Progress has been slow in defining molecular requirements for human B lymphopoiesis in part because of differences from experimental animals and also because of the lack of culture conditions that efficiently support the process. We recently found that human CD10+ lymphocytes were produced when CD34+ hematopoietic stem and progenitor cells were cultured in contact with human mesenchymal stem cells (hMSC). Further investigation revealed that it occurred even when progenitors were separated from hMSC by membrane filters. Experiments with neutralizing antibodies suggested that important heat labile factors produced by hMSC are unlikely to be IL-7, TSLP, CXCL12 or hemokinin-1. Further manipulation of culture conditions revealed that optimal lymphopoiesis required careful selection of fetal calf serum lots, maintenance of high cell densities, as well as recombinant cytokines (SCF, FL and G-CSF). G-CSF was particularly important when adult bone marrow rather than umbilical cord blood derived CD34+ cells were used to initiate the cultures. These improved methods should facilitate identification of molecules that can be used to speed regeneration of the humoral immune system following chemotherapy and might suggest ways to inhibit growth of B lineage malignancies.

Immune system is a complex network that clears pathogens, toxic substrates, and cancer cells. Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens. The innate immune system elicits an early-phase response to various stimuli, whereas the adaptive immune response is tailored to previously encountered antigens. During immune responses, B cells differentiate into antibody-secreting cells, while naïve T cells differentiate into functionally specific effector cells [T helper 1 (Th1), Th2, Th17, and regulatory T cells]. However, enhanced or prolonged immune responses can result in autoimmune disorders, which are characterized by lymphocyte-mediated immune responses against self-antigens. Signal transduction of cytokines, which regulate the inflammatory cascades, is dependent on the members of the Janus family of protein kinases. Tyrosine kinase 2 (Tyk2) is associated with receptor subunits of immune-related cytokines, such as type I interferon, interleukin (IL)-6, IL-10, IL-12, and IL-23. Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing. This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anticancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

STAP-2 is an adaptor molecule regulating several signaling pathways, including TLRs and cytokine/chemokine receptors in immune cells. We previously reported that STAP-2 enhances SDF-1α-induced Vav1/Rac1-mediated T-cell chemotaxis. However, the detailed mechanisms of STAP-2 involvement in enhancing T-cell chemotaxis remain unknown. In the present study, we demonstrate that STAP-2 directly interacts with Pyk2, which is a key molecule in the regulation of SDF-1α/CXCR4-mediated T-cell chemotaxis, and increases phosphorylation of Pyk2. Pyk2 itself can induce STAP-2 Y250 phosphorylation, and this phosphorylation is critical for maximal interactions between STAP-2 and Pyk2. Finally, SDF-1α-induced T-cell chemotaxis is inhibited by treatment with Pyk2 siRNA or AG17, an inhibitor of Pyk2, in Jurkat cells overexpressing STAP-2. Taken together, the Pyk2/STAP-2 interaction is a novel mechanism to regulate SDF-1α-dependent T-cell chemotaxis.

Adult T-cell leukemia/lymphoma (ATLL) patients have an extremely poor prognosis, partly due to their immunosuppressive state. The majority of ATLL patients have leukemic cells with phenotype similar to Tregs, prompting suggestions that ATLL cells themselves have immunosuppressive functions. In this study, we detected CD39 expression on ATLL cells, particularly frequent on aggressive subtypes. CD39 and CD73 convert extracellular adenosine triphosphate (ATP) into adenosine, a key player in Tregs' immunosuppression. In vitro culture, both CD39+ ATLL cells and normal Tregs converted rapidly extracellular ATP to AMP, which was disturbed by CD39 inhibitors, and was negated in the CD39 knockout MJ cell line. The proliferation of cocultured CD4+/CD8+ normal T cells was suppressed by CD39+ MJ cells, but not by CD39 knockout MJ cells. Supplemented ATP was exhausted by an EG7-OVA T-cell line with stable CD39 induction, but not by mock. When these cell lines were subcutaneously transplanted into murine flanks, Poly(I:C) peritoneal administration reduced tumor size to 1/3 in mock-transplanted tumors, but not in CD39 induced tumors. Overall, we found that ATLL cells express CD39 at a high rate, and our results suggest that this helps ATLL cells escape antitumor immunity through the extracellular ATPDase-Adenosine cascade. These findings will guide future clinical strategies for ATLL treatment.

