Lineage determination in the immune system

Underlying all immune responses are the developmental programs that give immune cells their identities. Developmental controls determine the kinds of effector mechanisms that are available to different classes of cells, the degree of amplification that they can achieve through clonal expansion, the signals that they will respond to, and the limits that will be set on their abilities to alter function in different environmental conditions. As all these cell types are ultimately derived from common hematopoietic precursors, they each become distinct from other cell types at developmental choice points that define a time course of branching lineage relationships. This volume showcases striking new advances that illuminate the mechanisms governing several particularly interesting choice points where divergent immune cell identities are established. Development in the immune system raises a number of issues that are not usually addressed in classic embryonic developmental systems. One is the mobility of the cells, both in their functional roles and in their traffic to diverse inductive microenvironments, which introduces a stochastic element. Another is the question of when the irreversible aspects of immune-cell differentiation end and physiologically reversible ‘effector responses’ begin. The cells that mediate both adaptive and innate branches of immunity are specialized for the ability to mobilize finely tuned response programs based on the combinations of signals that they detect in the environment. This responsiveness involves major inducible changes in gene expression as well as effects on migration, morphology, and proliferation or death. All mature immune cells have sophisticated machinery for powerful signal-dependent responses. The behavior of a mature immune cell in a given situation can be seen as the superimposition of an environmentally modulated gene expression program on top of the core gene expression program dictated by that cell’s developmental history. But what determines the stability of the core program? The mechanisms involved are turning out to be more interesting, and more complex, than might have been suspected earlier, as described by the reviews in this volume. One aspect is the highly branched nature of immune cell developmental pathways beyond initial fate choice. The best known cases are the divergence of different T-cell receptor (TCR) αβ T-cell subtypes during intrathymic positive selection, and the multiple T-helper (Th) subsets of conventional CD4+ TCRαβ+ T cells generated during responses to antigen. But multibranched pathways are now also recognized for TCRγδ+ cells, unconventional T cells, NK cells, and dendritic cells. This raises the Question how firm is the boundary between a ‘lineage decision’ and an ‘activation state’ in these branch points. Effector subsets of CD4+ T cells, such as Th1 and Th2 cells, were originally thought to be irreversibly polarized, terminal fates; however, as additional subsets have been defined, evidence has also accumulated for plasticity and for the potential for interconversion among these effector fates. Whereas different variants of T, NK, and dendritic cells are increasingly recognized as distinct, other branches of immune cell development appear now to be less clearly separated. It was thought for many years that the precursors of innate immune cells and the precursors of all adaptive immune cells were completely separate, having diverged much earlier in the hematopoietic hierarchy. This might suggest that once the initial lympho/myeloid decision was made, completely different sets of regulatory molecules could program the choices made at lower levels of the hierarchy. However, it is now clear that myeloid and lymphoid development can be closely intertwined, as discussed from several points of view in contributions in this volume (1-6). Not only can lymphoid and myeloid cells be derived from overlapping precursor subsets (under appropriate conditions), but also for developmental decisions, they can share use of the same lineage-defining transcription factors. These shared regulatory molecules participate in different combinations of factors to promote distinct cell fate choices across the adaptive/innate divide, such as PU.1 in dendritic cells, and B cells, or Id2 and PLZF (Zbtb16) in NK cells, myeloid cells, iNKT cells, and some TCRγδ cells (4, 7-10). It is the combination that specifically defines an ‘adaptive’ or ‘innate’ immune cell program, rather than any ‘master gene’ itself. Thus, access to a cell fate can probably be restricted by control over any member of the combination. This volume highlights a number of key branch points in the lympho-hematopoietic developmental decision tree, where new evidence illuminates the molecules that exert control. T-lineage examples are prominent, as T-cell development has long been recognized to include multiple tiers of fate choices. Here these choices are discussed in the context of mechanisms used for B-cell, NK cell, dendritic cell, and progenitor cell fate determination as well. The reviews here cover lymphoid/myeloid lineage choice among hematopoietic precursors; B-cell developmental checkpoints, both in normal cells and as they are distorted in malignancy; specification of dendritic cells; and divergence among dendritic cell subsets. A series of reviews then consider molecules that act at a succession of branch points along the T-cell developmental pathway: mechanisms separating T/NK precursors from myeloid cells, T from NK cells, αβ T cells from γδ T-cells and different γδ T-cell subsets from one another, mechanisms creating divergences between conventional TCRαβ+ T cells and ‘innate-like’ iNKT cells, and finally mechanisms specifying and maintaining distinctions among different mature effector T-cell subsets. Focusing on the most mature and best-defined cell types, contributors describe in detail the biochemical mechanisms of action of the controlling transcription factors themselves and how they work on cis-regulatory elements of the key target genes in the differentiation pathways. These are models of the level of understanding that it is hoped the earlier, more complex developmental choices will eventually receive. An area that is unfortunately underrepresented in this collection is the diversification and terminal maturation of B cells, which involves an important developmental switch that is based on the balance between two sets of mutually antagonistic regulators. However, the relationships between ‘adaptive immune’ lymphoid programs for B and T cells and the ‘innate immune’ differentiation programs used in dendritic cells, natural killer cells, monocytes, and granulocytes are a recurrent theme. Contributions in this volume address these relationships both at the cell biological level and in terms of the roles of factors like PU.1, E proteins, signal transducer and activator of transcription (STAT) factors, GATA-3, PLZF, Notch, ThPOK, T-bet, and the newly appreciated factor Bcl11b, as summarized below. B cells and T cells share undisputed central roles in adaptive immunity, also sharing many similar features of cell biology and the unique use of recombination activating gene-mediated recombination to distinguish them from all other cell types, including even their lymphoid relatives the NK cells. The discovery of common lymphoid precursors marked by interleukin-7 receptor (IL-7R) expression (11) was a watershed for the field, as the first prospective isolation of individual progenitors with defined partial restrictions on potential. It was also deeply appealing as evidence that the similarities between T and B cells might result from a uniquely shared inheritance. However, problems have arisen with the notion that B and T cells emerge from a precursor that first loses all non-lymphoid potentials before choosing between T and B fates. These problems have commanded most attention with respect to the T-cell pathway, where isolation of an ordered succession of precursors from the thymus is fairly easy, and many independent labs have observed what these cells do when tested in different environmental conditions (reviewed here in 2, 3). It is seen here that B-cell potential is completely extinguished soon after the precursors enter the thymus, and yet a robust myeloid potential continues to be preserved for multiple cell divisions into T-cell specification. This myeloid potential is conditional, as it is restricted by environmental signals in the thymus, but it is quite evident upon transfer of the cells to permissive conditions in vitro, so that the later stage when myeloid potential is also lost can be clearly defined. There is also molecular evidence to explain how the normal gene regulatory features of early T-cell precursors in the thymus would still give access to myeloid but not B-cell programs. These T-lineage precursor assays thus imply that T and B-cell potential segregate before T-cell precursors lose myeloid or NK potential. However, evidence both old and new has been adduced to show that in vivo, under unperturbed conditions, the cells that seed the thymus may very well have a history of IL-7R expression, resembling the common lymphoid precursors. Even though these cells can undergo myeloid differentiation in vitro, they are poorer at generating myeloid cells in vivo than stem cells or dedicated myeloid precursors (12, 13). The meaning of this discrepancy has been sharply debated. The authors in this volume address this problem from several points of view. Yang et al. (2) review the observed sequence of lineage commitment stages for T-cell precursors, combining discussion of underlying transcriptional requirements with an emphasis on the environmental dependence of all these fate decisions. Thus, T-lineage competence depends first of all on the ability of the candidate precursors to enter the thymus, where the all-important Delta-like 4 Notch ligand drives the inductive signaling that triggers and sustains the T-cell program. These authors point to the crucial importance of chemokine receptor regulation in controlling this process, thus determining the range of inductive signals a cell would ever actually see in vivo. Kawamoto et al. (3) focus on mechanisms that sustain and limit intrinsic myeloid competence, relating these to a broader hematopoietic context. They argue that a conditional myeloid potential may be a basic feature of hematopoiesis, a latent but useful program option with deep evolutionary roots that can be given up only when T, B, or erythroid cells become fully committed to another fate. Kondo (1) reviews the early lymphoid precursor debate from a vantage point based on B and myeloid cell development, which both occur in the bone marrow. Here again, there is a conflict between the range of potentials that ‘lymphoid precursors’ show in vivo and in vitro. Kondo notes that in fact the bone marrow microenvironment is quite diversified and that regulated migration into particular zones within it is crucial for cells to receive appropriate signals for the lymphoid and myeloid programs. He cites results that inhibition of the appropriate chemokine receptor signals may be sufficient to reveal the latent myeloid potential in lymphoid precursors. Thus, even if lymphoid precursors express myeloid-permissive transcription factors, a strong bias for lymphoid lineage