BCR signaling plays a critical role in purging the self-reactive repertoire, or in rendering it anergic to establish self-tolerance in the periphery. Differences in self-reactivity between human ...naive and IgM(+) memory B cells may reflect distinct mechanisms by which BCR signaling dictates their survival and death. Here we demonstrate that BCR stimulation protected naive B cells from apoptosis with induction of prosurvival Bcl-2 family proteins, Bcl-x(L) and Mcl-1, whereas it rather accelerated apoptosis of IgM(+) memory B cells by inducing proapoptotic BH3-only protein Bim. We found that BCR-mediated PI3K activation induced the expression of Mcl-1, whereas it inhibited Bim expression in B cells. Phosphorylation of Akt, a downstream molecule of PI3K, was more sustained in naive than IgM(+) memory B cells. Abundant expression of T cell leukemia/lymphoma 1 (Tcl1), an Akt coactivator, was found in naive B cells, and enforced expression of Tcl1 induced a high level of Mcl-1 expression, resulting in prolonged B cell survival. In contrast, Galectin-1 (Gal-1) was abundantly expressed in IgM(+) memory B cells, and inhibited Akt phosphorylation, leading to Bim up-regulation. Enforced expression of Gal-1 induced accelerated apoptosis in B cells. These results suggest that a unique set of molecules, Tcl1 and Gal-1, defines distinct BCR signaling cascades, dictating survival and death of human naive and IgM(+) memory B cells.
Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα ...coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.
•C/EBPα is needed for transition from stem/progenitor cells to common dendritic cell progenitors.•C/EBPα is dispensable in later stages of dendritic cell maturation.
Abstract 768
In murine hematopoiesis, hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) have been identified within the LSK (Lin− Sca-1+ c-kit+) fraction of bone marrow cells ...(Morrison SJ and Weissman IL Immunity 1994). We have proposed that the first commitment step at the myeloid versus lymphoid bifurcation occurred outside the LSK fraction, where myeloid or lymphoid lineage-committed progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) are prospectively isolated (Akashi et al Nature 2000). However, recent studies revealed that the initial commitment step might occur within LSK fraction. Adolfsson et al showed that a fraction of LSK cells expressing Flt3 at a high level have lost megakaryocyte/erythroid (MegE) potential and were largely primed for granulocyte/monocyte (GM) and lymphoid cells. This population was named lymphoid-primed multipotent progenitors (LMPPs) (Adolfsson et al Cell 2005). In our hands, by utilizing mice harboring a fluorescent reporter for GATA-1 transcription factor, we demonstrated that the initial upregulation of GATA-1 transcription factor was observed within MPP population, which was defined as CD34+ LSK fraction, and GATA-1+ MPPs were capable of generating only myelo-erythroid cells but lacked lymphoid potential (Arinobu et al Cell Stem Cell 2007). This result strongly suggested that the initial myelo-erythroid commitment occurs at the MPP stage.
To isolate the earliest myelo-erythroid LSK progenitors in normal mice without utilizing the GATA-1 reporter system, we conducted expression profiling of GATA-1+ MPP population by cDNA microarray analyses and following validation by FACS analyses. We found that a cell-surface antigen CD41 was specifically expressed at a high level in GATA-1+ MPP cells, indicating that CD41hi MPP cells might be the corresponding population to GATA-1+ MPP cells.
CD41hi MPPs gave rise exclusively to GM and MegE colonies in vitro, but no lymphoid colonies were observed even under lymphoid-inducible conditions such as co-culture with OP9 or OP9-DL1 stromal cell layer. In vivo, CD41hi MPPs showed differentiation potential into GM cells, erythroid cells and platelets, lacking B or T lymphoid cell-producing potential in congenic transplantation assay. While the GM cell-reconstitution potential of LMPPs and CMPs peaked around day10 and day4 after transplantation, respectively, CD41hi MPPs had their peak around 3 weeks and relatively long-lasting reconstitution potential, presumably reflecting their immaturity. To further evaluate differentiation potential more directly and quantitatively, CD41hi MPPs with LMPPs or CMPs were injected together into one individual recipient mouse. In this head-to-head competitive assay system, CD41hi MPPs generated a plenty of mature GM cells, whose numbers were nearly ten times of those produced from the original CMPs or LMPPs in a reconstitution setting. Thus CD41hi MPPs possess potent restricted-lineage differentiation potential into GM and MegE lineages.
In addition, the gene expression analysis by single-cell quantitative real-time PCR and cDNA microarray revealed that the CD41hi MPP population expresses both GM- and MegE-affiliated genes, but not lymphoid genes, reflecting their lineage restriction. More importantly, each single CD41hi MPP cell expresses either one or both of GATA-1 and PU.1 at a low level, suggesting that the promiscuous expression of these transcription factors might play a critical role in myelo-erythroid lineage commitment (Miyamoto et al Developmental Cell 2002).
Furthermore, to evaluate the functional importance of CD41hi MPP population, we created the systemic infectious model mice, mimicking the severe peritonitis. CD41hi MPP pool as well as GMP expands dramatically in response to an increased demand for GM cells, while LMPP does not; thus, CD41hi MPPs compose physiologically important pathway in myelo-erythropoiesis.
