CYTOKINES IN LYMPHOPOIESIS
The many cytokine pathways that regulate lymphoid development, differentiation, and function are too numerous and complex for a full description here. However, the cytokine receptors of the common gamma (γc) chain family should be mentioned particularly because of their biologic importance in lymphopoiesis and their clinical relevance in primary immune deficiency disease. The γc subunit is a signaling component of six different cytokine receptors, interleukin (IL)-2,83 IL-4,84,85 IL-7,86,87 IL-9,88 IL-15,89 and IL-21,90 all of which act on different stages and pathways involved in lymphopoiesis.51,91,92 All six γc-dependent receptors are unique in their activation of the Janus kinase 3 (JAK3) tyrosine kinase, a molecule that directly interacts with γc to mediate signaling.93 In addition to the γc subunit, each of these receptors are comprised of an α subunit through which specific ligands bind; IL-2R and IL-15R also share a common β subunit.51
Null mutations of γc result in severe combined immunodeficiency (SCID) syndromes in mice and humans. However differences in the specific lineages affected reveal important species differences in cytokine dependency.51 The most important of these differences is in the requirement for IL-7 signaling in human and murine B-cell development. Adult murine B-cell development has an absolute requirement for IL-7 to IL-7R interaction and subsequent downstream signaling involving the γc subunit of the IL-7R and JAK3.94 In contrast, IL-7 is not essential for human B-cell development. X-linked SCID patients with mutations in the γc cytokine-receptor subunit exhibit profound thymic hypoplasia and an absence of NK cells but normal or elevated numbers of B cells.51 SCID patients with mutations in JAK395,96 or the IL-7R97 also have normal numbers of blood B cells. Although B-cell numbers are normal, B-cell function in patients with γc-deficient SCID is not normal and patients are hypogammaglobulinemic, presumably partly as a result of the role of IL-4 in B-cell function and the absence of T-cell interactions in antibody production. These collective results indicate IL-7 is not essential for at least the numerically normal development of human B cells.
NK cells are absent in patients with γc-deficient and JAK3-deficient SCID, but are normal in IL-7Rα deficiency.65,97,98 NK cells are also absent in mice deficient in IL-15,99 IL-15Rα,100 or IL-2Rβ (a subunit shared by IL-2R and IL-15R),101 demonstrating the essential role of IL-15, but not IL-7, in NK cell development. Although no null mutations for IL-15 or its receptor have been described in humans, a familial NK cell deficiency has been described in humans in which the response to IL-15 and IL-2 appears to be subnormal.102
The production of both B and NK cells in patients with IL-7Rα deficiency, shows that in humans IL-7 is not required for the earliest stages of lymphoid commitment or growth of CLPs. This point is further supported with the finding that multilymphoid CD34+CD38negCD7+ progenitors in human cord blood do not express IL-7Rα,53 and that early lymphoid progenitor subsets are preserved in the marrow of γc and JAK3-deficient patients.103 In contrast to B cells and NK cells, however, T-cell development is absolutely dependent on IL-7 in both mice and humans.91 In both species, mutations of any portion of the IL-7 signaling pathway, that is, γc, IL-7Rα, or JAK3, completely prevents T-cell development.51 IL-2, in contrast, although an important cytokine in proliferation and function of mature T cells, is not essential for thymopoiesis; mutations in IL-2,104 IL-2Rα, or IL2Rβ105 result in functional T-cell defects, but T cells are not absent.
TRANSCRIPTIONAL REGULATION IN LYMPHOPOIESIS
The hierarchical differentiation pathways that lead irreversibly to the diverse array of functionally specialized mature lymphocytes are regulated by groups of genes expressed and repressed in a complex, precisely orchestrated sequence. As with cytokine regulation, our understanding of which transcriptional factors control each stage of differentiation has been developed using a combination of gene expression analyses in isolated progenitors and precursors, and an examination of the functional consequences of genetic mutations in mice and humans. The review in this chapter focuses on genes that regulate the earliest commitment decisions in the production of lymphoid progenitors; regulation of later differentiation stages in each lineage is discussed in Chaps. 75 to 77, respectively.
