Early Lymphoid Cells Research Articles

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

SummaryIt is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

Benign TdT-positive cells in pediatric and adult lymph nodes: a potential diagnostic pitfall

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.

Ikaros establishes an epigenetic program required for proper two-signal activation of naive B cells

Abstract While there are many different types of autoimmune diseases, a common feature of chronic inflammatory diseases is the production of autoantibodies by B-cells. We and others have shown in mouse models that loss-of-function mutations in the transcription factor Ikaros (Ik) result in a hyper-reactive (HR) B-cell phenotype. In particular, naïve B-cells containing a targeted deletion in the Ikzf1 zinc finger domain (Ikzf1ΔF4/ΔF4) produce large quantities of autoantibodies and proliferate in the absence of a costimulatory signal. Ik is required for early lymphoid cell development and has been shown to regulate gene expression directly or through the recruitment of chromatin remodeling complexes. However, the epigenetic and transcriptional regulation by Ik in mature B-cells and its role in B-cell HR remain unknown. We hypothesized that Ik functions to establish and maintain an epigenetic program required for proper two-signal activation in naïve B-cells. We have generated whole transcriptome profiles of wildtype and Ikzf1ΔF4/ΔF4 cells and defined the gene expression program required for B-cell activation. A comparison of these profiles revealed distinct deficiencies in genes associated with the HR phenotype. Specifically, a variety of cell signaling pathways are derepressed in Ik-mutant B-cells. In addition, we have performed Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), over the course of B-cell activation, to explore the altered chromatin structure in Ik-mutant B-cells. Together these results revealed transcriptional pathways required for proper B-cell activation and demonstrate how mutations in Ik contribute to B-cell HR.

The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development

Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early Tcell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish Tcell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated Tcell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, Tcell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate.

Targeted deletion of the Hoxa cluster affects B lymphopoiesis through depletion of early lymphoid progenitors.

It is well established that Hoxa genes play a critical role in the proliferative capacity of adult hematopoietic stem and progenitor cells, but the importance of Hoxa genes in later stages of hematopoietic differentiation is less clear. Previously, we observed that B-cell numbers were reduced in adult mice in which Hoxa deletion was induced. In the current study, we investigated the requirement of Hoxa genes at different stages of B-cell development. Using an MxCre-inducible conditional knock-out mouse model, we showed that immature B-cell fractions and early lymphoid progenitors were markedly reduced in the absence of Hoxa, whereas mature B-cell populations were found at levels comparable to controls. Deletion of Hoxa genes in B-cell lineage-committed cells, however, did not affect B-cell development despite sustained Hoxa gene expression in immature CD19+ B-cell subsets. Together, these results suggest that the effect of Hoxa on B-cell development originates in early lymphoid progenitor cells.

A Transient Developmental Hematopoietic Stem Cell Gives Rise to Innate-like B and T Cells

The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage-tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients but does not persist into adulthood insitu. These HSCs are fully multipotent, yet they display both higher lymphoid cell production and greater capacity to generate innate-like B and Tlymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs (drHSCs) define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance,with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.

The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model

Abstract Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

Blinatumomab for the treatment of acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a potentially fatal disease that involves clonal expansion of early lymphoid progenitor cells. Much of drug development for ALL treatment involves targeting antigens of the clonal cell surface. Blinatumomab belongs to an emerging class of anti-cancer therapeutics referred to as bispecific T-cell engaging antibodies. The Food and Drug Administration approved its use in relapsed or refractory adult Philadelphia chromosome-negative B-cell precursor ALL in December of 2014. Blinatumomab contains both an anti-CD3 and anti-CD19 arm, allowing for the juxtaposition of CD3+ T-cells to malignant CD19+ B-cells, thereby resulting in granzyme- and perforin-mediated B-cell apoptosis. Preclinical studies suggest that blinatumomab's efficacy is related to the effector-to-target ratio and to the difference between its affinity for CD19 and CD3. Preclinical and early phase clinical studies have allowed for the characterization of the pharmacokinetics of blinatumomab, including the determination of its short half-life. The metabolic pathway has not been fully characterized but is thought to be similar to that of other antibodies. Phase I and II studies led to the identification of an ideal stepwise dose, involving long-term continuous intravenous infusion (CIVI), to optimize its efficacy and reduce the risk of certain toxicities. A high remission rate and duration were noted among a relapsed/refractory population of patients. The results of clinical trials have identified cytokine release syndrome and neurotoxicity, among others, as serious drug-related toxicities, leading to the institution of a Risk Evaluation and Mitigation Strategy. Blinatumomab represents a significant addition to the treatment options for ALL, but it is not without its limitations, of which are its short-half life, necessitating long-term CIVI, and the eventual emergence of CD19-negative clones. Continual development of the agent involves assessing its role in the frontline setting and in combination with chemotherapy.

