Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.
ABSTRACT: Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T(-)B(+)NK(-) SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid development.
Project description:Expression of the transcription factor Zbtb1 is required for normal lymphoid development. We report in the present study that Zbtb1 maintains genome integrity in immune progenitors, without which cells undergo increased DNA damage and p53-mediated apoptosis during replication and differentiation. Increased DNA damage in Zbtb1-mutant (ScanT) progenitors was due to increased sensitivity to replication stress, which was a consequence of inefficient activation of the S-phase checkpoint response. Increased p53-mediated apoptosis affected not only lymphoid but also myeloid development in competitive bone marrow chimeras, and prevention of apoptosis by transgenic Bcl2 expression and p53 deficiency rescued lymphoid as well as myeloid development from Zbtb1-mutant progenitors. Interestingly, however, protection from apoptosis rescued only the early stages of T cell development, and thymocytes remained arrested at the double-negative 3 developmental stage, indicating a strict requirement of Zbtb1 at later T cell developmental stages. Collectively, these results indicate that Zbtb1 prevents DNA damage in replicating immune progenitors, allowing the generation of B cells, T cells, and myeloid cells.
Project description:Specialized niche environments specify and maintain stem and progenitor cells, but little is known about the identities and functional interactions of niche components in vivo. Here, we describe a modular system for the generation of artificial hematopoietic niches in the mouse embryo. A circumscribed tissue that lacks niche function but is physiologically accessible for hematopoietic progenitor cells is functionalized by individual and combinatorial expression of four factors, the chemokines Ccl25 and Cxcl12, the cytokine Scf and the Notch ligand DLL4. The distinct phenotypes and variable numbers of hematopoietic cells in the resulting niches reveal synergistic, context-dependent and hierarchical interactions among niche effector molecules. The surprisingly simple rules determining niche outcomes enable the in vivo engineering of artificial niches conducive to the presence of distinct myeloid or T or B lymphoid lineage precursors. The dataset comprises 24 samples divided into eight sample groups each representing a different lymphoid progenitor cell type isolated from wild-type (+/-) or transgenic (-/-) thymic niches. -/-, Foxn1-deficient genotype; +/-, Foxn1 heterozygous phenotype; DP, CD4/CD8 double-poisztive thymocytes; DN3, CD4/CD8-negative stage 3 thymocytes; SP4, CD4 single-positive thymocytes; SP8, CD8 single-positive thymocytes; B IgM-, IgM surface negative B cells; B IgM+, IgM surface positive B cells; B IgM- -/-, IgM surface negative B cells from Foxn1-deficient genotype.
Project description:Innate lymphoid cells (ILCs) play a central role conferring protection at the mucosal frontier. In this study, we have identified a requirement of the transcription factor Zbtb1 for the development of ROR?t+ ILCs (ILC3s). Zbtb1-deficient mice lacked NKp46+ ILC3 cells in the lamina propria of the small and large intestine. This requirement of Zbtb1 was cell intrinsic, as NKp46+ ILC3s were not generated from Zbtb1-deficient progenitors in bone marrow chimeras and Zbtb1-deficient ROR?t+ CCR6-NKp46- ILC3s didn't generate NKp46+ ILC3s in co-cultures with OP9-DL1 stroma. In correlation with this impairment, Zbtb1-deficient ILC3 cells failed to upregulate T-bet expression, and to acquire IFN-? production characteristic of NKp46+ cells. Finally, absence of NKp46+ILC3 cells combined with the absence of T-cells in Zbtb1-deficient mice, led to a transient susceptibility to C. rodentium infections. Altogether, these results establish that Zbtb1 is essential for the development of NKp46+ ILC3 cells.
Project description:Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3(-/-) stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3(-/-) stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3(-/-) stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.
Project description:<h4>Background</h4>The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRalphabeta lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRalphabeta prematurely at the double negative stage and abnormal TCRalphabeta populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice.<h4>Methodology and principal findings</h4>To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRalphabeta diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORgammat-positive CD4(+)8(+) (double positive, DP) stage to accumulate either as CD4(-)8(-) (double negative, DN) or as CD8alpha(+) T cells in lymph nodes or gut epithelium. Likewise, DN TCRalphabeta cells in lymphoid tissue of female mice were not derived from DP thymocytes.<h4>Conclusion</h4>The results further support the hypothesis that the premature expression of the TCRalphabeta can divert DN thymocytes into gamma delta lineage cells.
Project description:Studies of adult human hematopoiesis have until now relied on the expression of CD10 to define lymphoid commitment. We report a novel lymphoid-primed population in human bone marrow that is generated from hematopoietic stem cells (HSC) prior to the onset of CD10 expression and B cell commitment, and is identified by high levels of the homing molecule L-selectin (CD62L). CD10-CD62Lhi progenitors have full lymphoid (B/T/NK) potential, and show reduced myeloid and absent erythroid potential. Genome-wide gene expression analysis demonstrates that the CD10-CD62Lhi population represents an intermediate stage of differentiation between CD34+CD38- HSC and CD34+lin-CD10+ progenitors marked by down-regulation of TAL1 and MPL, upregulation of E2A, CD3E and IL2RG expression, and absent B cell commitment or RAG1/2 expression. Immature CD34+CD1a- thymocytes are also CD62Lhi and L-selectin ligands are expressed at the cortico-medullary junction, suggesting a possible role for L-selectin in human thymic homing. These studies identify the earliest stage of lymphoid priming in human bone marrow. Freshly harvested human bone marrow was ficoll purified, enriched for CD34+, and then FACS sorted. It was then sorted into 3 samples CD34+CD38-, CD34+ CD10- CD62L++, and CD34+ CD10+, with 3 independent biological replicates
Project description:An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69(+) thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1 (-/-) genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1 (flox/flox) mice crossed to pLck-Cre, pKeratin14-Cre, or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO), and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components.
Project description: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.
Project description:Mitochondrial metabolism is a tightly regulated process that plays a central role throughout the lifespan of hematopoietic cells. Herein, we analyze the consequences of the mitochondrial oxidative phosphorylation (OXPHOS)/metabolism disorder associated with the cell-specific hematopoietic ablation of apoptosis-inducing factor (AIF). AIF-null (AIF-/Y ) mice developed pancytopenia that was associated with hypocellular bone marrow (BM) and thymus atrophy. Although myeloid cells were relatively spared, the B-cell and erythroid lineages were altered with increased frequencies of precursor B cells, pro-erythroblasts I, and basophilic erythroblasts II. T-cell populations were dramatically reduced with a thymopoiesis blockade at a double negative (DN) immature state, with DN1 accumulation and delayed DN2/DN3 and DN3/DN4 transitions. In BM cells, the OXPHOS/metabolism dysfunction provoked by the loss of AIF was counterbalanced by the augmentation of the mitochondrial biogenesis and a shift towards anaerobic glycolysis. Nevertheless, in a caspase-independent process, the resulting excess of reactive oxygen species compromised the viability of the hematopoietic stem cells (HSC) and progenitors. This led to the progressive exhaustion of the HSC pool, a reduced capacity of the BM progenitors to differentiate into colonies in methylcellulose assays, and the absence of cell-autonomous HSC repopulating potential in vivo. In contrast to BM cells, AIF-/Y thymocytes compensated for the OXPHOS breakdown by enhancing fatty acid ?-oxidation. By over-expressing CPT1, ACADL and PDK4, three key enzymes facilitating fatty acid ?-oxidation (e.g., palmitic acid assimilation), the AIF-/Y thymocytes retrieved the ATP levels of the AIF +/Y cells. As a consequence, it was possible to significantly reestablish AIF-/Y thymopoiesis in vivo by feeding the animals with a high-fat diet complemented with an antioxidant. Overall, our data reveal that the mitochondrial signals regulated by AIF are critical to hematopoietic decision-making. Emerging as a link between mitochondrial metabolism and hematopoietic cell fate, AIF-mediated OXPHOS regulation represents a target for the development of new immunomodulatory therapeutics.
Project description:We previously reported that deletion of Foxo1, via Ncr1-iCre mice from the expression of NKp46 onward, led to enhanced natural killer (NK) cell maturation and effector function. In this model, however, the role of Foxo1 in regulating NK cell specification and early development remains exclusive. Herein, we utilized a murine model of hematopoietic-specific deletion of Foxo1 before lymphoid specification, by crossing mice carrying floxed Foxo1 alleles (Foxo1 fl/fl) with Vav1-iCre mice, to revisit the role of Foxo1 on NK cell specification and early development. The data showed that hematopoietic-specific deletion of Foxo1 resulted in increased proportion and numbers of common lymphoid progenitors (CLP) (Lin-CD127+c-Kit+Sca-1+), pre-pro NK b cells (Lin-Sca-1+c-Kit-CD135-CD127+), as well as committed Lin-CD122+ cells and CD3-CD19-NKp46+ NK cells in bone marrow. Hematopoietic-specific deletion of Foxo1 also promoted NK cells proliferation in a cell-intrinsic manner, indicated by increased Ki-67 expression and more expansion of NK cell after ex vivo stimulation with IL-15. The reason for Foxo1 suppressing NK cell proliferation might be its direct transcription of the cell-cycle inhibitory genes, such as p21 cip1, p27 kip1, p130, Gadd45a, and Ccng2 (cyclin G2) in NK cells, supported by the evidence of decreased mRNA expression of p21 cip1, p27 kip1, p130, Gadd45a, and Ccng2 in Foxo1-deficient NK cells and direct binding of Foxo1 on their promoter region. Furthermore, hematopoietic-specific deletion of Foxo1 resulted in increased ratio of mature NK subsets, such as CD11b+CD27- and CD43+KLRG1+ NK cells, but decreased ratio of immature NK subsets, such as CD27+CD11b- and CD27+CD11b+ NK cells, consistent with the findings in the murine model of Ncr1-iCre mediated Foxo1 deletion. Conclusively, Foxo1 not only acts as a negative checkpoint on NK cell maturation, but also represses NK cell specification and proliferation. The relative higher expression of Foxo1 in CLP and early NK precursors may also contribute to the later NK cell proliferation and responsiveness, which warranties another separate study in the future.