Dendritic cell potentials of early lymphoid and myeloid progenitors

It has been proposed that there are at least 2 classes of dendritic cells (DCs), CD8alpha(+) DCs derived from the lymphoid lineage and CD8alpha(-) DCs derived from the myeloid lineage. Here, the abilities of lymphoid- and myeloid-restricted progenitors to generate DCs are compared, and their overall contributions to the DC compartment are evaluated. It has previously been shown that primitive myeloid-committed progenitors (common myeloid progenitors [CMPs]) are efficient precursors of both CD8alpha(+) and CD8alpha(-) DCs in vivo. Here it is shown that the earliest lymphoid-committed progenitors (common lymphoid progenitors [CLPs]) and CMPs and their progeny granulocyte-macrophage progenitors (GMPs) can give rise to functional DCs in vitro and in vivo. CLPs are more efficient in generating DCs than their T-lineage descendants, the early thymocyte progenitors and pro-T cells, and CMPs are more efficient DC precursors than the descendant GMPs, whereas pro-B cells and megakaryocyte-erythrocyte progenitors are incapable of generating DCs. Thus, DC developmental potential is preserved during T- but not B-lymphoid differentiation from CLP and during granulocyte-macrophage but not megakaryocyte-erythrocyte development from CMP. In vivo reconstitution experiments show that CLPs and CMPs can reconstitute CD8alpha(+) and CD8alpha(-) DCs with similar efficiency on a per cell basis. However, CMPs are 10-fold more numerous than CLPs, suggesting that at steady state, CLPs provide only a minority of splenic DCs and approximately half the DCs in thymus, whereas most DCs, including CD8alpha(+) and CD8alpha(-) subtypes, are of myeloid origin. (Blood. 2001;97:3333-3341)     

Single-cell analysis of early B-lymphocyte development suggests independent regulation of lineage specification and commitment in vivo

To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early B-lineage cells from Pax-5–deficient animals crossed to a B-lineage–restricted reporter mouse, allowing us to identify B-lineage–specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promoter-controlled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified B-lineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5–deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5– and Ebf-1–deficient progenitors, it was possible to identify a set of B-cell–restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.     

Activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathway is involved in myeloid lineage commitment

Common lymphoid progenitors (CLPs) are lymphoid-lineage-committed progenitor cells. However, they maintain a latent myeloid differentiation potential that can be initiated by stimulation with interleukin-2 (IL-2) via ectopically expressed IL-2 receptors. Although CLPs express IL-7 receptors, which share the common gamma chain with IL-2 receptors, IL-7 cannot initiate lineage conversion in CLPs. In this study, we demonstrate that the critical signals for initiating lineage conversion in CLPs are delivered via IL-2 receptor beta (IL-2R beta) intracellular domains. Fusion of the A region of the IL-2R beta cytoplasmic tail to IL-7R alpha enables IL-7 to initiate myeloid differentiation in CLPs. We found that Shc, which associates with the A region, mediates lineage conversion signals through the mitogen activated protein kinase (MAPK) pathway. Because mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitors completely blocked IL-2-mediated lineage conversion, MAPK activation, specifically via the MEK/ERK pathway, is critically involved in the initiation of this event. Furthermore, formation of granulocyte/macrophage (GM) colonies by hematopoietic stem cells, but not by common myeloid progenitors (CMPs), was severely reduced in the presence of MEK/ERK inhibitors. These results demonstrate that activation of MEK/ERK plays an important role in GM lineage commitment.     

Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors

OBJECTIVE: The aim of this study was to determine the lineage origin of interferon-alpha-producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs. MATERIALS AND METHODS: Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-alpha production after virus challenge. RESULTS: Transplantation of 1 x 10(4) common myeloid progenitors, 1 x 10(4) common lymphoid progenitors or 2.5 x 10(4) granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology. CONCLUSION: Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin.     

Rac GTPase isoforms Rac1 and Rac2 play a redundant and crucial role in T-cell development

Rac GTPases have been implicated in the regulation of diverse functions in various blood cell lineages, but their role in T-cell development is not well understood. We have carried out conditional gene targeting to achieve hematopoietic stem cell (HSC)- or T-cell lineage-specific deletion of Rac1 or Rac1/Rac2 by crossbreeding the Mx-Cre or Lck-Cre transgenic mice with Rac1(loxp/loxp) or Rac1(loxp/loxp);Rac2(-/-) mice. We found that (1) HSC deletion of both Rac1 and Rac2 inhibited production of common lymphoid progenitors (CLPs) in bone marrow and suppressed T-cell development in thymus and peripheral organs, whereas deletion of Rac1 moderately affected CLP production and T-cell development. (2) T cell-specific deletion of Rac1 did not affect T-cell development, whereas deletion of both Rac1 and Rac2 reduced immature CD4(+)CD8(+) and mature CD4(+) populations in thymus as well as CD4(+) and CD8(+) populations in spleen. (3) The developmental defects of Rac1/Rac2 knockout T cells were associated with proliferation, survival, adhesion, and migration defects. (4) Rac1/Rac2 deletion suppressed T-cell receptor-mediated proliferation, IL-2 production, and Akt activation in thymocytes. Thus, Rac1 and Rac2 have unique roles in CLP production and share a redundant but essential role in later stages of T-cell development by regulating survival and proliferation signals.     

Lymphoid progenitors in normal mouse lymph nodes develop into NK cells and T cells in vitro and in vivo

We have identified a population of normal mouse LN cells, termed LN lymphoid progenitor (LNLP), resembling common lymphoid progenitor (CLP) in the bone marrow. LNLPs lack lineage markers and express CD127, low levels of CD117 (c-Kit), and Sca-1, but lack fms-related tyrosine kinase 3. They efficiently differentiate in vitro into natural killer (NK) cells and T cells, but not mature B cells. LNLPs injected into nonirradiated lymphopenic mice that have no LN develop into mostly splenic T cells with low numbers of NK cells and B cells. When injected into irradiated mice, they generate NK cells and T cells, but not B cells, in the LN. By contrast, bone marrow CLPs develop into mostly B cells with very small numbers of T and NK cells in recipients' spleen and LN. LNLPs have NK and T-cell potentials, but little B-cell potential, and they can develop into NK cells within the LN of normal mice, but their contribution to the T-cell lineage is unknown.     

Reconstitution of early lymphoid proliferation and immune function in Jak3-deficient mice by interleukin-3.

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through gamma(c)-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7-responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3(-/-) mice. Newborn Jak3(-/-) mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4(+), CD8(+), and B220(+)/IgM(+) splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3-treated Jak3(-/-) mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3-treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7Ralpha(+)/IL-3Ralpha(+) cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R(+) lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.     

Control of murine Ly6C(high) monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6

The atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. Here we report that mice lacking D6 expression in the nonhematopoietic compartment have a selective increase in the number of Ly6C(high) monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6C(high) monocytes accumulate in increased number in secondary lymphoid organs of D6(-/-) mice in a CCR2-dependent manner. Ly6C(high) monocytes derived from D6(-/-) mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses, and partially protect mice from the development of GVHD. Thus, control of CCR2 ligands by D6 regulates the traffic of Ly6C(high) monocytes and controls their immunosuppressive potential.     

Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7- lympho-myeloid thymic progenitors

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34(+)lin(-) thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7(-). CD34(+)lin(-) thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7(hi) fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7(int) (CD10(+)) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7(-) fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34(+)lin(-)CD7(-) thymocytes also expressed early T lymphoid genes Tdt, pTalpha, and IL-7Ralpha and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7(-) lympho-myeloid thymic progenitor.     

Reduced production of B-1-specified common lymphoid progenitors results in diminished potential of adult marrow to generate B-1 cells

B-1 B cells have been proposed to be preferentially generated from fetal progenitors, but this view is challenged by studies concluding that B-1 production is sustained throughout adult life. To address this controversy, we compared the efficiency with which hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs) from neonates and adults generated B-1 cells in vivo and developed a clonal in vitro assay to quantify B-1 progenitor production from CLPs. Adult HSCs and CLPs generated fewer B-1 cells in vivo compared with their neonatal counterparts, a finding corroborated by the clonal studies that showed that the CLP compartment includes B-1- and B-2-specified subpopulations and that the former cells decrease in number after birth. Together, these data indicate that B-1 lymphopoiesis is not sustained at constant levels throughout life and define a heretofore unappreciated developmental heterogeneity within the CLP compartment.     

A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-), PAX5(+), TdT(+), IgH rearranged)/myeloid (CD11b/Mac1(+), c-fms(+), lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model.     

A partial deficiency of interleukin-7R alpha is sufficient to abrogate T-cell development and cause severe combined immunodeficiency

Both in vitro and in vivo studies established that interleukin 7 (IL-7) is essential for differentiation of immature T cells and B cells but not natural killer (NK) cells in the mouse. In humans, although both T-cell and B-cell progenitors express the functional IL-7 receptor that consists of IL-7R alpha and the gamma common (gamma c) chain, this lymphocyte receptor system is critical for T lineage but not for B lineage development. Indeed, complete gamma c deficiency like IL-7R alpha deficiency results in the arrest of T-cell but not B-cell development (T(-)B(+) SCID). However, partial deficiency of gamma c caused by missense mutations results in a T(+)B(+) phenotype and a delay of clinical presentation. It was therefore plausible to assume that partial deficiency of IL-7R alpha, like partial gamma c deficiency may lead to a milder clinical and immunologic phenotype. A P132S mutation in the IL-7R alpha was identified in 3 patients with severe combined immunodeficiency (SCID) within an extensively consanguineous family. Substitution of proline with serine in the extracellular portion of IL-7R alpha did not affect IL-7R alpha messenger RNA (mRNA) and protein expression, but severely compromised affinity to IL-7, resulting in defective signal transduction. In response to IL-7 stimulation, Jak-3 phosphorylation was markedly reduced in both patient cells as well as in COS cells reconstituted with mutant IL-7R alpha. Surprisingly, this partial deficiency of IL-7R alpha resulted in a severe phenotype, including markedly reduced circulating T cells while sparing B-cell numbers similar to gamma c chain deficiency. However, unlike the previously reported cases, serum immunoglobulins were virtually absent. Further, unlike gamma c deficiency, NK cell numbers and function was preserved. Despite the partial deficiency, clinical presentation was indistinguishable from a complete gamma c deficiency, including severe and persistent viral and protozoal infections and failure to thrive. Unlike partial gamma c deficiency, a partial deficiency of IL-7R alpha results in an arrest of T-cell development, leading to typical severe combined immunodeficiency. This underscores the critical role of IL-7R alpha chain in the differentiation of T cells. (Blood. 2000;96:2803-2807)     

Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators

Recently, a collection of surface markers was exploited to isolate viable Lin(-) TdT(+) cells from murine bone marrow. These early pro-B cells were enriched for B-lineage lymphocyte precursor activity measured by short-term culture and had little responsiveness to myeloid growth factors. Early precursors can be propagated with remarkably high cloning frequencies in stromal cell-free, serum-free cultures, permitting this analysis of direct regulatory factors. Expression of the interleukin-7 receptor (IL-7Ralpha) chain marks functional precursors and IL-7 is necessary for progression beyond the CD45RA(+) CD19(-) stage. Efficient survival and differentiation were only observed when stem cell factor and Flt-3 ligand were also present. IL-7-responsive CD19(+) precursors are estrogen resistant. However, B-lineage differentiation was selectively abrogated when highly purified Lin(-) precursors were treated with hormone in the absence of stromal cells. In addition, early stages of B lymphopoiesis were arrested by limitin, a new interferon (IFN)-like cytokine as well as IFN-alpha, IFN-gamma, or transforming growth factor beta (TGF-beta), but not by epidermal growth factor (EGF). Lin(-) TdT(+) early pro-B cells are shown here to be CD27(+) AA4.1(+/-)Ki-67(+) Ly-6C(-) Ly-6A/Sca-1(Lo/-)Thy-1(-)CD43(+) CD4(+/-)CD16/32(Lo/-)CD44(Hi) and similar in some respects to the "common lymphoid progenitors" (CLP) identified by others. Although early pro-B cells have lost myeloid differentiation potential, transplantation experiments described here reveal that at least some can generate T lymphocytes. Of particular importance is the demonstration that a pivotal early stage of lymphopoiesis is directly sensitive to negative regulation by hormones and cytokines.     

IL-7R-dependent survival and differentiation of early T-lineage progenitors is regulated by the BTB/POZ domain transcription factor Miz-1

T cells originate from early T lineage precursors that have entered the thymus and differentiate through well-defined steps. Mice deficient for the BTB/POZ domain of zinc finger protein-1 (Miz-1) almost entirely lack early T lineage precursors and have a CD4(-)CD8(-) to CD4(+)CD8(+) block causing a strong reduction in thymic cellularity. Miz-1(ΔPOZ) pro-T cells cannot differentiate in vitro and are unable to relay signals from the interleukin-7R (IL-7R). Both STAT5 phosphorylation and Bcl-2 up-regulation are perturbed. The high expression levels of SOCS1 found in Miz-1(ΔPOZ) cells probably cause these alterations. Moreover, Miz-1 can bind to the SOCS1 promoter, suggesting that Miz-1 deficiency causes a deregulation of SOCS1. Transgenic overexpression of Bcl-2 or inhibition of SOCS1 restored pro-T cell numbers and their ability to differentiate, supporting the hypothesis that Miz-1 is required for the regulation of the IL-7/IL-7R/STAT5/Bcl-2 signaling pathway by monitoring the expression levels of SOCS1.