The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.

How hematopoietic stem cells (HSCs) produce particular lineages is insufficiently understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was markedly induced with lymphoid lineage specification. HSCs from Satb1-deficient mice were defective in lymphopoietic activity in culture and failed to reconstitute T lymphopoiesis in wild-type recipients. Furthermore, Satb1 transduction of HSCs and embryonic stem cells robustly promoted their differentiation toward lymphocytes. Whereas genes that encode Ikaros, E2A, and Notch1 were unaffected, many genes involved in lineage decisions were regulated by Satb1. Satb1 expression was reduced in aged HSCs with compromised lymphopoietic potential, but forced Satb1 expression partly restored that potential. Thus, Satb1 governs the initiating process central to the replenishing of lymphoid lineages. Such activity in lymphoid cell generation may be of clinical importance and useful to overcome immunosenescence.

Plasticity of supporting cells in a stem cell factory.

In this issue of Immunity, Omatsu et al. (2010) provide new information on the CXCL12 abundant reticular (CAR) cells that support hematopoietic stem cells and lymphoid progenitors in bone marrow. CAR cells can convert to adipocytes and osteoblasts, additional cells known to regulate hematopoiesis.

Estrogen-inducible sFRP5 inhibits early B-lymphopoiesis in vivo, but not during pregnancy.

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen-induced soluble Frizzled-related proteins (sFRPs), and particularly sFRP5, suppress B-lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B-lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B-cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B-lineage-committed progenitors was observed in the BM of sfrp5-null pregnant females. We concluded that, although high sFRP5 expression inhibits B-lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho-hematopoiesis in the maternal BM, but the maternal-fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.

The canonical Wnt pathway shapes niches supportive of hematopoietic stem/progenitor cells.

Considerable information has accumulated about components of BM that regulate the survival, self-renewal, and differentiation of hematopoietic cells. In the present study, we investigated Wnt signaling and assessed its influence on human and murine hematopoiesis. Hematopoietic stem/progenitor cells (HSPCs) were placed on Wnt3a-transduced OP9 stromal cells. The proliferation and production of B cells, natural killer cells, and plasmacytoid dendritic cells were blocked. In addition, some HSPC characteristics were maintained or re-acquired along with different lineage generation potentials. These responses did not result from direct effects of Wnt3a on HSPCs, but also required alterations in the OP9 cells. Microarray, PCR, and flow cytometric experiments revealed that OP9 cells acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells, including the expression of angiopoietin 1, the c-Kit ligand, and VCAM-1. In contrast, the production of decorin, tenascins, and fibromodulin markedly increased. We found that at least 1 of these extracellular matrix components, decorin, is a regulator of hematopoiesis: upon addition of this proteoglycan to OP9 cocultures, decorin caused changes similar to those caused by Wnt3a. Furthermore, hematopoietic stem cell numbers in the BM and spleen were elevated in decorin-knockout mice. These findings define one mechanism through which canonical Wnt signaling could shape niches supportive of hematopoiesis.

CD86 is expressed on murine hematopoietic stem cells and denotes lymphopoietic potential.

A unique subset of CD86(-) HSCs was previously discovered in mice that were old or chronically stimulated with lipopolysaccharide. Functionally defective HSCs were also present in those animals, and we now show that CD86(-) CD150(+) CD48(-) HSCs from normal adult mice are particularly poor at restoring the adaptive immune system. Levels of the marker are high on all progenitors with lymphopoietic potential, and progressive loss helps to establish relations between progenitors corresponding to myeloid and erythroid lineages. CD86 represents an important tool for subdividing HSCs in several circumstances, identifying those unlikely to generate a full spectrum of hematopoietic cells.

Functional diversity of stem and progenitor cells with B-lymphopoietic potential.

Technical advances have made it possible to separate hematopoietic tissues such as the bone marrow into ever smaller populations, complicating our understanding of immune system replenishment. Patterns of surface marker expression and transcription profiles as well as results obtained with reporter mice suggest that lymphopoietic cells are not closely synchronized, and there is considerable cell to cell variation. Loss of differentiation options is gradual, and ultimate fate can be established at different stages of lineage progression. For example, individual hematopoietic stem cells can be biased such that some are very poor sources of lymphocytes as contrasted to ones with balanced outputs. Still other hematopoietic stem cells are effective at generating B and T cells but are defective with respect to expansion and difficult to distinguish from early lymphoid progenitors. That diversity carries forward to later events, and similar appearing cells in the immune system can arise from alternate differentiation pathways. In fact, new categories of lymphoid progenitors are still being discovered. Heterogeneity provides adaptability as hematopoiesis can be dramatically altered during infections, influencing numbers and types of cells that are produced.

Early events in lymphopoiesis: an update.

PURPOSE OF REVIEW: Cells of the immune system are replaced in large numbers throughout life, and the underlying mechanisms have been extensively studied. Whereas the pace of discovery in this area is unprecedented, many questions remain, particularly with respect to lymphocyte formation. RECENT FINDINGS: While transcription factors have long been a focus of investigation, microRNAs are also being implicated in lymphopoiesis. Lymphocytes are normally replaced in correct proportion to other blood cells, but ratios change dramatically during infections. Long-standing issues relating to T versus B lineage divergence remain but have been enriched with remarkable new findings about thymus seeding. There are indications that at least some age-related changes in lymphopoiesis may be reversible. Finally, knowledge obtained from studies of mice is slowly being extended to humans. SUMMARY: We can now appreciate that new lymphoid progenitors are drawn from a heterogeneous collection of hematopoietic stem cells through asynchronous patterns of gene expression. Complex interactions then occur between the gene products, preparing lymphoid progenitors to respond to environmental cues. Whereas unique markers describe the process of lymphocyte formation in humans, fundamental information now available should suggest ways to promote rebound from chemotherapy or transplantation and reverse declines associated with aging.

Chronic exposure to a TLR ligand injures hematopoietic stem cells.

Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation, and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential, would be consistent with age- or TLR-related injury. In contrast, HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.

Leptin signaling maintains B-cell homeostasis via induction of Bcl-2 and Cyclin D1.

Regulation of apoptosis and cell cycle progression plays an essential role in the maintenance of B-cell homeostasis, because a fine balance of survival and expansion is critical for preventing lymphocytic disorders. Although remarkable progress in understanding B-cell development has been achieved, much less is known concerning niches that are critical to the maintenance of B-cell homeostasis. Leptin has recently been recognized to be important for modulating the immune responses, but it has remained unclear how leptin signaling influences B-cell physiology. A variety of lymphocytic malignancies have been reported to be linked to leptin, and therefore it is necessary to elucidate the mechanisms involved. Here we demonstrate that leptin promotes B-cell homeostasis by inhibiting apoptosis and by inducing cell cycle entry through the activation of expressions of B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1. We further show that leptin can induce Bcl-2 and cyclin D1 expression by two pathways, including the direct activation of their promoters and suppression of microRNAs (miRNAs) that target their putative 3'untranslated regions. Amplification of these leptin-modulated miRNAs inhibited B lymphoma cell growth. These findings provide insights into mechanisms for leptin regulation of the humoral immune system and suggest new therapeutic strategies for leptin receptor expressing malignancies.

A differentiation pathway for B1 cells in adult bone marrow.

The recent description of a Lin(-)AA4.1(+)CD19(+)B220(Lo/-) B1-specified progenitor (B1P) population in adult marrow adds support for the argument that these unique B cells arise from a distinct lineage. However, the origins of B1P were not investigated and their developmental relationships to conventional B2 cells remain unclear. We now report that B1P development is IL-7Ralpha-dependent, and negatively regulated by Bruton tyrosine kinase. Lymphoid characteristics of B1P were further studied with recombination activating gene (RAG)-1/GFP knock-in, RAG-1/Cre reporter, and VEX transgenic mice. Our results reveal that they are heterogeneous with respect to lymphocyte affiliation. RAG-1(+) early lymphoid progenitors and Lin(-)Sca-1(+)cKit(Lo)IL-7Ralpha(+) common lymphoid progenitors from adult marrow efficiently generated CD19(+)CD45R/B220(Lo/-) cells in vitro and in vivo. Moreover, early lymphoid progenitors and common lymphoid progenitors produced significant numbers of peritoneal CD11b(+)CD5(+) B1a and CD11b(+)CD5(-) B1b cells in vivo. Finally, 2-step transplantation experiments established a differentiation pathway between conventional lymphoid progenitors, B1P, and mature B1 lymphocytes. Thus, our findings indicate that at least some B1P can be produced in adult bone marrow from primitive B2 progenitors, and suggest a developmental relationship between the major categories of B lymphocytes.

Many roads, one destination for T cell progenitors.

The thymus manufactures new T cells throughout life but contains no self-renewing potential. Instead, replenishment depends on recruitment of bone marrow-derived progenitors that circulate in the blood. Attempts to identify thymic-homing progenitors, and to assess the degree to which they are precommitted to the T cell lineage, have led to complex and sometimes conflicting results. As described here, this probably reflects the existence of multiple distinct types of T cell lineage progenitors as well as differences in individual experimental approaches.

The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice.

Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity, more information about their characteristics will improve our understanding of their development and stage-related functions. Here, using microarray technology, we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1(-) c-kit(Hi) Sca1(+) HSC-enriched fraction, but sharply down-regulated with activation of the Rag1 locus, a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors, ESAM expression showed intimate correlation with HSC activity. The ESAM(Hi) population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity, and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2(+) c-kit(+) lymphohematopoietic cells in the E9.5-10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore, ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly, ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life.

Loss of Bright/ARID3a function promotes developmental plasticity.

B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested that it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely, and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.

Asynchronous RAG-1 expression during B lymphopoiesis.

Changes in cell surface markers and patterns of gene expression are commonly used to construct sequences of events in hematopoiesis. However, the order may not be as rigid as once thought and it is unclear which changes represent the best milestones of differentiation. We developed a fate-mapping model where cells with a history of RAG-1 expression are permanently marked by red fluorescence. This approach is valuable for appreciating lymphoid-lineage relationships without need for irradiation and transplantation. Hematopoietic stem cells (HSC) as well as myeloid and dendritic cell progenitors were unlabeled. Also as expected, most previously identified RAG-1(+) early lymphoid progenitors in bone marrow and all lymphoid-affiliated cells were marked. Of particular interest, there was heterogeneity among canonical common lymphoid progenitors (CLP) in bone marrow. Labeled CLP expressed slightly higher levels of IL-7Ralpha, displayed somewhat less c-Kit, and generated CD19(+) lymphocytes faster than the unlabeled CLP. Furthermore, CLP with a history of RAG-1 expression were much less likely to generate dendritic and NK cells. The RAG-1-marked CLP were lineage stable even when exposed to LPS, while unlabeled CLP were redirected to become dendritic cells in response to this TLR4 ligand. These findings indicate that essential events in B lymphopoiesis are not tightly synchronized. Some progenitors with increased probability of becoming lymphocytes express RAG-1 while still part of the lineage marker-negative Sca-1(+)c-Kit(high) (LSK) fraction. Other progenitors first activate this locus after c-Kit levels have diminished and cell surface IL-7 receptors are detectable.

Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated interleukin-6 production by myeloid cells in bone marrow.

The Src homology (SH)2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP(-/-) animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP(-/-) mice. Lineage-negative marrow (Lin(-)) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP(-/-) mice. Furthermore, culture supernatants of SHIP(-/-) Lin(-) cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP(-/-) Lin(-) cells contained more IL-6 transcripts than wild-type Lin(-) cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP(-/-) Lin(-) cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin(-) bone marrow cells reproduced the phenotype of SHIP(-/-) bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

A possible contribution of retinoids to regulation of fetal B lymphopoiesis.

We recently found that all trans retinoic acid (ATRA) accelerated B lymphocyte formation. In the current study, we address the question whether retinoids account for the rapid lymphopoiesis that is characteristic of fetal progenitors. Surprisingly, addition of ATRA to fetal liver cultures actually reduced B lymphopoiesis. A pan-retinoid receptor antagonist selectively suppressed lymphocyte formation from fetal and adult progenitors, suggesting some normal contribution of retinoids to this process. Consistent with this role, B lymphopoiesis was compromised in the marrow of mice with prolonged vitamin A deficiency. Recently identified B1 progenitors from adult marrow were similar to adult B2 progenitors in that their differentiation was stimulated by ATRA. The inhibitory response observed with fetal cells was seen when adult progenitors were exposed to high doses in culture or when adult mice were treated with ATRA for 2 wk. In addition to explosive lymphocyte generation, fetal progenitors tend to be less IL-7 dependent than their adult counterparts, but ATRA did not make fetal progenitors IL-7 independent. We conclude that all known categories of B lineage progenitors are responsive to retinoids and probably regulated by these compounds under physiological conditions. Retinoids may account in part for rapid differentiation in fetal life, but not all unique features of fetal progenitors.

Lymphoid precursors are directed to produce dendritic cells as a result of TLR9 ligation during herpes infection.

Hematopoietic stem and progenitor cells were previously found to express Toll-like receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19+ B lineage cells and had augmented competence to generate DCs. TNFalpha mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNFalpha-/- mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. Common myeloid progenitors are normally a good source of DCs, but this potential was reduced by TLR9 ligation. Thus, alternate differentiation pathways may be used to produce innate effector cells in health and disease.

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals.

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.

Soluble frizzled-related protein 1 is estrogen inducible in bone marrow stromal cells and suppresses the earliest events in lymphopoiesis.

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally simulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker(-) c-kit(high) ScaI(+), hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen-inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-alpha was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized beta-catenin and blocked early lymphoid progression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differentiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels.