Knowledge Graphs in Practice: Characterizing their Users, Challenges, and Visualization Opportunities.

This study presents insights from interviews with nineteen Knowledge Graph (KG) practitioners who work in both enterprise and academic settings on a wide variety of use cases. Through this study, we identify critical challenges experienced by KG practitioners when creating, exploring, and analyzing KGs that could be alleviated through visualization design. Our findings reveal three major personas among KG practitioners - KG Builders, Analysts, and Consumers - each of whom have their own distinct expertise and needs. We discover that KG Builders would benefit from schema enforcers, while KG Analysts need customizable query builders that provide interim query results. For KG Consumers, we identify a lack of efficacy for node-link diagrams, and the need for tailored domain-specific visualizations to promote KG adoption and comprehension. Lastly, we find that implementing KGs effectively in practice requires both technical and social solutions that are not addressed with current tools, technologies, and collaborative workflows. From the analysis of our interviews, we distill several visualization research directions to improve KG usability, including knowledge cards that balance digestibility and discoverability, timeline views to track temporal changes, interfaces that support organic discovery, and semantic explanations for AI and machine learning predictions.

Isolation and expansion of murine γδ T cells from mouse splenocytes.

Elucidation of the function of gamma delta T cells (γδ T cells) requires robust models that show how γδ T cells are commonly involved in inflammation, since very little is known about the factors that promote and control their development and function. There are few studies of murine γδ T cells primarily because these cells have proven difficult to isolate, expand and characterize. Here, we describe a simple method that utilizes key expansion elements to isolate and expand murine CD4-CD8-CD3+ γδ T cells typically found in secondary lymphoid tissues. Expansion of γδ T cells reached 150-fold by day 8 of culture, depended on exogenous IL-2, αCD3, and αCD28, and supported efficient and reproducible in vitro differentiation. These studies showed high production of cytokines IFNγ and Granzyme B, with the novel finding of IL-24 upregulation as well. Expression analysis of expanded γδ T cells, after treatment with IL-2, revealed high levels of Granzyme B, Granzyme D, and IFNγ. Lactate dehydrogenase (LDH) cytotoxicity assays showed that expanded γδ T cells were effective at inducing >90% cytolysis of murine MC38 colon cancer, E0771 breast cancer, and B16 melanoma cells at 10:1 effector to target ratios. These findings indicated that murine γδ T cells can be successfully isolated, expanded, and used to perform preclinical therapy studies.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

BACKGROUND: Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. METHODS: Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. RESULTS: We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. CONCLUSION: The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1-/- or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown. Human HepG2 hepatocytes that express CEACAM1 and form BC in vitro were compared with CEACAM1-/- cells and CEACAM1-/- cells expressing Ser508Ala null or Ser508Asp phosphorylation mimic mutations or to phosphorylation null mutations in the tyrosine ITIMs known to be phosphorylated by the tyrosine kinase Src. CEACAM1-/- cells and the Ser508Asp and Tyr520Phe mutants strongly retained lipids, while Ser508Ala and Tyr493Phe mutants had low lipid levels compared with wild-type cells, indicating that the ITIM mutants phenocopied the Ser508 mutants. We found that the fatty acid transporter CD36 was upregulated in the S508A mutant, coexpressed in BCs with CEACAM1, co-IPed with CEACAM1 and Src, and when downregulated via RNAi, an increase in lipid droplet content was observed. Nuclear translocation of CD36 associated kinase LKB1 was increased sevenfold in the S508A mutant versus CEACAM1-/- cells and correlated with increased activation of CD36-associated kinase AMPK in CEACAM1-/- cells. Thus, while CEACAM1-/- HepG2 cells upregulate lipid storage similar to Ceacam1-/- in murine liver, the null mutation Ser508Ala led to decreased lipid storage, emphasizing evolutionary changes between the CEACAM1 genes in mouse and humans.

Potent immunomodulatory effects of an anti-CEA-IL-2 immunocytokine on tumor therapy and effects of stereotactic radiation.

While anti-CEA antibodies have no direct effect on CEA-positive tumors, they can be used to direct potent anti-tumor effects as an antibody-IL-2 fusion protein (immunocytokine, ICK), and at the same time reduce the toxicity of IL-2 as a single agent. Using a fusion protein of humanized anti-CEA with human IL-2 (M5A-IL-2) in a transgenic murine model expressing human CEA, we show high tumor uptake of the ICK to CEA-positive tumors with additional lymph node targeting. ICK treated CEA-positive tumors exhibit significant tumor eradication. Analysis of tumor-infiltrating lymphocytes shows a high frequency of both CD8+ and CD4+ T cells along with CD11b positive myeloid cells in ICK treated mice. The frequency of tumor-infiltrating FoxP3+ CD4+ T cells (Tregs) is significantly reduced vs anti-CEA antibody-treated controls, indicating that ICK did not preferentially stimulate migration or proliferation of Tregs to the tumor. Combination therapy with anti-PD-1 antibody did not improve tumor reduction over ICK therapy alone. Since stereotactic tumor irradiation (SRT), commonly used in cancer therapy has immunomodulatory effects, we tested combination SRT+ICK therapy in two tumor model systems. Use of fractionated vs single high dose SRT in combination with ICK resulted in greater tumor inhibition and immunity to tumor rechallenge. In particular, tumor microenvironment and myeloid cell composition appear to play a significant role in the response rate to ICK+SRT combination therapy.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

A conserved GXXXG motif in APH-1 is critical for assembly and activity of the gamma-secretase complex.

The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.