restriction may arise by programming cells to migrate in vivo to sites where lymphoid but not myeloid development is supported. Thus, the range of competence of the cells in vivo is partly a result of intrinsic regulatory state, but also partly a result of the cells’ ability to choose their own homing sites, where cell non-autonomous mechanisms then come forcefully into play. B-cell developmental paradigms have led the field for over a decade (14-17). B cells depend for their specification on a small group of transcription factors, two of which, EBF1 and Pax5, are B-lineage specific, so that hematopoietic precursors lacking these factors show selective B-cell defects. It was through analysis of these factors’ effects that the distinction first emerged clearly between positive regulation of lineage-specific genes (‘specification’) and exclusion of alternative fates (‘commitment’) (18). More widely shared factors like the E-protein E2A gain B-cell specific roles through incorporation in a network organized around the core of these two highly specific factors (19-21), as reviewed here by Zandi et al. (6). EBF1 and Pax5 and the expression of B-cell target genes in precursors are among the earliest signs of competence to generate B-cell progeny. Zandi et al. describe how lineage priming, i.e. initial onset of B-cell gene expression, substantially precedes commitment, and then the separate mechanisms through which actual B-lineage commitment is established. One crucial regulator used in B-cell development is shared with myeloid cells, namely PU.1. This Ets-family transcription factor works not only to promote myeloid and dendritic cell development but also to set up a pool of competent precursors for lymphoid development, and much later, to regulate aspects of B-cell responses to antigen (4). Carotta et al. now review these innate and adaptive functions, highlighting a dose-dependent role for PU.1 in dendritic cell development that is explained in part by exquisitely sensitive PU.1 regulation of the Flt3 (Flk2) cytokine receptor gene. Dendritic cells are highly diverse and programmed through distinctive, signal-dependent pathways. Watowich and Liu (22) reveal the web of mechanisms through which instructive signaling from cytokine and Toll-like receptors blends with cell-intrinsic transcription factor activities to direct dendritic cell subtype programs. In this complex network, there is clear evidence that distinct, cytokine-activated STAT factors and interferon-response factors play individualized, instructive roles both to foster certain fates and to block others. This recently appreciated network echoes aspects of the complex STAT-dependent networks governing CD4+ T-cell effector fates, described later in the volume. PU.1 is perhaps the main transcription factor that all immune cells, adaptive and innate, most distinctively share, for at least some parts of their developmental histories. In contrast, the next sections of this volume focus on the factors whose activities cause the discontinuities between one immune cell type and there were a ‘master for the genes that distinguish B and T cells from innate immune cells, E would probably the of This of transcription factors, including and to lymphoid developmental potential as well as to developmental choices and within lymphoid fates. As reviewed by and these factors and their regulated play roles in up precursors with lymphoid potential in the first to the myeloid and to separate B and T cells from their innate relatives like NK cells and most dendritic cells. However, with like iNKT cells T also appear to use E-protein for their and set the of E in the context of the broader networks of regulatory genes in B and T cells. T cells, an factor is et al. review the complex activities of this which is crucial for the pool of progenitor cells within the thymus and is then used and T-cell from the own as well as the dependence of in T-cell development from early where it can act as a between the T-cell fate and other fates for precursors, such as NK cells and even cells. These features of function have made it a to but role in T-cell development is now to and molecular T-lineage precursors, the most closely fate is that of NK cells. many NK cells the thymus, there is NK developmental potential among early T-cell precursors and a set of NK cells that in the thymus as and the regulatory that determine these NK fates are reviewed by et al. It has been known for a decade that E-protein Id2 is important for but a number of other transcription factors and Id2 are now clearly in distinct aspects of the T-cell transcription factor has been as a factor that sets the boundary between and gene expression programs as well as a factor that controls a commitment within the T-lineage known to be for regulation of later stages of T-cell development and this newly recognized early role interesting for role in T-lineage features in in this collection The nature of this factor and the evidence for role in NK development are reviewed in detail by Liu et al. The evidence they that may the regulatory function that is crucial to distinguish the T-cell fate from the fate. These authors also describe the functional of mature T cells that have been to a partial by of in vivo. A that T NK lineage choice may be based on the of to is by et al. The of the reviews on early T-cell development, from the group on regulatory gene expression and analysis of to argue for a program in early T-cell A boundary also is to an which is by a of from the later, on gene and selection, when the genes are In this the from the T-cell program to alternative fates depend on the specific regulatory in the initial is sharply in vivo before the boundary between the It is or to the of potential as well as to the to commitment within the T-cell development program The to commitment is closely to the earliest point of between at least some of the T cells that can as TCRγδ cells and that will as cells. The lineage divergence has become a focus of attention in as TCRγδ cells have been to a diverse of cell TCRγδ cells are distinct from each other in use of Notch E and their and other regulatory factors including and and and also distinct in their expression of the effector or as reviewed here by et al. These authors consider the mechanisms that separate TCRγδ differentiation pathways from differentiation pathways as well as models for TCRγδ selection, some of which appear to use highly of and gene in their at the underlying problem of the nature of TCRγδ they this is a program with branches or it is in fact a collection of distinct regulatory programs. The next major intrathymic is the between CD4+ and cells, as a result of positive The discovery that was a and regulator of the CD4+ cell fate was one of the major advances in this field As for many of the factors involved in T-cell development, however, is turning out to play a range of and these are described in detail by In is important for of two of the most mature subsets of TCRγδ cells and to regulate the cytokine expression of the mature TCRγδ is also to or some of the of iNKT cells. The iNKT fate is a with much of an cell type in response in of adaptive use of genes and for positive There are different subsets of these cells as well and an increasingly of the roles they can play in immune responses. As reviewed by et al. iNKT cells other T-cell have a set of positive signaling requirements and a for the factor PLZF as well as use of the E-protein Id2 for their terminal which them from conventional T cells. However, like conventional CD4+ T cells they are diverse in function and and et al. review the evidence that cells by iNKT cells may undergo to conventional CD4+ T cells the work on NK, and iNKT cells of type of functional and even the use of a at all are of in different at the adaptive/innate immune system The part of the volume into the sophisticated that are now how particular transcription factors and particular transcription factor target genes actually their roles in these effector fate decisions. et al. focus on the molecular mechanisms involved in activating expression of the and which distinguish not only Th1 and CD4+ conventional T cells but also subsets of mature TCRγδ cells and subsets of iNKT cells et al. the regulatory that are now recognized to to the of these and the transcription factors that act at each of these They show how the of these cis-regulatory sites even in effector cells is still to under the of certain cytokine signaling pathways. to another view of this time from the point of the transcription and the killer and regulator as a for biochemical They detail the combination of positive and regulatory effects of this factor in terms of the different particular factors that with it through distinct to or specific This much of what is to be at the level with other transcription factors as well in to the changes discussed by et al. These ultimately into a broader of transcription factor to define different CD4+ T-cell effector and a for this network is out at the end of this volume by and The to not only for the of effector cells into regulatory T and but also for the plasticity that to a degree of interconversion in particular examples of which are in both of the This gene network and the between the transcription factors within the cells and the cytokine signals that the cells do or do not receive from their in part on the regulated expression of cytokine receptors and signal genes It is this set of regulatory relationships that the effector response of mature T cells in the The reviews in this volume address only a of the immune cell lineage commitment field, but they several central points. One is how the combination of particular signals from the with a cell’s transcription factor regulatory to promote development along a particular or to an Another point is that in many immune cell development pathways are branched and diversified even after major aspects of lineage commitment, at least including dendritic cell, or T-cell fates. These developmental pathways appear to out distinct that are not more or less mature but in fact different for the between these subtypes are and they appear to use mechanisms less than the lineage exclusion mechanisms separating major immune cell classes or the mechanisms response However, factors’ like E and their or PLZF can be used in to separate subtypes within different major cell from these reviews is that in the at both early development and later effector development use of overlapping but sets of transcription factors. The use of transcription factors to work in different pathways points to for developmental roles not based on target genes of a given transcription but rather on the distinct set of sites to which that transcription factor is in a given developmental through with other factors. These examples a to models of developmental choice that are based on a between two ‘master different pathways discussed in this can see development from hematopoietic stem cells use of multiple combinations of transcription factors that not mutually but which can work or in different The reviews in this volume a of the of distinct cell types that can be generated as a result and the and highly regulated they can be used in the immune A of is to all the authors their time and to the reviews in this volume. The in this were with from from the and and from the at