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270 Part IV: Molecular and Cellular Hematology Chapter 18: Hematopoietic Stem Cells, Progenitors, and Cytokines 271

arguing that the defects in these disorders might affect a CLP; however, directs marrow stem cells into immature CD4+/CD8+ T cells and
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this does not prove the existence of a cell common for all. Work using inhibits B-lymphocyte development. Overexpression of Notch1 in
cell sorting for CD10+/CD34+/Thy−/c-Kit−/Lin− human marrow cells RAG-deficient precursors also results in differentiation to the T-cell lin-
revealed the capacity to develop into T, B, NK, and lymphoid dendritic eage, although only to the immature CD4−/CD8− stage, indicating that
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cell progenitors, but the report did not demonstrate a common pro- Notch cannot substitute for pre–T-cell-receptor signaling. 310
genitor capable of giving rise to each lineage on a clonal level. B-Lymphocyte Progenitors B-cell progenitors include the pro-B
More recent work, based upon the importance of IL-7 for all single- cell, the earliest cell irreversibly committed to the lineage, which are
lineage lymphoid progenitor cells, and on the severe lymphopenia seen CD34+/CD10+/CD38+/CD19+/CD20+, the pre-B cell, which displays
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when the gene was eliminated in mice, has indicated that the IL-7R the initial stages of immunoglobulin rearrangement, expresses immu-
marks a CLP that can be used in flow cytometry to isolate a population noglobulin heavy chains in their cytoplasm and are CD34−/CD10+/
of IL7R+/Lin−/Thy−/Sca /Kit cells that engrafts all of lymphopoiesis CD19+/CD20+/CD38−, and immature B cells which begin immu-
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but no myelopoiesis in congenic mice. For example, the injection of noglobulin light-chain production, express cell-surface IgM, and are
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2000 such CD45.1+ cells plus 1 × 10 whole-marrow CD45.2+ cells into CD10+/CD19+/CD20+. Pro-B cells can be detected in a simple colony-
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lethally irradiated CD45.2 recipients lead to 3 to 20 percent CD45.1+ forming assay. Normally, B-cell precursor development occurs in con-
B and T lymphocytes, which disappear after approximately 6 months. tact with the hematopoietic microenvironment, mediated by precursor
In contrast, this strategy never results in the appearance of CD45.1+ cell integrin β β and stromal cell VCAM or matrix FN. A number of
1 1
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myeloid cells. When limiting dilution studies were performed, approx- cytokines affect B-cell progenitor proliferation, including IL-7,
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imately 1 in 20 such cells could give rise to short-term B-lymphopoie- insulin-like growth factor (IGF)-1, SDF-1, and SCF, although
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sis when injected intravenously, and an equal number could give rise based on genetic knockout studies, B cells are absolutely dependent only
to T-lymphopoiesis when injected into the thymus. In colony-forming on IL-7 and SDF-1. 22
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assays using IL-7, SCF, and FL, approximately 20 percent of such cells A number of cytokines also inhibit B-cell precursor development,
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gave rise to pre-B and pro-B cell colonies in vitro. Thus, given the low including interferon (IFN) α/β, IFN-γ, IL-4, and TGF-β. The
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likelihood of proper homing when injected into mice, it is almost cer- role of these and other inhibitory cytokines in B lymphopoiesis is com-
tain that the CLP exists and is IL7R+/Lin−/Thy−/Sca /Kit . When a plex, as in some situations a cytokine can inhibit one stage and stimulate
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genetic expression analysis was performed comparing HSCs to CLPs, another stage of development, and some might act indirectly.