Abstract A34: Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL).

Abstract B-precursor acute lymphoblastic leukemia (B-pre ALL) is characterized by malignant proliferation and accumulation of early lymphoid precursor cells in the bone marrow, blood and lymphoid organs, due to acquired mutations in early B-cells. Around 20% of children and the majority of adults relapses after the treatment and the poor outcome following relapse did not significantly changed over the past twenty years. For this reason, many research efforts are currently devoted to the development of targeted therapies to limit side effects of chemotherapy and to increase treatment efficacy for poor prognosis patients. Constitutively active phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is a common feature of leukemia patients, where it negatively influences response to therapeutic treatments. Thus, targeting the PI3K/AKT /mTOR pathway is an attractive therapeutic strategy. Rapamycin, an allosteric inhibitor of mTORC1, acutely inhibits mTORC1 but not mTORC2 activity. Prolonged suppression of mTORC1 also results in disruption of a negative feedback loop and consequently results in hyperphosphorylation of Akt through activation of IRS1 and PI3K. mTOR inhibitors are currently in clinical trial for a range of hematological malignancies including ALL, mostly in combination with conventional chemotherapy. RAD001, a mTORC1 inhibitor, has been shown to affect cell cycle progression and survival with a potentially relevant therapeutic efficacy. However, prolonged inhibition of mTORC1 often leads to AKT re-phosphorylation at Ser473which may counteract RAD001 activity. A new class of ATP competitive mTOR inhibitors, such as Torin-2, have revealed that this new class of inhibitors potently targets mTORC1 and mTORC2. Together, these features have generated hope that the new generation of ATP-competitive mTOR inhibitors will exhibit broader clinical efficacy relative to the rapalogs. We therefore hypothesized that dual inhibition of mTORC1 and mTORC2 by Torin-2 would provide a superior outcome in ALL as compared to inhibition of mTORC1 alone with RAD001 in B-pre-ALL. We tested the capability of Torin-2 to prevent AKT reactivation after mTORC1 and mTORC2 inhibition. Furthermore we explored if dual targeting of mTORC1 and AKT may achieve results similar to those obtained with Torin-2 alone. We used a panel of B-pre-ALL to test the drugs by MTT Assay and Western Blotting after different time exposures of the cells. Cell cycle, apoptosis and autophagy were analyzed by flow cytometry, Western Blotting and fluorescent staining. In all the B-pre-ALL cell lines Torin-2 showed a powerful cytotoxic activity, inhibiting the growth of each cell line in a dose-dependent manner, with an IC50 in the nanomolar range as assessed by MTT assays. The major proteins along the PI3K/AKT/mTOR signaling pathway where heavily dephosphorylated after 2 hrs of drug exposure. This inhibition lasted up to at least 48 hrs at variance to RAD001, that already after 24 hrs was unable to prevent AKT reactivation. However the association of RAD001 with MK-2206, an allosteric AKT inhibitor, prevented AKT reactivation and reached a significant cytotoxicity. Our data suggest an interesting cytotoxicity of Torin-2 in B-pre-ALL acting on both mTORC1 and mTORC2 as assessed by their substrate inhibition. Torin-2 alone suppresses feedback activation of PI3K/AKT, whereas RAD001 requires the addition of MK-2206 to achieve the same efficacy. These two pharmacological options targeting PI3K/Akt/mTOR at different points of the signaling pathway cascade might represent a new therapeutic potential for treatment of B-pre-ALL patients. Citation Format: Alice Cani, Carolina Simioni, Alberto M. Martelli, Giorgio Zauli, Silvano Capitani, Luca M. Neri. Therapeutic potential of the novel mTOR inhibitor Torin-2 to overcome AKT reactivation in B-precursor acute lymphoblastic leukemia (B-pre ALL). [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A34.

Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. DisclosuresNo relevant conflicts of interest to declare.

Adult NK lineage development in humans is strengthened upon emergency

Emergency conditions such as infection demand rapid production and activation of innate immune cells to combat noxious extrinsic agents. In mice, pathogen recognition through Toll like receptors (TLR) and the resulting pro-inflammatory cytokine release induce the expansion of bone marrow hematopoietic stem cells and instruct early differentiation fates so immediate innate cell development is guaranteed. Accordingly, recent work suggests that human primitive cell populations are also capable of microbial components discrimination through TLR, a mechanism that facilitates their differentiation to myeloid lineage cells. We have now found that early lymphoid progenitor cells from adult bone marrow display TLR9 and its ligation promotes the quick development of NK lineage cells by using mechanisms that involve IL-15R up-regulation. Strikingly, this phenomenon is not obvious in their neonatal counterparts, suggesting that contribution of neonatal seminal cells to the emergent cell production in response to viral signals may differ from that of adult cells.

Clonal allelic predetermination of immunoglobulin-κ rearrangement

Although most genes are expressed biallelically, a number of key genomic sites--including immune and olfactory receptor regions--are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

AbstractAbstract 1449Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Functional Analysis of Pegasus: The ‘Atypical' Member of the Ikaros Gene Family.

Abstract 3645Poster Board III-581The Ikaros family of zinc finger transcription factors are an important class of hematopoietic regulators involved in the development of the immune system. However, the in vivo role of its most divergent ‘atypical' member, Pegasus, remains unknown. Therefore, we have utilised zebrafish to explore the function of Pegasus. Bioinformatics analysis identified a single pegasus orthologue in zebrafish. Wholemount in situ hybridisation revealed an initial broad distribution of maternally-derived pegasus transcripts during early zebrafish embryogenesis, which was replaced by specific expression in the brain, eye, thymus, gut and pancreas during late embryogenesis. Morpholino-mediated gene knockdown of zebrafish pegasus resulted in a reduction in cells expressing markers for hematopoietic stem cells (lmo2), granulocytes (mmp9), macrophages (fms), as well as early (ikaros) and late (rag1) lymphoid cells while, conversely, those expressing early (spi1) and late (l-plastin) myeloid markers were increased. No difference was noted in the early erythroid (gata1) compartment, although mature red blood cells appeared reduced, as judged by o-dianisidine staining. During emergency hematopoiesis (elicited by LPS injection), numbers of granulocytes (mpo+) and macrophages (lyz+) were both significantly further reduced. In addition, morphants also showed anterior defects, including reduced forebrain, smaller eyes and otoliths, which corresponded with a reduction of expression of the brain markers pax2.1, krox20 and dlx1. Gut expression of ifabp also appeared affected. Collectively, these experiments suggest a clear role for pegasus in hematopoiesis, as well as novel functions in brain and gut development. Disclosures:No relevant conflicts of interest to declare.

The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

Characterization of Early Human B Cell Development by Gene Expression Profiling.

