Eduardo Vadillo

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis

Development of B-lymphoid cells is a highly ordered multi-step process that, in adult mammals, starts in bone marrow in a pool of self-renewing multipotential hematopoietic stem cells, which gradually commit to the lymphoid lineage and advance through high regulated differentiation pathways until formation of mature functional cells. Over the last few years, exceptional advances have been recorded in identifying primitive progenitors that lay the foundations of the lymphoid program while losing myeloid potential, along with patterns of transcriptional activity controlling lineage fate decisions and environmental cues that influence the differentiation pathway during normal hematopoiesis. Multicolor flow cytometry, controlled cell cultures, genetic marking systems, microarray technologies and xenotransplantation approaches are being extensively used to address fundamental questions on this regard. Of special interest is the stem cell research with relevance to hierarchy and early events in malignant lymphopoiesis, and to new insights into perspectives that may allow progress in means to protect and sustain the immune system during chemotherapy, inflammation, infection, and following hematopoietic transplantation. In this book chapter, we focus on the hierarchical structure of the early lymphoid system, the current knowledge about intrinsic and microenvironmental factors regulating the differentiation of lymphoid progenitors, and the emerging research to understand malignant lymphoid development.

Bone Marrow Cells in Acute Lymphoblastic Leukemia Create a Proinflammatory Microenvironment Influencing Normal Hematopoietic Differentiation Fates.

B-cell acute lymphoblastic leukemia (B-ALL) is a serious public health problem in the pediatric population worldwide, contributing to 85% of deaths from childhood cancers. Understanding the biology of the disease is crucial for its clinical management and the development of therapeutic strategies. In line with that observed in other malignancies, chronic inflammation may contribute to a tumor microenvironment resulting in the damage of normal processes, concomitant to development and maintenance of neoplastic cells. We report here that hematopoietic cells from bone marrow B-ALL have the ability to produce proinflammatory and growth factors, including TNFα, IL-1β, IL-12, and GM-CSF that stimulate proliferation and differentiation of normal stem and progenitor cells. Our findings suggest an apparently distinct CD13+CD33+ population of leukemic cells contributing to a proinflammatory microenvironment that may be detrimental to long-term normal hematopoiesis within B-ALL bone marrow.

Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

TLR Stimulation of Bone Marrow Lymphoid Precursors from Childhood Acute Leukemia Modifies Their Differentiation Potentials

Acute leukemias are the most frequent childhood malignancies worldwide and remain a leading cause of morbidity and mortality of relapsed patients. While remarkable progress has been made in characterizing genetic aberrations that may control these hematological disorders, it has also become clear that abnormalities in the bone marrow microenvironment might hit precursor cells and contribute to disease. However, responses of leukemic precursor cells to inflammatory conditions or microbial components upon infection are yet unexplored. Our previous work and increasing evidence indicate that Toll-like receptors (TLRs) in the earliest stages of lymphoid development in mice and humans provide an important mechanism for producing cells of the innate immune system. Using highly controlled co-culture systems, we now show that lymphoid precursors from leukemic bone marrow express TLRs and respond to their ligation by changing cell differentiation patterns. While no apparent contribution of TLR signals to tumor progression was recorded for any of the investigated diseases, the replenishment of innate cells was consistently promoted upon in vitro TLR exposure, suggesting that early recognition of pathogen-associated molecules might be implicated in the regulation of hematopoietic cell fate decisions in childhood acute leukemia.

Early Hematopoietic Differentiation in Acute Lymphoblastic Leukemia: The Interplay Between Leukemia-Initiating Cells and Abnormal Bone Marrow Microenvironment

By virtue of their self-renewal and tightly regulated multi-lineage differentiation properties, hematopoietic stem cells (HSCs) generate the whole blood system throughout postnatal life. During malignant hematological disorders, including acute leukemias, a number of intrinsic and extrinsic cues influence the hematopoietic differentiation pathway and cooperate to make aberrant cell fate decisions concomitant with cell transformation. The cellular origin of these disorders is a fundamental matter in question. In keeping with the hierarchical model of tumor evolution, a conspicuous and unique leukemic stem cell (LSC) population is most likely the foundation of acute and chronic myeloid leukemias. In contrast, all B-cell differentiation stages in acute lymphoblastic leukemia (ALL) function as leukemia-initiating cells (LICs), are endowed with primitive stem cell properties and are apparently responsible for the long-term maintenance of tumor growth within the bone marrow (BM) and for relapse of the disease following remission. Furthermore, LICs reveal the ability to create irregular BM microenvironments that may result in proinflammatory scenarios with a permissive role by allowing leukemic cell development at the expense of normal hematopoiesis. This chapter outlines the recent findings contributing to the understanding of malignant hematopoiesis through the biology of early stem and progenitor cells in the context of abnormal microenvironments within leukemic BM. By unraveling the role of leukemic precursor cells in the initiation of local inflammatory processes leading to hematopoietic instability, we may learn about additional mechanisms co-participating in the etiology and maintenance of this pathological condition.