the latter demonstrated a down-modulation of many molecules asso- A number of transcription factors are required for mature B cell
ciated with HSCs, such as the cell-surface receptors c-Mpl, β -integrin, function, including PU.1, nuclear factor-κB (NF-κB), early B-cell factor
1
and Tie2, and the transcription factors HOXA9 and EGR1, and upreg- (EBF), interferon regulatory factor 4 (IRF4), and Oct2, many of which
ulation of the IL-7R and the recombination activating protein recombi- bind to the promoters and enhancers involved in immunoglobulin
nation activating gene (RAG) 2. 300 gene expression. In contrast to these relatively later stage effects, E2A is
T Lymphocyte/NK Cell, T Lymphocyte, and NK Cell Progenitors required for commitment to the lineage. The marrow of E2A-deficient
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Simple colony-forming assays for mixed T/NK cell progenitors have mice is devoid of CD19 B cells, as well as most B-cell lineage-specific
not been developed, but the existence of the bipotent progenitor can be genes, including RAG1/2, Pax5, EBF, and VpreB. Moreover, no immu-
inferred from studies in which CD44+CD25−FcγRII/III− fetal thymic noglobulin rearrangement is detectable, and there are no IL-7 respon-
cells are cultured with genetically marked, deoxyguanosine treated fetal sive cells. Reintroduction of E2A into the marrow cells of null mice
thymic lobes under 70 percent oxygen at 37°C. Without cytokine sup- reconstitutes pre–B-cell development. 322
plementation, such cultures yield primarily CD3+/Thy1+ T cells, but E2A sits upon a hierarchy of B-cell lineage-specific genes and tran-
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if IL-2 plus IL-15 are added, the NK cell potential (CD3−/NK1.1+) of scription factors ; E2A directly regulates the expression of RAG1, λ5,
these cells is realized, and if IL-7 is also added, the number of single cells D-J , V-Jκ, and the transcription factor EBF, the latter, in turn, regulat-
H
that yield cells of both lineages increases significantly. As this type of ing VpreB, mb-1, D-J , V-Jκ, and the transcription factor Pax5, which,
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H
readout is possible from single day 12 fetal thymus cells, such studies in turn, regulates CD19 and LEF1 and shuts down genes associated with
establish that bipotent T/NK cell progenitors exist. alternate lineages, such as M-CSF-R (monocytic), myeloperoxidase
E box–binding proteins consisting of HEB, E2–2, and the E2A gene (neutrophilic), GATA1 (MEP), and pTα (T lymphocytic). As noted ear-
products E12 and E47 form a distinct subgroup within the large family of lier in the section “Notch Ligands”, a critical condition for B-cell com-
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basic helix-loop-helix transcription factors. Heterodimers or homod- mitment is the absence of Notch signaling.
imers form between family members through their helix-loop-helix
(HLH) region, and through their basic regions bind to canonical E-box Myeloid Progenitors
DNA sequences and thereby affect gene expression, including T-cell Common Myeloid Progenitors Flow cytometry has also been exten-
targets such as CD4 and the pre-Tα, and assist in recombination sively used to purify myeloid progenitors; an IL-7R−/Lin−/c-Kit+/
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of the γδ T-cell receptors. It is now clear that E2A is required for the Sca-1− population of murine marrow cells, which by virtue of being
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transition from the bipotent T/NK progenitor to committed T-cell pro- Sca1− excludes HSCs, develop into all myeloid lineages. Based on
genitors as its genetic elimination leads to preservation of the former expression of CD34 and the Fcγ RII/III, three distinct subpopulations
but elimination of the latter. 301 can be identified by further flow cytometry, IL-7Rα−/Lin−/c-Kit+/
Another important subgroup of HLH proteins is the Id family, Sca-1−/CD34+/FcRγ , IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34−/FcRγ , and
lo
lo
hi
which contain an HLH region but lack a DNA binding domain, thereby IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34+/FcRγ . When tested in colony-
acting as a sink for functional HLH proteins and thus negatively regu- forming assays in the presence of SCF, FL, IL-11, IL-3, GM-CSF, EPO,
lating the function of E proteins. Id proteins appear to be essential for and TPO, each cell population yielded distinct mature cell types.
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NK cell development, as genetic elimination of Id2 leads to a profound IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34+/FcRγ cells give rise to all mye-
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loss of NK cell progenitors and forced overexpression of Id3 leads to a loid colony types, including CFU-Mix, BFU-E, CFU-megakaryo-
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shift of T/NK cells preferentially into the NK cell lineage. 308 cyte (CFU-MK), MEP, CFU-GM, CFU-granulocyte (CFU-G), and
It is also clear that Notch activation plays a vital role in T-cell CFU-macrophage (CFU-M), consistent with that expected for the CMP.
hi
lineage commitment from the CLP. Overexpression of active Notch1 In contrast, IL-7Rα−/Lin−/c-Kit+/Sca-1−/CD34+/FcRγ cells form
Kaushansky_chapter 18_p0257-0278.indd 270 9/19/15 12:06 AM