The family of signal-transducing adapter proteins (STAPs) has been reported to be involved in a variety of intracellular signaling pathways and implicated as transcriptional factors. We previously cloned STAP-2 as a c-Fms interacting protein and explored its effects on chronic myeloid leukemia (CML) leukemogenesis. STAP-2 binds to BCR-ABL, upregulates BCR-ABL phosphorylation, and activates its downstream molecules. In this study, we evaluated the role of STAP-1, another member of the STAP family, in CML pathogenesis. We found that the expression of STAP-1 is aberrantly upregulated in CML stem cells (LSCs) in patients' bone marrow. Using experimental model mice, deletion of STAP-1 prolonged the survival of CML mice with inducing apoptosis of LSCs. The impaired phosphorylation status of STAT5 by STAP-1 ablation leads to downregulation of antiapoptotic genes, Bcl-2 and Bcl-xL. Interestingly, transcriptome analyses indicated that STAP-1 affects several signaling pathways related to BCR-ABL, JAK2, and PPARγ. This adapter protein directly binds to not only BCR-ABL, but also STAT5 proteins, showing synergistic effects of STAP-1 inhibition and BCR-ABL or JAK2 tyrosine kinase inhibition. Our results identified STAP-1 as a regulator of CML LSCs and suggested it to be a potential therapeutic target for CML.

Anamorsin (AM) is an anti-apoptotic molecule cloned by us as a molecule that confers resistance against apoptosis induced by growth factor deprivation. AM-deficient mice are embryonic lethal, which impedes detailed analyses of the roles of AM in various types of adult cells. To overcome the embryonic lethality, we generated AM conditional knockout (AMflox/flox) mice and cell type-specific genetic modification became possible using the Cre-loxP system. CD19-Cre/AMflox/flox mice with AM deleted specifically in CD19+ B cells exhibited less B220+ B cells in their spleen, peripheral blood, and lymph node compared with control CD19-Cre mice. Using flow cytometry to categorize bone marrow and spleen cells into B cell subsets, we observed significantly less follicular type I cells, which are the most mature follicular B cells, compared with control CD19-Cre mice. These data suggest that AM has an important role in the generation of mature B cells.

Graft-versus-host disease (GVHD) is the most frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT), and is one of the major causes of non-relapse mortality. Transferred mature lymphocytes are thought to be responsible for GVHD based on the findings that mice transplanted with lymphocyte-depleted bone marrow (BM) cells from MHC-mismatched donors do not develop GVHD. However, we found that overexpression of signal-transducing adaptor protein (STAP)-2 in lymphoid cells could induce GVHD after lymphocyte-depleted BM transplantation. To examine the function of STAP-2, which has been shown to play an important role in development and function of lymphocytes, in GVHD, we transplanted BM cells from STAP-2 deficient, or Lck promoter/IgH enhancer-driven STAP-2 transgenic (Tg) mice into MHC-mismatched recipients. Unexpectedly, mice transplanted with lymphocyte-depleted BM cells from STAP-2 Tg mice developed severe acute GVHD with extensive colitis and atrophy of thymus, while no obvious GVHD developed in mice transplanted with the wild type or STAP-2 deficient graft. Furthermore, mice transplanted with lymphocyte-depleted BM cells from the syngeneic STAP-2 Tg mice developed modest GVHD with colitis and atrophy of thymus. These results suggest that STAP-2 overexpression may enhance survival of allo-, and even auto-, reactive lymphocytes derived from engrafted hematopoietic progenitor cells in lethally irradiated mice, and that clarification of the mechanism may help understanding induction of immune tolerance after HSCT.

Chronic myeloid leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We recently reported that signal-transducing adaptor protein 1 (STAP-1) is upregulated in CML stem cells (LSCs) and functions to reduce the apoptosis of CML LSCs by upregulating the STAT5-downstream anti-apoptotic genes. In this study, we demonstrate the detailed molecular interactions among BCR-ABL, STAP-1, and signal transducer and activator of transcription 5 (STAT5). Studies with deletion mutants have revealed that STAP-1 interacts with BCR-ABL and STAT5a through its SH2 and PH domains, respectively, suggesting the possible role of STAP-1 as a scaffold protein. Furthermore, the binding of STAP-1 to BCR-ABL stabilizes the BCR-ABL protein in CML cells. Since STAP-1 is highly expressed in CML cells, we also analyzed the STAP-1 promoter activity using a luciferase reporter construct and found that NFATc1 is involved in activating the STAP-1 promoter and inducing STAP-1 mRNA expression. Our results demonstrate that STAP-1 contributes to the BCR-ABL/STAT5 and BCR-ABL/Ca2+/NFAT signals to induce proliferation and STAP-1 mRNA expression in CML cells, respectively.

CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process.

Macrophages are highly plastic in their pro-inflammatory/anti-inflammatory roles. Type I and II interferons (IFNs) are known to modulate macrophage activation. Tyrosine kinase 2 (Tyk2) has an intimate relationship with type I and II IFN signaling. Animal studies have shown that Tyk2 knock-out (KO) in mice is associated with reduced inflammatory responses in various mouse models of diseases. To investigate the role of Tyk2 in inflammation in more detail, we intraperitoneally injected heat-killed Propionibacterium acnes (P. acnes) to Tyk2 KO mice. P. acnes-induced acute peritoneal inflammation, assessed by neutrophil infiltration, was reduced in Tyk2 KO mice. The reduction was accompanied with diminished productions of inflammatory cytokines and an enhanced production of anti-inflammatory IL-10. Unexpectedly, pre-treatment of wild-type mice with the neutralizing antibodies for IFNs did not affect P. acnes-induced neutrophil infiltration. A neutralizing antibody for the IL-10 receptor in Tyk2 KO mice restored P. acnes-induced peritoneal inflammation. Enhanced production of IL-10 from Tyk2 KO peritoneal cells was suppressed by either the cyclooxygenase inhibitor diclofenac or protein kinase A inhibitor H-89. The level of prostaglandin E2 (PGE2) in the steady-state peritoneal cavity in Tyk2 KO mice was higher than that in wild-type mice. Tyk2 KO macrophages showed an enhanced CREB phosphorylation induced by P. acnes plus PGE2. Taken together, these results showed that Tyk2 deficiency potentiates the PGE2-protein kinase A-IL-10 pathway in macrophages, and thereby contributes to potentiation of the immunosuppressive phenotype.