Accordingly, CD41hi MPP population might represent the earliest branch point for myelo-erythroid development, which resides upstream of the conventional CMP. Based on these data, we propose to redefine the true CMP as CD41hi MPP population. This CD41hi earliest myelo-erythroid progenitor should be useful to investigate the molecular mechanisms for hematopoietic lineage fate decision.
No relevant conflicts of interest to declare.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is caused by natural killer (NK) cells upon recognition of antigen-bound IgG
via
FcγRIIIa. This mechanism is crucial for cytolysis of ...pathogen-infected cells and monoclonal antibody (mAb)-mediated elimination of cancer cells. However, there is concern that mAb-based cancer therapy induces ADCC against non-target cells expressing antigens. To date, no strategy has been reported to enhance the selectivity of ADCC to protect non-target cells expressing antigens. Here, we introduce a model inhibitor which specifically blocks ADCC of anti-EGFR mAbs towards EGFR/folate receptor α (FRα) double positive cells. This inhibitor recruits mAbs on the FRα of the cell surface independent of Fab antigen recognition. The resulting ternary and/or quaternary complexes formed on the cell surface suppress signal transduction of FcγRIIIa in NK cells, consequently leading to more specific ADCC.
ADCC is caused by NK cells upon recognition of antigen-bound IgG
via
FcγRIIIa.
ADCC is caused by NK cells upon recognition of antigen-bound IgG
via
FcγRIIIa.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is caused by natural killer (NK) cells upon recognition of ...antigen-bound IgG
via
FcγRIIIa. This mechanism is crucial for cytolysis of pathogen-infected cells and monoclonal antibody (mAb)-mediated elimination of cancer cells. However, there is concern that mAb-based cancer therapy induces ADCC against non-target cells expressing antigens. To date, no strategy has been reported to enhance the selectivity of ADCC to protect non-target cells expressing antigens. Here, we introduce a model inhibitor which specifically blocks ADCC of anti-EGFR mAbs towards EGFR/folate receptor α (FRα) double positive cells. This inhibitor recruits mAbs on the FRα of the cell surface independent of Fab antigen recognition. The resulting ternary and/or quaternary complexes formed on the cell surface suppress signal transduction of FcγRIIIa in NK cells, consequently leading to more specific ADCC.
During mammalian embryogenesis, hematopoietic stem and progenitor cells (HSPCs) originate from mesoderm-derived endothelial cells in the aorta-gonad-mesonephros (AGM) region and placenta (PL). Later, ...HSPCs expand in fetal liver (FL) and migrate to bone marrow (BM) shortly before birth. Understanding global transcriptional regulation governing HSPC emergence from embryonic stem/induced pluripotent stem cells is necessary to devise clinical applications, such as novel transplantation approaches. In this study, to assess transcriptional dynamics during development, we performed cap analysis of gene expression on 10 developmental murine HSPC populations isolated from the AGM region, PL, FL, and BM and identified 15,681 transcripts across HSPC ontogeny. We performed microarray analysis of AGM-derived HSPCs at 9.5 and 10.5 days postcoitum (dpc) and identified 40 differentially expressed genes, 23 confirmed as significantly changed by real-time polymerase chain reaction. We conclude that a transcriptional switch point occurs in HSPC ontogeny between 9.5 and 10.5 dpc in the AGM region.
Mechanisms underlying differentiation of embryonic hematopoietic stem/progenitor cells (HSPCs) remain unclear. In mouse, intra-aortic clusters (IACs) form in the aorta-gonad-mesonephros region and ...acquire HSPC potential after 9.5 days postcoitum (dpc). In this study we demonstrate that Twist1 is highly expressed in c-Kit+CD31+CD34+ IACs, which are equivalent to embryonic HSPCs, compared with adult HSPCs. Progenitor activities of colony-forming unit (CFU) of granulocytes and macrophages, CFU of macrophages, burst-forming unit of erythroid, and B lymphopoiesis were impaired in IACs of Twist1−/− relative to wild-type embryos. Microarray analysis and real-time polymerase chain reaction showed downregulated expression of Myb and Gata2 transcripts in Twist1−/− IACs. Chromatin immunoprecipitation and promoter binding assays indicated that Twist1 directly binds the Myb and Gata2 promoters in 10.5-dpc IACs. We conclude that Twist1 is a novel transcriptional regulator of HSPC differentiation through direct binding to promoter regions of key regulators of the process.
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•Twist1, a hematopoietic transcription factor, is highly expressed in embryonic HSPCs.•Twist1 functions in embryonic HSPC differentiation through binding to Myb and Gata2 promoter regions and activates their transcription.
