huCD4+Tconv and CD8+ cells was diminished supporting the improved xGVHD outcomes. These findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced GVHD following "MUD" and MHC-mismatched mouse HSCT without loss of GVL activity. Moreover, utilizing ex-vivo expanded huTregs from a mobilized PB donor and added back to donor PB ameliorated xGVHD. In total, these studies support the notion that in vivo or ex-vivo manipulation of donor Tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes.

Hematopoietic Stem Cell Heterogeneity.

Hematopoietic stem cells (HSCs) maintain lifelong production of mature blood cells and regenerate the hematopoietic system after cytotoxic injury. Use of expanding cell surface marker panels and advanced functional analyses have revealed the presence of several immunophenotypically different HSC subsets with distinct self-renewal and repopulating capacity and bias toward selective lineage differentiation. This chapter summarizes current understanding of the phenotypic and functional heterogeneity within the HSC pool, with emphasis on the immunophenotypes and functional features of several known HSC subsets, and their roles in steady-state and emergency hematopoiesis, and in aging. The chapter also highlights some of the future research directions to elucidate further the biology and function of different HSC subsets in health and disease states.

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

New therapeutic approaches for protecting hematopoietic stem cells in aplastic anemia.

Aplastic anemia (AA) is an immune-mediated and life-threatening form of acquired bone marrow failure (BMF), characterized by development and expansion of self-reactive T cells. These T cells cause continuous destruction of hematopoietic stem cells (HSCs), progenitors, and mature blood cells, leading to severe and if left untreated fatal marrow hypoplasia and pancytopenia. Standard treatment options for patients with AA include: (1) immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine A which targets self-reactive T cells, or (2) matched sibling or unrelated BM transplant (BMT). The IST treatment is often not effective due to poor response to therapy or disease relapse after IST. Also, BMT is not an option for many patients due to their age, comorbidities, and the lack of histocompatible donor. This necessitates development and testing of novel approaches to reduce severity of AA and to efficiently treat patients with refractory and relapsed AA. Immune-mediated AA was reproduced in animals, including mouse lymphocyte infusion models, which are used to study further etiology and pathophysiology of AA and test new drugs and approaches in treating and managing AA. In these mouse models the immune correlates and pathologic features of AA are strikingly similar to features of severe human AA. In this article we (a) briefly review standard and developing approaches for treating AA and (b) describe development and testing of novel treatment approach with a potential to safely reduce BM hypoplasia and significantly decrease the loss of HSCs in mouse lymphocyte infusion model of AA.

Hematopoietic progenitor cell regulation by CD4+CD25+ T cells.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) possess the capacity to modulate both adaptive and innate immune responses. We hypothesized that Tregs could regulate hematopoiesis based on cytokine effector molecules they can produce. The studies here demonstrate that Tregs can affect the differentiation of myeloid progenitor cells. In vitro findings demonstrated the ability of Tregs to inhibit the differentiation of interleukin-3 (IL-3)/stem cell factor (colony-forming unit [CFU]-IL3)-driven progenitor cells. Inhibitory effects were mediated by a pathway requiring cell-cell contact, major histocompatibility complex class II expression on marrow cells, and transforming growth factor-beta. Importantly, depletion of Tregs in situ resulted in enhanced CFU-IL3 levels after bone marrow transplantation. Cotransplantation of CD4(+)FoxP3(+)(gfp) Tregs together with bone marrow was found to diminish CFU-IL3 responses after transplantation. To address the consequence of transplanted Tregs on differentiated progeny from these CFU 2 weeks after hematopoietic stem cell transplantation, peripheral blood complete blood counts were performed and examined for polymorphonuclear leukocyte content. Recipients of cotransplanted Tregs exhibited diminished neutrophil counts. Together, these findings illustrate that both recipient and donor Tregs can influence hematopoietic progenitor cell activity after transplantation and that these cells can alter responses outside the adaptive and innate immune systems.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

OBJECTIVE: Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. MATERIALS AND METHODS: EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. RESULTS: The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. CONCLUSIONS: These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors.

OBJECTIVE: FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. MATERIALS AND METHODS: FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. RESULTS: Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. CONCLUSIONS: The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.

Murine hematopoietic stem cells and multipotent progenitors express truncated intracellular form of c-kit receptor.

The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors.

Noncanonical Wnt signaling promotes apoptosis in thymocyte development.

The Wnt-beta-catenin signaling pathway has been shown to govern T cell development by regulating the growth and survival of progenitor T cells and immature thymocytes. We explore the role of noncanonical, Wnt-Ca(2+) signaling in fetal T cell development by analyzing mice deficient for Wnt5a. Our findings reveal that Wnt5a produced in the thymic stromal epithelium does not alter the development of progenitor thymocytes, but regulates the survival of alphabeta lineage thymocytes. Loss of Wnt5a down-regulates Bax expression, promotes Bcl-2 expression, and inhibits apoptosis of CD4(+)CD8(+) thymocytes, whereas exogenous Wnt5a increases apoptosis of fetal thymocytes in culture. Furthermore, Wnt5a overexpression increases apoptosis in T cells in vitro and increases protein kinase C (PKC) and calmodulin-dependent kinase II (CamKII) activity while inhibiting beta-catenin expression and activity. Conversely, Wnt5a deficiency results in the inhibition of PKC activation, decreased CamKII activity, and elevation of beta-catenin amounts in thymocytes. These results indicate that Wnt5a induction of the noncanonical Wnt-Ca(2+) pathway alters canonical Wnt signaling and is critical for normal T cell development.

Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue.

Wnt5a is a member of the Wnt family of secreted glycoproteins that play essential organizing roles in development. Similar to other Wnt members, Wnt5a can upregulate cell proliferation and has been proposed to have oncogenic function. Here we report that Wnt5a signals through the noncanonical Wnt/Ca++ pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation in a cell-autonomous manner. Wnt5a hemizygous mice develop myeloid leukemias and B cell lymphomas that are clonal in origin and display loss of Wnt5a function in tumor tissues. Furthermore, analysis of human primary leukemias reveals deletion of the WNT5A gene and/or loss of WNT5A expression in a majority of the patient samples. These results demonstrate that Wnt5a suppresses hematopoietic malignancies.

The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function?

Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.

Mouse Pum1 and Pum2 genes, members of the Pumilio family of RNA-binding proteins, show differential expression in fetal and adult hematopoietic stem cells and progenitors.

Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.

Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing.

Engraftment failure following allogeneic bone marrow (BM) transplantation is of clinical concern particularly involving T-cell-depleted inoculum and transplantations for aplastic anemia. Immune resistance by lymphoid and natural killer (NK) populations with "barrier" function is well established. Major histocompatibility complex (MHC)-identical marrow allografts were examined to investigate effector pathways in non-NK-mediated resistance. Barrier function was examined in cytotoxic normal and deficient B6 (H-2(b)) recipients primed to donor minor histocompatibility antigen (MiHA) prior to BM transplantation. Host resistance was sensitively evaluated by colony-forming unit (CFU) assays to directly assess for donor progenitor cell (PC) and peripheral chimerism. B6 host CD8(+) T cells but not CD4(+) or NK1.1(+) cells effected rejection of primitive (CFU-HPP [high-proliferative potential]) and lineage-committed (CFU-IL3/GM [interleukin 3/granulocyte macrophage]) allogeneic donor progenitors. To address complementation by the cytotoxic pathways existing in singly deficient (perforin or FasL) recipients, cytotoxically double (perforin plus FasL) deficient (cdd) recipients were used. Resistance in B6-cdd recipients was comparable to that of wild-type B6 recipients and was also dependent on CD8(+) T cells. A "triple" cytotoxic deficient model, involving transplantation of TNFR1(-/-) (tumor necrosis factor receptor 1) progenitor grafts did not diminish the ability of B6-cdd recipients to reject allografts. Finally, injection of anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) monoclonal antibody (mAb) in B6-cdd recipients also failed to inhibit rejection of TNFR1(-/-) marrow grafts. In total, these studies demonstrate that CD8(+) host T cells can effectively resist MHC-matched MiHA-mismatched donor PCs via alternative effector pathway(s) independent of perforin-, FasL-, TNFR-1-, and TRAIL-dependent cytotoxicity. Therefore, inhibition of these effector pathways in sensitized recipients is unlikely to result in stem cell engraftment following PC allografts.

Cloning and comparative sequence analysis of PUM1 and PUM2 genes, human members of the Pumilio family of RNA-binding proteins.

Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.

The dual specificity JKAP specifically activates the c-Jun N-terminal kinase pathway.

The involvement of dual specificity phosphatases (DSPs) in the mitogen-activated protein kinase (MAPK) signaling has been mostly limited to the inactivation of MAPKs by the direct dephosphorylation of the TXY motif within their activation loop. We report the cloning and characterization of a murine DSP, called JNK pathway-associated phosphatase (JKAP), which lacks the regulatory region present in most other MAP kinase phosphatases (MKPs) and is preferentially expressed in murine Lin(-)Sca-1(+) stem cells. Overexpression of JKAP in human embryonic kidney 293T cells specifically activated c-Jun N-terminal kinase (JNK) but not p38 and extracellular signal-regulated kinase 2. Overexpression of a mutant JKAP, JKAP-C88S, blocked tumor necrosis factor-alpha-induced JNK activation. Targeted gene disruption in murine embryonic stem cells abolished JNK activation by tumor necrosis factor-alpha and transforming growth factor-beta, but not by ultraviolet-C irradiation, indicating that JKAP is necessary for optimal JNK activation. JKAP associated with JNK and MKK7, but not SEK1, in vivo. However, JKAP did not interact with JNK in vitro, suggesting that JKAP exerts its effect on JNK in an indirect manner. Taken together, these studies identify a positive regulator for the JNK pathway and suggest a novel role for DSP in mitogen-activated protein kinase regulation.