Signal-transducing adaptor protein-2 (STAP-2) was discovered as a C-FMS/M-CSFR interacting protein and subsequently found to function as an adaptor of signaling or transcription factors. These include STAT5, MyD88 and IκB kinase in macrophages, mast cells, and T cells. There is additional information about roles for STAP-2 in several types of malignant diseases including chronic myeloid leukemia, however, none have been reported concerning B lineage lymphocytes. We have now exploited gene targeted and transgenic mice to address this lack of knowledge, and demonstrated that STAP-2 is not required under normal, steady-state conditions. However, recovery of B cells following transplantation was augmented in the absence of STAP-2. This appeared to be restricted to cells of B cell lineage with myeloid rebound noted as unremarkable. Furthermore, all hematological parameters were observed to be normal once recovery from transplantation was complete. Furthermore, overexpression of STAP-2, specifically in lymphoid cells, resulted in reduced numbers of late-stage B cell progenitors within the bone marrow. While numbers of mature peripheral B and T cells were unaffected, recovery from sub-lethal irradiation or transplantation was dramatically reduced. Lipopolysaccharide (LPS) normally suppresses B precursor expansion in response to interleukin 7, however, STAP-2 deficiency made these cells more resistant. Preliminary RNA-Seq analyses indicated multiple signaling pathways in B progenitors as STAP-2-dependent. These findings suggest that STAP-2 modulates formation of B lymphocytes in demand conditions. Further study of this adapter protein could reveal ways to speed recovery of humoral immunity following chemotherapy or transplantation.

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.

Signal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. Notably however, the biological functions of STAP-1 in other cells are not known. We aimed to investigate the functions of STAP-1 in invariant natural killer T (iNKT) cells and iNKT cell-dependent hepatitis.

Signal-transducing adaptor protein (STAP)-2 is one of the STAP family adaptor proteins and ubiquitously expressed in a variety types of cells. Although STAP-2 is required for modification of FcεRI signal transduction in mast cells, other involvement of STAP-2 in mast cell functions is unknown, yet. In the present study, we mainly investigated functional roles of STAP-2 in IL-33-induced mast cell activation. In STAP-2-deficient, but not STAP-1-deficient, mast cells, IL-33-induced IL-6 and TNF-α production was significantly decreased compared with that of wild-type mast cells. In addition, STAP-2-deficiency greatly reduced TLR4-mediated mast cell activation and cytokine production. For the mechanisms, STAP-2 directly binds to IKKα after IL-33 stimulation, leading to elevated NF-κB activity. In conclusion, STAP-2, but not STAP-1, participates in IL-33-induced mast cells activation.

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34+CD38+CD33+ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3+ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.

The signaling elicited by the cytokine interleukin-17A (IL-17) is important for antimicrobial defense responses, whereas excessive IL-17 production leads to autoimmune diseases such as psoriasis and multiple sclerosis. IL-17-induced stabilization of mRNAs has been recognized as a unique and important feature of IL-17 signaling. Previously, we demonstrated that IL-17 signaling protein ACT1 is required to counteract constitutive inhibitor of nuclear factor kappa B zeta (IκB-ζ) mRNA degradation by the ribonuclease Regnase-1. However, information about the mechanism of mRNA stabilization in IL-17-stimulated cells remains insufficient. In the present study, we aimed to clarify the mechanism in more detail and identify an agent that can inhibit IL-17-induced mRNA stabilization. Experiments using small interfering RNA and an inhibitor of TANK-binding kinase 1 (TBK1) revealed that TBK1 was required for IκB-ζ mRNA stabilization through Regnase-1 phosphorylation. Intriguingly, this TBK1-mediated phosphorylation of Regnase-1 was suppressed by the addition of dimethyl fumarate (DMF), an electrophilic small molecule that has been used to treat IL-17-related autoimmune diseases. Confocal microscopic observation of the cellular localization of ACT1 revealed that DMF treatment resulted in the disappearance of ACT1 nuclear dots and perinuclear accumulation of ACT1. These results suggested that DMF is a small molecule that compromises IL-17-induced activation of the ACT1-TBK1 pathway, thereby inhibiting IL-17-induced mRNA stabilization.