Lymphoid Cells

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Lymphoid Cells Una Chen International Senior Professional Institute (ISPI e.V.), Giessen, Germany

INTRODUCTION For researchers interested in cell-based therapy, it is a great challenge to learn how to expand lymphoid cells and their precursor cells ex vivo under defined conditions. Lymphocytes are defined by their cell surface receptor e BCR (B Cell Receptor, or immunoglobulin, Ig) for B cells and TCR (T Cell Receptor) for T cells. Precursor B cells bear a pre-B receptor, which contains lambda 5 in mice (‘physi’ in humans), and precursor T cells bear a pre-TCR-alpha. No biochemically and genetically defined surface receptors for the progenitors of lymphoid precursors have been reported. It is not entirely clear how committed stem cells differentiate into lymphoid cells, which then mature into effector cells. Many theories have been postulated. Differentiation and maturation involve both antigen-independent and antigen-dependent processes. For the first process, I will explore the sequential commitment model proposed in references [1,2]. For my treatment of the second process, I am indebted to many colleagues who helped me to summarize current views. Ligand-receptor interactions must play an important role in causing lymphoid cells to proliferate and differentiate. The term ligand means all external signals, including those provided by stroma cells and cytokines during the antigen-independent stage and those provided by Ag (antigens) and APCs (antigen-presenting cells), or accessory cells during the antigen-dependent stage. The process of turning on and turning off transcription factors at each stage of differentiation as a consequence of ligand-receptor interaction is one of the key elements controlling both the phenotype and the function of cells. Transcription factors known to be important at various stages of lymphopoiesis, due to the notion that the growth of lymphoid (precursor) cells can be manipulated at will by using genetic tools, have been discussed in the last three editions. There are examples of expansion of normal lymphoid (precursor) cells in culture. All are based on the support of stroma cell lines and cytokines. On removal of these elements, cells either differentiate toward terminal effector cells or die. Recently, the post transcriptional regulations of various pathways are shown to be conducted by the miRNAs (micro RNAs), which are the regulators of networks. I have arbitrarily defined seven stages of lymphopoiesis to discuss in detail. For each one, I discuss the available cellular and molecular markers, the current understanding of cellular phenotype, the potential of these cells to expand ex vivo, and if possible, what could be done in the future. Both somatic and genetic manipulations are possible in animal models. By combining modern cellular and molecular technologies and available mutant mice, one should be able to grow normal lymphoid (precursor) cells at every stage of lymphopoiesis. Despite the efforts of many, human stroma cell lines that can reproducibly support the growth of different stages of lymphoid (precursor) cells are still under development. The inducible Principles of Tissue Engineering. http://dx.doi.org/10.1016/B978-0-12-398358-9.00050-1 Copyright Ó 2014 Elsevier Inc. All rights reserved.

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PART 12 Hematopoietic System regulation of stage-specific and lymphoid-specific transcription and post-transcriptional factors in controlling the growth of normal lymphoid cells seems to be the key element. The aim of this chapter is to help the reader to understand lymphocytes and their precursors. The intention of research is to learn how these cells might be manipulated to make them useful in cell-based, somatic gene/molecular therapy. I attempt to address the possibility of growing normal lymphoid cells and their stem cells e of mouse and human origin e in a controllable manner. That is, cells should proliferate ex vivo without becoming malignantly transformed and with little or no differentiation. Three main types of stem cells can be distinguished: totipotent, pluripotent, and multipotent. Totipotent and pluripotent stem cells usually divide symmetrically to give rise to two daughter stem cells, with the same properties and identical phenotype to their parent. Under appropriate conditions, totipotent and pluripotent stem cells can differentiate into any other form of stem cell. The known stem cell lines that seem to be close to totipotent are ESCs (embryonic stem cells) and, more recently, iPCs (induced pluripotent cells). Multipotent stem cells, which exist for the lifetime of an organism, undergo primarily asymmetric divisions. One daughter cell is another stem cell like its parent, whereas the other daughter cell is a more highly differentiated cell that performs a tissue-specific function. Due to this unique property, multipotent stem cells are ideal vehicles for cell-based, somatic, gene/molecular therapy. They will carry the transgene for the lifetime of the organism, and they will maintain expression of the transgene in the differentiated cells that they spawn.

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Both lymphoid and lymphoid precursor cells are the progeny of HSCs (hematopoietic stem cells). One of the main tasks here is to review whether lymphoid cells and their progenitors possess stem cell-like properties. Are they self-renewing? And with available culture conditions and technology, can they expand ex vivo under controllable growth conditions? Unlimited growth of lymphoid cells and their progenitors is well documented. Lymphoid leukemia cells develop either spontaneously or after infection with viruses. These cell lines are useful for studying lymphoid cells but not for cell-based therapy, because they develop tumors when reintroduced into the organism. Thus, this chapter is devoted to exploring the possibility of growing normal lymphoid cells that can be engineered in culture and reimplanted into syngeneic or autologous hosts for therapeutic purposes. Other mouse normal cell types, expandable at will using genetic approaches, are available: tet-on (tetracyclineinducible) glial stem cells, mesenchymal stem cells, tet-off (tetracycline-regulated) ectodermal progenitor cells, lung stem cells, as well as tet-on and tet-off pancreatic beta islet cells, summarized in Chen [3].

PROPERTIES OF LYMPHOCYTES Lymphoid precursor cells are bipotential progenitors of pre-T and pre-B cells. The development of lymphoid precursor cells is independent of the presence of Ag (Antigen). It is controversial whether lymphoid precursor cells divide asymmetrically with self-renewal. Lymphocytes and their precursors are similar to cells of other lineages, in that they proliferate, differentiate, migrate, communicate with other cells, age, and die. However, lymphocytes also possess the following unique properties that distinguish them from other cells: 1) 2) 3) 4) 5) 6) 7)

Formation of receptor genes by VDJ recombination, Requirement for a thymus-like environment to generate mature CD4þ/8þ T cells, Requirement for somatic education and selection by Ag, Somatic hypermutation to generate more diversity in B cells, Immunologic memory, Ig heavy-chain CSR (Class Switch Recombination) in B cells, Kappa light-chain editing somatically to generate new specificities of B cell receptor,

CHAPTER 50 Lymphoid Cells 8) Secretion of various cytokines by CD4þT helper cells, Treg (T regulatory) cells, in communicating and regulating other lymphoid cell populations [4]. Development of techniques to expand ex vivo, to manipulate genetically, and to re-implant lymphocytes and their precursors into host animals requires lymphocyte engineers.

LYMPHOCYTE ENGINEERING: REALITY AND POTENTIAL Two models describe the sequence of cell commitment during lymphopoiesis: stochastic and inductive. In this section, I use an inductive model in an attempt to explain lymphocyte commitment during development of the organism and in the adult.

Inductive model of sequential cell commitment of hematopoiesis This model is divided into two parts. The first part deals with the Ag-independent stage, starting with the differentiation from the null cells (fertilized egg to ESCs, or iPS cells) to mesoderm and then to lymphoid precursor cells. A significant portion of the first part of this model was expanded from Bailey’s hypothesis of developmental progress [4a] and modified according to Brown et al. [1,2].

ANTIGEN-INDEPENDENT STAGE OF LYMPHOPOIESIS e PART 1 Because multipotent stem cells are committed to differentiate, it is hypothesized that the differentiation sequence is genetically determined. Cells within the sequence are precommitted in how they are determined. Cells within the sequence are pre-committed in their ability to respond to various inducers of differentiation; and on encountering appropriate factors or a suitable microenvironment, they proliferate and mature along a particular pathway. It is proposed that multipotent stem cells that do not receive a signal to differentiate into mature end cells progress to the next stage in the sequence of development. Alternatively, cells that do not receive a signal die. Only cells that receive proper signals will differentiate further toward mature end cells. Because commitment is gradual, there is a continuous spectrum of multipotent stem cells. Thus, some of them may be able to respond to inducers of one sort or another. In terms of self-renewal, multipotent stem cell populations continually occupy each potential for proliferation at any given time and therefore respond to the requirement for each cell type. Some daughter cells will differentiate into the next stage. The continual development of stem cells may not be diverted entirely toward one cell type, even in the presence of differentiation factors for that type. Because stem cells divide as they respond, some cells are still able to progress to the next stage of commitment.

ANTIGEN-DEPENDENT STAGE OF LYMPHOPOIESIS e PART 2 The second part of this model deals with the Ag-dependent stage, after receptors are expressed on the surface of lymphocytes. There are two main lineages of lymphocytes: T cells and B cells. T cells can mature into CD8þ cytotoxic T cells and CD4þ helper/regulatory T cells. Based on their function and the spectrum of cytokines produced, CD4þ helper T cells can be divided into Th1, Th2 and more recently, Th17 subpopulations. Treg cells are defined by the expression of Foxp3 and CD25 markers, and the regulatory function. The main functions of CD4þ T cells are to recognize cell-bound antigens, and to communicate with and help cytotoxic T and B cells to perform their duties as effector cells. Both lineages generate memory cells. How and when lymphoid cells become committed to differentiate into effector cells or into memory cells remains a mystery.

Diagrams to explain this model Fig. 50.1 is a simple diagram to explain this model. HSCs are multipotent stem cells derived from the mesoderm. Originally they are descended from common stem cells (null, or 0, cells),

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1078 FIGURE 50.1 Proposed sequence of cell commitment during lymphopoiesis and stages of hematopoiesis and lymphopoiesis at which somatic and/or genetic engineering of lymphocytes and their precursor cells might be feasible. Solid block arrows point to the cell stages that might be targeted. The first part of the theory is the sequential commitment model of hematopoiesis of Brown et al. [1,2], modified to include the pattern of expression of transcription factors. The cells depicted diagonally to the right represent HSCs (hematopoietic stem cells) committed to multipotent precursor cells or HPCs (hematopoietic progenitor cells), which gradually lose pluripotency, as expressed by the numbers 5, 4, 3, and 2 inside the stem cells. Number 5, for example, means that the HPCs at this stage have the potential to differentiate into five different types of precursor cells. The committed monopotent precursor cells can then differentiate toward mature end cells, such as Me (megakaryocytes), E (erythrocytes), G (neutrophils/granulocytes), M (monocytes), and finally L (lymphocytes). HSCs are derived from very primitive stem cells (null cells, or 0 cells), such as fertilized eggs and/or ESCs, ES (embryonic stem cells), and/or iPSCs, iPS (induced pluripotent stem cells). In the second part of the theory, lymphoid precursor cells differentiate into mono-potential precursor B-cells (B) and monopotent precursor T cells (T). After the expression of cell surface receptors and on encountering Ag (antigen), B cells can differentiate to PFCs (plaque-forming cells) or memory B cells. T cells can become CD8þ or CD4þ cells after education and maturation in the thymus. CD8þ memory T cells can be generated at a certain stage of differentiation. CD4þTh1þmemory T cells are also described. Foxp3þ (Forkhead box P3), CD25þ Treg (T regulatory)-cells are rediscovered and APCs (antigen processing cells), including dendritic cells and macrophage, seem to play a role in directing peptide-antigen recognition and activation of Th1, vs Th2, vs Th17þ cells. Th17þ cells can be further distinguished into two populations: IL10-, Th17e1 cells and IL10þ, Th17e2 cells. Lipid antigen is directed to TCR via the CD1 molecule pathway. The transcription factors are expressed and are known to affect differentiation. The data come mostly from phenotypic expression in loss/gain of function approaches. The known transcription factors are cited in Chen 2007 and not repeated here. The microRNA (miRNA) pathway, which involves ARS2 (arsenate resistance protein 2), affects engraftment or reconstitution potential of HSC. Individual miRNA’s, which shown to repress the expression of HSC relevant genes and to affect the differentiation of lymphoid cells and other lineages from stem cells, are indicated: miR-221 / miR-222 regulate c-KIT expression, which is thought to influence stem cell homeostasis. MiR-221 / miR-222 are also suggested to repress pax5/þ expression, implied in the migration destination of PreB-cells/B-cells to peripheral lymphoid tissues (Melchers, personal communication, unpublished). MiR-196b is expressed in HPC and regulates mRNAs encoding the homeobox (HOX) family, possibly in cooperation with another, not yet well defined,

CHAPTER 50 Lymphoid Cells such as fertilized eggs, and from ESCs or iPS. The cells diagonally to the right (Fig. 50.1) represent cells committed to hematopoietic lineages, which gradually lose their multipotentiality during embryogenesis. This concept is expressed with numbers (5, 4, 3, and 2) inside the progenitor cells on the diagonal. For example, HSCs (non-cycling) enter the proliferating stage to become the hematopoietic progenitor cells (HPCs, cycling). The number 5 on a cell, for instance, means that the HPCs at this stage have the potential to differentiate into five different types of precursor cells. The committed, monopotent precursor cells then differentiate toward mature end cells, such as Me (megakaryocytes), E (erythrocytes), G (neutrophils/granulocytes), M (monocytes), or L (lymphocytes). Lymphoid precursor cells then differentiate into monopotential precursors for B and T cells. After expression of Ag receptors on the cell surface and on encountering Ag, B cells differentiate into plasma cells, i.e., PFCs (plaque-forming cells), or memory B cells. T cells become CD8þ or CD4þ after thymic education, maturation, and negative and positive selection. Memory T cells are generated later during differentiation. Fig. 50.2 shows the markers and events that occur during the development of pre-T cells e the process of maturation, education, and apoptosis of mature CD4þ or CD8þ T cells from CD4þ8þ T cells. Genes encoding transcription factors and other important genes known to be expressed specifically in lymphopoiesis and that affect function, i.e., loss of function (knockout, knock-down) or gain-of-function (ectrophic expression by transducing with retroviral vectors containing transgene of interests) are cited in the chapter of Chen [4]. Recently post-transcriptional events, especially the miRNA pathways, have been shown to play important roles in targeting the mRNAs encoding for genes relevant to the differentiation and function of the precursor/mature cells of the immune system. Some are described in the Fig. 50.1.

Some comments on this model Very little is known about how totipotent and pluripotent stem cells differentiate into ectoderm, mesoderm, and endoderm or how mesoderm differentiates into hematopoietic lineages. These are the central issues of embryogenesis, and this part of the model is intentionally very sketchy. Why and when multipotent, lineage-specific stem cells divide asymmetrically or progress to the next stage of commitment are unknown. The underlying rules may be stochastic or inductive. But the progression of the stage of commitment is more likely due to the turning on and off of genes encoding transcription factors, and the post-

= miR-10 family members. MiR-126 represses the mRNAs encoding polo-like kinase 2 (PLK2) and HOXA9, promotes the expansion of lymphoid-myeloid committed progenitor cells [16]. The production of miRNAs by Dicer is required for efficient T cell, as well as B cell development. miR-146, and miR-223 target the developmental stage from lymphoid committed stage to T cell development, and miR-181 repress the development from lymphoid committed stage to pro-B stage [58]. During the T cell development in the thymus, the miR-17e92 cluster is shown to target mRNAs encoding for PTEN (phosphatase and TENsin homologue) and BIM (BCL-2-Interacting Mediator of cell death). The repression of mRNAs coding for BIM by miR-17e92 cluster also affects the differentiation of pre-B to B cells. MiR-181a is shown to target mRNAs encoding for phosphatases such as DUSP5 (DUal-specificity protein phosphatase 5), DUSP6, SHP2 (SH2-domain-containing protein tyrosine phosphatase 2) and PTPN22 (protein tyrosine phosphatase, non-receptor type 22). In the periphery, T cell differentiation is modulated by several miRNAs: miR-155 is shown to have dual roles in T cells: to repress mRNAs of MAF (Macrophage-Activating Factor) which leads to in favor of the differentiation of CD4þTh0 cells towards Th1 cells; to repress mRNAs of SOCS1 (suppressor of cytokine signaling 1) which is implicated in the survival of Treg cells. MiR-148a is shown to repress mRNAs encoding for BIM, affecting the commitment of Th1 memory (Haftmann and Radbruch, personal communication, unpublished). MiR-326 is shown to target mRNAs encoding ETS1 and resulting in promoting the differentiation of CD4þTh0 cells toward Th17 cells. miRNAs are implied to maintain immune tolerance of Treg cells to self- tissues, thereby preventing autoimmunity. During the B cell development, besides the role of miR-17e92 cluster on BIM (above), miR-150 is shown to repress mRNAs encoding for MYB, affecting the development of ProB cells. In the periphery B cell development, Beside affecting two steps of T cells development (above), miR-155 targets mRNAs encoding for AID (activation-induced cytidine deaminase) and PU.1 which promotes class switch and antibody production [16].

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1080 FIGURE 50.2 Going down the Toll mine. Toll-like receptors (TLRs) provide a repertoire for sensing pathogen-derived molecules during the innate immune response. TLRs in endosomal membranes detect bacterial and viral nucleic acids. The relative contribution of each TLR to the innate immune response is not yet known because pathogens contain multiple ligands specific for several different TLRs. The signaling pathways associated with each TLR are different, although they share common components. Specific signals may emanate from the adapter proteins recruited by each TLR (MyD88, Mal, Trif, and Tram). One important question concerns how the immune response is tailored to each pathogen according to the activation of specific signaling pathways triggered by different pathogen products (O’Neill et al., 2003). Besides dsRNA, TLR3 was shown to be able to sense ssRNA such as poly(I) (polyinosinic adid) [105]. HSV-1, herpes simplex virus 1; LPS, lipopolysaccharide; VIPER (Viral inhibitor peptide of TLR4) from A46 protein of vaccinia virus [104], RSV, respiratory syncytial virus (corrected following the erratum, Science, postdate 4 June 2004); MMTV, mouse mammary tumor virus; Porin (bacterium, not influenza). (From O’Neill [62] with permission. The figure has been modified following the erratum in Science, postdate 4 June 2004), i.e., Porin is a bacterial component and not viral and is shifted from the grey region upward to the green region. I have also included the IRF family transcriptional factors as targets for TLR 5 members signaling [64]).

transcriptional events regulating the epigenesis, and expression, degradation of mRNAs and the translation machinery. Brown and coworkers originally formulated their model based on data from the differentiation pattern of several myeloid cell lines. Their scheme of the development of lymphoid lineages was purely hypothetical in the original model, and the development of mature lymphoid cells (in Part 2, Ag-dependent step) was not included. It was proposed that precursor B cells become committed before precursor T cells. I have modified this step to the pre-commitment to lymphoid precursor cells. Also, I have changed the commitment of lymphoid precursor cells to occur after the myeloid precursor cells. However, the committed lymphoid precursor cells should still be viewed as hypothetical, because there are many examples supporting the common lineage development of B cells and myeloid cells and not of precursor T cells. Committed lymphoid precursor cells are not clearly identified and isolated (details in Stage 3, later).

CHAPTER 50 Lymphoid Cells There is ambiguity in the original model; for example, the issue of the timing of each point of decision was not addressed. The original hypothesis implied that stem cells that do not receive the appropriate signals would proceed ’spontaneously’ to the next stage in the differentiation sequence. Based on published data and on observations on the abortive development of lymphoid cells from blood islands derived from EBs (embryoid bodies) [4,5] and on the essential requirement of cytokines to maintain and to propagate stem cells derived from transgenic fetal liver, I tend not to be in favor of spontaneous progression; appropriate inductive signals for the transcriptional activation of lineage-committed genes are stringently required. Moreover, because programmed cell death plays an important role in the fate of cells during embryogenesis, I have included apoptosis as another important factor. This model has gained support from the phenotypes of mice in which genes encoding transcription factors have been deleted by knockout, knock-down, the so-called loss-of-function approach, and from the phenotype of cells in which transgenes are transduced using retroviral vectors, the so-called gain-of-function approach. Transcription factors controlling the sequential commitment of HSCs have been summarized [4]. In this chapter, I shall include the individual/clusters of miRNAs, which are reported to play critical roles in regulating the differentiation pathways and some functions of lymphoid (precursor) cells.

CRITERIA FOR ENGINEERING DEVELOPMENTAL STAGES OF LYMPHOPOIESIS I attempt to explore the feasibility of engineering lymphoid cells and their stem cells at several defined stages of differentiation. The criteria for deciding on feasibility are: Intrinsic self-renewal of HSCs, HPCs, and more differentiated cells, The availability of cell surface markers for identification and purification, The supply of recombinant growth factors and appropriate stroma cells, The existence of favorable cell culture conditions that promote growth instead of differentiation, 5) The possibility of educating lymphocytes to become antigen-specific memory cells, 6) The possibility of introducing, either genetically or somatically, genes of interest along with inducible promoter and regulatory sequences, 7) The possibility to introduce siRNAs / miRNAs of interests into the cells in culture or by genetic manipulation of mice to show the phenotype of targeting the mRNAs encoding the gene products. 1) 2) 3) 4)

Technical advances make the engineering of HSCs, HPCs, and lymphoid cells attractive for further manipulation, such as introducing new genes or reprogramming cells to iPS or manipulating the gene products and function post-transcriptionally via miRNAs.

STAGES OF LYMPHOPOIESIS FOR ENGINEERING Fig. 50.1 shows a schematic diagram of the stages at which lymphoid cells and lymphoid precursor cells might be engineered. These stages are now discussed individually.

Stage 1: 0 cells Null (0) cells include the fertilized eggs, which are totipotent, the blastocyst-derived Embryonic Stem Cell (ESC) lines, and somatic cells reprogrammed into induced Pluripotent Stem Cells (iPS), both of which are pluripotent. Mouse ES cell lines of different strain origin have been available for a long time, and they possess the property of differentiating into different somatic lineages in vitro and in vivo, homologous recombination, and germline transmitted through the crossing and breeding of progeny. ES cells from many other animal species are also available. Establishment of human ES-like cell lines has been reported since 1998, and many

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PART 12 Hematopoietic System In Vitro Fertilization (IVF) units can derive such cell lines. Cellular reprogramming of mouse somatic cells to ES-like, or iPS was reported, when embryonic fibroblasts were infected with retroviral vectors containing four transcription factors: Oct4, Sox2, Klf4 and c-Myc (mOSKM) [6,7]. Using similar strategy, human iPS cells (h-iPS) were derived using human OSKM transcription factors [8] or when Klf4 and c-Myc were replaced with transcriptional factors, Nanog and Lin28 [9]. Other technologies and delivery methods have been rapidly applied to this field; integrating viral vector system in combination with Dox-inducible expression, transient or excisable methods have been reported. More encouraging approaches include non-integrated RNA viral vectors (such as the Sendai virus [10], or more recently temperature-sensitive Sendai virus (Yamada, et al., personal communication, unpublished)), recombinant proteins incorporating cell-penetrating peptide moieties [11,12], improved episomal plasmid vectors [13], modified synthetic mRNAs of hOSKM [14,14a], are shown to be able to reprogram human skin fibroblasts and other cell types to become h-iPS cells. Some strategies are labor intense, but have reasonable efficiency and no genomic alternation which allow potential clinical application. More strategies to derive iPSs are developing. Through differentiation in vitro, several lineage-committed cells, such as those from bone and cartilage, have been shown to be obtainable from hES cells and h-iPS. There are conflicting reports about the differentiation potential of hES/h-iPS cells into lymphoid lineages in vitro (see later).

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Due to its potential to be an unlimited source for ’personalized’ transplantation or autotransplantation for treating blood diseases, many groups are investigating whether iPSs could differentiate into lymphoid lineages in vitro. Some investigators suggested that iPSs might retain epigenetic memory; i.e., they are prone to differentiate preferentially into the original somatic cell types from which they are derived. This might distinguish iPSs from ES cells. Strictly speaking, the only way to prove that an hES/iPS cell line is truly pluripotent is to demonstrate germline transmission in human beings e a task that should not even be considered. Similarly, genetic and cellular engineering at this level should be limited to studies in vitro. ES/iPS cells are stem cell lines that divide symmetrically. Unlike lineage-committed stem cells, which are rare, ES/iPS cells are almost an exception, being readily available in abundance. mES cells can proliferate without differentiation if feeder cells and/or cytokines such as LIF (leukemia inhibitory factor) are provided, whereas the hES/h-iPS cell lines established so far are feeder-cell-dependent, and LIF has no supportive effect. They can be induced to differentiate by removing the feeder cells (and cytokines). A few surface molecules are available for characterizing ES/iPS cells. Antibodies against various components that have been used to characterize such cells, such as those against stage- specific embryonic antigen-l (SSEA-l), enable one to distinguish undifferentiated from differentiated mES cells. mES/m iPS cells are SSEA1þ, SSEA3, SSEA4, TRA-1e60, TRA-1e81, Oct4, Oct6þ, APþ (alkaline phosphatase), telomeraseþ; hES/h-iPS cells are SSEA1, SSEA3þ, SSEA4þ, TRA1e60þ, TRA1e81þ, Oct4þ, APþ, telomeraseþ. Stem cell genomics, or the systems biology approach to stem cells, is being used. It considers the complex nature of cells and aims at a comprehensive understanding of stem cell mechanisms. The gene expression profiles of different stem cell types are being determined using DNA microarrays. These molecular signatures are stored and available in the stem cell database and allow a comparison of different stem cell types. On the basis of the obtained molecular signature, candidate genes, such as transcription factors, are subjected to the loss-of-function approach by knocked-out, knocked-in, or knocked-down by RNAi/siRNA, and the effects on the expression of downstream genes are determined with DNA microarrays. In this way, gene regulatory networks can be rapidly identified. More miRNAs pathways identified to regulate networks are characterized.

CHAPTER 50 Lymphoid Cells This approach has been used to identify gene regulatory networks implicated in the selfrenewal and differentiation of mES cells. Based on a microarray time course study of retinoic acid-induced ES cell differentiation, 169 genes that are downregulated in the course of differentiation were found, and 40 of them were investigated further. ES cell cultures infected with lentiviral vectors containing the EGFP gene in addition to one of these 40 genes (RNAi/siRNA knock-down or overexpression) were investigated in a competition assay, where respective infected cells were mixed with not infected cells. In this way, higher or lower proliferation rates of the infected cells could be determined based on measured EGFP fluorescence. Changes in ES cell number could be due to a function of the respective gene in cell cycle progression, cell survival, or prevention of lineage-specific differentiation. Downstream effects of changes of gene expression were determined with DNA microarrays. Such an approach also applies to the study of other cells, such as hES cells, hEC cells, h-iPS cells, HSCs, pre-B cells of mouse and, human origins, as well as mouse pre-T cells [15], and Treg cells (ref. cited in [4]). In addition to microarray studies for stem cells, the extent of post-transcriptional modifications within the stem cell transcription has been reported. Thousands of genes expressed in HSCs and ES cells undergo alternative splicing. Using combined computational and experimental analyses, the frequency of alternative splicing has been found to be especially high in tissue-specific genes, as compared to ubiquitous genes. Negative regulation of constitutively active splicing sites can be a prevalent mode for generation of splicing variants, and alternative splicing is generally not conserved between orthologous genes in human and mouse (ref. cited in [4]). The study of miRNAs has become a major subject in recent years. miRNAs are highly conserved, small, single-stranded non-coding RNAs, encoded by genomic DNA and are most commonly transcribed by RNA polymerase II. They function by directly binding to the 3’ untranslated regions (UTRs) of specific target mRNAs, leading to the repression of protein expression and the promotion of target mRNA degradation. The initial discovery was made in plants and worms, and later they were found in mammals. At least 700 different miRNAs have been identified in the human genome, and > 100 different miRNAs are expressed by immune cells. miRNAs have the potential to influence the molecular pathways that control the development and function of the immune system. The pathways of miRNAs are shown to be the regulators of networks. In addition, several post-transcriptional regulatory mechanisms that affect miRNA processing have been identified. The processes of miRNA biogenesis, target mRNA repression and function have been reviewed [16e18] and are beyond scope of this chapter. Many genes encoding lymphoid-specific functions have been knocked out, miRNA/siRNA knock-down technology has been applied, and more studies are under way to develop specific deletions or replacements of genes. Vectors with inducible promoters are being used to introduce genes in a controllable, tissue-specific manner. Fig. 50.1 includes various individual or clusters of miRNAs, which have been shown to target the mRNAs encoded by genes critical for the regulation of differentiation to lymphoid lineages from HSCs.

Growth requirement for differentiation of ES/iPS cells to lymphoid cells Under appropriate conditions, ES/iPS cells can differentiate in vitro. Many different culture systems are available for studying the development of lineage commitment. Because ES/iPS cells are pluripotent, optimal culture conditions would enable the formation of mature cells and tissues and even of organs in well-organized three-dimensional structures in vitro. That is, one should be able to generate an artificial fetus in culture without any external cells or other factors. In fact, organogenesis from ES/iPS cell culture is still in its infancy. Nevertheless, with the culture conditions and system that we have developed, mature and embryonic cells in

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PART 12 Hematopoietic System some form of tissue-like organization, though never become the functional and true organs, of certain orderly structures (gut-like, epidermal-like, and thymus-like structures), can be observed. Many genes have been identified that play important roles in the differentiation of ES/iPS cell to mesoderm and then to derivatives such as HSCs (see figure legends [4]). Many researchers have attempted but failed to study the differentiation of lymphopoietic precursor cells obtained from mES cells. Many have observed the development of yolk sac blood islands in culture; these presumably contain HSCs. But only myeloid cells could be produced in the culture systems reported. Only a few systems allow the generation of lymphoid precursor cells, and these were mostly B cells, but also T and B cells, in a quantity sufficient for further manipulation [4,5]. The reasons for these variations are unclear. It is partially due to the origin and pluripotentiality, hence the fragility and variability, of ES cells but partially due to experimental manipulations, different culture conditions [5,18a], and the transient expression of genes relevant to the developmental stage of hemato-lymphopoiesis from mesoderm equivalent at the yolk sac stage. Homeodomain transcription factors, HoxB4 (see later) is an example of such genes. Even in the hands of a single researcher, the potential of lymphopoiesis from the same ES cell lines varies greatly among different experiments, and why this is so remains a mystery. The culture conditions vary, depending on which lineages are chosen for study. For example, methylcellulose culture is preferred for myeloid lineages but is inferior for lymphoid lineages. Several culture conditions that favor the differentiation of ES / iPS cells to lymphoid lineages are available.

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Through the work of many groups, it has become clear that lymphoid precursor cells can be obtained from mouse ESCs in culture. However, the development of mature B plasma cells and mature T cells requires a two-step procedure e a two-step culture procedure for plasma cells, and culture and animal implantation for mature B and T cells. When chimeric bone marrow cells from embryoid body-implanted nude mice, which contain cells of ESC origin, were injected into the host mice, the bone marrow cells were shown to repopulate the primary and secondary lymphoid organs, i.e., bone marrow, spleen, and lymph nodes. The data indicate that HSCs and HPCs derived from ESC can be transferred. However, when injected into the host animal, such as lethally irradiated adult mice instead of un-irradiated nude mice, dissociated cells from blood island of the EB, instead of the whole EB, killed the mice when intravenial injection was performed, and failed to repopulate the host immune system or to achieve a stable engraftment when intra-peritoneal injection was performed [5]. The work on the expression of (transcriptional) molecules such as HoxB4, Cdx4, and especially, the transduction of transgenic expression under the inducible tet-promoter using retroviral vector, has been shown to understand the mechanism and pathway. HoxB4 is a homeodomain-containing transcription factor with diverse roles in embryonic development and the regulation of adult stem cells. This gene has a double life and can act in opposite ways when expressed by different cells, promoting the proliferation of stem cells while activating the apoptotic pathway in some embryonic structures. It is implicated in the self-renewal of definitive HSCs, being transiently expressed in blood developmental stages somewhere between the yolk sac stage and the fetal body stage and also being expressed in human cervical carcinoma. Persistent overexpression of HoxB4, however, inhibits the development of blood lineage. It was examined to show its effect on hemopoiesis using a variety of tools, including retroviral transgenic expression. Expression of Homeodomain transcription factors, HoxB4 in primitive progenitors, and when combined with culture on stromal cells, induces a switch to the definitive HSC phenotype. These progenitors engraft lethally irradiated adult mice and contribute to hematopoiesis in primary and secondary hosts. Thus, HoxB4 expression

CHAPTER 50 Lymphoid Cells promotes the transition from primitive cells to become HSCs. Overexpression a high level of expression, of HoxB4 is shown to perturb the differentiation and to predispose the manipulated cells to leukemogenesis. HoxB4 may affect cell growth in a dose-dependent manner and may disturb the differentiation into lymphopoiesis. The manipulation of essential genes has partially overcome the problems of variability in the development of hematopoietic cells from mesoderm-derived cells, such as yolk sac cells and EBs, and increased engraftment into irradiated adult mice. Combining HoxB4 gainof-function and caudal gene family, Cdx4 gain-of-function has been shown to increase the multilineage haematopoietic, including lymphoid, engraftment of lethally irradiated adult mice. Others that have been tried are: mMix, Stat 5, a signal transducer under similar inducible conditions to express in mES-derived HSC and to grow similarly on OP9 stroma cells, shown to increase the induction of preferable myeloid, rather than lymphoid, differentiation. BCR/ ABL, a chronic myeloid leukemia-associated oncoprotein, is shown to transform a subset of HSC between erythryo- and lymphoid-myeloid lineages (ref. cited in reference [4]). The combined in vitro and in vivo culture system described earlier using nude mice as the hosts can also be employed to study thymic stroma as well as well-organized, highly differentiated cells of many other lineages, including gut epithelia, skin with hair follicles, bone, muscle, neurons, and glia. One essential requirement for successfully applying this combined system is that the cells should first differentiate in culture, until the EB developed before the subcutaneous implantation. Otherwise, tumors may grow, due to the expression of the acylated dimeric iLRP (immature 32e44 kDa Laminin Receptor Protein) and other, still-to-be-identified OFA (OncoFetal antigen) positive in early to mid-gestation [19]. The development of endogenic thymic epithelial cells as well as stroma cells inside the ES-embryonic bodies also explain partially the findings of an efficient development of T cells in the absence of exogenous stroma cells. Stroma cell line RP.0.10 is able to support both T- and B-lymphoid precursor cells derived from ESCs in some labs. Stroma cell lines OP9 and S17 were originally reported to support B precursor cells and myeloid but not T precursor cells derived from ESCs. Many other stroma cell lines do not support the differentiation of ESCs to lymphoid precursor cells. The S17 stroma cell line was compared to many stroma cell lines for the production of cytokines and stroma function, i.e., the stem cell support function (ref. cited in reference [4]) (see also later, Stage 2; there were no correlations in the assays. Thus a yet-to-be-identified extracellular matrix molecule of S17 cell lineage may play a role in supporting the function of HPCs. With the insertion of genes involved in Notch-Delta signaling pathways, it was shown that the OP9-derived cell line, OP9-DL1 (Delta-like 1), increases efficient T cells, which appeared from sources such as fetal liver [20], human cord blood [21], ES cells [22] and recently ES / iPS (see later). The study using switch cultures shows that while necessary to induce and sustain T cell development, Notch/Delta signaling is not sufficient for T-lineage specification and commitment but, instead, can be permissive for the maintenance and proliferation of uncommitted progenitors that are omitted in binary-choice models. Moreover, it was demonstrated that a three-dimensional structure to allow the interaction of precursorsupporting cells plays an essential role for it to function well (see also Stage 4). Other candidate molecules and cells that may support the lymphopoiesis include the following: dlk, an epidermal growth factor-like molecule of 66 kDa in stroma cell lines was reported to influence the requirement for IL (interleukin)-7 in supporting mouse pre-B lymphopoiesis, and dlk is suggested to play an important role in the bone marrow HPC microenvironment. Another high-molecular-weight CD166 (HCA/ALCAM) glycoprotein was shown to express in human HPCs as well as stroma cells. This molecule is involved in adhesive interaction between HPCs and stroma cells in the most primary blood-forming organs.

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PART 12 Hematopoietic System Rethinking the seemingly conflicting results and the inconsistent report of differentiation of ES/iPS cells to lymphocytes, I wonder if culturing ES/iPS-derived lymphoid progenitor cells on stroma cell lines derived from the most primitive stroma environment from the fetus (such as paraaortic cells) may be a better choice for obtaining consistent results, rather than using the existing stroma cell lines. Indeed, stroma cell lines from such a source have been derived. The field of hESC/iPS (human ESCs/induced pluripotent) cells represents a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means of treating various diseases. hESCs/iPSs are also an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. They are also a potential source of HSCs for therapeutic transplantation and can provide a model for human hematopoiesis. The first stable hES cell line was reported in 1998 [23]. Now many centers can establish hES cell lines, and clinical trials for treating spinal injury and Macular Dystrophy are proceeding in the US and Europe. The first human iPS cell line was reported in 2007 [8], and many other labs could derive such somatic reprogrammed stem cell lines using better strategies. The current goal is to establish clinical grade hES/iPS cell lines for potential therapeutic purposes. Many clinical grade hES cell lines are available. However, so-called clinical grade, personalized, stem cell lines e the human iPS cell lines e still need to be derived in the clean air condition. Hopefully in due time, the limiting steps such as the sources for establishing Good Manufacturing Practices (GMP) grade non-integrating vectors/ synthetic RNAs, other essential technical tools, raw materials required for culturing of transfected or transduced somatic cells, and the isolators instead of GMP facility, will become widely available and affordable. 1086

More attempts to differentiate hESCs to HSCs in vitro have been reported recently [24e29b]. From such studies, hES cells have been shown to differentiate into HSCs, using various protocols and different supportive stroma cell lines, with or without cytokines. It seems that the differentiation into mesoderm-derived lineages and then to MHE (mesodermal-hematoendothelial) lineage can occur, resembling human yolk sac development. The cellular and molecular kinetics of the stepwise differentiation of hESCs to primitive and definitive erythromyelopoiesis from hEBs in serum-free clonogenic assays have been demonstrated [27]. Hematopoiesis initiates from CD45þ hEB cells, with the emergence of MHE colonies. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis. A second wave of a definitive type of colonies of erythroid, GranulocyteMacrophage (GM), and multi- lineage Colony Forming Cells (CFCs) follows. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and Cdx4. These data indicate that the earliest events of embryonic and definitive hematopoiesis can be differentiated from hESCs in vitro. However, differentiating hEBs further into HSCs, and lymphohematopoietic lineages is variable and unpredictable. Vodyanik et al. [25] reported that hES cells could differentiate to CD34þ cells using mouse stroma OP9 (better than S17 and MS-5) co-culture system. However, when cultured on stroma MS-5 (but not OP9, S17) in the presence of stem cell factor, Flt3-L, IL7, and IL3, such CD34þ cells could differentiate into natural killer cells and pre-B-lymphoid cells which expressed mRNAs for VpreB and Ig-alpha (CD79a or mb-1) components of pre-B cell receptor complex, as well as myeloid lineages, but not T lymphoid cells. In another report, the hES-derived CD34þ cells were implanted into fetal sheep intraperitoneally and differentiated in primary

CHAPTER 50 Lymphoid Cells and secondary hosts, the cells could differentiate into many cell types but no lymphoid cells could be detected [29a]. Martin et al. [109] claimed that natural killer (NK) cells could be derived, but not T or B lymphoid cells under the culture condition and protocol used. The expression of transcription factor HoxB4 appears to play a role in causing cells to become lymphoid cells in the mouse system and would explain the occasional determination of precursor cells to commit to lymphoid lineages. It implies right away that can apply to study the lymphohemopoiesis in the human system. Indeed, Bowles et al., (2006) have reported such an attempt. Culture of hES cells on mouse feeder cells or in cell-free culture conditions result in low levels of differentiated HPCs. Transgenic stable HoxB4-hESC clones were generated by the lipofectamin transfection method. In vitro differentiation of hESCs, as EBs, in serumcontaining media without cytokines led to the sequential expansion of erythroid, myeloid, and monocytic progenitors from day 10 of culture. These cells retained the capacity to develop into blood elements during culture. Overexpression of HoxB4 considerably augments the development of three myeloid lineages of hESCs; however, no lymphoid progeny were reported. Theoretically, lentiviral vector introducing HoxB4 under a tet-on system should be a comparable approach for further study. However, another report does not support this notion to show a functional role of HoxB4 in HSCs in their combined in vitro and in vivo system, using Server Combined ImmunoDeficiency (SCID) mice [26]. It is interesting that hES-derived HSCs failed to reconstitute i.v. transplanted host because of cellular aggregation causing fatal emboli formation, which is similar to our attempt using mEB-derived cells. Direct femoral injection allowed host survival and resulted in multilineage hematopoietic repopulation. However, hES-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs, which correlated with a distinct gene expression pattern of hESC-derived HSCs that included HoxA and HoxB gene clusters. Transduction of HoxB4 had no effect on the repopulating capacity of hES-derived cells. A yet-to-be-identified molecular program is postulated to contribute to the atypical behavior of hES-derived cells in vivo. The following more recent reports contribute to the positive aspects of the debate. Using sequential in vitro co-culture of EGFP-expression hESC on stroma OP9 for 10 days, and engraftment into the kidney capsule of SCID mice, or RAG2/ mice, containing fragments of human thymic- and fetal liver tissues, the so-called SCID-hu(Thy/Liv) / RAG2-hu(Thy/Liv) mice, Galic et al. [28] demonstrated that T lymphoid lineage with the phenotype of EGFP-expressing cells could be found in various stages of thymocyte maturation, including immature CD4þCD8þ, and mature CD4þCD8, and CD8þCD4 cells. When such thymocytes were harvested and cultured with plate-bound anti-CD3 plus soluble anti-CD28 stimulating conditions, the cells were shown to be responsive to the stimulation by increasing CD25 expression. The reporter EGFP transgene continued to express at high frequency in the hosts throughout thymopoiesis in 3e5 weeks of experimental time. Timmermans et al. (2009) used four steps of differentiation protocols by co-culturing hESC with stroma OP9, and OP9-DL1. After 7e9 weeks of differentiation, they could find T lymphoid cells, with the phenotype of CD4þCD8betaþ and TCRgamma-deltaþ cells. CD3þ T cells was stimulated with PHA, and shown to be able to proliferate and to produce IFNg. Thus, conflicting reports continue to surface, giving the impression that the differentiation into lymphohemopoiesis seems to be, if not more rare, as rare as variable as the in vitro differentiation system using mES cells. The question of the variability among different hES cell lines and clones, similar to the observation using mES cells, are also reported. Derived, somatic, tissue-committed, stem, precursor cells remain rare. The study of individual gene expression using hES cells and its relationship with the group of genes and the final outcome of the cell fate is a challenge and will require more research in the future. Combining multiple genes, such as mMix, Cdx4, and HoxB4, with a gain-of-function approach might be a possible way of increasing the lymphohematopoiesis derived from hES cells.

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PART 12 Hematopoietic System In addition to hES cells, many groups are investigating whether iPSs could differentiate into lymphoid lineages in vitro, using the iPSs derived from blood cells as well as fibroblasts. Below are some selected examples of potentially promising experiments. Wada et al. [110] used a three step system for m-iPS differentiation in vitro: EB formed after 5 days differentiation: in suspension culture, single cells were harvested and co-cultured with Flt3L (Flt3 Ligand) and stroma OP9 for eight days, then harvested every six days and co-cultured with Flt3L, IL7, and stroma OP9 for B cells, or stroma OP9-DL1 for T cells. The results showed that mouse B cells and embryonic fibroblast derived iPS could differentiate into T cells by showing TCRVbeta rearranged, CD4CD8þ were generated, Treg could be detected upon TGFbeta stimulation, but no B cells could be detected. The data challenged the suggestion that iPS tend to retain epigenetic memory. Carpenter et al. [111] reported differentiation into pre-B cells by co-culturing human dermal fibroblast derived iPS cells with stroma OP9 for 10 days. CD34þ cells were harvested, then co-cultured with stroma MS-5 for a further 21 days. The differentiated cells had the phenotype of CD45þ CD19þ CD10þ; and expressed mRNAs encoding for Pax5, IL7R-like, and VpreB receptor; exhibited partial genomic D fragment-J fragment of Heavy chain (D-JH) rearrangements, but no full Variable region of Heavy chain -J fragment of Heavy chain (VHJH) rearrangement. Such cells were negative for surface IgM and CD5 expression.

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For the purpose of treating patients infected with HIV, Ye et al. (personal communication, unpublished) developed HIV resistant h-iPS by preparing iPS cells from patients CD34þ cells and peripheral blood mononuclear monocytes using Sendai viral vector. iPS cells were knocked out both wild-type CCR5 alleles or replaced them with the CCR5d32 mutation using BAC based homologous recombination and differentiate them into HSC by the spin Embryoid Body (EB) method without any stroma cells in the culture. The HSCs are w50% CD34þ and w25% CD34þ/CD43þ HSC, HIV resistant, and could be used for further characterization, differentiation into mature blood cells including monocytes and lymphocytes, and for autologous transplantation. In order to completely remove the residual Sendai viral vector containing the MOSK which was shown to interfere with the differentiation potential of iPS in vitro, Yamada et al. (personal communication, unpublished) derived h-iPS cells from blood T cells or fibroblasts, which were reprogramd using temperature sensitive (ts)-Sendai viral vector containing tsA58 (SV40Tag)MOSK. Such iPS could differentiate in culture to HSC by co-culturing with stroma OP9, and further cultured at the presence of stroma OP9-DL1 plus SCF, IL7, Flt3L, T lymphoid lineages could be detected. Tumor antigen-specific CD3þ, CD4þ or CD8þ lymphocytes from precursor T cells could be generated and expanded. Antigen-specific T cells derived from iPS were also obtained by Nishimura et al. (personal communication). Vizcardo et al. (personal communication) could establish Mart1-T-iPS cells from Mart1 (Melanoma antigen recognized by T cells 1)-specific CTL cells. These iPS cells were differentiated in vitro into CD4þCD8þMart-1þ precursor T cells. In the mouse system they could expand proT cells with solid phase DL-1 and IL7. The h-Mart1-T-iPS was shown to differentiate to proT-cell stage in vitro and to expand using a set of human cytokines. They are trying to induce mature T cells by transplantation into NSG (NOD-SCID common gamma/) mice and in vitro.

Stage 2: HSCs and HPCs The terms hematopoietic stem cells, progenitor cells, and precursor cells are used loosely in the literature. Hematopoietic stem cells and HPCs are defined as two populations of stem cells sharing similar surface markers. HSCs are rather quiescent, non-cycling stem cells, whereas HPCs are cycling cells. In mice, c-kitþ, thy-1low, lin, and scalþ bone marrow cells are defined as HSCs or HPCs. HSCs and HPCs in bone marrow are heterogeneous in size and in selfrenewal capacity. Moreover, they have finite life spans. In human bone marrow,

CHAPTER 50 Lymphoid Cells CD34þHLA-lin-thy-1low rhodamine123low stem cells are generally defined as HSCs and HPCs. However, CD34 is also expressed in other lineages, such as vascular endothelial cells and muscle precursor cells. It is well established that mouse HSCs and HPCs can be isolated, cultured, and transduced by retroviruses and used to repopulate animal hosts. In addition to bone marrow, they can be isolated from fetal liver, cord blood, fetal blood, yolk sac, and para-aortic spanchnopleura.

DO HSCS AND HPCS SELF-RENEW? The self-renewal of HSCs and HPCs was carefully examined (ref. cited in reference [4]). By transferring bone marrow stem cells from one host to the other, it was found that the pool of donor bone marrow stem cells was smaller on transfer, and it was concluded that self-renewal is limited for somatic bone marrow stem cells. However, the results could be due to a dilution effect of the donor stem cells, if a certain absolute number of stem cells were required for the successful implantation of donor stem cells in the bone marrow. Using fluorescence in situ hybridization (FISH) and telomerase assays, it was shown that telomere DNA length predicts the age and replication capacity of human fibroblasts. DNA telomere length has been correlated with the replication potential of HSCs and HPCs. In human CD34þ, CD71low, and CD45RAlow bone marrow stem cells, the length of telomeric DNA in HSCs and HPCs is correlated with the age of the donor. Stem cells in adults have shorter telomeres than those found in fetal cells, but both have the same telomerase activity. When the HPCs were expanded with cytokines without the support of stroma cells, there was a loss of telomeric DNA in culture. The loss of telomeric DNA in HSCs and HPCs from older people and cultured stem cells was interpreted as meaning that their replication potential is finite. An alternative interpretation would be that the culture conditions that allow stem cells to grow in the presence of cytokines may not favor the maintenance of telomerase activity and hence not the self-renewal of HSCs and HPCs. Work on global miRNA expression profiling of human CD34þ HSC / HPC from Bone Marrow (BM), mobilized Peripheral Blood Cells (PBC) harvests, and umbilical cord blood have helped to identify several miRNAs expressed by the stem cell population. Genetic manipulation of the mice also helped to identify the miRNAs pathway related factors. Arsenate-resistance protein 2 (ARS2) is one of the factors identified to be involved in post-transcriptional regulatory mechanisms that affect miRNA processing. ARS2 expressed by HPC and is a component of the RNA cap-binding complex that promotes processing of pri-miRNA transcripts [30]. Mice deficient in ARS2 have bone marrow failure, possibly owing to defective HSC function, such the engraftment or reconstitution potential of HSC. Individual miRNAs, which shown to repress the expression of HSC relevant genes and to affect the differentiation of lymphoid cells and other lineages from stem cells are indicated in Fig. 50.1: miR-221 and miR-222 were shown to inhibit c-Kit expression in HSC and HPC, leading to impaired cell proliferation and engraftment potential of human CD34þ cells in NOD-SCID mice [31]. miR-221 and miR-222 are also suggested to play a role influencing cell migration at the Pre-B cell stage. miR-196 and miR-10 were found to be located in the HOX loci; directly repress HOX family expression, thus regulating HSC homeostasis. Particularly, miR-196b was found to express in mouse HPC, regulated by transcription factor Mll (Mixed lineage leukemia), and to modulate HSC homeostasis and lineage commitment by repressing HOX family [32]. miR-126 is thought to act on suppressor PlK2 (polo-like kinase 2), shown to increase colony formation of bone marrow M-L progeny in vitro [33]. More work is needed to study the role of miRNAs in the long-term engraftment potential of HSC in vivo. Many culture systems for studying the biology of mouse HSCs and HPCs are available. Many mouse stroma cell lines support the growth of HPCs. Studies on human stem cells use mouse stroma cell lines, such as S17 and AFTO24 among many others, or mixed primary stroma populations, called MSCs (mesenchymal stem cells), isolated from human bone marrow or

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PART 12 Hematopoietic System other sources, to support purified HSCs and HPCs or unseparated bone marrow cells. The establishment of heterogeneous human stroma cell lines with the help of a plasmidcontaining SV40 T antigen under the control of an inducible metallothionein promoter has been reported, but their ability to support lymphopoiesis could not be demonstrated. Mixed human stroma cells, or MSCs, and cell lines that support human long-term culture-initiating cells also exist. However, these human stroma cell lines could not be shown to support the development of human lymphocytes from bone marrow stem cells. MSCs have become a promising field, and many clinical trials are under way. The expansion of human MSCs in culture presents a challenge. They proliferate poorly in media supplemented with human serum, and they require the presence of selective lots of Fetal Calf Serum (FCS). Human MSCs are not immunogenic in an allogenic system in vitro, nor are they rejected in vivo. This makes them candidates for allogenic cellular therapy. MSCs have immunomodulatory effects: they inhibit T cell proliferation in mixed lymphocyte cultures, prolong skin allografts survival in baboons and decrease graft versus host disease (GvHD) when cotransplanted with hematopoietic cells. MSC induce their immunosuppressive effect, at least in part via a soluble factor and in part due to the activation of CD4þCD25hFoxp3þ Treg cells with suppressor activity through production of prostaglandin E2, transforming growth factor beta 1 and by direct cell contact [34]. Interestingly, MSCs have been shown to express Foxp3 [35]. A report indicated that prolonging the culture of MSCs runs the risk of them becoming tumor cells [36]. However, others have claimed that over several years of study in treating GvHD [37], no tumor has been observed (Le Blanc, personal communication, unpublished). Clinical trials which use MSCs to treat familial hemophagocytic lymphohistiocytosis [38], autoimmune disease such as scleroderma, multiple sclerosis, Crohn’s disease and other diseases are ongoing [39]. 1090

Major problems in growing HSCs and HPCs are the variability of culture conditions, the efficiency of differentiation of the cultured cells to lymphoid lineages, and the poor reproducibility in the hands of different investigators. Also contributing to problems is the failure to report specific reagents and ingredients used as well as undescribed procedures, stroma cell conditions, batches of serum, and growth factors required in each system. Of course, the intrinsic multipotentiality of HSCs and HPCs also contributes significantly to variability. Many have claimed that a cocktail of cytokines alone could promote the differentiation of myeloid lineages from HSCs and HPCs in mouse and human systems (ref. cited in reference [4]). A few have claimed the development of B cells at various stages of maturation, but no mature T cells could be found in such culture conditions, however, they are not reproducible in other’s laboratories. The maintenance of long term cultures of self-renewing stem cells with potency for lymphopoiesis, especially T lymphopoiesis, requires stroma cells, additional cytokines, a threedimensional structure to support the precursor cell-stroma cell interaction (also see Stage 3), and other culture conditions yet to be defined. Recently, several groups reported that solid phase-Delta-L-1, either fixed on tissue culture dish surface, or integrated into mouse stroma OP9 can mimic the thymic stroma requirement for growing human and mouse T cells from early progenitor cells including iPS derived HSC (see stage 1, above). Several culture systems seem to be promising for expansion ex vivo and potential differentiation of human and mouse HSCs. Human-based supporting cell lines, such as an endothelial-like cell line, ECV304/T24, were used to generate split-function amphotropic packaging cell lines. This manipulated cell line, APEX, was used for transduction and for support of the growth of HSPs [40]. The development of supporting human cell lines for lymphopoiesis is essential for the production of large quantities of cultured cells for manipulation and re-implantation. Ruedl et al. [41] reported that transduction of mouse HPS with retroviral vector containing gene coding for NUP (NUcleoPorin) 98 and HOXB4 fusion can expand bone marrow derived HPCs for several weeks in culture, with the expansion of cells of ca.1016 fold. The expanded cells can differentiate into all lineages including T and B cells, mimic the self-renewal property

CHAPTER 50 Lymphoid Cells of primary HSC-HPS. An additional important finding is that such immortalized cells do not produce tumor in transplanted mice. This study opens interesting questions, such as whether this viral vector containing NUP98-HOXB4 fusion protein can immortalize the HSCs of embryoid bodies differentiated from mES cells in culture, and show similar potential to differentiate into all lympho-hemapoietic lineages. Another question is whether this viral vector can immortalize human HSCs-HPCs, and if not, whether the counterpart of the NUP98-HOXB4 fusion protein exists and exercises similar effects in human HSCs.

SELECTED EXAMPLES OF THE CLINICAL APPLICATION OF HUMAN HSCS AND HPCS The use of human HSCs and HPCs as the cell base for gene therapy is a complicated issue. In most clinical protocols, HPCs are transduced ex vivo with retroviral vectors and re-implanted into patients. One example which has undergone phase I clinical trial for decades is the treatment of ADA (Adenosine DeaminAse) deficiency. ADA deficiency exists in all cells examined. However, T cells of these patients are selectively missing, causing a SCID symptom. Despite decades of study, the mechanism by which the ADA defect causes this specific deficiency of T cells remains unknown. Possible mechanisms have been postulated, such as the apoptotic pathway of CD95 (Fas/apo-1)-induced cell death. An ongoing, improved ADA gene therapy protocol has been described. This is a modified protocol running with more than 20 patients, using a combination of low-dose chemotherapy with busulfan/fludarabine and a single transplantation with ADA-retroviral-transduced HPC. No ADA enzyme replacement was included in this modified protocol to treat the patients. The low dose (one quarter of the full dose) chemotherapy is to condition or to make space in the bone marrow for the transplanted, gene corrected HPC. No side effect has been reported of the gene-transferred patients, they live normally, and more important, no patient died so far. This improved treatment was conducted first in Milan [42], then in several centers in UK and USA [43]. CD34þ HPCs and progeny PBL cells were collected from five ADA-SCID children before transplantation and in their myeloid and lymphoid progenies up to 47 months after transplantation and a genome-wide analysis of RISs (retroviral vector integration sites) were performed [44,45]. The data revealed similar patterns of integration into the human genome before and after transplantation, with a preference for gene-dense regions, promoters, and transcriptionally active genes. The occurrence of insertion sites proximal to proto-oncogenes or genes controlling cell growth and self- renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of cell progeny revealed highly polyclonal T cell populations and shared RISs among multiple lineages, indicating that they were derived from the engrafted HPCs. Besides ADA-deficient SCID, several clinical trials of gene correction therapy using CD34þ stem cells have been conducted, trying to restore immune cells in patients with SCID-Xl (X-linked severe combined immune deficiency), CGD (chronic granulomatous disease), WAS (Wiskott-Aldrich Syndrome). The study of the SCID-Xl gene therapy clinical trials has been reported in Paris, in which HPCs are the target of MLV (murine leukemia virus)-derived retroviral vectors containing IL (interleuken) receptor gamma common chains. In this trial, no chemotherapy was administered, with the expectation that the progeny lymphoid cells would have a very high selective survival in the patients, and marrow conditioning to facilitate higher engraftment of HPC would not be needed. Immune function has been restored in nie of 10 treated children with the transduction of gamma common gene transfer in CD34þ cells. The distribution of both TCR V beta family

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PART 12 Hematopoietic System usage and TCR V beta CDR3 (complementarity-determining region 3) length revealed a broadly diversified T cell repertoire. Further retroviral integration site analysis showed that insertion sites were shared by progenic T and B cells, granulocytes, monocytes, and the transduced CD34þ. This finding demonstrates the initial transduction of very primitive multipotent progenitor cells with self-renewal capacity. These results provide evidence in the setting of a clinical trial that CD34þ cells maintain both lympho-myeloid potential as well as self-renewal capacity after ex vivo manipulation [108]. A trial in the US followed using similar techniques achieved immune restoration in another 10 patients [46]. However, due to the developed leukemia-like T lympho-proliferative disorder of a total of five of the 20 treated children in both trials, the trials were on hold [47e50]. Patients who had been clinically stable with good immune function developed a relatively abrupt onset of escalating levels of circulating T lymphocytes, with thymic mass and organomegaly. They were treated with chemotherapy and four have remained in complete remission with continued restored immunity, but one died from the leukemia. The research now focuses on the basic biology of the oncogenicity of the vector-related insertion site LMO2 of the affected T cells and of using the Lmo2-TLL transgenic mice model. The integrations are found to reside within FRA11E, a common fragile site known to correlate with chromosomal break points in tumors. The fragile sites attract a non-random number of MLV integrations. This explains the mechanism of four of the leukemia cases. The other leukemia is attributed to the transplanted IL2RG gene [48].

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The reasons why this leukemic complication occurred in five of 20 XSCID gene-corrected patients, but none of more than 20 ADA-deficient SCID gene treated patients are not clear. The reasons possibly involve the role of the common gamma gene product (a trans-membrane protein capable of providing intracellular signaling), the nature of the HPCs of the XSCID patients, effects of the common gamma-deficiency on the susceptibility to transformation and the rapidity of immune reconstitution in the XSCID patients [43]. New approaches using safer integrating mouse SIN (Self INactivate) retroviral vectors, HIV-based lentiviral vector, or direct correction of the defective gene underlying the immune deficiency diseases are being developed, which might lead to safer and effective gene therapy.

Stage 3: Lymphoid precursor cells The characterization of lymphoid lineages e committed precursor cells defined in terms of cell surface markers and functional assays of precursors and progenies e is still a challenge. One described cell type that is closest to the definition of lymphoid precursor cells is the CD4low precursor population isolated from adult thymus (ref. cited in reference [4]). Because this population of cells is restricted in its potential to differentiate into myeloid cells, it is preferentially committed to differentiate into T and B cells. These cells can also differentiate into thymic dendritic cells. The molecular markers of this population have not been well characterized. It would be interesting to see whether this population of cells can differentiate into myogenic cells, because it is possible to obtain differentiated myogenic cells from the adult thymus. The thymus contains lymphoid and MSCs, which can develop into other lineages, such as bone, cartilage and lung. Little is known about the culture conditions for growing this population of cells. It is possible that stroma cell lines such as S17 could be used to expand the CD4low population of cells in culture, because it has been possible to use S17 and cytokines to maintain para-aortic splanchnopleuralderived precursor cells possessing T cell lineage potency in culture (in reference [4]). A lymphoid lineage-preferable transcriptional factor, lkaros, was thought to be an important molecular marker of lymphoid precursor cells at this specific stage of development. lkaros was subsequently shown to have multiple effects on HSCs, lymphoid cells, T precursor cells, and others.

CHAPTER 50 Lymphoid Cells Using the transgenic approach, it is possible to trace lymphopoiesis between common lymphoid precursor and alpha-beta T cells. A transgenic mouse system is applied using pTalpha (pre-T cell receptor alpha) promoter to drive the human CD25 (hCD25) surface marker as a reporter. It marked intra- and extra-thymic lymphoid precursors but not myeloid cells. The extrathymic precursors were characterized as a common lymphoid precursor population (no. 1) expressing CD19-B220þThy1þCD4þ cells using clonogenic assays. The earliest intrathymic precursors were CD4lowCD8CD25CD44þc-Kitþ cells and Notch-1 mRNAþ. By using the regulatory sequences from the gene encoding pre-T cell receptor alpha to drive hCD25 reporter and to produce transgenic mice, another common lymphoid precursor population (no. 2) was identified, which was B220þc-Kit. In short term culture, population no. 2 cells could be derived from the no. 1 subset and contained cells that in clonogenic assays were characterized as bipotent T and B precursors. Mature alpha-beta T cells were produced when transgenic bone marrow cells were injected i.v.; thymocytes were cultured using a thymic organ culture system. The no. 2 subset may represent the most differentiated population with T cell potential before commitment to the B cell lineage. The human counterpart of the mouse CD4low population is unknown. The expansion of CD34þ, CD31þ human bone marrow B-progenitor cells and partial differentiation to B-precursors in serum-free culture medium in the presence of mixed primary human stroma cells and IL7 for a limited duration has been shown to be possible (ref. cited in reference [4]). A few such CD34þ precursor cells could differentiate into NK cells and T precursors when subjected to a secondary culture condition, with the thymic environment provided. The three-dimensional structure and the expression of Notch/Delta signaling of the thymic environment have been shown to play critical roles for supporting the differentiation of cells to the T cell lineages (also, in Stage 2) [51e54]. 1093

Stage 4: Precursor T cells Mouse precursor T cells are identified to be thy-lþ, CD117low, CD3, pTþ (ref. cited in reference [4]). They can be derived from fetal and adult blood. Using markers such as the CD25, CD44, and TIS 21 (TPA-inducible sequence 21), pre-T cells can be classified into four subpopulations. They are the quiescent CD44þCD25TIS21þ early cells and CD44CD25þTIS21þ cells prior to TCR-beta selection. After selection, the cells are proliferating CD25þCD44þTIS-21low precursor T cells and CD25low CD44TIS21low cells. By transgenic overexpression of TIS21 in precursor T cells and HPC, it can inhibit the expansion of thymocytes. By transgenic overexpression of TIS21 in precursor T-cells and HPC, it can inhibit the expansion of thymocytes (Konrad and Zuniga-Pflucker, 2005). Thus, somatic and genetic manipulations would become easier with cloning and identifying such “quiencenting genes” and their ability to express at stage-specific matter. Other genes expressed which might play a role in the function of cells at this stage are the cell cycle control genes such as the D3 cycline of D type, the preTCR (pre-T Cell Receptor gene) and the BCL2A1 (antiapoptotic A1 gene). PreTCRþ cells are selected to survive and differentiate further, whereas preTCR cells are selected to die. The induction of the BCL2A1 gene will induce pre-T cell survival by inhibiting activation of caspase-3. The knock-down of BCL2A1 expression can compromise survival, even in the presence of a functional preTCR. However, the overexpression of preTCR-induced BCL2A1 can contribute to T cell leukemia in mice and humans. Both OLIG2 and LMO1 were overexpressed in large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2-LMO1 mice revealed upregulation of Notch1. Two genes considered to be downstream of Notch1 e Deltex1 (Dtx1) and preTCR-alpha e are also upregulated. The established OLIG2-LMO1 leukemic cell line was suppressed by a gamma-secretase inhibitor, suggesting that Notch1 upregulation is important for the proliferation of OLIG2-LMO1 leukemic cells. Thus Notch-Delta signaling is

PART 12 Hematopoietic System critical to trigger the T cell development program. In addition, commitment to the T cell lineage is also shown to depend on BCL1b, while initiation of the T cell differentiation program begins earlier with the induction of TCF-1 (Tcf7 gene product) and GATA-3 [107]. T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: they either differentiate or they die. These fates are determined by signals that originate from the TCR. CD4þCD8þ thymocytes undergo one of three fates in the thymus: positive selection, negative selection (TCR-mediated apoptosis), or death by neglect. Only 5% of developing thymocytes are exported as mature T cells. Negative selection of thymocytes that express TCRs with high affinity for self-peptide-Major HistoCompatible antigen (MHC) deletes potentially self-reactive thymocytes, generating a largely self-tolerant peripheral T cell repertoire. Most negative selection is thought to occur in the thymic medulla, for this contains two types of specialized antigen-presenting cell e DCs (dendritic cells) and thymic epithelial cells (TECs). Medullary TECs transcribe genes that are normally expressed in peripheral tissues. Negative selection can occur before or after, and is thus independent of, positive selection and in thymocytes at all stages of development. Negative selection in response to high-affinity ligands might be due to increased TCR occupancy or a slower off rate (kinetic proof-reading). Although discrepancies exist between blocking experiments and genetically deficient mice, TCR signal and a second co-stimulatory signal might be required for negative selection. The kinetics of MAPK (mitogen-activated protein kinase) signaling might determine positive vs. negative selection signals. ERK (extracellular signal regulated kinase) is induced more rapidly during negative selection, which might determine the triggering of transcriptional factors, NUR77 and NF-B (ref. cited in [4]). The recent view on roles of TCR signaling required for activating T cells and for generating memory T cells has been reviewed [55].

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In the thymus, two major T cell lineages e alpha-beta T cells and gamma-delta T cells developed from common lymphoid precursors. Their differentiation requires outside signaling. Development of alpha-beta T cells is driven in its early stages by signaling from the pre-TCR, most likely in a ligand-independent fashion, and later, by signals delivered by alpha-beta TCRs binding to their ligands e MHC molecules. Similarly, gamma-delta T cells require TCR signaling for their differentiation. However, most gamma-delta TCRs remain orphan receptors, and it is not clear whether ligands are required for gamma-delta TCR signaling in the development of gamma-delta T cells. Work with transgenic and knockout mice affecting the TCR pathways suggests that TCR signaling ensures the developmental progression towards mature alpha-beta T and gammadelta T lineages and that the strength of TCR signal instructs lineage fate; i.e., a stronger TCR signal results in gamma-delta T lineage commitment, and a weaker TCR signal results in alpha-beta T lineage commitment. This decision of alpha-beta vs. gamma-delta fate choice during thymus development is called the first TCR-controlled checkpoint. Distinct molecular programs have been revealed to govern this lineage decision choice. ERK, Egr (early growth response), Id3 (inhibitor of differentiation 3) were identified to be potential molecular switches operating downstream of TCR which determine lineage choice. Removal of Id3 was sufficient to redirect gamma-delta TCR transgenic cells to the alpha-beta T cell lineage, even in the presence of a strong TCR signal. However, in TCR non-transgenic Id3 knockout mice, the overall number of gamma-delta T cells was increased due to an outgrowth of a Vgamma1Vdelta6.3 subset, suggesting that not all gamma-delta T cells depend on this molecular switch for lineage commitment. Thus, two or more lineages not sharing a common molecular program might exist in the gamma-delta T cell lineage. In addition to signaling through TCR, signaling from Notch and CXCR4 receptors, cooperate with the TCR in controlling the development of alpha-beta T cells and gamma-delta T cells [56]. Precursor T cells are not self-renewing. Apoptosis occurs easily, and only some cells of this compartment differentiate into double-positive mature T cells. A scale-up expansion of this

CHAPTER 50 Lymphoid Cells population presents a challenge. Fetal thymus organ culture systems and suspension cultures with dissociated TECs and, a cell line, OP9, which requires a very high concentration of FBS, (20%, in the culture medium) transgenic with notch-ligand such as DL1 (Delta-Like-1), for studying this particular population are available (ref. cited in reference [4]). It is possible to expand transiently para-aortic splanchnopleurally-derived precursor T cells in culture but such cells perish quickly. Variations in the physical culture environment, such as the O2 level of the incubator where the fetal organ culture was set up, were shown to help the survival of (precursor) T cells. Free radicals promote molecules that lead to the apoptotic pathway. Thus, reducing the concentration of local O2 and increasing the level of N2 in the incubator, or supplying the cultured cells with a high density of stroma cells, favors the survival of (precursor) T cells. To engineer the thymic niche in vitro, Shukla and Zandstra (personal communication, unpublished) have engineered an artificial thymus niche containing an immobilized ligandhydrogel system containing Notch signaling ligands: DL4 (Delta-like 4) and Jagged-1 which are essential for T cell development. This system might allow a robust and scalable generation of mouse proT cells from HSCs and iPS (stage 2, above). It is also possible to grow mouse pre-T cells by using solid phase DL4, and cytokines such as IL7, SCF (Rolink, personal communication, unpublished). Beaudette-Zlatanova et al. [57] described a hTEC (human thymic epithelial cell) line culture system to support T lymphopoiesis from hHSCs. The cells were immortalized by infection with an amphotropic retrovirus from a cell line containing the HPV E6E7 early genes. The parental cell line, expressing low levels of DL1 and DL4, permits HPCs to differentiate to a B cell lineage. This cell line was engineered to overexpress mouse DL1, called TEC-DL1. In co-cultures with HPCs from cord blood or BM, TEC-Dl1 cells promote the generation of CD7þCD1aþ T-lineage-committed cells, most of them being CD4þCD8þ cells. CD3þ(lo) cells were detected within the Double Positive (DP) and Single Positive (SP) CD4 and CD8 populations. The CD3þ(lo) SP cells expressed lower levels of IL2R and IL7R. However, this cell line is not sufficient to generate CD3þ/high mature single positive T cells. OP9DL1 (and OP9) cell lines have been shown to be able to support the differentiation of T cells (and B cells), however due to its mouse origin, its clinical application for cell therapy is challenged by the regulatory authorities. Thus, this hTEC line might become a promising candidate for clinical application. The role of miR-142, -181, and -223 in mouse hematopoiesis has been studied [58] and it was found that ectopic overexpression of miRNA-181 in mouse HSPCs resulted in an increase in B lymphopoiesis, and miRNA-142 and -223 resulted in small but significant increases in T lymphopoiesis. However, the relevant target mRNAs were not determined. The analysis of human and mouse HSCs/HPCs showed large differences in miRNA expression. In human BM, miRNA-181 is expressed more weakly than miRNA-146, which was found to be expressed strongly in all mouse hematopoietic tissues. miRNA-142 is not expressed in human HSPs [59]. The in silico model indicates that miRNA-181 and miRNA-146 might block differentiation very early in human lymphopoiesis. The production of miRNAs by Dicer is required for efficient T cell and B cell development. miR-146, miR-223, and miR-150 target the developmental stage from lymphoid committed stage to T cell development in mouse. T cell development is controlled by complex protein signaling networks, which are subjected to regulation by miRNAs (Fig. 50.1). The expression patterns of miRNAs are found to vary between T cell subsets and stages of development. Mature miRNAs of a given miRNA species can have several variants, which can vary in length at either the 30 or 50 end, or can contain mutated sequences. Proliferating T cells express genes with shorter 30 UTRs than those in resting T cells, rendering these mRNAs less susceptible to regulation by miRNAs due to the

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PART 12 Hematopoietic System loss of miRNA binding sites. These findings suggest that miRNA-mediated regulation of mRNA targets in T cells is a dynamic process that is influenced by a broad range of factors [16]. Only a few selected individual mRNAs which play a role in early T cell development will be mentioned in this section. Xiao et al. [60] generated mice with elevated miR-17e92 expression in lymphocytes. These mice developed lympho-proliferative disease and autoimmunity, and died prematurely. Lymphocytes from these mice showed increased proliferation and reduced activation-induced cell death. The miR-17e92 cluster was shown to target mRNAs encoding for the pro-apoptotic protein BIM (BCL-2-interacting mediator of cell death) and the tumor suppressor, PTEN (phosphatas and TENsin homologue) during DN (double negative) thymocyte stage of development by increasing T cell survival. This mechanism likely contributed the disease and death of miR-17e92 transgenic mice, and to lymphoma development in patients carrying amplifications of the miR-17e92 coding region. The strength of TCR signaling influences whether thymocytes are positively or negatively selected during thymic development, and specific miRNAs have been implicated in this process, for example, Li et al. [103] showed that miR-181a enhanced TCR signaling strength by targeting multiple protein phosphatases, such as DUSP5 (DUal-specificity protein phosphatase 5), DUSP6, SHP2 (SH2-domain-containing protein tyrosine phosphatase 2) and PTPN22 (protein tyrosine phosphatase, non-receptor type 22), which lead to elevated steady state levels of phosphorylated intermediates and a reduction of the TCR signaling threshold. They showed that inhibiting miR-181a expression in the immature T cells reduced sensitivity and impaired both positive and negative selection. The miR-181a was also shown to increase the expression in mature T cells augmented the sensitivity to peptide antigens. 1096

Stage 5: Mature T cells and memory T cells Using cultures of mature T cells in the presence of APCs and recombinant cytokines, one can study the growth and differentiation of T cells from a variety of sources. Long term mouse and human T cell clones are also available. T cells can be maintained as clones in culture far longer than B cells. Human T cells (mainly NK cells) grown in the presence of cytokines in a short term culture have been re-implanted into autologous cancer patients. Most CD8þ and CD4þ T cells have a short lifespan. Memory T cells have been well studied, but in reality they do not exist in abundance and can be demonstrated in vivo only by repeated priming with antigen. They are very difficult to define at the cellular and molecular levels. There is no isotype 30 -end downstream of the TCR constant region for class switching to occur. Somatic hypermutation of the TCR-beta gene has never been claimed, and it is debatable whether the TCR-alpha gene is hypermutable. Memory T cells are thus defined using criteria such as accelerated cellular responses, distinct pathways of lymphocyte recirculation in vivo, distinct DNA motifs of TCR genes, cytokine- producing pattern and diminishing expression of surface markers (ref. cited in [4]), and functional and antigen requirements. In mice, memory CD4þ T cells are CD45RO, L-selectin (MEL-14)low. This is the equivalent of human CD45RO, keeping in mind that the CD45R family (A, B, C, and O) may not be the best marker to define naı¨ve versus memory T cells. Because foreign antigens do not always quickly elicit unprimed T cells, memory T cells must exist, but the commitment, the mechanism of development, and the maintenance of these cells are unknown. Memory T cells are thought to be generated, either when T cells acquire specificity to kill, or to help during thymic education, or they are generated during the mature stage.

MEMORY CD8þ T CELLS Because cell-bound antigen on APCs (see later for details) and more than one signal are required for educating T cells to perform effector functions instead of becoming tolerated,

CHAPTER 50 Lymphoid Cells work on the generation of CD8þ memory T cells has been mainly performed in vivo, using viruses. Is it possible to prevent cytotoxic T cells from performing their function by releasing granzymes. If this is the case, what happens to committed CD8þ T cells? Do they die, become anergic, or become memory cells? A study addressing the avoidance of granzyme B-induced apoptosis in target cells is interesting in this regard (ref. cited in reference [4]). Dephosphorylation of cdc2 was shown to be a critical step in granzyme B-induced apoptosis in the targets of cytotoxic T cells. A nuclear kinase encoded by the wee1 gene was transiently expressed and shown to induce phosphorylation of the tyrosine residues of cdc2 kinase, and that in turn provoked mitosis and the rescue of target cells. Because cytotoxic T cells are subject to being killed by their colleagues, the apoptosis pathway in these cytotoxic T cells could be similar to that of the target cells. The priming, clonal expansion, and differentiation into memory T cells can be achieved. The expansion of memory T cells can be demonstrated in culture. Besides viral peptide antigens, recent other examples are the use of mycobacterial glycolipids as the antigen, the antigen processed by CD1b, CD1e (ref. in [4]). Primed T cells are harvested from a human adult and stimulated in vitro with antigen with hIL2. The T cell clone can demonstrate the killing of cells infected with M. (Mycobacterium) tuberculosis (see later). It is interesting that DNA methylation may contribute to regulation of mouse T effector cell function (ref. cited in [4]). In Dnmt1/ (the maintenance DNA methyl-transferase), silencing of IL4, IL5, IL13, and IL10 in CD8 T cells was abolished, and expression of these Th2 cytokines increased drastically as compared with that of control CD8 T cells. Th2 cytokine expression also increased in Dnmt1/ CD4 T cells, but the increase was less than for CD8 T cells. As a result, both Dnmt1/ CD4 and CD8 T cells expressed high and comparable amounts of Th2 cytokines. Loss of Dnmt1 had more subtle effects on IL2 and IFN expression and did not affect the normal bias for greater IL2 expression by CD4 T cells and greater IFN expression by CD8þ T cells or the exclusive expression of perforin and granzyme B by the CD8 T cells. Dnmt1 and DNA methylation seem to be necessary to prevent cell-autonomous Th2 cytokine expression in CD8 T cells, but were not essential for maintaining proper T cell subsetspecific expression of Th1 or CTL effectors. Thus, transcription factors and DNA methylation are complementary and non-redundant mechanisms by which the Th2 effector program is regulated.

MEMORY CD4þ T CELLS Like memory CD8þ T cells, the generation of memory CD4þ T cells has been studied in vivo. This population of cells has been well characterized in mice. Effector memory CD4þ cells in mouse spleen against the soluble protein antigen KLH (keyhole limpet hemocyanin) and other protein antigen were found to be CD45RO, L-selectin, CD44þ and to produce elevated levels of IL4 (ref. cited in reference [4]). Whether this population of cells can be expanded in culture remains a question. Because T cells from IL2 knockout mice survive much longer than those of conventional mice, they may be useful for studying the development of memory T cells. Moreover, molecules such as Fas and Fas ligands of the apoptotic pathways play critical roles in determining the fate of cells after activation. The Bcl-2/bcl-x family seems to function by helping the survival of cells via action on the intermediate steps of apoptosis. Genes controlling the cell death pathway may play critical roles in the development of, and the subsequent genetic manipulation of, memory T cells. It is important to improve the conditions for growing CD8þ T cells in vitro, because for tumor therapy, the generation of tumor-specific CD8þ T cells is a critical step. Although surface markers and the life span of a population of T cells equivalent to those of the mouse system have been documented, ethical and safety considerations have prevented a systematic study of human memory T cells in vivo. The possibility of producing human CD4þ T cells on a large scale has been reported. By stimulating peripheral, resting T cells with cytokines such as IL2, anti-CD28, and solid phase anti-CD3, survival and proliferation of CD4þ, but not CD8þ, T cells could be greatly

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PART 12 Hematopoietic System promoted. What remains to be shown are the specificity and function of these cells and how they are related to the regulation of the Bcl-2/bcl-x family and CD95 (Fas/apo-1) ligand and whether they are candidate memory CD4þ T cells or abortive T cells.

ROLE OF APC, TOLL-LIKE RECEPTOR, IN THE ACTIVATION OF CD4þ, Th1 VS. Th2 PATHWAYS The recognition and stimulation of antigen epitopes by T cells requires that antigen molecules first be processed to become fragmented epitope and presented properly by APCs. The APCs identified are macrophages, dendritic cells, and B cells when in the process of TeB cell cooperation. T cells will only recognize the antigenic epitope when it is embedded in MHC (major histocompatibility complexes, H2 in mouse and HLA in humans), which enables the immune system to distinguish its own cells from foreign cells. Foreign antigens are great engineers. The nature of the antigen to be processed by the APCs decides which processed antigenic epitopes are to be presented by which kind of T cells and how to influence the reaction of the immune system. For example, when cytosol enters the host, viral proteins will be digested by proteasomes to become fragmented epitopes and be presented on the surface of APCs in complex with class I MHC. Their interaction with the CD8þ T cells will enable them to become cytotoxic T cells to perform cell-mediated immunity.

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Soluble bacterial toxins (with exceptions, see next section), phagocytized into acidic vesicles in APCs, will be processed by the vesicular proteases, and the fragmented epitopes will be presented on the surface of APCs in complex with class II MHCs. In macrophages, foreign proteins within the acidic phagocytic vesicles will be digested and presented on their surface in complex with class II MHC, similar to APCs. These epitopes are recognized by the CD4þ helper T cells and will cause immune responses, such as the activation of phagocytes and antibody production by activated B cells. Two major subsets, Th1 and Th2, were originally recognized in the population of CD4þ T cells, though more recently Treg, Th17 cells were also described. Th1 has been shown to secrete IFN-gamma and to help cell-mediated responses. Th1 stimulation results in local inflammatory responses, including the activation of macrophages, and the production of complement-fixing and -opsonizing antibodies IFN-gamma and IL12 drive the differentiation of IFN-gamma-producing Th1. Th2 is shown to secrete IL4, IL3, IL5, IL13, and IL25 and to help the generation of antibody-mediated responses. IL4 is also required for Th2 cell differentiation. Th2 activation leads to the production of IgG and IgE and to the activation of basophil and eosinophils to fight against allergens and large mucosal parasites [61]. It has been a puzzle as to how Th1 vs. Th2 cells recognize and distinguish the epitopes of foreign antigen on APCs, since both require the presence of class II MHCs. The mammalian equivalent of TLRs (Toll-like receptors) derived from Drosophila ’Toll’ was discovered and helped to address this question. TLRs are found on the surface of APCs. More than 11 different receptors have been shown to bind to various cellular components of microorganisms. The binding of most of these components to the preferred TLRs produces a differential signaling cascade that stimulates an immune response in favor of Th1 over Th2. Stimulation of TLR7, and TLR8 in humans by synthetic imidazoquinoline compounds leads to activation of the Th2 pathway; while stimulation of TLR4 by MPL (monophosphoryl lipid A) and LipoPolySaccharide (LPS) or viral peptides such as VIPER [104], and stimulation of TLR9 by unmethylated CpG of bacterial DNA lead to activation of the Th1 pathway. TLR11 and several other TLRs are also used for activation of Th cells, but by which specific pathways remains unknown [62,63], and other refs. cited in reference [4]. As shown in Fig. 50.2, signaling by TLRs involves five adaptor proteins: MyD88, MAL, TRIF, TRAM and SARM. Apart from NF-kappa-B activation and IFN-gamma signaling, MyD88 was shown to activate the transcription factors IRF1, IRF5 and IRF7. MAL and TRAM act as bridging adaptors, with MAL

CHAPTER 50 Lymphoid Cells recruiting MyD88 to TLR2 and TLR4, and TRAM recruiting TRIF to TLR4 to allow for IRF3 activation. The fifth adaptor, SARM, was shown to negatively regulate TRIF [64].

THE ROLE OF CD1 IN THE T CELL RECOGNITION OF LIPID ANTIGEN Besides peptide-protein antigen, which stimulates the Th1, Th2 pathway via TLR on APCs, lipid antigen activates T cells by CD1 molecules, independently of TLR. Studies have shown that CD1 molecules are lipid antigen-presenting molecules that offer the lipid antigens to the TCR of T cells, resembling MHC presentation of peptides, a MHC class III molecule. They have no structural homology with TLR, though they are also present on APCs. Some lipids are recognized by the immune system as classical antigens, resembling peptides associated with MHCs, whereas other lipids trigger the TLR innate receptors. A panel of T cell clones with different lipid specificities isolated from M. tuberculosis was established. A novel lipid antigen belonging to the group of diacylated sulfoglycolipids, Ac2SGL(2-palmitoyl or 2-stearoyl 3-hydroxyl-phthioceranoyl-20 -sulfate-alpha-alpha’-D-trehalose) was identified. Ac2SGL is mainly presented by CD1b, after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release IFN-gamma, efficiently recognize M. tuberculosis-infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis but is independent of the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate vaccine against tuberculosis. Another molecule which has been isolated, PIM6 (hexa-mannosylated phosphatidylmyo-inositols) is another molecule isolated, stimulates CD1b-restricted T cells after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase, and soluble CD1e, one of the CD1 family members, is required for the processing. Recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6 into dimannosylated forms: PIM2, which was stimulatory to specific CD1b-restricted T cells. CD1 molecules, after exiting from the endoplasmic reticulum, travel to the cell surface via the secretory pathway before being re-internalized into the endosomal compartments. CD1a molecules undergo cycles of internalization into early sorting endosomes followed by early recycling endosomes, whereas CD1c molecules travel to early recycling endosomes and, to a lesser extent, to late endosomes and lysosomes. In contrast, CD1b and human CD1d molecules recycle in late endosome, lysosome compartments where they can co-localize with CD1e. It was debatable whether CD1e participates in presentation of lipid antigens by other CD1 molecules. Recent studies showed that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect was also shown in APCs from CD1e transgenic mice, which was caused by the capacity of CD1e to facilitate rapid formation of CD1-lipid complexes, and to accelerate their turnover. Thus CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses through glycolipid editing. (ref. cited in [4,65,66]).

INNATE IMMUNITY, T REGULATORY CELLS, AND Th17 CELLS Another heterogeneous population of T cells has been re-identified, the so-called Treg (Tregulatory) or suppressor cells. They can be innate or induced. The subset of Treg cells in mouse can be identified using different surface markers and the secreted cytokines. They include: 1) Naturally occurring T cells possessing CD4þCD25þNrp1þFoxp3þTGFbetaþ, and 2) Cells induced in the periphery following antigen exposure, such as CD4þ Treg1 cells possessing CD4þCD25/þFoxp3/þIL10þIFNgammaþTGFbetaþIL5þ; Th3 / Th17 cells

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PART 12 Hematopoietic System possessing CD4þCD25þFoxp3?TGFbetaþIL4þ/IL10þ/ and CD8þTreg cells possessing CD8þCD25lowfoxp3-IL10þTGFbetaþ/ (ref. cited in [4]). The CD4þTreg cell subsets can be further classified on the basis of their phenotype, cytokines secreted (such as newly described IL35) function, migration preference to lymphoid or to nonlymphoid tissues [61]. The cells express Foxp3 (fork-head/winged helix transcription factor), which appears to play a key role in supporting their action [67,68]. Microarray study of Treg cells isolated from different tissues of mice transgenic with Foxp3-GFPþ revealed an even more diverse heterogeneity of cell populations (later [69]). The expression of Foxp3 in Treg cells is not stable, several studies have reported the plasticity of Treg in diseases and in inflammation, shown rather complicated mechanisms exerted and influenced by (micro)-environment. It is also important to keep in mind differences in Foxp3 expression observed in mouse vs. human T cells. In mouse, Foxp3 appears to be a reliable marker for either thymic-derived or induced Treg cells. However, nearly all human CD4þ T cells transiently express Foxp3 during activation, and this is not associated with acquisition of regulatory function. Human MSC have also been shown to express Foxp3 (in Stage 2). Thus, Foxp3 alone is not a reliable marker for human Treg cells, further complicating analyses of their function and stability [61]. In addition, not all antibodies against markers used to characterize mouse Treg work for human Treg, CD45RA marker is used in conjunction with Foxp3 to better classify human Treg phenotypes [106]. So far, three classes of human Treg have been identified based on Foxp3 and CD45RA: (1) Resting Tregs (rTreg) are CD45RAþ, Foxp3low; (2) CD4þ. Activated Treg (aTreg) cells are CD4þ, CD45RA, Foxp3high; and (3) cytokine-secreting non-Tregs are CD4þ, CD45RA, Foxp3lo. The way that the cytokines which are secreted fit into this classification is not clear. 1100

When stimulated, rTreg can increase Foxp3 expression, convert to an aTreg, and proliferate in vitro. However, aTreg rapidly die following proliferation. Furthermore, aTreg inhibit rTreg conversion to aTreg, controlling the balance of active Treg. Naı¨ve CD4þT cells and non-Treg CD4þ cells are also capable of converting to aTreg, but less so than rTreg. Interestingly, CD4þ non-Treg cells have higher levels of Th17 factors (RORgamma, RORalpha, IL17, later), showing a bias toward developing a Th17 phenotype. APCs are essential in the activation of Treg cells; the immature APCs can support the differentiation of Treg cells. Targeting of antigens to immature dendritic cells has been shown to result in antigen-specific T cell tolerance in vivo. The mechanism of suppression could be multiple and is not entirely known. One possibility is to tolerate the APCs, and the other is via the activation of Treg. Treg cells can down-regulate Th1, and Th2 responses. Th17 cells are rather resistant to Treg suppression (later). Suppression of CD8þT effector cells by Treg could occur to prevent the development of effector function, and could interfere with the execution during the effector phase. Direct cell contact through binding of cell surface molecules such as CTLA4 (cytotoxic T cell-associated antigen 4) on Treg cells to CD80 and CD86 molecules on T cytotoxic cells results in the suppression. The local production of suppressive cytokines such as TGFbeta, IL10, and IL35 also results in suppression. It requires the appropriate co-localization of Treg and T effector cells in different tissue, and may involve interference with the T cell receptor signaling that triggers transcription factors important in regulating effector cell function [61,70]. Treg are usually anergic (do not respond or proliferate to antigen stimulation), IL2 dependent and proliferate poorly in culture. Naı¨ve CD4þ T cells can be converted into Treg cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells or by changing culture conditions, such as adding TGFbeta or reducing IL2, or at the presence of MSC [34]. Treg cell populations induced in low dose antigen conditions could subsequently be expanded by delivery of higher- or immunogenic-dose antigen [70,71].

CHAPTER 50 Lymphoid Cells For a better understanding of transcriptional control in Treg cells, Feuerer et al. [69] compared gene expression profiles of a broad panel of Treg from various origins or anatomical locations of mice such as spleen, peripheral lymph nodes (cervical, axillary, inguinal) of in vivo converted Foxp3-GFPþ cells, and lamina propria T cells from Foxp3-GFP mice [68]. The data showed that Treg generated by different means form different sub-phenotypes and could be identified by particular combinations of transcripts. None of which fully encompassed the entire Treg signature. Molecules involved in Treg effector function, chemokine receptors, and transcription factors were differentially represented in these sub-phenotypes. Treg from the gut are different to Treg converted by exposure to TGFbeta in vitro, however, they resembled a CD103þKlrg1þ sub-phenotype preferentially generated in response to lymphopenia. The study of the mechanisms of human MSC-mediated allo-suppression has suggested a sequential process of Treg cell induction involving direct MSC contact with CD4þ cells followed by prostaglandin E2 and TGFbeta1 expression (Stage 2). In vitro, Treg cells could be generated by T cell stimulation in the presence of high doses of TGFbeta [72]. However, the fast in vitro conversion process resulted in Treg with unstable Foxp3 expression that correlated with lack of demethylation of the Foxp3 locus. This in vitro conversion method was suboptimal where Treg cell activation was limited for an initial 18 hour culture period; exogenously TGF-beta must be continuously added into the culture and required the inhibition of PI3K (Phosphatidyl Inositol 3-Kinase), Akt/PKB (Protein Kinase B), or protein kinase mTOR (mammalian target of rapamycin) [73]. Thus, how to conduct largescale production of stable Treg cells in vitro is still developing. Polansky et al. [74] found that the in vivo priming with suboptimal antigen resulted in complete demethylation of the TSDR (Treg-specific demethylated region) within the Foxp3 locus, and stable Foxp3 expression. Daniel et al. [75] described an efficient protocol applying the combination of everolimus, a rapamycin analog, and IL2-IL2 antibody complexes to achieve highly effective antigen-driven conversion of naı¨ve CD4þTh cells into Treg and their expansion in transgenic mice. The mechanism of the role of everolimus in enhancing Treg conversion was possibly by interfering with T cell co-stimulation, reducing cell division and thereby activation of DNA methyltransferase1; and by reducing T cell activation through the ATP-gated P27 receptor controlling Ca2þ influx. The resulting Treg cells exhibit increased stability of Foxp3 expression even when generated in TGFbeta-containing media in culture. This protocol constitutes an important tool to achieve immunological tolerance by Treg vaccination. Whether it can be applied to therapeutic usage remains to be investigated. Th17 (T helper 17) cells were first described in mice [76,77] then in humans [78] and other groups) to be a CD4þ T cell subset characterized by production of IL17, which is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Studies in RA (rheumatoid arthritis) have shown that cytokines secreted by Th17 cells can be further divided: IL17A (originally described), IL17F (a total of six members), IL22, and IL21 [79]. Besides differentiate to Th1, Th2 and Treg, naı¨ve CD4þ T helper cells can be induced to differentiate to Th17 lineage according to the local cytokine milieu. The differentiation towards Th17 and Treg phenotypes is in a mutually exclusive manner. Each phenotype is characterized by unique signaling pathways and transcription factors, such as T-bet for Th1, GATA-3 for Th2, Foxp3 for Treg and RORalpha (Receptor-related Orphan Receptor) and RORgammat for Th17 cells [80e82]. Very little is known about the pathways that control the expression of IL17 in humans. Evans et al. [78] showed that the factors that determine the expression of IL17 in human CD4þ T cells are different from mice. IL6 and IL21 were unable to induce IL17 expression in either naı¨ve or activated T cells, and TGFbeta actually inhibited IL17 expression. The expression of IL17 was maximally induced from pre-committed precursors present in human PBL by cellcell contact with Toll-like receptor-activated monocytes / APCs in the context of TCR ligation.

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PART 12 Hematopoietic System Although the pro-inflammatory activity of Th17 cells can be beneficial to the host during infection, uncontrolled or inappropriate Th17 activation has been linked to several autoimmune, including RA, multiple sclerosis, psoriasis, lupus, inflammatory bowel disease, asthma, and might also be involved in tumor and transplant rejection. In the absence of the Th1 in vivo, Th17 cells are capable of rejecting cardiac allografts. A report suggests that Th17 cells might be involved in the auto-inflammatory disorder AOSD (adult-onset Still’s disease) [83]. Addition of inflammatory signals to a Treg inducing environment leads to Th17 development. Established Treg can be converted to Th17 cells under inflammatory conditions. These findings might explain some of the dys-regulation seen in autoimmune diseases [84,85]. T cell differentiation is modulated by several miRNAs: miR-148a is shown to repress mRNAs encoding for BIM, affecting the commitment of Th1 memory (Haftmann and Radbruch, personal communication, unpublished). miR-155 is shown to have a role in B cells (later), and dual roles in T cells: 1) To repress mRNAs of MAF (Macrophage-Activating Factor) which leads to in favor of the differentiation of CD4þTh0 cells towards Th1 cells; 2) To repress mRNAs of SOCS1 (Suppressor Of Cytokine Signaling 1) which is implicated in the survival of Treg cells.

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The miR-155 maps within, and is processed from an exon of its precursor, called bic. In activated B, T cells, and APC, bic/miR-155 shows greatly increased expression. Bic/miR-155 knockout mice are immune-deficient and developed lung pathology with age. Bic/miR-155 was shown to modulate levels of transcription factor c-Maf in CD4þT cells, contributing to bias towards Th2 differentiation [86,87]. Global analysis of the network of genes regulated by Foxp3 has identified the miR-155, which is highly expressed in Tregs, as a direct target of Foxp3. The miR-155 knockout mice have reduced numbers of Treg in the thymus and periphery. However, there was no evidence for defective suppressor activity in miR-155/ Treg cells, either in vitro or in vivo, suggesting that additional unidentified miRNAs control Treg function [88]. Lu et al. [89] have shown that miR155 deficiency in Treg cells results in increased SOCS1 expression accompanied by impaired STAT5 activation in response to limiting amounts of IL2. Drosha and Dicer, the two RNaseIII enzymes, mediate the stepwise maturation of miRNAs. Chong et al. [90] found that miRNA biogenesis is indispensable for the function of Treg cells, mice specific deletion of either Drosha or Dicer phenocopies lack a functional Foxp3 gene or Foxp3þ cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease later in life. In order to analyze the role of miRNAs in the development and function of Treg cells, Zhou et al. [91] have crossed a Treg cell-specific, Foxp3-GFP-hCre bacterial artificial chromosome transgenic mouse to a conditional Dicer knockout mouse. The peripheral Treg cells showed altered differentiation and dysfunction. Dicer-deficient Treg cells failed to remain stable, as a subset of cells down-regulated the FoxP3, whereas the majority expressed altered levels of multiple genes and proteins including Neuropilin 1, glucocorticoid-induced TNFR, CTLA4. A significant percentage of the Treg cells possessed a memory Thelper cell phenotype including increased levels of CD127, IL4, and IFNgamma. Dicer-deficient Treg cells lost suppression activity in vivo. The mice rapidly developed fatal systemic autoimmune disease resembling the foxp3 knockout phenotype. Th17 cells have been identified as important mediators of inflammatory disease. Du et al. [92] found that miR-326 promotes Th17 cell development in vitro and in vivo by targeting ETS1, a negative regulator of Th17 differentiation, resulting in promoting the differentiation of CD4þTh0 cells toward Th17 cells. The expression of miR-326 was correlated with disease severity in patients with MS (Multiple Sclerosis) and mice with EAE (Experimental

CHAPTER 50 Lymphoid Cells Autoimmune Encephalomyelitis). In vivo silencing of miR-326 resulted in fewer Th17 cells and mild EAE, its overexpression led to more Th17 cells and severe EAE.

Stage 6: Precursor B cells Mouse and human precursor B cells have been characterized extensively. Surface markers and molecular events of precursor lymphoid to precursor B cells at intermediate stages of development have been defined (Table 50.1, Fig. 50.3) [93]. The markers for peripheral B cells at different stages are summarized in Table 50.1 [94]. More updated markers are summarized by Alinikula and Lassila [95]. For the reason of simplicity, such newly assigned markers are not included in the tables of this chapter. TABLE 50.1 Expression of cellular and molecular markers during early stages of B cell development Progenitor B stage

Precursor B stage

Human B cell CD34 CD38 gL CD10 CD19 lambda-like/Vpre-B Rag 1 TdT VH/Cmu V-kappa/C-kappa mbl/B29 cyto-mu

I + nd  nd  + + +   +/ 

II + + + +  + + + + + +/ 

III + + + + + + + + + + + +/

Mouse B cell B220 CD43 HSA BP1 lambda-5/Vpre-B Rag 1e2 TdT mbl D-JH VH-D-JH

A + +   +/ +/ +/ +/

B + + +  + + + + +

C + + + + + + + + + +(out)

CD43 c-Kit CD25 IL-7Ralpha CD19 gL lambda5/Vpre-B Rag 1e2 TdT cyto-mu DHeJH VHeD-JH VLeJL

Pro-B

Pre-B I

+ (+)/  +  + + + + (+)   

+ (+)  + + + + + +  +  

Pre-B  + + + + + +  + + + + C’ + + + + + + +/ +

+(on) Pre-B II

Large +/  + + + + +   + + + 

D +  + + + +  +

Small   + (+) + + + +  + + + +

Data in this table are partially derived from Martensson et al. [102], Matthias and Rolink [94], see Chen [4].

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PART 12 Hematopoietic System

TABLE 50.2 Expression of cellular markers during periphery stages of B cell development Stage periphery B cell marker

CD21 CD23 CD93 IgM IgD

Immature

Mature

t1B cell

t2B cell

t3B cell

MZB cell

Follicular B cell

  þ þ (þ)

þ þ þ þ þ

þ þ þ þ þ

þ (þ)  þ (þ)

þ þ  þ þ

t: transitional; peripheral immature B cells can be further classified into three transitional stages. MZ: marginal zone. This table is modified from Matthiasand Rolink [94]. More markers are described recently by Alinikula and Lassila [95].

MOUSE PRE-B CELL LINES

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In the mouse system, with the help of several stroma cell lines and recombinant growth factors such as IL7 (in reference [4]), it becomes feasible to expand mouse pre-B cells without gross differentiation. On release from the stroma cells and IL7 and in the presence of the bacterial mitogen LPS, some pre-B cell lines differentiate into plasma cells. Pre-B cell clones have been established from various lymphoid organs of wild-type, transgenic, and knockout mice. Thus, both somatic and genetic manipulation of these cell lines becomes possible. Some pre-B cell lines have been used to repopulate SCID mice and RAG-2 knockout mice. Injected cells migrate to the bone marrow, lymph nodes, peritoneal cavity and spleen. Plasma cells, mature B cells, and pre-B cells were detected in the host. The percentage of cells that mature into various B compartments seems to vary from experiment to experiment. However, several questions remain. Do these pre-B cell lines retain the capacity to expand? Are these cells self-renewing in vivo, as stem cells must be? The critical experiment of repeatedly transferring donor pre-B cells from one host to another, to show that the implanted cells are still precursor B cells, has not yet been done. Pax5 signatures the commitment of lymphoid precursors to B cell lineage. Pre-B cells established from Pax5/ mice differentiated into T cells, myeloid cells, dendritic cells and osteoclasts but not mature B cells in SCID mice. The data indicate that the microenvironment, which plays a role in keeping pre-B cells committed to the B cell lineage, may be missing in the Pax5/ mice. BAFF (B cell activation factor, BLYS) belongs to the Tumor Necrosis Factor (TNF) family, and its receptors have a critical role in the transition from immature to mature B cells (ref. cited in references [4,94,95]). Due to its potential to develop into mature B- lymphocytes and also into antibody-secreting plasma cells, which can be performed either in vitro or in vivo, mouse pre-B cells have been subjected to genetic modification to become a vehicle to generate and secrete human antibodies against a variety of infectious antigens in SCID mice. The genetically modified pre-B cells can be subjected to T cell-dependent stimuli, inducing somatic hypermutation in the human antibody constructs. In this way, affinity-matured antibodies can be generated, allowing the development of therapeutic antibodies with the pharmacological efficacies.

CURIOSITIES OF GROWING HUMAN PRECURSOR B CELLS The question of whether human precursor B cells can expand ex vivo is still open. Several reports claim it is possible by using either primary mixed human or mouse stroma cells (ref. cited in reference [4]). When subjected to mixed stroma cells and cytokines such as IL7, most human bone marrow cells expand for a limited period and then either perish or

CHAPTER 50 Lymphoid Cells

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FIGURE 50.3 Surface markers and sorting strategy for isolation of thymic subsets. (a) Synopsis of cellular populations and surface markers used for isolation of lymphocyte developmental stages. Top: B cell development; bottom: T cell development. Cellular stages are depicted as circles. Markers used for separation of the stages appear in the upper boxes; Ig and TCR gene loci rearrangement status appear in the lower boxes. Arrows connect corresponding stages between B- and T cell development. SL, surrogate L chain. (b) Cell-sorting strategy for separation of T cell precursors. To obtain DN thymocytes, CD4þ and CD8þ cells were removed by complement-mediated lysis from thymus single-cell suspension. Remaining lymphocytes were stained with a panel of lineage markers (B220, NK.1.1, CD3, CD4, CD8), and negative cells (R2 gate in top left panel) were gated and analyzed for CD25 and CD44 surface expression (bottom left). For the isolation of DP and SP subsets, single-cell suspension was stained with CD4 and CD8 mAbs (top right); SP cells were sorted according to gates R2 for CD4þ and R3 for CD8þ. DP thymocytes (gate R4 in top right panel) were further resolved into large (R5) and small (R6) subsets (bottom right). From Hoffmann et al. [93] with permission. (c) Similarity scores of ordered gene lists between human and mouse precursor B cells (open bars) and between corresponding stages of mouse B and T cell development (filled bars). Pool I, II and III are mouse Pre-BI, large Pre-BII, Small Pre-BII, stages shown in (a). (Modified from Hoffmann et al. [15]. Fig. 4A.)

PART 12 Hematopoietic System differentiate. The culture conditions established seem to be appropriate only for the short term expansion of pre-B cells. However, no normal human pre-B cell lines have been established. This distinguishes mouse and human precursor B cells. Global gene screen using microarrays on cells from stages of precursor mouse B, human B and mouse T developments has shown that gene expression patterns differ substantially between human and mouse B cell development. Among 644 genes which were differentially expressed in four early stages of human B cell development, only 48, 86, and 75 genes could be identified, which are upregulated in both human and mouse pre-BI, large pre-BII, and small pre-BII cells, respectively (see Fig. 50.3a for stage definition). A comparison of mouse B and T cell development reveals that gene expression patterns of early mouse B and T precursors are most similar, but not mouse B and human B precursors. However, in more differentiated precursors, human and mouse B precursor have a more similar gene expression profile (panel C) [93]. This finding might partially explain why the culture conditions that are optimal for growing mouse precursor B cells are not optimal for growing human precursor B cells.

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The study of human pre-B cells cannot be separated completely from the study of human HSCs and HPCs, given the complexity of the cell types involved and the difficulty in establishing human stroma cell lines. The establishment of human stroma cell lines supporting the growth of human pre-B cells is critical. The failure lies partially in the inability of adherent cells derived from human bone marrow (stroma cells) to proliferate well under normal culture conditions with conventional sera. Methods for immortalizing human cells include transfecting plasmids or retroviruses containing oncogenes such as SV40 T antigen. Dando et al. [40] have done that with human fetal bone marrow, and established many stroma cell lines; a few cell lines were partially characterized for supporting the growth and expansion of cord blood stem cells. However, no further work was performed to test these cell lines using other cell types. Cell proliferation is a prerequisite for the stable integration of transgenes into chromosomes and for immortalization. A breakthrough for human HSCs and pre-B cells would be to establish stroma cell lines for B lymphopoiesis and to optimize the conditions for the growth of stroma cells, as has been done for human MSCs (ref. cited in reference [4]). MSCs have been shown to expand to large quantity when selected lots of FCS are used. The topic of expanding MSCs for clinical trials was discussed in Stage 1 (above).

THE ROLES OF miRNAs IN PRECURSOR B CELLS The roles of miRNAs in controlling the development of ProB to PreB to B cells are thought to involve the modulation of key factors that control transcription factor networks, V(D)J recombination, selection of BCR, Ig). Overexpression of miR-181 causes a skewing of hematopoiesis towards the development of B cells, leading to a two- to three-fold increase in the number of B cells and no increase in T cells or myeloid cells, but the target mRNA of miR-161 was not identified [58]. Mice with a conditional deletion of Dicer in early progenitor B cells had a block at the pro-B to pre-B transition [96]. The proapoptotic molecule BIM was highly upregulated. B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2 (B cell lymphoma 2). The V(D)J recombination program in developing B cells of Dicer-deficient mice was investigated, and intact Ig gene rearrangements in IgH and IgKappa loci were found, but increased sterile transcription and usage of D(H) elements of the DSP family in Ig Heavy chain (IgH), and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene. Besides affects T cell development (above), the repression of mRNAs coding for the proapoptotic factor BIM (also called BCL2-like 11) by miR-17e92 cluster also affects the differentiation of pro-B to pre-B cells. The changes observed by gene expression profiling of Dicer-

CHAPTER 50 Lymphoid Cells deficient B cell precursors were similar to those observed in B cells lacking the miR-17e92 family. Constitutive expression of miR-150 causes a block at the pro-B to pre-B cell transition [112], and this inhibition depends on dys-regulation of c-Myb expression. Mice deficient in miR-150 have an accumulation of B-1 B cells in the spleen and the peritoneal cavity, with a relative decrease in the number of short life B-2 B cells. The miR-150-deficient mice have increased levels of antibody secretion both at baseline and following T cell dependent antigenic stimulation [97]. Overexpression of miR-34a causes an increase in cells at the pro-B to pre-B cell transition. miR-34a was shown to target transcription factor Foxp1, which regulates expression of the Rag1 and Rag2 (recombination-activating genes) (in [113]). Because miR-34a is a p53-induced miRNA [98], this repression might link the regulation of DNA damage responses with regulation of RAG proteins. Besides influence stem cell homeostasis, miR-221 and mir-222 are suggested to repress Pax5/þ expression, implied in the migration destination of Pre-B/B cells to peripheral lymphoid tissues (Melchers, personal communication, unpublished).