In the chronic phase of chronic myeloid leukemia (CML-CP), leukemic stem cells do not necessarily depend on the BCR-ABL tyrosine kinase activity for their growth and survival and thus resistant to ...tyrosine kinase inhibitors (TKIs). In this study, we aimed to identify the initial progenitor population that is getting switched on BCR-ABL growth signaling and tried to elucidate the underlying molecular mechanisms of BCR-ABL dependent cell growth. We thus intensively analyzed the involvement status of CML clones in each developmental stage at diagnosis. To identify the hematopoietic stem or progenitor cell stage that is responsible for CML clone expansion, bone marrow cells from 13 newly-diagnosed CML-CP patients were analyzed by FACS, and purified stem/progenitor populations were tested for the t(9;22) involvement by FISH. Gene expression signature of each purified population was also evaluated by cDNA microarray.
CD34+CD38- HSC fraction was markedly diminished in all CML-CP patients compared to healthy volunteers (<1% of CD34+ cells in patients vs. ∼10% in volunteers), whereas CD34+CD38+ myeloid progenitors expanded. Interestingly, the t(9;22) positive ratios in the HSC fraction were greatly diversified among patients (Figure 1). Of note, in 4 patients, the involvement of t(9;22) positive clone was less than 10%, suggesting that their leukemic stem cells have not outgrown normal HSCs. Among CD34+CD38+ myeloid progenitors, common myeloid progenitors (CMPs) robustly expanded and were composed more than 90% of t(9;22) positive clone in all cases. Downstream of CMPs, megakaryocyte/erythrocyte progenitors (MEPs) but not granulocyte/macrophage progenitors (GMPs) were dominantly involved in leukemia (t(9;22) positive ratio; 96.0+/-5.5% vs. 56.3+/-37%). The expression level of BCR-ABL is not different among these progenitor populations. These observations collectively suggest that in CMP-CP, BCR-ABL signaling becomes effective on cell proliferation especially at the CMP stage. Display omitted
Gene expression analysis of stem/progenitor populations in CML patients revealed that IRF8 and GFI1, transcription factors playing critical roles in myeloid differentiation and cell proliferation, were down-regulated specifically in CMPs as compared with that in normal controls (Figure 2). In order to substantiate the role of IRF8 and GFI1 in CML pathogenesis, we used a CML mouse model established by enforced retroviral expression of BCR-ABL. As in analysis of CML patients, BCR-ABL expressing CMPs but not stem/multipotent progenitor cells acquired growth advantage over normal counterparts. Importantly, the expression of IRF8 and GFI1 became undetectable after BCR-ABL transduction in the expanding CMPs. Display omitted
Our observations revealed that, in CML-CP hematopoiesis, BCR-ABL dependent cell proliferation initiates at the CMP stage, and is accompanied with the down–regulation of IRF8 and GFI1. Because IRF8 knockout mice develop myeloproliferative disorders, and because CMPs expand in GFI1 null mice, the attenuation of these molecules could be downstream effector of BCR-ABL dependent myeloid cell growth. Taken together, the reactivation of these molecules might be useful to develop alternative therapeutic strategies for CML-CP, for example, with TKI-resistant BCR-ABL mutants.
Miyamoto:Kyushu University Hospital: Employment.
Adult T-cell leukemia (ATL) is an aggressive peripheral T-cell neoplasm with extremely poor prognosis. It typically develops due to long-term infection with human T-cell leukemia virus type 1 ...(HTLV-1). Among HTLV-1 antigens, the viral regulatory protein Tax is widely recognized as a target of cytotoxic T lymphocytes (CTLs), and Tax-specific CTLs are often activated in ATL patients following hematopoietic stem cell transplantation (HSCT). This strongly suggests the presence of Tax expression in vivo and potential contributions of CTL to GVL effects. Based on these findings, we developed an anti-ATL therapeutic vaccine consisting of autologous dendritic cells (DC) pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL that were previously identified from human leukocyte antigen (HLA)-A2, A24 or A11-possessing ATL patients post-HSCT (Cancer Res. 2004, J Virol. 2005). In preliminary experiments, the DC induced with a conventional method showed matured phenotype and produced interleukin (IL)-12 in two of three ATL patients tested.
After approval by the institutional ethics committees, we conducted a phase I clinical trial of anti-ATL immunotherapy for pretreated ATL patients in stable condition in order to evaluate whether HTLV-1 Tax targeting immunotherapy is safe and feasible. The vaccine protocol consists of three subcutaneous injections of peptide-pulsed DC at 2-week intervals in a total 8-week observation period to assess adverse events. Three acute-type patients enrolled and completed the course of the study. Modest clinical symptoms, such as injection site reaction, fever, and skin damage, were observed in all cases. DC vaccine-related toxicities were grade 1 or 2 and were considered relatively mild, and non-hematological toxicity was acceptable. In addition, significant reduction in sIL-2R levels were observed at least in one patient, and the HTLV-1 proviral loads were maintained below 60 copies /1000 PBMCs during the monitored period after administration of DC in the all patients. Enhanced proliferative responses of Tax-specific CTLs were also observed in vitro after therapy. Clinical effects, as evaluated by RECIST, were partial remission in two patients and stable disease in one patient. Durable remissions and improvements in performance status were observed in all three patients without the requirement for new treatment for 4-10 months after treatment with Tax-targeted DC vaccination. These results indicate that Tax-specific DC vaccination is safe and well tolerated, and may be promising as a new therapeutic strategy for ATL patients.
No relevant conflicts of interest to declare.
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