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Lymphocyte development from hematopoietic stem cells
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Lymphocyte development from hematopoietic stem cells Motonari Kondo*, David C Scherer†, Angela G King‡, Markus G Manz§ and Irving L Weissman# The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition. Addresses Departments of Pathology and Developmental Biology, Stanford University School of Medicine, B259 Beckman Center, Stanford, California 94305, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] # e-mail: [email protected] Correspondence: Irving L Weissman
the lymphoid or the myeloid lineage. Whereas lymphoid lineage cells (T, B and NK cells) play a role in both the adaptive and innate arms of the immune system, myeloid lineage cells (i.e., macrophages and granulocytes) function solely in the innate arm. T and B lymphocytes are the central components of adaptive immunity. Each individual T cell and B cell expresses an antigen receptor that specifically recognizes an unique non-self determinant. The number of distinct T and B cell clones is large enough to recognize virtually any molecule. The incredible diversity required in this recognition system is achieved by genetic rearrangement at the antigen receptor loci: the T-cell receptor gene (TCR) in T cells, and the immunoglobulin gene in B cells [2,3]. Another lymphoid lineage cell, the natural killer (NK) cell, is important in innate immunity. NK cells function to kill virally infected cells and cancer cells.
Current Opinion in Genetics & Development 2001, 11:520–526 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations CLP common lymphoid progenitor CMP common myeloid progenitor DC dendritic cell ES embryonic stem GM-CSF granulocyte/macrophage colony-stimulating factor IL interleukin HSC hematopoietic stem cells LIF leukemia inhibitory factor MPP multipotent progenitor NK natural killer ST-HSC short-term HSC TCR T-cell receptor
Thus, the lymphocyte lineage has evolved to provide specialized immune surveillance functions, and is developmentally and functionally distinct from the myeloid lineage. In this review, we briefly summarize recent findings on the characteristics of HSCs and the regulation of commitment to specific subsets of lymphocytes.
Hematopoietic stem cells The first experimental evidence to indicate the existence of HSCs was the discovery of the multi-lineage colony formation activity of bone-marrow cells in irradiated mouse spleens (CFU-S) [4]. These CFU-S-initiating cells in the bone marrow were proposed to be HSCs — progenitors with the essential characteristics of self-renewal and differentiation potential for all types of blood cells.
Introduction The immune system has evolved to defend against the invasion of foreign organisms by distinguishing self from non-self. The immune system can be divided functionally into two arms: the adaptive and the innate. In an adaptive immune response, specific determinants on a non-self antigen induce the clonal expansion of a subset of immune cells that recognize and eliminate the invading organism, while at the same time generating immunologic memory that protects the host from future infection by the same pathogen. Innate immunity defends the body against invaders through a more generalized recognition system, without the generation of immunologic memory.
The availability of multi-parameter fluorescence-activated cell sorting has made it possible to purify homogenous cell populations according to their cell-surface expression of specific molecules. For example, in mice lineage marker negative (Lin–) bone-marrow cells expressing stem-cell antigen 1 (Sca-1+) and low levels of Thy-1.1 (Thy-1.1lo) have been purified (~0.05% of total nucleated bone marrow cells) using a multi-color cell sorter and shown to contain HSCs at a very high frequency [5]. Further studies have demonstrated that, within this population, c-Kit+ and CD34–/lo cells are HSCs that have long-term reconstitution activity in vivo [6,7].
The cells that comprise the immune system are continually generated from self-renewing progenitors in the bone marrow called hematopoietic stem cells (HSCs) [1]. On receiving differentiation signals, HSCs commit to either
Importantly, when transplanted into a lethally irradicated mouse, a single purified HSC can give rise to cells of all hematopoetic lineages for over six months. In addition, a phenotypically identical donor-derived population that
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maintains all of the requisite functional qualities of HSCs can be re-purified from transplanted hosts. This confirms the existence of ‘true’ HSCs in the bone marrow, as was proposed originally in the early 1960s [6,8].
Transcription factors in HSC maintenance Several transcription factors have been proposed to have a critical role in the maintenance of HSCs and/or very early hematopoiesis [9•]. However, the molecular mechanisms that regulate self-renewal activity are poorly understood. The zinc-finger transcription factor GATA-2 is indispensable for early hematopoiesis, as GATA-2–/– mice die at embryonic day 10–11 with severe anemia. GATA-2 null hematopoietic progenitors induced by in vitro differentiation from genetargeted embryonic stem (ES) cells proliferate poorly and form small colonies with extensive cell death, suggesting that GATA-2 is necessary for the proliferation and survival of blood cell progenitors [10]. In the presence of LIF (leukemia inhibitory factor), ES cells can proliferate without undergoing terminal differentiation. Studies investigating the molecular mechanisms that regulate this phenomenon have revealed that LIFinduced activation of the transcription factor STAT3 is indispensable for self-renewal activity in ES cells [11]. It therefore seems likely that cytokines and/or cell-surface molecules expressed by bone-marrow stromal cells are necessary for maintaining the self-renewal activity of HSCs; however, the identity of these self-renewal factors remains to be determined. Therefore, when HSCs are purified and cultured in vitro they begin to differentiate immediately, even in the presence of bone-marrow-derived stromal cells. It will be of great interest to uncover what molecular interactions in vivo are necessary to maintain HSCs in an undifferentiated state so that their propagation in vitro can be achieved. Several recent studies have found that HSCs have the potential to differentiate into cells outside the hematopoietic lineage. These studies suggest that bone-marrow cell populations either enriched for or composed entirely of HSCs can give rise to neuronal cells [12,13], skeletal muscle cells [14], cardiac muscle cells [15•] and hepatocytes [16••] in vivo. These unexpected results have opened new lines of investigation into defining the extent of developmental plasticity of HSCs, and have broadened the potential therapeutic use of these cells for regenerating tissues outside the hematopoietic system.