Early Differentiation of Human CD11c

Reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem/progenitor cells within the bone marrow. Although in the last decade the use of dialyzable leukocyte extracts (DLE) as supportive therapy in both infectious and malignant settings has increased, its activity on the earliest stages of human hematopoietic development remains poorly understood. Here, we have examined the ability of DLE to promote replenishment of functional lymphoid lineages from CD34+ cells. Our findings suggest that DLE increases their differentiation toward a conspicuous CD56+CD16+CD11c+ NK-like cell population endowed with properties such as IFNy production, tumor cell cytotoxicity, and the capability of inducing γδ T lymphocyte proliferation. Of note, long-term coculture controlled systems showed the bystander effect of DLE-stromal cells by providing NK progenitors with signals to overproduce this cell subset. Thus, by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment, DLE may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional CD11c+ NK cells in a partially TLR-related manner. Unraveling the identity and mechanisms of the involved DLE components may be fundamental to advance the NK cell-based therapy field.

Newcastle Disease Virus: Potential Therapeutic Application for Human and Canine Lymphoma

Research on oncolytic viruses has mostly been directed towards the treatment of solid tumors, which has yielded limited information regarding their activity in hematological cancer. It has also been directed towards the treatment of humans, yet veterinary medicine may also benefit. Several strains of the Newcastle disease virus (NDV) have been used as oncolytics in vitro and in a number of in vivo experiments. We studied the cytolytic effect of NDV-MLS, a low virulence attenuated lentogenic strain, on a human large B-cell lymphoma cell line (SU-DHL-4), as well as on primary canine-derived B-cell lymphoma cells, and compared them to healthy peripheral blood mononuclear cells (PBMC) from both humans and dogs. NDV-MLS reduced cell survival in both human (42% ± 5%) and dog (34% ± 12%) lymphoma cells as compared to untreated controls. No significant effect on PBMC was seen. Cell death involved apoptosis as documented by flow-cytometry. NDV-MLS infections of malignant lymphoma tumors in vivo in dogs were confirmed by electron microscopy. Early (24 h) biodistribution of intravenous injection of 1 × 1012 TCID50 (tissue culture infective dose) in a dog with T-cell lymphoma showed viral localization only in the kidney, the salivary gland, the lung and the stomach by immunohistochemistry and/or endpoint PCR. We conclude that NDV-MLS may be a promising agent for the treatment of lymphomas. Future research is needed to elucidate the optimal therapeutic regimen and establish appropriate biosafety measures.

Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow

BACKGROUND AND AIMS Childhood acute leukemias (AL) are characterized by the excessive production of malignant precursor cells at the expense of effective blood cell development. The dominance of leukemic cells over normal progenitors may result in either direct suppression of functional hematopoiesis or remodeling of microenvironmental niches, contributing to BM failure and AL-associated mortality. We undertook this study to investigate the contents and functional activity of hematopoietic stem/progenitor cells (HSPC) and their relationship to immune cell production and risk status in AL pediatric patients. METHODS Multiparametric flow cytometry of BM aspirates was performed to classify AL on the basis of lineage and differentiation stages and to analyze HSPC and immune cell frequencies. Controlled co-culture systems were conducted to evaluate functional lineage potentials of primitive cells. Statistical correlations and inter-group significant differences were established. RESULTS Among 113 AL BM aspirates, 26.5% corresponded to ProB, 19.5% to PreB and 32% contain ProB and PreB differentiation stages, whereas nearly 9% of the cases were T- and 13% myeloid-lineage leukemias. We identified ProB-ALL as the subtype endowed with the highest relative contents of HSPC, whereas T-ALL and PreB-ALL showed a critically reduced size of both HSC and MLP compartments. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis at disease debut. CONCLUSIONS HSPC abundance at initial diagnosis may aid to predict the clinical course of ALL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies.