Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and β-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias.

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Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics.

BackgroundThe efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T–B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL+ effector B cells and their impacts on osteoclast differentiation.MethodsPeripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells.ResultsRANKL expression was accentuated in CD80+CD86+ B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21.Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3+RANKL+ effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL+ effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro.ConclusionsOur current findings have shed light on the generation mechanism of pathogenic RANKL+ effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0957-6) contains supplementary material, which is available to authorized users.

IntroductionB-cell receptor (BCR) signaling guides critical cell fate decisions in B cells during ontogeny. 1,2 BCRs can generate tolerogenic signals to purge or silence B cells that bind to self-antigens, and immunogenic signals to expand B cells that are specific for foreign antigens. Thus, BCR signaling must be properly regulated at the various stages of B-cell development, as aberrant regulation of BCR signaling potentially leads to autoimmunity and B-cell malignancies.On BCR ligation by antigens, the Src-family protein tyrosine kinase (PTK) Lyn and Syk are initially activated. Syk propagates the signal by phosphorylating downstream signaling molecules, causing the activation of critical signaling intermediates phosphoinositol 3-kinase (PI3K) and phospholipase C (PLC)␥2. PI3K activates Akt kinase, which is important for B-cell survival. 3 PLC␥2 activation induces the release of intracellular Ca 2ϩ and the activation of protein kinase C (PKC), which cause the activation of mitogen-activated protein kinases (MAPKs; ERK, JNK, and p38 MAPK) and of transcription factors, including NF-B and NF-AT. These molecules regulate further downstream molecules that are responsible for determining B-cell fates such as survival, growth, and differentiation. 1,2 Casitas B-lineage lymphoma (Cbl) proteins are E3 ubiquitin ligases that regulate signals of various receptors by promoting the ubiquitination of signaling components. 4,5 Tyrosine phosphorylation of Cbl proteins is critical for their function. 6 Mammalian Cbl proteins consist of 3 members, c-Cbl, Cbl-b, and Cbl-3, among which c-Cbl and Cbl-b are expressed in hematopoietic cells. 7 In B cells, Cbl proteins associate with Syk and B-cell linker (BLNK), and negatively regulate BCR signaling. 8,9 B cell-specific ablation of c-Cbl/Cbl-b proteins in mice causes aberrant BCR signaling as well as impaired B-cell anergy, culminating in the development of systemic lupus erythematosus (SLE)-like disease. 10 In addition, c-Cbl is hypophosphorylated on tyrosine in advanced stages of chronic lymphocytic leukemia (CLL). 11 These findings suggest that Cbl-mediated regulation of BCR signaling is critical for the fate decisions of self-reactive and malignant B cells.Adaptors are noncatalytic molecules that integrate the spatial and temporal assembly of multiprotein complexes involved in the survival, growth, and differentiation of B cells. We previously showed that the B lymphocyte adaptor molecule of 32 kDa (Bam32)/DAPP1 regulates BCR signaling/internalization and B-cell survival. 12,13 The SH3KBP1 (SH3-domain kinase-binding protein 1) gene, which is also known as CIN85 (c-Cbl interacting protein of 85 kDa), encodes an adaptor that is independently identified by several groups and contains 3 SH3 domains, a proline-rich region, and a coiled-coil domain. [14][15][16][17] Early studies showed that in nonimmune cells, CIN85 regulates the clathrindependent internalization of receptor tyrosine kinases (RTKs) such as epidermal growth factor receptors (EGFRs). 18,19 The formation of the ternary...

Our results suggest that the activated Syk-mediated TRAF6 pathway leads to aberrant activation of B cells in SLE, and also highlight Syk as a potential target for B-cell-mediated processes in SLE.

The findings elucidate a model in which Btk not only plays a fundamental role in the regulation of BCR signalling, but may also mediate crosstalk with cytokine signalling pathways through regulation of IL-21-induced phosphorylation of STAT1 in the nuclei of human B cells. Btk appears to have pathological relevance in RA.

Activation of the transforming growth factor (TGF) α/epidermal growth factor receptor (EGFR)-mediated signaling pathway is a common mechanism for dysregulated growth of head and neck squamous cell carcinoma (HNSCC). c-Cbl-interacting protein of 85 kDa (CIN85) is an adaptor protein that facilitates EGFR internalization. Little is known, however, about a role of CIN85 in EGFR signaling as well as its relevance to tumor development and progression of HNSCC. Here, we demonstrate that CIN85 is highly expressed in HNSCC tumor samples compared with adjacent normal tissues, and this overexpression is significantly correlated with advanced clinical stage. The experiments using CIN85-overexpressing and knockdown HNSCC cell lines showed that CIN85 promotes HNSCC growth and facilitates EGFR internalization without apparently affecting phosphorylation of EGFR. Moreover, CIN85 promoted TGF-α-induced activation of Ras and phosphorylation of downstream molecules such as c-Raf, MEK, and extracellular signal-regulated kinase, leading to expression of c-Myc that is critical for sustained proliferation of HNSCC. Taken together, these findings suggest that CIN85 not only controls EGFR internalization but also promotes the EGFR-mediated tumor development and progression, and thus, CIN85 may serve as a potential therapeutic target in a subset of HNSCC.