Stage 7: Memory B cells B cells with surface Ig isotypes such as IgG, IgA, and IgE, which possess higher affinities for antigen, are generally defined as memory B cells. In human beings, CD38þ, CD20þ germinal center B cells can be distinguished from memory CD38, CD20þ B cells and CD38þ, CD20 plasma cells. In mice, though still debating, CD38high, CD27þ, IgD memory B cells are thought to be different from long-live and short lived plasma cells, both are CD38þCD138þ [95]. The mechanisms of memory B cell generation are unknown and have been the subject of debate for decades. Memory B cells are mature B cells that have encountered antigen that have been activated but not tolerated and have switched to higher-affinity isotypes. IgM-secreting plasma cells, Syndecan-1/CD138þ, are terminal cells destined to die. It is not known how memory B cells develop in vivo. In vitro, at least two systems may generate memory B cells: the germinal center-like culture system and the suspension culture system, wherein resting B cells are activated by LPS plus anti-mu. These B cells are activated, alive, proliferating, and nonabortive, but they are not plasma cells.

A SYSTEM STIMULATING GERMINAL CENTERS IN VITRO TO CULTURE B CELLS In a culture environment that mimics the germinal center in lymphoid organs, B cells survive better and live longer. A germinal center-like environment is a culture system that provides cytokines and supporting cells from either purified follicular dendritic cells (FDCs) from mouse spleen (ref. cited in [4]) or stroma cells (L cells transfected with CD40 ligand called CD154, or fibroblasts). Both human and mouse B cells survive for two weeks instead of three to four days, and absolute cell numbers increase two- to three-fold. The in vitro germinal center culture system developed was designed to study the differentiation of mouse B cells into plasma cells rather than to maintain long term B cells in vitro. Mouse FDCs are non-proliferating, terminally differentiated cells. However, other studies reported that the primary mouse FDCs could be partially replaced by fibroblast cell lines expressing CD154. The cytokines, required to maintain mature B cells in growth phase and differentiation, are controversial. For mouse B cells, combined IL2, IL4, and IL5 induce differentiation into mature cells; for further differentiation into plasma cells, IL6 seems to be essential. For human B cells, IL2 plus ILI0, combined IL3, IL6, IL7, and combined IL2, IL6, IL10, and more recently discovered cytokines IL21 (later), have been reported to play roles in plasma cell

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PART 12 Hematopoietic System differentiation. Cellular interactions, including those mediated by CD40 and CD154, are critical in the generation of both memory B cells and plasma cells. If CD154 is removed in the secondary culture, human B cells differentiate into plasma cells. These memory-like cells are neither cell lines nor cell clones; rather, they are a mixed B cell type with a limited life span (up to a few weeks), and they preferentially switch to certain Ig isotypes, such as IgG and IgA. The data suggest that down-regulation of the J chain may not be essential during the development of memory B cells. IL21, a cytokine affecting T cells, NK cells, and B cells, signaling through IL21 receptor and the common cytokine receptor gamma chain, has been shown to play a role in stimulating the differentiation of mouse (refs cited in reference [4]). In the mouse, IL21 is shown to promote differentiation of B cells into CSR (Ig class switch recombination) and plasma cells, using IL21-transgenic mice and hydro-dynamics-based gene delivery of IL21 plasmid DNA in vivo and in vitro. IL21 induces expression of Blimp-1 and Bcl-6, which play a role in the development of autoimmune disease. When human B cells were stimulated through the BCR, anti-IgM, a minimal proliferation, IgD down-modulation, and small numbers of plasma cells were shown by IL21 stimulation. In contrast, after anti-CD40 activation of human B cells, extensive proliferation, CSR (Class Switch Recombination), and plasma cell differentiation were demonstrated by IL21 stimulation. On cross linking BCR and CD40, IL21 induced the largest numbers of plasma cells. IL21 drove both CD27þ memory cells and naı¨ve cord blood B cells to differentiate into plasma cells. In the latter, the effect of IL21 was more potent than the combination of IL2 and IL10. IL21 co-stimulation induced the expression of Blimp-1 and AICD (activation-induced cytidine deaminase), required for CSR, secreted IgG from B cells, but did not induce somatic hypermutation. IL2 enhanced the effects of IL21, whereas IL4 inhibited IL21-induced plasma cell differentiation. 1108

A SUSPENSION CULTURE SYSTEM FOR STIMULATING B CELLS WITH LPS PLUS ANTI-MU Systems for the short-term culture of primary splenic lymphocytes have long existed (in [4]). These systems are valuable for studying the proliferation and differentiation of B cells, T and B cell interaction, the priming of B cells by antigen, and the mechanism of memory B cell generation. If B cells could be kept alive and not tolerated but could be prevented from becoming IgM secretory plasma cells, they might become memory B cells. One example is the finding that when stimulated with a bacterial mitogen, LPS, some B cells die and some proliferate and become plasma cells (in [4]). When stimulated with anti-mu, most B cells die right away, some proliferate and exhibit growth arrest at G1 phase and then die two days later, and none become plasma cells. When stimulated with LPS plus anti-mu, most B cells proliferate and none become plasma cells. This non-plasma-cell phenomenon has been known as an anti-differentiation effect, and it was postulated to be a way to generate memory B cells in culture. Through the efforts of many including us, the molecular mechanism of this anti-differentiation phenomenon became clear. In the presence of the two stimuli, B cells proliferate maximally, over 90% being in the cell cycle, but IgM secretion is turned off. The block has been shown to be primarily at the level of nuclear RNA processing of the mum-to-mus switch. Inducible nuclear factors binding to the pre-mRNA secretory polyA site have been reported, though the nature of these factors remains unclear.

TRANSCRIPTION FACTORS AND miRNAs ACTIVE IN THE DEVELOPMENT OF PLASMA CELLS On activation of B cells, many transcription factors (Oct-2, OBF-1, Blimp-1, or PRDI-BF1 in human, Xbp1, IRF4, Bcl6, etc.) become engaged in the production and secretion of Ig genes. Extensive discussion on the transcriptional factors controlling the differentiation of B cells has been summarized in reference [4]. Here I will just briefly mention some key players.

CHAPTER 50 Lymphoid Cells Oct-2 and Blimp-l transcription factors play a role in the decision to switch from mum to mus. Blimp-l is described as a cofactor of transcription factor PU.1 and was shown to bind to multiple Ig-enhancer motifs and the J chain regulatory element. It is crucial for the transcription of mu, kappa, and J chains. Oct2 is POU-domain-containing transcription factor, binds to the mu intron enhancer octamer motif, on which they can form a ternary complex with the coactivator OBF1, and is essential in transcriptional activation of the mu chain. It was shown that Oct2 and the J chain are highly expressed in LPS-stimulated B cells and are diminished in LPSþ anti-mustimulated B cells. It has been shown that Blimp-l is highly expressed in LPS-stimulated B cells and is diminished in LPSþ anti-mu-stimulated B cells. On the other hand, sterile gamma chain is highly expressed in the latter system. Transfection of Blimp-l into LPSþ anti-mu-activated B cells provoked them to become IgM-secreting plasma cells. The data indicated that transcription factors such as Oct2 and Blimp-l are tightly regulated in plasma cell development. If one postulates that LPSþ anti-mu-stimulated B cells represent some stage in memory B cell development, then the down-regulation of Oct2 and Blimp-l reflects the specific transcriptional regulation when B cells make the commitment to the memory cell pathway instead of the plasma cell pathway. The identification of such candidate transcription factors that control memory B cell commitment provided a powerful genetic tool to manipulate the turning on and off of these lineages at will. The human Blimp-1, PRDI-BF1, is a DNA-binding protein involved in post-induction repression of INFbeta gene transcription in response to viral infection. In terminal differentiation of B cells, it has an essential function in driving differentiation and therein silences multiple genes. Microarray experiments of the expression of Blimp-1 indicate that it regulates a large set of genes of plasma cell expression signature. Blimp-1 affects numerous aspects of plasma cell maturation, ranging from migration, adhesion, and homeostasis to antibody secretion. It regulates Ig secretion by affecting the nuclear processing of the mRNA transcript and by affecting protein trafficking by regulating genes that impact the activity of the endoplasmic reticulum. The differentiation events that Blimp-1 regulates appear to be modulated, depending on the activation state of the B cell, and hint at the complexity of Blimp-1 and the genetic program that it initiates to produce a pool of plasma cells. There is a concerted activation-suppression of transcriptional factors during B cell differentiation. In (human) germinal center B cells, a set of genes has been shown to be involved in the differentiation: down-regulation of Bcl-6, which is implied to transform B- cells, activation of Blimp-1/PRDI-BF1, modulation of Myc, and the upregulation of the Mad1 and Mad4 transcription factors. Transcription factor E47 is required for CSR, at least in part, via expression of AICD (activation-induced cytidine deaminase). Id2 has been identified as a negative regulator of E47. Bach2 is a B cell-specific transcription repressor interacting with the small Maf proteins, whose expression is high only before the plasma cell stage. It is critical for CSR and somatic hypermutation of Ig genes. Mitf (microphthalmia-associated transcription factor) is highly expressed in naı¨ve B cells, where it antagonizes the process of terminal differentiation through the repression of IRF-4. Xbp1 (X-box binding protein 1) is shown to be essential for the differentiation of B cells into plasma cells. By using microarray analyses, a set of genes have been defined whose induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or Xbp1. Xbp1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Xbp1 is essential to coordinate diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells. Aiolos, a member of Ikaros family, was shown to be required for the generation of long-lived high affinity plasma cells resident in the bone marrow. Recent results on the network of transcriptional factors regulating plasma cell differentiation, have been described by Alinikula and Lassila [95].

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PART 12 Hematopoietic System In the periphery B cell development, miR-155, besides affecting two steps of T cells development (above), is shown to be upregulated following B cell activation in the germinal center. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high affinity IgG1 antibodies [86,87]. Global gene expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function. Among many of the miR-155-mediated regulation of targets are PU.1 and AID (ActivationInduced cytidine Deaminase) and PU.1. PU.1. contributes to class switch and antibody production. When Pu.1 is overexpressed in B cells, fewer IgG1 cells are produced [99]. B cells perform somatic hypermutation and CSR of the Ig locus to generate antibody diversity in affinity and function. These somatic diversification processes are catalyzed by AID, a potent DNA mutator whose expression and function are highly regulated. AID also promotes chromosomal translocations. AID was regulated post-transcriptionally by miR-155. The miR-155 was upregulated in mouse B cells undergoing CSR and it targeted a conserved site in the 3’-untranslated region of the mRNA encoding AID. Disruption of this target site in vivo resulted in quantitative and temporal deregulation of AID expression, along with functional consequences for CSR and affinity maturation. Thus, miR-155 does so in part by directly down-modulating AID expression [100]. Mice carrying a mutation in the putative miR-155 binding site in the 3’-untranslated region of AID, designated Aicda(155) mice, were generated [101]. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Thus, miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID. 1110

CONCLUDING REMARKS AND PROSPECTS FOR LYMPHOCYTE ENGINEERING I have discussed and summarized the current understanding of the expansion of lymphoid cells and their precursors ex vivo at certain stages of lymphopoiesis. I have tried to address the feasibility of expanding lymphoid cells under controlled growth conditions; that is, we need to expand untransformed, non-malignant cells. In general, in order to maintain the status of cell survival and growth without apoptosis and differentiation, cytokines and cell contact with feeder cells are required. Fundamental questions regarding the engineering of lymphoid cells and their precursors for therapeutic purposes remain and can be traced to our current understanding of the immune system. Do we ask too much for the survival in culture of cells that are programmed to die? From extensive studies in gene- manipulated mice, it is possible to generate antigen-specific memory T cells; it remains a puzzle that there is no good systematic study of human memory cells in culture, although the surface markers have been defined. If there is a massive programmed cell death occurred during the development from HSCs to lymphoid precursors and from pre-T cell to T cell maturation, I wonder whether it is realistic to try to produce enough HPCs and precursor lymphoid cells for therapeutic purposes. In clinical protocols such as ADA trials, expansion of cells ex vivo for the purpose of reinfusion into patients is limited to as few passages as possible in order to avoid mutation and contamination in vitro. Bioreactors for large-scale production of cells in liquid suspension using cytokines are available. However, they are not designed for co-culturing of stem cells with stroma cells, additional micro-particles in culture allowing the attachment of stroma cells in such particles, are needed. With advances in culture technology and bioreactors and with increased supply of recombinant cytokines, it becomes possible to obtain a quantity sufficient for re-implantation from 10 ml of bone marrow cells. However, under these conditions, very few cells engage in lymphopoiesis. Thus, to grow the HPCs consistently and to favor

CHAPTER 50 Lymphoid Cells lymphopoiesis, there is a great need for a better way to grow human stem cells using human stroma cell lines. The current study using transcription factors such as HoxB4, Cdx4, and mMix; small molecules, miRNAs, to manipulate the behavior of hESCs or h-iPS in vitro may be a promising approach. In view of the massive apoptosis at several stages of lymphopoiesis, it is amazing that mouse precursor B cells can grow normally, become lines and clones, and retain the potential to differentiate in vitro and in vivo. It is still a puzzle why the human precursor B cells cannot grow in culture yet. To grow cells from other stages of lymphopoiesis, it might be advantageous to use cells from the many available mutated or knockout mice. The future of cell-based immune therapy lies in the ex vivo expansion of cells. Because the techniques to establish ligand-regulatable vectors are available, the derived cell lines will become available and will become valuable resources for many purposes. Other areas remain to be improved, including the search for novel markers of true HSCs and precursor lymphocytes; better sources of HSCs and HPCs, such as cord blood, fetal tissues, hES cells, h-iPS cells, improved retroviral/lentiviral vectors and large-scale culture systems for expansion of HPCs and precursor lymphoid cells. With advances in genetic tools and mutated-mice technology, it is possible to turn on or off the transcription factors, miRNAs that control the differentiation of cells. With the discovery and understanding of Toll-like molecules on APCs to stimulate Th1 vs. Th2 vs. Treg vs. Th17, the approaches to fight against autoimmune diseases, allergy, inflammation, and transplantation rejection will become accessible. Thus, immunology will continue to be a very exciting field.

Acknowledgments The author wishes to thank Alexander Aiuti, Betty Padovan, Hans-Martin Jack, Michael Lohoff, Ton Rolink, Claudia Haftmann, Andrea Radbruch, Raju Pareek, and all our other colleagues for providing information including unpublished observation, fruitful discussions during the revision of this chapter. The author also wishes to thank Reinhard Hoffmann, Fritz Melchers, Patrick Matthias, Ton Rolink, for giving permission to use figures and tables. This work is supported by funds from third-party agencies.

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