Initiation of lymphoid commitment from HSCs Loss of pluripotent differentiation
During maturation, HSCs first lose the ability to selfrenew while still maintaining their full developmental potential. The population of cells in mouse bone marrow at this developmental stage has been isolated and termed short-term (ST)-HSCs, because on transplantation they self-renew for approximately 6 weeks. Therefore, like their
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multipotent progenitor (MPP) progeny, ST-HSCs only transiently give rise to all hematopoietic lineages [7]. From our current understanding of early hematopoiesis, the next developmental step after cells pass through the ST-HSC/MPP stages is commitment to either the lymphoid or myeloid lineage. This developmental checkpoint is the first stage at which the cells lose pluripotent developmental potential [17]. Recently, clonogenic lymphoid- and myeloid-lineage committed progenitors have been identified in mouse bone marrow. Common lymphoid progenitors (CLPs) are the most immature lymphoid-committed progenitors identified to date. Importantly, CLPs can give rise to all classes of lymphocytes (T, B and NK cells) at the clonal level [18]. Common myeloid progenitors (CMPs) can give rise to all myeloid lineages (granulocytes, monocytes, erythrocytes and megakaryocytes) but still maintain the potential for B-cell lineage differentiation at an extremely low frequency [19]. Of significant interest is the finding that both CLPs and CMPs can give rise to dendritic cells (DCs) [20•,21•], as discussed below. Although the developmental pathways for lymphoid and myeloid lineages are considered to be mutually exclusive, alternative commitment pathways do exist. For example, B/macrophage bi-potent progenitors, whose existence has been suggested in fetal liver, are also present in adult mouse bone marrow [22]. Their contribution to B and macrophage development in vivo, however, remains to be determined. Factors in lymphoid lineage commitment
Although we now have a more complete map of the developmental hierarchy of specific cell populations during lymphopoiesis, the molecular mechanisms that regulate lymphoid lineage commitment are not clear. One potentially important lymphoid commitment factor is the zinc-finger transcription factor Ikaros, as Ikaros gene knockout mice have no lymphocytes [23]. Given that Ikaros is ubiquitously expressed in various hematopoietic cells including HSCs (albeit with stage-specific isoform displays) [24], however, it seems unlikely that it is a master gene for the initiation of lymphoid lineage commitment. In addition, Ikaros-deficient bone-marrow cells have reduced myeloid reconstitution activity in irradiated mice [25], indicating that they have a more generalized role in blood cell development. It is unclear whether a single master gene or a combination of several factors initiates lymphoid lineage commitment from HSCs or MPPs. Currently, it seems likely that there are more steps required to become lymphoid cells than myeloid cells from HSCs. This hypothesis stems from many experimental observations. For example, HSCs form various types of myeloid colonies in standard methylcellulose assays, whereas B-cell colony formation is rarely seen.
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To induce B-cell differentiation from HSCs in vitro, a twostep culture system or long-term stromal cell culture (over 2 weeks) is required [26,27]. This indicates that even if a single gene is responsible for lymphoid lineage commitment, the induction of this gene (or activation of this gene product) only happens under very specific conditions. It is therefore of central importance to determine the geneexpression profiles of cells representing each distinct stage of lymphopoiesis.
Biology of lymphoid lineage commitment Commitment to a specific cell lineage is generally thought to be unidirectional [28]; however, developing cells may have more plasticity than we observe under physiological conditions. In this regard, we and our coworkers [29•] have discovered recently that CLPs maintain a latent myeloid differentiation potential that can be initiated by signaling through exogenously expressed cytokine receptors, specifically those for interleukin 2 (IL-2) and granulocyte/macrophage colonystimulating factor (GM-CSF). Under normal conditions, myelo-monocytic cytokine receptors expressed at low levels on HSCs are not present on CLPs. This suggests that one key event that allows CLPs to become restricted to the lymphoid lineage is the downregulation of cytokine receptors that can initiate the intracellular myeloid lineage differentiation program maintained in CLPs. Our study sheds light on a couple of important issues regarding the molecular basis of lymphoid and myeloid lineage commitment [29•]. First is the issue of how a progenitor cell interprets competing lymphoid and myeloid developmental cues. As mentioned above, a close lineage relationship between B cells and macrophages has been proposed because some immature B-cell lines can be induced to change morphologically from lymphocytes to macrophages (e.g. [30]). But the converse developmental switch (from monocytes or macrophages to lymphocytes) has not been reported; therefore, it is possible that, before irreversible lymphoid commitment has been achieved, any event that can initiate a myeloid differentiation program dominates, leading to a myeloid rather than lymphoid outcome for that cell. If this proposed hierarchy holds true, it seems likely that the downregulation of myelo-monocytic cytokine receptors such as GM-CSF receptors is genetically programmed to occur at the onset of lymphoid lineage commitment [29•]. Second, the lymphoid-committed cells that still have multi-lineage developmental potential (T cell/B cell/NK or T cell/NK) may commonly possess latent myeloid differentiation activity. This is supported by our recent finding that proT cells in the thymus that have not yet fully committed to the T lineage also harbor a latent granulocyte/macrophage differentiation potential that can be induced by signaling through exogenously expressed IL-2 receptors. Importantly, T-lineage-committed preT cells
(CD44–CD25+ TN thymocytes; see Figure 1) cannot divert their cell fate in response to IL-2 receptor signaling (AG King et al., unpublished observation). In addition, B-cell-committed pre-proB cells do not convert to the granulocyte/macrophage lineage in this system [29•]. It is still quite possible, however, that pre-pro B cells do maintain latent myeloid differentiation potential that is activated through other mechanisms [31]. Interestingly, CLPs and proT cells maintain DC differentiation potential, whereas preT and pre-proB cells do not [21•]. This suggests that the coincidental loss of DC potential and latent myeloid differentiation potential is linked genetically. In our experimental model, CLPs and proT cells can only change cell fate from lymphoid to myeloid if the conversion factor (IL-2 or GM-CSF) is added in the first 2 days of culture, again suggesting that irreversible lymphoid lineage commitment does not occur in a single step (AG King et al., unpublished observation; [29•]). We currently view the lymphoid commitment process as having two distinct phases. The first phase is the initiation of lymphoid lineage commitment, represented by CLPs and proT cells, in which the downregulation of receptors that promote myelopoiesis has occurred. The second phase is the induction of irreversible lymphoid lineage commitment, represented by pre-proB and preT cells. The irreversible commitment of developing cells to a specific lymphoid lineage must therefore require the expression of an additional factor(s). The strongest candidate for an irreversible commitment factor is Pax5, a B-lineagespecific paired domain transcription factor [32••]. B-cell development in Pax5-deficient mice is blocked at the proB-cell stage in which immature B cells have completed immunoglobulin heavy chain (IgH) D to J rearrangement but not V to DJ rearrangement. Importantly, these Pax5–/– proB cells have gained the capability to differentiate into T, NK and some myeloid cells, but not into erythroid cells. Unlike their wild-type counterparts, Pax5–/– proB cells express myeloid-specific genes, including M-CSF receptor and myeloperoxidase, suggesting that one function of Pax5 in B-cell development is the transcriptional inactivation of genes that promote the development of alternate lineages. It is still unclear whether Pax5 acts in concert with other factors to irreversibly commit cells to the B lineage. To address this issue we are testing whether the ectopic expression of Pax5 can block myeloid cell differentiation in HSCs and lineage-restricted progenitors (CLPs and CMPs).
Correlation between maturation status of developing lymphocytes and antigen receptor gene rearrangement To better understand the commitment process, it is necessary to correlate the known developmental steps with the acquisition of irreversible lymphoid lineage commitment. For T and B cells, tracking the status of
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Figure 1 Current model of hematopoiesis in adult mouse bone marrow. Two major lineages, lymphoid and myeloid, are separable at a progenitor level. Common lymphoid progenitors can differentiate into T, B and NK cells, as well as into DCs. Common myeloid progenitors can differentiate into all types of myeloid cells, as well as into DCs. In this figure, we refer to proT1 and proT2 cells as c-KithighCD44+CD25– and c-KithighCD44+CD25+ TN thymocytes (CD3–CD4–/loCD8–), respectively. PreT cells are c-Kit–/loCD44–CD25+ TN thymocytes. ProT1 cells have a very low myeloid differentiation potential in vitro. Recent immigrants into the thymus from the bone marrow have not been identified. The intermediate populations between CLPs and NK1.1+ NK cells have not yet been characterized although the presence of a T/NK bipotent progenitor (proT2 cells in this figure) is suggested. CLP, proT1 and proT2 have a latent myeloid differentiation potential that can be initiated by signaling through exogenously expressed cytokine receptors, such as IL-2 and GM-CSF receptors. CMPs show a very low B-cell readout frequency in stromal cell culture in vitro (<1 in 2500).
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antigen receptor gene rearrangement has proved to be a useful yardstick by which to measure developmental progress in these lineages. Although pre-proB cells in the bone marrow have not yet initiated immunoglobulin heavy chain gene rearrangement, they have already committed to the B lineage [33]. As discussed above, our data support this hypothesis as we found that pre-proB cells do not convert from the lymphoid to myeloid lineage through ectopic IL-2 receptor signaling. This suggests that immature lymphocytes undergo irreversible lymphoid commitment before the initiation of antigen receptor gene rearrangement. While this may hold true for B-cell development, it is not the case for T cells. Specifically, we have examined the TCR-β gene rearrangement status in granulocyte/macrophage colonies derived from proT cells and have found that roughly 10% had TCR-β D to J rearrangement (AG King et al., unpublished observation). In contrast, we did not detect V to DJ rearrangement in any proT-derived granulocyte/macrophage colony. Significantly, DJ rearrangement of the TCR-β gene has been shown to occur in both T- and B-lineage cells but never in myeloid cells [3]. This finding confirms our initial lineage-conversion studies with CLPs, which showed that lymphoid-lineage-committed cells truly maintain a latent myeloid differentiation potential.
Although the lineage conversion that we observe in our experimental system does not represent a physiological developmental process, this discovery may help to explain the finding that some acute myeloblastic leukemia cells have TCR gene rearrangement [34].
Origin of dendritic cells As opposed to the lymphoid and myeloid lineages, the origin of dendritic cells (DCs) is not at all clear. In mice, DCs in primary and secondary lymphoid organs have been subdivided into at least two major populations according to their expression of CD8α and Mac-1 (CD8α+ Mac-1– or CD8α– Mac-1+ DCs). All thymic DCs and about 30% of DCs in secondary lymphoid organs are CD8α+ DCs, whereas about 70% of DCs in secondary lymphoid organs are CD8α–. On transplantation, early T-cell progenitors isolated from the thymus give rise almost exclusively to CD8α+ Mac-1– DCs in the thymus and secondary lymphoid organs [35]. Accordingly, it has been assumed that all CD8α+ Mac-1– DCs are of lymphoid origin (called ‘lymphoid DCs’) and all CD8α– Mac-1+ DCs are of myeloid origin (called ‘myeloid DCs’). The concept of different lineal origins of DCs seems to be supported by mice deficient in the transcription factors RelB [36], PU.1 [37] and Ikaros [38], as these mice lack CD8α– DCs but not CD8α+ DCs.
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In addition, it has been shown that CD8α– DCs and CD8α+ DCs differentially regulate immune responses. Importantly, CD8α+ DCs have been found to kill CD4 T cells via Fas/Fas-ligand-induced apoptosis [39], suggesting that these DCs are involved in maintaining not only central but also peripheral tolerance. However, the concept of independent lineal origins of CD8α+ Mac-1– lymphoid DCs and CD8α– Mac-1+ myeloid DCs has been challenged by findings in other mutant mice. Indeed, mice deficient in PU.1 reportedly lack both CD8α+ and CD8α– DCs [40], and two recent studies show that there is a developmental dissociation of CD8α+ DC and T cells in c-Kit–/–/γc–/– and Notch1–/– mice [41,42]. Both mutant animals are completely deficient in T cells, but show normal percentages of CD8α+ DCs in thymus and spleen. Although the complete absence of thymocyte progenitors was not formally proved in these mice, it seems likely that this developmental stage may not be required for thymic CD8α+ DC development. In an attempt to clarify the question of DC origin, we and our co-workers [20•,21•] have shown recently that myeloid lineage committed progenitors (CMPs) and lymphoid committed progenitors (CLPs) in mouse bone marrow give rise to both CD8α+ and CD8α– DCs in secondary lymphoid organs and CD8α+ DCs in the thymus. The DC developmental potential is preserved during early T-cell differentiation but not B-cell differentiation from CLPs, and during granulocyte/macrophage but not megakaryocyte/erythrocyte development from CMPs. In addition, our data suggest that most of the CD8α+ and CD8α– DCs in secondary lymphoid organs and about half of the thymic DC are of myeloid origin. Our data directly show that CD8α does not indicate the lineal origin of DCs but rather marks either a functional subset or a developmental stage of DCs. In this regard, it has been shown that Langerhans cells upregulate CD8α expression on activation and migration into the draining lymph node [43]. Moreover, on receiving inflammatory signals marginal-zone DCs that are primarily CD8α– move to the T-cell areas where CD8α+ DCs predominate [44]. As yet, there is no direct evidence to show that CD8α– DCs are the direct precursors of CD8α+ DCs, and in fact the results of a recent study assessing DC turnover rates argue against this hypothesis [45]. At present, we do not know whether the ontogeny of DCs is important in determining their function or whether DC function is controlled predominantly by the microenvironments in which they develop and become activated. The production of CD8α+ and CD8α– DCs from both lymphoid- and myeloid-restricted precursors seems to reflect some level of developmental redundancy. This hypothesis needs to be confirmed, however, through careful comparisons of the functional characteristics of lymphoid- and myeloid-derived DCs.
Control of cell differentiation by cytokine receptor signaling The functional role of cytokines in hematopoiesis is of significant interest and has been the subject of many studies [46]. At issue is whether cytokines can instructively promote commitment to a specific lineage, or whether their function is limited to supporting cell survival and stimulating cell proliferation during development [28]. Mice deficient in IL-7 have a severe reduction in B and T lymphocytes, demonstrating that this cytokine is of central importance in lymphopoiesis [47]. IL-7 is not required to initiate lymphoid lineage commitment, however, as IL-7 knockout mice produce proT and proB cells and have normal NK cell development. Further studies have revealed that IL-7 receptor signaling plays an important role in promoting gene rearrangement in the immunoglobulin heavy chain (IgH) and TCR-γ loci [48,49]. In contrast, investigations into the role of cytokines in myeloid cell development have so far suggested only a supportive role [28]. While this holds true for the experimental systems used to date, we have recently discovered that signaling through exogenously expressed IL-2 and GM-CSF receptors can initiate myeloid lineage commitment in otherwise lymphoid-committed progenitors (CLPs and proT cells) [29•]. This finding raises the issue of how signaling through these receptors induces the latent myeloid differentiation activity in CLPs and proT cells. Because we use an overexpression system, it is possible that the activation of a myeloid differentiation pathway is simply caused by unusually strong cytokine receptor signaling; however, we do not observe lineage conversion in CLPs when we overexpress and stimulate through either the Epo receptor or the IL-7 receptor. Hence, it is more likely that IL-2 and GM-CSF receptor signaling activate unique signaling pathway(s) that are not downstream of the IL-7 receptor. We are currently searching for the signal transduction pathways that are required to initiate lineage switching from lymphoid to myeloid in CLPs.
NK cell differentiation The molecular regulation of NK cell differentiation is still not clear, mainly because the developmental intermediates of NK cell maturation (pro or preNK cells) have not been identified in vivo. In spite of this, several mouse models have shed some light on the factors involved in generating a functional NK cell compartment. Two transcriptional regulators, a basic helix–loop–helix Id family protein, Id2 [50•], and a winged helix-turn-helix Ets family protein [51], are requisite for NK cell development. In addition, IL-15induced signaling through functional IL-15 receptors composed of IL-15Rα, IL-2Rβ and γc chains is also indispensable for the development of NK cells [52]. The STAT5 transcription factor may be important in regulating the effects of IL-15, as an IL-2Rβ mutant that lacks the region
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necessary for STAT5 activation does not rescue impaired NK cell development in IL-2Rβ-deficient mice [53].
Conclusions and perspectives The first step in understanding complex developmental pathways is to identify, at the clonal level if possible, the cells that represent each successive developmental step in vivo. Our laboratory and many others have put enormous effort into achieving this goal for the hematopoietic system and have identified a number of distinct cell populations that represent discreet developmental steps in hematopoiesis. Currently, the principal effort in the field of hematopoiesis has been focused on determining the gene-expression profiles of these populations in the hopes of gaining insight into the molecular mechanisms that control the unique developmental characteristics of each cell type. Recently, the characterization of 21,076 full-length cDNAs derived from a variety of mouse tissues has been reported [54••], and soon the complete sequence of the mouse genome will be available. Many laboratories are taking advantage of the explosion of genetic information being generated to elucidate the geneexpression profile of distinct cell populations using many different methods, including DNA microarrays. Geneexpression profiling of hematopoietic progenitors and their lineal descendants will rapidly advance our understanding of the molecular regulatory networks that control the development of all blood cells including lymphocytes.
Acknowledgements We apologize to those whose work was not cited owing to space limitations. M Kondo and MG Manz are fellows of the Irvington Institute for Immunological Research and Deutsche Krebshilfe, Dr Mildred-ScheelStiftung für Krebsforschung, respectively. AG King and DC Scherer are supported by the United States Public Health Service grant CA09302 awarded by the National Cancer Institute, and California Division, American Cancer Society Fellowship #2-42-99, respectively. Our research was supported by USPHS grants AI47458, CA86065 and CA4255.
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Muller-Sieburg CE, Whitlock CA, Weissman IL: Isolation of two early B-lymphocyte progenitors from mouse marrow: a committed pre-pre-B-cell and a clonogenic Thy-1-lo hematopoietic stem cell. Cell 1986, 44:653-662.
28. Metcalf D: Stem cells, pre-progenitor cells and lineage-committed cells: are our dogmas correct? Ann New York Acad Sci 1999, 872:289-304. 29. Kondo M, Scherer DC, Miyamoto T, King AG, Akashi K, Sugamura K, • Weissman IL: Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature 2000, 407:383-386. CLPs have been characterized as the bone-marrow cells in mouse that can give rise to all classes of lymphocyte but not to any type of myeloid cell. By IL-2 and GM-CSF signaling through exogenously expressed receptors, CLPs change cell fate from lymphoid to myeloid. The authors conclude that cytokine receptor expression regulates the differentiation potential of CLPs. The data also suggest the possible ‘instructive’ role of cytokines in hematopoiesis. 30. Hara H, Sam M, Maki RA, Wu GE, Paige CJ: Characterization of a 70Z/3 pre-B-cell derived macrophage clone. Differential expression of Hox family genes. Int Immunol 1990, 2:691-696. 31. Klinken SP, Alexander WS, Adams JM: Hemopoietic lineage switch: v-raf oncogene converts Emu-myc transgenic B-cells into macrophages. Cell 1988, 53:857-867. 32. Nutt SL, Heavey B, Rolink AG, Busslinger M: Commitment to the •• B-lymphoid lineage depends on the transcription factor Pax5. Nature 1999, 401:556-562. Pax5 is an essential transcription factor in B cell development. The authors show that proB cells from Pax5 knockout mice have multiple-lineage differentiation potential. As Pax5–/– proB cells show promiscuous lineagespecific gene expression, it is likely that Pax5 plays a critical role in definitive B lineage commitment by repressing the expression of genes that are necessary for non-B lineage cell development. 33. Allman D, Li J, Hardy RR: Commitment to the B-lymphoid lineage occurs before DH-JH recombination. J Exp Med 1999, 189:735-740. 34. Cheng GY, Minden MD, Toyonaga B, Mak TW, McCulloch EA: T-cell receptor and immunoglobulin gene rearrangements in acute myeloblastic leukemia. J Exp Med 1986, 163:414-424. 35. Wu L, Li CL, Shortman K: Thymic dendritic cell precursors: relationship to the T-lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med 1996, 184:903-911. 36. Wu L, D’Amico A, Winkel KD, Suter M, Lo D, Shortman K: RelB is α– dendritic essential for the development of myeloid-related CD8α α+ dendritic cells. Immunity cells but not of lymphoid-related CD8α 1998, 9:839-847. 37.
Guerriero A, Langmuir PB, Spain LM, Scott EW: PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells. Blood 2000, 95:879-885.
38. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K: Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 1997, 7:483-492.