B cells develop from hematopoietic stem cells (HSC) in the bone marrow (BM) through a number of distinct stages before they migrate to the periphery as naïve mature B lymphocytes. These developmental stages can be identified by expression of cell surface antigens and Ig gene rearrangement status. The aim of this study was to characterize the earliest steps of normal human B cell development by gene expression profiling. Immunomagnetic selection and subsequent fluorescence-activated cell sorting (FACS) were used to isolate five populations from adult human BM: CD34+CD38− (HSC), CD34+CD10+CD19− early lymphoid progenitor cells (ELP), CD34+CD10+CD19+IgM− progenitor B cells (pro-B), CD34−CD10+CD19+IgM− precursor B cells (pre-B) and CD34−CD10+CD19+IgM+ immature B cells (IM). Total RNA was extracted from the purified cell populations, amplified and hybridized to Lymphochip cDNA microarrays. Six independent experiments from different donors were performed for each cell population. Expression of the genes encoding the selection markers confirmed the validity of the approach. Interestingly, genes necessary for the V(D)J-recombination such as RAG-1, RAG-2, TdT and ADA showed higher gene expression in the ELP population than in the HSC. In contrast, the transcription factors E2A, EBF, and Pax-5, which are all essential for early B-cell development, were first turned on in pro-B cells, in accordance with the B-cell lineage commitment. The ELP did not express B, T or NK lineage markers, except for a higher expression level of CD2 in the ELP population than in the four other cell populations. Taken together, the expression pattern of CD2 and the V(D)J-recombination genes in the ELP population, indicate that these cells have developed a lymphocyte potential, but are not fully committed to B-lineage cells. Hierarchical cluster analysis of the 758 differentially expressed genes (differences in relative expression by a factor of two or more and with maximum10% FDR) revealed a pattern that clearly separated the five consecutive cell populations. Furthermore, we created expression signatures based on information from Gene Ontology (GO) http://source.stanford.edu/cgi-bin/source/sourceSearch. One of the clearest distinctions between the gene expressions of the five developmental populations involved genes associated with proliferation, and showed that the HSC and IM populations are relatively indolent while the pro-B and pre-B populations comprised high expression levels of nearly all the proliferation associated genes. Finally, we examined in further detail the transitions between HSC, ELP and pro B cells. We found 25 genes to be differently expressed in the ELP population in comparison to the HSC and pro-B populations, including IGJ, BCL2 and BLNK. To identify combinations of markers that could better discriminate the ELP population, we also performed a gene pair class separation test. This resulted in 68 gene pairs with score above 10 that were denoted very good discriminators. For several of the markers the differences in gene expression were verified at the protein level by five colour FACS analysis. Taken together, these results provide new insight into the molecular processes that take place in the early human B cell differentiation, and in particular provide new information regarding expression of genes in the ELP population.

The Heat Is on

It is common knowledge that atherosclerosis is a multifactorial disease, and the disease-promoting role of classic risk factors for atherosclerosis, such as high serum cholesterol levels, diabetes, hypertension, smoking, and various types of infections, has been proven in innumerable clinical studies. Despite the fact that we are dealing with a chronic disease, however, there is a surprising scarcity of reports dealing with the very first stages of atherogenesis, ie, those that are not yet leading to clinical symptoms. It is now evident that inflammatory/immunologic processes play a major role in the development of atherosclerosis. The correlation between the presence and amount of inflammatory surrogate markers and the occurrence of sonographically demonstrable early arterial changes becomes more significant the earlier in the course of the disease that these analyses are performed. Although complicated atherosclerotic lesions, as the name implies, are composed of many different cellular and extracellular constituents, the earliest lesions are characterized by intimal infiltration by mononuclear cells often still without the presence of foam cells, especially in those instances in which risk factors other than high serum cholesterol levels are operative. In this context, it is important to remember that the first intima-infiltrating cells in early, clinically nonapparent lesions are lymphoid cells, followed by blood-borne monocytes, and finally smooth muscle cells immigrating from the media.1 Later, both the scavenger receptor–expressing macrophages and smooth muscle cells may transform into foam cells accompanied by fibroblast and myofibroblast proliferation with collagenous and noncollagenous extracellular matrix protein deposition leading to the paradigmatic hardening of the arterial wall. The antigenic specificity of early intima-infiltrating lymphoid cells is still to be determined. Article p 886 In this issue of Circulation , Park et al2 address the possible role of heat shock protein (HSP) 27 in atherogenesis. HSPs have risen to prominence in atherosclerosis …

A Lymphoid Progenitor Propagates AML in a Mouse Model of CALM/AF10 Positive Leukemia.