The complex interplay between groups of genes involved in hematopoietic differentiation has been likened to a multidimensional network whose “regulatory space” is formed by a dynamic balance between certain transcriptional regulators.106 Expression analysis of multiple genes in defined progenitor populations demonstrates levels of promiscuity at early stages of hematopoiesis, that preclude assignment of any unique gene expression pattern to each stage.106,107,108 As differentiation proceeds, a more specific “genetic fingerprint” for each lineage develops.
Regulation of Early Lymphoid Commitment
Ikaros Although no single gene has been identified as a lymphoid-specific master regulator, several transcription factors have been shown to be essential for the early stages of lymphopoiesis. The gene Ikaros, which encodes a family of DNA-binding zinc finger proteins, was identified in murine knockout studies as essential for all fetal lymphopoiesis.109,110 However, in the postnatal setting, the role of Ikaros is more complex and less specific. Adult Ikarosnull mice completely lack B cells, and although T cells are produced, their differentiation is abnormal.111 A murine study has suggested that Ikaros is not required for the initial lymphomyeloid versus myeloerythroid commitment decision, and that not only lymphoid differentiation, but also certain fate choices in the myeloerythroid pathway are affected by Ikaros.112 As the expression of two key lymphoid cytokine receptors, FLT3 and IL-7Rα, is dependent on Ikaros, and as these markers are used to isolate murine LMPP and CLP, respectively, it is still not completely clear at which exact lymphoid progenitor stage Ikaros exerts its effects.112 In addition to lymphoid progenitors, Ikaros isoforms are also expressed in HSCs, and myeloid lineages in mice112,113,114,115,116 and humans.116,117 Although Ikaros may act as a typical transcription factor in some settings, Ikaros also affects gene expression through its role in chromatin formation.118
Pu.1 The transcription factor PU.1 is essential for normal B- and T-lymphocyte development, but its effects are highly dose dependent. At high levels of PU.1, key myeloid regulatory genes are upregulated and macrophage differentiation is induced preferentially over lymphoid differentiation.119 Low-level expression of PU.1, however, is essential for lymphopoiesis.120,121 Mice in which PU.1 is completely absent lack B cells and have abnormal fetal thymopoiesis. However, studies with mice in which PU.1 is deleted specifically in B-lineage cells show that PU.1 is not essential for B-cell differentiation beyond the pre-B stage.122 It is likely that the critical role for PU.1 in murine lymphopoiesis lies in its upregulation of expression of the receptor for IL-7, which as mentioned above, is a key cytokine in both B and T lymphopoiesis in mice.120
E2A E2A (encoded by TCF3) generates two basic helix-loop-helix proteins, E12 and E47, through differential splicing.123 Murine studies suggest that E2A is necessary for lymphoid priming of multipotent progenitors and that the E2A proteins prime expression of a number of lymphoid-associated genes.124 There is a dose-dependent requirement for E2A expression in the development of LMPP and CLP.124 Both B- and T-lineage commitment are severely reduced in the absence of E2A, but Ikaros and PU.1 expression are normal.124,125,126 E2A affects B lymphopoiesis in part through upregulation of early B-cell factor (EBF)127 and T lymphopoiesis through upregulation of expression and function of the key T-cell specification factor Notch 1.128
Regulation of B-Cell Commitment
The transcription factors Ikaros, PU.1, E2A, EBF, and Pax5 are essential for normal B-cell differentiation. Mice that have functional deletions in any one of these genes have severely abnormal B-cell development; however, of these genes, only EBF and Pax5 are B-cell specific within the hematopoietic system.
