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Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

The mouse thymus supports T-cell development, but also contains non–T-cell lineages such as dendritic cells, macrophages, and granulocytes that are necessary for T-cell repertoire selection and apoptotic thymocyte clearance. Early thymic progenitors (ETPs) are not committed to the T-cell lineage, as demonstrated by both in vitro and in vivo assays. Whether ETPs realize non–T-cell lineage potentials in vivo is not well understood and indeed is controversial. In the present study, we investigated whether ETPs are the major precursors of any non–T-lineage cells in the thymus. We analyzed the development of these populations under experimental circumstances in which ETPs are nearly absent due to either abrogated thymic settling or inhibition of early thymic development by genetic ablation of IL-7 receptorα or Hes1. Results obtained using multiple in vivo approaches indicate that the majority of thymic granulocytes derive from ETPs. These data indicate that myelolymphoid progenitors settle the thymus and thus clarify the pathways by which stem cells give rise to downstream blood cell lineages.

Common myeloid progenitors (CMPs) were first identified as progenitors that were restricted to myeloid and erythroid lineages. However, it was recently demonstrated that expression of both lymphoid- and myeloid-related genes could be detected in myeloid progenitors. Furthermore, these progenitors were able to give rise to T and B lymphocytes, in addition to myeloid cells. Yet, it was not known whether these progenitors were multipotent at the clonogenic level or there existed heterogeneity within these progenitors with different lineage potential. Here we report that previously defined CMPs possess T-lineage potential, and that this is exclusively found in the Flt3+CD150– subset of CMPs at the clonal level. In contrast, we did not detect B-lineage potential in CMP subsets. Therefore, these Flt3+CD150– myeloid progenitors were T/myeloid potent. Yet, Flt3+CD150– myeloid progenitors are not likely to efficiently traffic to the thymus and contribute to thymopoiesis under normal conditions because of the lack of CCR7 and CCR9 expression. Interestingly, both Flt3+CD150– and Flt3–CD150– myeloid progenitors are susceptible to Notch1-mediated T-cell acute lymphoblastic leukemia (T-ALL). Hence, gain-of-function Notch1 mutations occurring in developing myeloid progenitors, in addition to known T-lineage progenitors, could lead to T-ALL oncogenesis.

T lymphopoiesis requires settling of the thymus by bone marrow–derived precursors throughout adult life. Progenitor entry into the thymus is selective, but the molecular basis of this selectivity is incompletely understood. The chemokine receptor CCR9 has been demonstrated to be important in this process. However, progenitors lacking CCR9 can still enter the thymus, suggesting a role for additional molecules. Here we report that the chemokine receptor CCR7 is also required for efficient thymic settling. CCR7 is selectively expressed on bone marrow progenitors previously shown to have the capacity to settle the thymus, and CCR7−/− progenitors are defective in settling the thymus. We further demonstrate that CCR7 sustains thymic settling in the absence of CCR9. Mice deficient for both CCR7 and CCR9 have severe reductions in the number of early thymic progenitors, and in competitive assays CCR7−/−CCR9−/− double knockout progenitors are almost completely restricted from thymic settling. However, these mice possess near-normal thymic cellularity. Compensatory expansion of intrathymic populations can account for at least a part of this recovery. Together our results illustrate the critical role of chemokine receptor signaling in thymic settling and help to clarify the cellular identity of the physiologic thymic settling progenitors.

Proper thymocyte development is required to establish T-cell central tolerance and to generate naive T cells, both of which are essential for T-cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4+ T and CD8+ T cells that exhibit an activated phenotype and increased apoptosis and readily produce cytokines upon T-cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for thymocyte development and the generation of quiescent naive T cells.

Aging in mice and humans is characterized by declining T-lymphocyte production in the thymus, yet it is unclear whether aging impacts the T-lineage potential of hematopoietic progenitors. Although alterations in the lymphoid progenitor content of aged mouse bone marrow (BM) have been described, irradiation-reconstitution experiments have failed to reveal defects in T-lineage potential of BM hematopoietic progenitors or purified hematopoietic stem cells (HSCs) from aged mice. Here, we assessed T-progenitor potential in unmanipulated recipient mice without conditioning irradiation. T-progenitor potential was reduced in aged BM compared with young BM, and this reduction was apparent at the earliest stages of intrathymic differentiation. Further, enriched populations of aged HSCs or multipotent progenitors (MPPs) gave rise to fewer T-lineage cells than their young counterparts. Whereas the T-precursor frequency within the MPP pool was unchanged, there was a 4-fold decline in T-precursor frequency within the HSC pool. In addition, among the T-competent HSC clones, there were fewer highly proliferative clones in the aged HSC pool than in the young HSC pool. These results identify T-compromised aged HSCs and define the nature and cellular sites of prethymic, age-related defects in T-lineage differentiation potential.

Early T-lineage progenitors (ETPs) arise after colonization of the thymus by multipotent bone marrow progenitors. ETPs likely serve as physiologic progenitors of T-cell development in adult mice, although alternative T-cell differentiation pathways may exist. While we were investigating mechanisms of T-cell reconstitution after bone marrow transplantation (BMT), we found that efficient donor-derived thymopoiesis occurred before the pool of ETPs had been replenished. Simultaneously, T lineage–restricted progenitors were generated at extrathymic sites, both in the spleen and in peripheral lymph nodes, but not in the bone marrow or liver. The generation of these T lineage–committed cells occurred through a Notch-dependent differentiation process. Multipotent bone marrow progenitors efficiently gave rise to extrathymic T lineage–committed cells, whereas common lymphoid progenitors did not. Our data show plasticity of T-lineage commitment sites in the post-BMT environment and indicate that Notch-driven extrathymic Tlineage commitment from multipotent progenitors may contribute to early T-lineage reconstitution after BMT.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.

Lymphocyte development is an excellent model system to study cell fate choices and the underlying transcriptional mechanisms. However, the earliest events have been poorly understood, due in part to the rarity of the relevant cellular intermediates. In this issue of Blood , Mansson and colleagues identify several subsets within the CLP and fraction A population (B220 + CLP phenotype) with differing lineage potentials. 1 The results indicate that B-lineage restriction initiates at much earlier developmental stages than previously thought and provide insight into the hierarchical transcriptional mechanisms involved in B-lineage commitment.

Abstract 3647 Notch1 encodes an essential transmembrane receptor that is frequently mutated in T-ALL. Notch signaling regulates cell fate decisions, proliferation, survival and metabolism by activating transcription; yet the mechanism by which Notch selectively activates different transcriptional targets is poorly understood. After ligand-induced proteolysis, the intracellular part of Notch (ICN) generates signals by entering the nucleus and forming a transcriptional activation complex with the transcription factor CSL and the co-activator Mastermind (MAML). This complex can bind DNA as a monomer [1-3], however it can also dimerize on sites in which a pair of head-to-head CSL binding sites is properly spaced (so-called paired sites) [4, 5]. Whether Notch dimerization is physiologically important is unknown. Using T cell development and transformation as models, we now report that dimeric Notch transcriptional complexes are required for leukemic transformation but dispensable for T cell fate specification from a multipotential precursor. The varying requirements for Notch dimerization within the T cell lineage result from the differential sensitivity of specific Notch target genes. One particularly important dimerization-dependent target of Notch, required for both T cell maturation and leukemogenesis, is the proto-oncogene c-Myc, whereas other well-characterized target genes, such as Hey1, show no dimerization-dependence. These findings identify functionally important differences in the responsiveness among Notch target genes attributable to the formation of higher-order complexes. Our data suggest that it may be possible to develop a new class of Notch inhibitors, which selectively block outcomes that are dependent on Notch dimerization (e.g., leukemogenesis). References: 1. Nam, Y., et al., J Biol Chem, 2003. 278: 21232. 2. Nam, Y., et al., Cell, 2006. 124: 973. 3. Wilson, J.J. and R.A. Kovall, Cell, 2006. 124: 985. 4. Bailey, A.M. and J.W. Posakony, Genes Dev, 1995. 9: 2609. 5. Nam, Y., et al., Proc Natl Acad of Sci USA, 2007. 104: 2103. Disclosures: No relevant conflicts of interest to declare.

Studies on the etiology of MDS have been hampered by the lack of relevant animal model systems. Numerous murine models of MDS/MPD overlap syndromes have been described, but MDS/MPD overlap syndromes are now felt to be clinically distinct from MDS. Likewise, a number of murine models of myeloid leukemia have been developed in which dysplastic features arise during conversion to frank leukemia. Here we describe the hematopoietic phenotype of the PolgD257A mitochondrial mutator mouse and demonstrate that this model system fulfills the myeloid dysplasia criteria outlined in the Bethesda Proposals (Blood, 100, 238, 2002). The PolgD257A allele eliminates the proofreading activity of Polg, the sole mitochondrial DNA (mtDNA) polymerase, and PolgA/A mice rapidly accumulate mtDNA mutations. Starting at 9 months of age, PolgA/A mice display multiple features of accelerated aging and develop a progressive and ultimately lethal anemia (Science, 309, 481, 2005). In data obtained at time of necropsy for other studies, the hemoglobin of young PolgA/A mice is identical to that of controls but by 10 months of age begins to fall below 10 g/dL. Overall, while Polg+/+ mice maintain a hemoglobin of greater than 10 g/dL through 30 months of age, the hemoglobin of PolgA/A drops below 5 g/dL as the mice succumb to their anemia at 13–16 months of age. As the hemoglobin of aging PolgA/A mice falls the MCV demonstrates a striking increase, from 53.6 fL to 69.8 fL. In order to confirm these intial findings, complete blood counts have been assessed prospectively in mice of all three genotypes (Polg+/+, Polg+/A, and PolgA/A; 5 mice per genotype) every other month up to 8 months of age and monthly thereafter. Full data up to 11 months of age, as well as 12 month data from all 5 PolgA/A mice, will be presented. As expected, up to eight months of age no significant difference in hemoglobin levels are apparent, but by eight months PolgA/A mice demonstrate a significant increase in MCV (51.5 fL) compared to either Polg+/A (44.9, p=0.0015) or Polg+/+ (44.5, p=0.0010) littermate controls. The disparity in MCV between PolgA/A mice and the other genotypes is progressive, with the MCV at 9 months increasing to 53.4 fL in PolgA/A mice (N=5), compared to 44.8 (p

Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.

IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

The essential role of Notch for T lineage commitment is well established, but it remains unclear where and on which progenitor subset(s) Notch signals act. Addressing these questions is critical to understand the regulation of lymphopoiesis in normal and lymphopenic settings, such as after bone marrow transplantation (BMT). In normal mice, a small number of progenitors settle in the thymus from the blood and expand to generate a pool of early T lineage progenitors (ETPs). At the population level, ETPs can give rise to T cells, NK cells, B cells, DCs and myeloid cells. We found that ETPs could be subdivided based on expression of the cytokine receptor Flt3, which is expressed on about 10% of ETPs and is inversely correlated with expression of Notch target genes. B lineage potential was restricted to ETPFlt3 positive cells. The Notch targets Hes-1, Hes-5, and Deltex1 were present at low levels in ETPFlt3 positive and high levels in ETPFlt3 negative cells. Induction of Notch signaling resulted in the rapid downregulation of surface Flt3 expression. In contrast, culture of ETPFlt3 negative cells in the absence of Notch ligands resulted in upregulation of Flt3. Although both ETP subsets were efficient T lineage progenitors, ETPFlt3 negative cells had a more rapid differentiation kinetics resembling DN2 thymocytes, consistent with a more advanced state of T lineage commitment relative to the Flt3 positive subset. In mice reconstituted with HSCs transduced with the pan-Notch inhibitor DNMAML1 (Maillard et al., Blood 2004), no ETPFlt3 negative and very few ETPFlt3 positive cells were observed, indicating that the generation of ETPs is Notch-dependent. These observations position the physiological Notch checkpoint either very early after thymic seeding and/or in a prethymic location.To further investigate if Notch-dependent prethymic T lineage commitment occurs in adult mice, we studied lymphoid reconstitution early after BMT. ETPs were absent in the thymus of recipients until ≥ 6 weeks post-BMT, despite the presence of donor-derived pre-T cells and double positive thymocytes as early as 2–3 weeks post-BMT. Instead, cells with pre-T cell characteristics were present in the spleen of BMT recipients in the first month post-BMT. These cells were absent when DNMAML1-transduced bone marrow was used as the source of HSCs, indicating that generation of extrathymic pre-T cells is Notch-dependent. Thus, extrathymic sites may be important for efficient lymphoid reconstitution after BMT. Altogether, our results indicate that both intrathymic and extrathymic Notch-dependent checkpoints regulate T lineage commitment during normal development and in the post-BMT setting.

T cells develop in the thymus, but are ultimately derived from hematopoietic stem cells (HSCs) that reside in the bone marrow. In order to produce T cells throughout adult life, the thymus must be periodically seeded by bone marrow progenitors via the blood. The identity of progenitors that seed the adult thymus is unknown. To determine which bone marrow progenitors that have access to they thymus, we analyzed the blood of adult mice (Schwarz & Bhandoola, Nature Immunology 2004). We found that the only progenitors in blood with T lineage potential were lineage negative cells with high expression of Sca-1 and c-Kit (LSK). Such LSK cells in blood were potent T lineage progenitors, with the capacity to expand over a million fold in the thymus. Like the corresponding population in the bone marrow, the blood LSK population was heterogeneous, containing HSCs and downstream multipotent progenitors (MPPs) including RAG-expressing early lymphoid progenitors (ELPs) and CD62L+ cells. In order to determine which of these LSK subsets can settle in the thymus, we developed a quantitative assay for thymic seeding in normal adult mice. We find that the fraction of LSK cells that settle in the thymus from the blood is extremely small. Of the estimated 3,000 to 4,000 LSK cells that pass through the thymic circulation each day, less than 10 cells are able to settle in the thymus. Our data suggest that any decrease in thymic seeding, as may occur in aging, would lead to a decrease in total thymic output.

We have shown that high IL-7 transgene (Tg) over-expression (39-fold at day 1 in thymic tissue) under the T cell specific, proximal lck promoter had a dose effect on TCRαβ that was accompanied by active B cell development in the thymus. To further characterize these affects in the thymus of IL-7 transgenic mice, we analyzed thymi from day 18 embryos and newborn Tg mice, as well as fetal thymic organ culture (FTOC) derived from using day 16 embryos. We show that arrested T-cell and increased B-cell thymic development is initiated during fetal development. Using mixed bone marrow chimeras and anti-IL-7 monoclonal antibody injection, we further demonstrate that abnormalities in thymic T and B cell development are non-cell autonomous and are due to IL-7 over-expression. Recently, it was shown that only the early thymocyte progenitor (ETP, c-kit+IL-7R−/lo) fraction within the DN1 subpopulation had a T-cell proliferative potential in contrast to the c-kit−IL-7R+ DN1 subset. Here we show that in Tg mice the ETPs were decreased, while the c-kit−IL-7R+ cells are increased in both percentage and absolute count when compared to normal controls. In order to explore the T vs. B ETP potential, we seeded re-aggregate thymic organ cultures with sorted lin−CD44+CD25−c-kit+IL-7R+ cells. While ETPs derived from normal controls were able to proliferate and produce 83% of DP thymocytes, ETPs sorted from Tg mice developed poorly (10-fold less) into DP cells (30%) and produced 14% of B220+ cells vs. 6% in controls. Moreover, sorted Pro/Pre B derived thymic B cells from Tg mice, but not BM-derived Pro/Pre B cells had the TCRβD-J rearrangement, suggesting a T-specific origin. Since the B-cell differentiation pathway in normal mice is selectively inhibited by thymic presentation of Notch ligands, we hypothesized that IL-7 down-regulates Notch signaling. To test this hypothesis, we analyzed thymocyte progenitors (DN1-DN4) in normal and Tg mice for the intra-cytoplasmic part of Notch, that is cleaved upon Notch/Notch-ligand activation. Notch staining was decreased in the lin−CD44+CD25inter representing the only DN2 population present in these Tg mice. These data favour a decrease of Notch signalling in mice with high IL-7 Tg over-expression, inducing a block in TCRαβ development, and skewing of thymic B cell development by T vs. B lineage subversion. These conclusions may have implications for IL-7 in the clinical setting.