The identification of cancer stem cells is a major step towards the understanding of the pathogenesis of solid and hematological neoplasias and might have direct implications for the development of innovative therapeutic strategies aiming at the eradication of the tumor propagating cell. Here we describe that acute myeloid leukemia (AML), induced by the CALM/AF10 fusion gene, is propagated by a transformed lymphoid progenitor in a murine bone marrow (BM) transplantation model of t(10;11)(p13;q14) positive AML. When mice were transplanted with BM cells retrovirally engineered to express the C/A fusion, all animals (n=13) died from AML showing DJ rearrangement of the heavy chain of the IgH locus after a median of 110 days post transplantation. Diseased mice showed an accumulation of myeloid Gr1+/Mac1+ cells in the peripheral blood and spleen and a multi-organ infiltration by myeloperoxidase and chloracetate esterase positive cells in immunohistochemical sections. In the leukemic mice only a minor population counting for 6.7 % (± 2.1) cells in the BM displayed the B220 lymphoid antigen and lacked myeloid markers (on average 9.4 % ± 3). The majority of cells expressed myeloid markers (on average 82.9 % (± 8.6) Mac1+ cells, 86.4 % (± 3.7) Gr-1+ cells). Additionally, in the leukemic mice an average of 26.0 % (± 8.6) and 32.5 % (± 13.2) of these cells co-expressed B220 and Mac1 or B220 and Gr1, respectively, compared to 2.1 % (± 0.7) and 1.3 % (± 0.3), respectively, in GFP controls. Importantly, in vitro only the B220+/Mac− cell population had growth potential at the single cell level (seeding efficiency 29 %) compared to the B220+/Mac+ (1%) and B220−/Mac+ cells (1%). When the frequency of leukemia propagating cells (LPC) of the three different populations isolated from primary leukemic mice was determined by limiting dilution transplantation and Poisson statistics the frequency of the LPC was more than 380 fold higher in the ‘B220+/Mac1−' population (1 in 36 cells) than in the ‘Mac1+/B220−' bulk population (1 in 13906 cells) and more than 12fold increased compared to the B220+/Mac+ cells (1 in 437 cells). In vitro a single B220+/Mac1− cell isolated from a leukemic mouse was able to give rise to the B220+/Mac1+ as well as the Mac1+/ B220− population, both populations showing the identical genomic DJ rearrangement at the IgH locus as the initial B220+/Mac1− cell, demonstrating its capacity to differentiate into the myeloid lineage at the single cell level. The B220+/Mac1− population displayed a CD43+/AA4.1+/HSA+/CD19−/IL-7R− phenotype, was promiscuous in its transcription profile with positivity for EBF, but also MPO and lacked Pax5. Taken together, this murine leukemia model indicates that AML can be propagated from an early transformed lymphoid progenitor cell. The transformation of an early lymphoid cell, which is re-directed into the myeloid lineage by appropriate oncogenes, could explain recurrent observations of immunoglobulin rearrangements in patients with AML and provide a rationale for therapies, aiming at the elimination of the leukemia propagating cell with lymphoid characteristics, but sparing normal HSCs.

Cell surface expression of a peptide encoded by the unrearranged TCR-Vβ8.2 gene

Germline transcription of T-cell receptor ( TCR) genes has been described in early lymphoid cells. The most common explanation for this phenomenon is that transcription of unrearranged Vβ genes directs gene usage during the rearrangement event. Germline transcription of the TCR-Vβ8.2 gene has been detected in a precursor T-cell line, C1-V13D, which shows no rearrangement at any of the TCR gene loci. This cell line also shows weak binding of specific anti-Vβ8.2 antibody to the cell surface, consistent with expression of a truncated TCRβ chain. RT-PCR has been used to confirm expression of spliced germline transcripts of TCR-Vβ8.2 in C1-V13D initiated from both leader ( L) 5.1 and L8.2. Transcripts initiated from L8.2 were also detectable in unspliced form. In order to test expression and subcellular localisation of any encoded peptides, amplified germline transcripts in both spliced and unspliced form were cloned into the pEGFP-N1 fusion vector for stable transfection and overexpression in C1-V13D. Cell surface expression of a fusion protein between EGFP and a Vβ peptide has been confirmed in C1-V13D but not in control COS-7 cells. Results presented here raise the possibility of a new pre-TCR structure specific to early lymphoid cells and based on TCR-Vβ8.2 gene expression.