Pax5 Pax5 is expressed specifically in B-lineage–committed progenitors and is required for normal expression of the B-lineage genes CD19 and CD79a.121 Pax5–/– mice are blocked at the pro-B cell stage, but express most early B-cell–related genes.129 Although Pax5 can activate a small subset of B-lineage genes, its main function in B-cell differentiation appears to be the suppression of T-cell and myeloid transcriptional programs at the murine pro-B–cell stage, thus enforcing commitment to the B lineage.121,129,130 Consistent with this role, PU.1, E2A, and EBF function earlier than Pax5 in B lymphopoiesis, and forced expression of Pax5 does not rescue the B-cell defect seen in EBF–/– mice or PU.1–/– mice.121
Ebf EBF (encoded by EBF1) is a helix-loop-helix zinc finger protein that activates a B-lineage transcriptional program, and induces B lymphoid in preference to myeloid development, in part by antagonizing the expression of genes encoding alternative lineages such as C/EBPα (CCAAT/enhancer binding protein), Id2, and PU.1,131 and, in part, by inducing Pax5 expression.121 Ebf1–/– lymphoid progenitor populations from mice lack the ability to generate B cells but retain the ability to generate T, NK, and myeloid cells.131 Overexpression of EBF in multipotent progenitors promotes B-cell production at the expense of myeloid differentiation.131 EBF and E2A function cooperatively in early B lymphopoiesis124; however, overexpression of EBF can rescue B-cell differentiation in E2A-deficient mice, including activation of Pax5.132 Pax5 overexpression however cannot rescue the B-cell defect in EBF–/– mice,121 demonstrating a critical, Pax5-independent role of EBF in early B-cell fate decisions.
Regulation of T-Cell Commitment
Notch Upon arrival into the thymus, multipotent progenitors from the marrow become rapidly committed to the T- and NK-cell pathways. The most important environmental cue for T-cell commitment is delivered by the thymic epithelium in the form of the Notch ligands, Delta-like 1 (DLL1) and Delta-like 4 (DLL4).133 Binding of one of these ligands to the Notch 1 receptor expressed on the surface of thymocyte precursors causes activation of intracellular Notch and a series of transcriptional programs turn on to switch lineage fate toward the T lineage at the expense of B-cell development.133 In mice, Notch is absolutely required for T-cell differentiation and proliferation, including β selection.134 Analogous to control of early B cell differentiation by E2A, Notch signaling activates a transcriptional network which includes factors critical for lineage specification (GATA-3, TCF-1), and commitment (BCL11b).135 However, although Notch signaling is necessary for murine thymopoiesis it is not sufficient for activation of the full complement of T-cell genes.136 The ability of hematopoietic progenitors to respond to Notch signaling and commit to T-lineage fate depends on a balance between positive and negative regulators. Combinations of at least four other transcription factors are required to initiate T-cell development: PU.1, Ikaros, Runx family factors, and E2A.124,133 In addition, leukemia-lymphoma–related factor (LRF/Pokemon, encoded by Zbtb7a) must be downregulated to allow Notch signaling to induce T-cell fate decisions.137 Notch signaling also plays important roles at later stages of thymocyte differentiation.133
The effects of Notch signaling have been extensively studied in mice, but the exact stages and processes regulated by Notch appear to differ between mice and humans. For example, using in vitro studies of human T-cell development, it appears that while Notch is essential for early thymocyte proliferation, it is not required for β selection or T-cell receptor αβ differentiation.138,139 As with so much of the information described in this chapter, the most important challenge that lies ahead is to translate the detailed mechanistic framework developed from murine studies into careful investigations of human lymphopoiesis.
GATA-3 GATA-3 is a key transcriptional factor for T-cell development, and is essential at various stages of differentiation. However, in addition to T cells, GATA-3 is also expressed in uncommitted HSCs, CLPs, and even in nonhematopoietic cells, and its effects are complex and highly dose-dependent.33,135,140
Tcf-1 TCF-1 (encoded by the TCF7 gene) is a transcription factor essential for T-cell development, and is directly activated by Notch signaling.141,142 In ETPs, TCF-1 promotes cell survival as well as activation of T-lineage specific genes, including Gata3 and Bcl11b.141,142 Induction of a T-cell specific transcriptional program by TCF-1 can occur even in the absence of Notch signaling; however, it cannot activate the essential T-lineage gene Ptcra,142 indicating that, as in B-cell development, T-cell specification occurs through both hierarchical and combinatorial transcription factor interactions.
Bcl11b BCL11B was identified as a transcription factor required for the normal generation of αβ T cells during β selection; however, upregulation of Bcl11b first occurs at the earlier CD4negCD8neg (double negative)-2 (DN2) stage, likely through transcriptional activation by TCF-1.135 In DN2 cells, Bcl11b appears to contribute minimally to the T-lineage specification program governed by Notch/E2A/GATA-3/TCF-1 activity, but rather is required for the suppression of stem/multipotent progenitor-associated genes, which marks the loss of myeloid potential and final commitment to the T-cell lineage.143