45. Kamath AT, Pooley J, O’Keeffe MA, Vremec D, Zhan Y, Lew AM, D’Amico A, Wu L, Tough DF, Shortman K: The development, maturation, and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000, 165:6762-6770. 46. Kondo M, Weissman IL: Function of cytokines in lymphocyte development. Curr Top Microbiol Immunol 2000, 251:59-65. 47.
von Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R: Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med 1995, 181:1519-1526.
48. Corcoran AE, Riddell A, Krooshoop D, Venkitaraman AR: Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 1998, 391:904-907. 49. Durum SK, Candeias S, Nakajima H, Leonard WJ, Baird AM, Berg LJ, Muegge K: Interleukin 7 receptor control of T-cell receptor-γγ gene rearrangement: role of receptor-associated chains and locus accessibility. J Exp Med 1998, 188:2233-2241. 50. Yokota Y, Mansouri A, Mori S, Sugawara S, Adachi S, Nishikawa S, • Gruss P: Development of peripheral lymphoid organs and natural killer cells depends on the helix–loop–helix inhibitor Id2. Nature 1999, 397:702-706. Members of the Id family are known as important regulators in lymphocyte development. In this paper, the authors demonstrate that Id2 plays an indispensable role in NK cell development by gene targeting. More strikingly, Id2–/– mice lack secondary lymphoid organs, such as lymph nodes and Payer’s patches. CD4+CD3-IL-7Rα+ cells that play an important role in the organization of lymph nodes are also impaired. Although CD4+ CD3– IL-7Rα+ cells have NK cell differentiation potential, it is not clear whether the absence of CD4+ CD3– IL-7Rα+ cells is solely responsible for the NK cell deficiency. 51. Barton K, Muthusamy N, Fischer C, Ting CN, Walunas TL, Lanier LL, Leiden JM: The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 1998, 9:555-563. 52. Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, Matsuki N, Charrier K, Sedger L, Willis CR et al.: Reversible defects in natural killer and memory CD8 T-cell lineages in interleukin 15-deficient mice. J Exp Med 2000, 191:771-780. 53. Fujii H, Ogasawara K, Otsuka H, Suzuki M, Yamamura K, Yokochi T, Miyazaki T, Suzuki H, Mak TW, Taki S, Taniguchi T: Functional dissection of the cytoplasmic subregions of the IL-2 receptor βc chain in primary lymphocyte populations. EMBO J 1998, 17:6551-6557. 54. Kawai J, Shinagawa A, Shibata K, Yoshino M, Itoh M, Ishii Y, •• Arakawa T, Hara A, Fukunishi Y, Konno H et al.: Functional annotation of a full-length mouse cDNA collection. Nature 2001, 409:685-690. As a part of genome project, the first set of full-length cDNAs from mice is reported. Together with the sequence of the human and mouse genomes, this study and others greatly accelerate advances in determining the changes in gene expression that determine cell fate.
Lymphocyte development from hematopoietic stem cells Motonari Kondo*, David C Scherer†, Angela G King‡, Markus G Manz§ and Irving L Weissman# The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition. Addresses Departments of Pathology and Developmental Biology, Stanford University School of Medicine, B259 Beckman Center, Stanford, California 94305, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] § e-mail: [email protected] # e-mail: [email protected] Correspondence: Irving L Weissman
the lymphoid or the myeloid lineage. Whereas lymphoid lineage cells (T, B and NK cells) play a role in both the adaptive and innate arms of the immune system, myeloid lineage cells (i.e., macrophages and granulocytes) function solely in the innate arm. T and B lymphocytes are the central components of adaptive immunity. Each individual T cell and B cell expresses an antigen receptor that specifically recognizes an unique non-self determinant. The number of distinct T and B cell clones is large enough to recognize virtually any molecule. The incredible diversity required in this recognition system is achieved by genetic rearrangement at the antigen receptor loci: the T-cell receptor gene (TCR) in T cells, and the immunoglobulin gene in B cells [2,3]. Another lymphoid lineage cell, the natural killer (NK) cell, is important in innate immunity. NK cells function to kill virally infected cells and cancer cells.
Current Opinion in Genetics & Development 2001, 11:520–526 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations CLP common lymphoid progenitor CMP common myeloid progenitor DC dendritic cell ES embryonic stem GM-CSF granulocyte/macrophage colony-stimulating factor IL interleukin HSC hematopoietic stem cells LIF leukemia inhibitory factor MPP multipotent progenitor NK natural killer ST-HSC short-term HSC TCR T-cell receptor
Thus, the lymphocyte lineage has evolved to provide specialized immune surveillance functions, and is developmentally and functionally distinct from the myeloid lineage. In this review, we briefly summarize recent findings on the characteristics of HSCs and the regulation of commitment to specific subsets of lymphocytes.
Hematopoietic stem cells The first experimental evidence to indicate the existence of HSCs was the discovery of the multi-lineage colony formation activity of bone-marrow cells in irradiated mouse spleens (CFU-S) [4]. These CFU-S-initiating cells in the bone marrow were proposed to be HSCs — progenitors with the essential characteristics of self-renewal and differentiation potential for all types of blood cells.
Introduction The immune system has evolved to defend against the invasion of foreign organisms by distinguishing self from non-self. The immune system can be divided functionally into two arms: the adaptive and the innate. In an adaptive immune response, specific determinants on a non-self antigen induce the clonal expansion of a subset of immune cells that recognize and eliminate the invading organism, while at the same time generating immunologic memory that protects the host from future infection by the same pathogen. Innate immunity defends the body against invaders through a more generalized recognition system, without the generation of immunologic memory.
The availability of multi-parameter fluorescence-activated cell sorting has made it possible to purify homogenous cell populations according to their cell-surface expression of specific molecules. For example, in mice lineage marker negative (Lin–) bone-marrow cells expressing stem-cell antigen 1 (Sca-1+) and low levels of Thy-1.1 (Thy-1.1lo) have been purified (~0.05% of total nucleated bone marrow cells) using a multi-color cell sorter and shown to contain HSCs at a very high frequency [5]. Further studies have demonstrated that, within this population, c-Kit+ and CD34–/lo cells are HSCs that have long-term reconstitution activity in vivo [6,7].
The cells that comprise the immune system are continually generated from self-renewing progenitors in the bone marrow called hematopoietic stem cells (HSCs) [1]. On receiving differentiation signals, HSCs commit to either
Importantly, when transplanted into a lethally irradicated mouse, a single purified HSC can give rise to cells of all hematopoetic lineages for over six months. In addition, a phenotypically identical donor-derived population that
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maintains all of the requisite functional qualities of HSCs can be re-purified from transplanted hosts. This confirms the existence of ‘true’ HSCs in the bone marrow, as was proposed originally in the early 1960s [6,8].
Transcription factors in HSC maintenance Several transcription factors have been proposed to have a critical role in the maintenance of HSCs and/or very early hematopoiesis [9•]. However, the molecular mechanisms that regulate self-renewal activity are poorly understood. The zinc-finger transcription factor GATA-2 is indispensable for early hematopoiesis, as GATA-2–/– mice die at embryonic day 10–11 with severe anemia. GATA-2 null hematopoietic progenitors induced by in vitro differentiation from genetargeted embryonic stem (ES) cells proliferate poorly and form small colonies with extensive cell death, suggesting that GATA-2 is necessary for the proliferation and survival of blood cell progenitors [10]. In the presence of LIF (leukemia inhibitory factor), ES cells can proliferate without undergoing terminal differentiation. Studies investigating the molecular mechanisms that regulate this phenomenon have revealed that LIFinduced activation of the transcription factor STAT3 is indispensable for self-renewal activity in ES cells [11]. It therefore seems likely that cytokines and/or cell-surface molecules expressed by bone-marrow stromal cells are necessary for maintaining the self-renewal activity of HSCs; however, the identity of these self-renewal factors remains to be determined. Therefore, when HSCs are purified and cultured in vitro they begin to differentiate immediately, even in the presence of bone-marrow-derived stromal cells. It will be of great interest to uncover what molecular interactions in vivo are necessary to maintain HSCs in an undifferentiated state so that their propagation in vitro can be achieved. Several recent studies have found that HSCs have the potential to differentiate into cells outside the hematopoietic lineage. These studies suggest that bone-marrow cell populations either enriched for or composed entirely of HSCs can give rise to neuronal cells [12,13], skeletal muscle cells [14], cardiac muscle cells [15•] and hepatocytes [16••] in vivo. These unexpected results have opened new lines of investigation into defining the extent of developmental plasticity of HSCs, and have broadened the potential therapeutic use of these cells for regenerating tissues outside the hematopoietic system.
Initiation of lymphoid commitment from HSCs Loss of pluripotent differentiation
During maturation, HSCs first lose the ability to selfrenew while still maintaining their full developmental potential. The population of cells in mouse bone marrow at this developmental stage has been isolated and termed short-term (ST)-HSCs, because on transplantation they self-renew for approximately 6 weeks. Therefore, like their
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multipotent progenitor (MPP) progeny, ST-HSCs only transiently give rise to all hematopoietic lineages [7]. From our current understanding of early hematopoiesis, the next developmental step after cells pass through the ST-HSC/MPP stages is commitment to either the lymphoid or myeloid lineage. This developmental checkpoint is the first stage at which the cells lose pluripotent developmental potential [17]. Recently, clonogenic lymphoid- and myeloid-lineage committed progenitors have been identified in mouse bone marrow. Common lymphoid progenitors (CLPs) are the most immature lymphoid-committed progenitors identified to date. Importantly, CLPs can give rise to all classes of lymphocytes (T, B and NK cells) at the clonal level [18]. Common myeloid progenitors (CMPs) can give rise to all myeloid lineages (granulocytes, monocytes, erythrocytes and megakaryocytes) but still maintain the potential for B-cell lineage differentiation at an extremely low frequency [19]. Of significant interest is the finding that both CLPs and CMPs can give rise to dendritic cells (DCs) [20•,21•], as discussed below. Although the developmental pathways for lymphoid and myeloid lineages are considered to be mutually exclusive, alternative commitment pathways do exist. For example, B/macrophage bi-potent progenitors, whose existence has been suggested in fetal liver, are also present in adult mouse bone marrow [22]. Their contribution to B and macrophage development in vivo, however, remains to be determined. Factors in lymphoid lineage commitment
Although we now have a more complete map of the developmental hierarchy of specific cell populations during lymphopoiesis, the molecular mechanisms that regulate lymphoid lineage commitment are not clear. One potentially important lymphoid commitment factor is the zinc-finger transcription factor Ikaros, as Ikaros gene knockout mice have no lymphocytes [23]. Given that Ikaros is ubiquitously expressed in various hematopoietic cells including HSCs (albeit with stage-specific isoform displays) [24], however, it seems unlikely that it is a master gene for the initiation of lymphoid lineage commitment. In addition, Ikaros-deficient bone-marrow cells have reduced myeloid reconstitution activity in irradiated mice [25], indicating that they have a more generalized role in blood cell development. It is unclear whether a single master gene or a combination of several factors initiates lymphoid lineage commitment from HSCs or MPPs. Currently, it seems likely that there are more steps required to become lymphoid cells than myeloid cells from HSCs. This hypothesis stems from many experimental observations. For example, HSCs form various types of myeloid colonies in standard methylcellulose assays, whereas B-cell colony formation is rarely seen.
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To induce B-cell differentiation from HSCs in vitro, a twostep culture system or long-term stromal cell culture (over 2 weeks) is required [26,27]. This indicates that even if a single gene is responsible for lymphoid lineage commitment, the induction of this gene (or activation of this gene product) only happens under very specific conditions. It is therefore of central importance to determine the geneexpression profiles of cells representing each distinct stage of lymphopoiesis.
Biology of lymphoid lineage commitment Commitment to a specific cell lineage is generally thought to be unidirectional [28]; however, developing cells may have more plasticity than we observe under physiological conditions. In this regard, we and our coworkers [29•] have discovered recently that CLPs maintain a latent myeloid differentiation potential that can be initiated by signaling through exogenously expressed cytokine receptors, specifically those for interleukin 2 (IL-2) and granulocyte/macrophage colonystimulating factor (GM-CSF). Under normal conditions, myelo-monocytic cytokine receptors expressed at low levels on HSCs are not present on CLPs. This suggests that one key event that allows CLPs to become restricted to the lymphoid lineage is the downregulation of cytokine receptors that can initiate the intracellular myeloid lineage differentiation program maintained in CLPs. Our study sheds light on a couple of important issues regarding the molecular basis of lymphoid and myeloid lineage commitment [29•]. First is the issue of how a progenitor cell interprets competing lymphoid and myeloid developmental cues. As mentioned above, a close lineage relationship between B cells and macrophages has been proposed because some immature B-cell lines can be induced to change morphologically from lymphocytes to macrophages (e.g. [30]). But the converse developmental switch (from monocytes or macrophages to lymphocytes) has not been reported; therefore, it is possible that, before irreversible lymphoid commitment has been achieved, any event that can initiate a myeloid differentiation program dominates, leading to a myeloid rather than lymphoid outcome for that cell. If this proposed hierarchy holds true, it seems likely that the downregulation of myelo-monocytic cytokine receptors such as GM-CSF receptors is genetically programmed to occur at the onset of lymphoid lineage commitment [29•]. Second, the lymphoid-committed cells that still have multi-lineage developmental potential (T cell/B cell/NK or T cell/NK) may commonly possess latent myeloid differentiation activity. This is supported by our recent finding that proT cells in the thymus that have not yet fully committed to the T lineage also harbor a latent granulocyte/macrophage differentiation potential that can be induced by signaling through exogenously expressed IL-2 receptors. Importantly, T-lineage-committed preT cells
(CD44–CD25+ TN thymocytes; see Figure 1) cannot divert their cell fate in response to IL-2 receptor signaling (AG King et al., unpublished observation). In addition, B-cell-committed pre-proB cells do not convert to the granulocyte/macrophage lineage in this system [29•]. It is still quite possible, however, that pre-pro B cells do maintain latent myeloid differentiation potential that is activated through other mechanisms [31]. Interestingly, CLPs and proT cells maintain DC differentiation potential, whereas preT and pre-proB cells do not [21•]. This suggests that the coincidental loss of DC potential and latent myeloid differentiation potential is linked genetically. In our experimental model, CLPs and proT cells can only change cell fate from lymphoid to myeloid if the conversion factor (IL-2 or GM-CSF) is added in the first 2 days of culture, again suggesting that irreversible lymphoid lineage commitment does not occur in a single step (AG King et al., unpublished observation; [29•]). We currently view the lymphoid commitment process as having two distinct phases. The first phase is the initiation of lymphoid lineage commitment, represented by CLPs and proT cells, in which the downregulation of receptors that promote myelopoiesis has occurred. The second phase is the induction of irreversible lymphoid lineage commitment, represented by pre-proB and preT cells. The irreversible commitment of developing cells to a specific lymphoid lineage must therefore require the expression of an additional factor(s). The strongest candidate for an irreversible commitment factor is Pax5, a B-lineagespecific paired domain transcription factor [32••]. B-cell development in Pax5-deficient mice is blocked at the proB-cell stage in which immature B cells have completed immunoglobulin heavy chain (IgH) D to J rearrangement but not V to DJ rearrangement. Importantly, these Pax5–/– proB cells have gained the capability to differentiate into T, NK and some myeloid cells, but not into erythroid cells. Unlike their wild-type counterparts, Pax5–/– proB cells express myeloid-specific genes, including M-CSF receptor and myeloperoxidase, suggesting that one function of Pax5 in B-cell development is the transcriptional inactivation of genes that promote the development of alternate lineages. It is still unclear whether Pax5 acts in concert with other factors to irreversibly commit cells to the B lineage. To address this issue we are testing whether the ectopic expression of Pax5 can block myeloid cell differentiation in HSCs and lineage-restricted progenitors (CLPs and CMPs).
Correlation between maturation status of developing lymphocytes and antigen receptor gene rearrangement To better understand the commitment process, it is necessary to correlate the known developmental steps with the acquisition of irreversible lymphoid lineage commitment. For T and B cells, tracking the status of
Lymphocyte development from hematopoietic stem cells Kondo et al.
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Figure 1 Current model of hematopoiesis in adult mouse bone marrow. Two major lineages, lymphoid and myeloid, are separable at a progenitor level. Common lymphoid progenitors can differentiate into T, B and NK cells, as well as into DCs. Common myeloid progenitors can differentiate into all types of myeloid cells, as well as into DCs. In this figure, we refer to proT1 and proT2 cells as c-KithighCD44+CD25– and c-KithighCD44+CD25+ TN thymocytes (CD3–CD4–/loCD8–), respectively. PreT cells are c-Kit–/loCD44–CD25+ TN thymocytes. ProT1 cells have a very low myeloid differentiation potential in vitro. Recent immigrants into the thymus from the bone marrow have not been identified. The intermediate populations between CLPs and NK1.1+ NK cells have not yet been characterized although the presence of a T/NK bipotent progenitor (proT2 cells in this figure) is suggested. CLP, proT1 and proT2 have a latent myeloid differentiation potential that can be initiated by signaling through exogenously expressed cytokine receptors, such as IL-2 and GM-CSF receptors. CMPs show a very low B-cell readout frequency in stromal cell culture in vitro (<1 in 2500).
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antigen receptor gene rearrangement has proved to be a useful yardstick by which to measure developmental progress in these lineages. Although pre-proB cells in the bone marrow have not yet initiated immunoglobulin heavy chain gene rearrangement, they have already committed to the B lineage [33]. As discussed above, our data support this hypothesis as we found that pre-proB cells do not convert from the lymphoid to myeloid lineage through ectopic IL-2 receptor signaling. This suggests that immature lymphocytes undergo irreversible lymphoid commitment before the initiation of antigen receptor gene rearrangement. While this may hold true for B-cell development, it is not the case for T cells. Specifically, we have examined the TCR-β gene rearrangement status in granulocyte/macrophage colonies derived from proT cells and have found that roughly 10% had TCR-β D to J rearrangement (AG King et al., unpublished observation). In contrast, we did not detect V to DJ rearrangement in any proT-derived granulocyte/macrophage colony. Significantly, DJ rearrangement of the TCR-β gene has been shown to occur in both T- and B-lineage cells but never in myeloid cells [3]. This finding confirms our initial lineage-conversion studies with CLPs, which showed that lymphoid-lineage-committed cells truly maintain a latent myeloid differentiation potential.
Although the lineage conversion that we observe in our experimental system does not represent a physiological developmental process, this discovery may help to explain the finding that some acute myeloblastic leukemia cells have TCR gene rearrangement [34].
Origin of dendritic cells As opposed to the lymphoid and myeloid lineages, the origin of dendritic cells (DCs) is not at all clear. In mice, DCs in primary and secondary lymphoid organs have been subdivided into at least two major populations according to their expression of CD8α and Mac-1 (CD8α+ Mac-1– or CD8α– Mac-1+ DCs). All thymic DCs and about 30% of DCs in secondary lymphoid organs are CD8α+ DCs, whereas about 70% of DCs in secondary lymphoid organs are CD8α–. On transplantation, early T-cell progenitors isolated from the thymus give rise almost exclusively to CD8α+ Mac-1– DCs in the thymus and secondary lymphoid organs [35]. Accordingly, it has been assumed that all CD8α+ Mac-1– DCs are of lymphoid origin (called ‘lymphoid DCs’) and all CD8α– Mac-1+ DCs are of myeloid origin (called ‘myeloid DCs’). The concept of different lineal origins of DCs seems to be supported by mice deficient in the transcription factors RelB [36], PU.1 [37] and Ikaros [38], as these mice lack CD8α– DCs but not CD8α+ DCs.
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In addition, it has been shown that CD8α– DCs and CD8α+ DCs differentially regulate immune responses. Importantly, CD8α+ DCs have been found to kill CD4 T cells via Fas/Fas-ligand-induced apoptosis [39], suggesting that these DCs are involved in maintaining not only central but also peripheral tolerance. However, the concept of independent lineal origins of CD8α+ Mac-1– lymphoid DCs and CD8α– Mac-1+ myeloid DCs has been challenged by findings in other mutant mice. Indeed, mice deficient in PU.1 reportedly lack both CD8α+ and CD8α– DCs [40], and two recent studies show that there is a developmental dissociation of CD8α+ DC and T cells in c-Kit–/–/γc–/– and Notch1–/– mice [41,42]. Both mutant animals are completely deficient in T cells, but show normal percentages of CD8α+ DCs in thymus and spleen. Although the complete absence of thymocyte progenitors was not formally proved in these mice, it seems likely that this developmental stage may not be required for thymic CD8α+ DC development. In an attempt to clarify the question of DC origin, we and our co-workers [20•,21•] have shown recently that myeloid lineage committed progenitors (CMPs) and lymphoid committed progenitors (CLPs) in mouse bone marrow give rise to both CD8α+ and CD8α– DCs in secondary lymphoid organs and CD8α+ DCs in the thymus. The DC developmental potential is preserved during early T-cell differentiation but not B-cell differentiation from CLPs, and during granulocyte/macrophage but not megakaryocyte/erythrocyte development from CMPs. In addition, our data suggest that most of the CD8α+ and CD8α– DCs in secondary lymphoid organs and about half of the thymic DC are of myeloid origin. Our data directly show that CD8α does not indicate the lineal origin of DCs but rather marks either a functional subset or a developmental stage of DCs. In this regard, it has been shown that Langerhans cells upregulate CD8α expression on activation and migration into the draining lymph node [43]. Moreover, on receiving inflammatory signals marginal-zone DCs that are primarily CD8α– move to the T-cell areas where CD8α+ DCs predominate [44]. As yet, there is no direct evidence to show that CD8α– DCs are the direct precursors of CD8α+ DCs, and in fact the results of a recent study assessing DC turnover rates argue against this hypothesis [45]. At present, we do not know whether the ontogeny of DCs is important in determining their function or whether DC function is controlled predominantly by the microenvironments in which they develop and become activated. The production of CD8α+ and CD8α– DCs from both lymphoid- and myeloid-restricted precursors seems to reflect some level of developmental redundancy. This hypothesis needs to be confirmed, however, through careful comparisons of the functional characteristics of lymphoid- and myeloid-derived DCs.
Control of cell differentiation by cytokine receptor signaling The functional role of cytokines in hematopoiesis is of significant interest and has been the subject of many studies [46]. At issue is whether cytokines can instructively promote commitment to a specific lineage, or whether their function is limited to supporting cell survival and stimulating cell proliferation during development [28]. Mice deficient in IL-7 have a severe reduction in B and T lymphocytes, demonstrating that this cytokine is of central importance in lymphopoiesis [47]. IL-7 is not required to initiate lymphoid lineage commitment, however, as IL-7 knockout mice produce proT and proB cells and have normal NK cell development. Further studies have revealed that IL-7 receptor signaling plays an important role in promoting gene rearrangement in the immunoglobulin heavy chain (IgH) and TCR-γ loci [48,49]. In contrast, investigations into the role of cytokines in myeloid cell development have so far suggested only a supportive role [28]. While this holds true for the experimental systems used to date, we have recently discovered that signaling through exogenously expressed IL-2 and GM-CSF receptors can initiate myeloid lineage commitment in otherwise lymphoid-committed progenitors (CLPs and proT cells) [29•]. This finding raises the issue of how signaling through these receptors induces the latent myeloid differentiation activity in CLPs and proT cells. Because we use an overexpression system, it is possible that the activation of a myeloid differentiation pathway is simply caused by unusually strong cytokine receptor signaling; however, we do not observe lineage conversion in CLPs when we overexpress and stimulate through either the Epo receptor or the IL-7 receptor. Hence, it is more likely that IL-2 and GM-CSF receptor signaling activate unique signaling pathway(s) that are not downstream of the IL-7 receptor. We are currently searching for the signal transduction pathways that are required to initiate lineage switching from lymphoid to myeloid in CLPs.
NK cell differentiation The molecular regulation of NK cell differentiation is still not clear, mainly because the developmental intermediates of NK cell maturation (pro or preNK cells) have not been identified in vivo. In spite of this, several mouse models have shed some light on the factors involved in generating a functional NK cell compartment. Two transcriptional regulators, a basic helix–loop–helix Id family protein, Id2 [50•], and a winged helix-turn-helix Ets family protein [51], are requisite for NK cell development. In addition, IL-15induced signaling through functional IL-15 receptors composed of IL-15Rα, IL-2Rβ and γc chains is also indispensable for the development of NK cells [52]. The STAT5 transcription factor may be important in regulating the effects of IL-15, as an IL-2Rβ mutant that lacks the region
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necessary for STAT5 activation does not rescue impaired NK cell development in IL-2Rβ-deficient mice [53].
Conclusions and perspectives The first step in understanding complex developmental pathways is to identify, at the clonal level if possible, the cells that represent each successive developmental step in vivo. Our laboratory and many others have put enormous effort into achieving this goal for the hematopoietic system and have identified a number of distinct cell populations that represent discreet developmental steps in hematopoiesis. Currently, the principal effort in the field of hematopoiesis has been focused on determining the gene-expression profiles of these populations in the hopes of gaining insight into the molecular mechanisms that control the unique developmental characteristics of each cell type. Recently, the characterization of 21,076 full-length cDNAs derived from a variety of mouse tissues has been reported [54••], and soon the complete sequence of the mouse genome will be available. Many laboratories are taking advantage of the explosion of genetic information being generated to elucidate the geneexpression profile of distinct cell populations using many different methods, including DNA microarrays. Geneexpression profiling of hematopoietic progenitors and their lineal descendants will rapidly advance our understanding of the molecular regulatory networks that control the development of all blood cells including lymphocytes.
Acknowledgements We apologize to those whose work was not cited owing to space limitations. M Kondo and MG Manz are fellows of the Irvington Institute for Immunological Research and Deutsche Krebshilfe, Dr Mildred-ScheelStiftung für Krebsforschung, respectively. AG King and DC Scherer are supported by the United States Public Health Service grant CA09302 awarded by the National Cancer Institute, and California Division, American Cancer Society Fellowship #2-42-99, respectively. Our research was supported by USPHS grants AI47458, CA86065 and CA4255.
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