Enhanced TAT-Cre Protein Transduction for Efficient Gene Recombination in T cells.

Genetic manipulation has increased our understanding of gene function and led to the discovery of new therapeutic targets. Cre/LoxP DNA recombination is widely used for genetic studies in mammalian cells. The direct delivery of Cre recombinase fused to protein transduction domains (PTDs), such as TAT, has been described as a valid alternative to the conditional, site-specific Cre expression in transgenic mice. However, efficiently conveying proteins into live cells, especially primary T cells, remains a major challenge. In this study, we show that one of our recently developed PTDs synthetic mimic greatly enhances the cellular uptake of the TAT-Cre fusion protein, enabling significantly smaller amounts of the protein to be used. We used this technique in primary mouse T cells to successfully delete, ex vivo, two essential genes involved in regulating T cell activation, Notch1 and Rbpjκ. Ex vivo gene deletion resulted in substantial protein reduction, comparable to that obtained in vivo when Cre-expressing Notch1-floxed (MxCre±Notch1fl/fl) mice were treated with polyinosinic-polycytidylic acid (polyl/C), but in considerably less time, and without altering normal cell physiology. These results highlight several key advantages that include the ability to use less expensive protein (TAT-Cre), a major reduction in total experimental time and labor, and fewer side effects on the treated cells. This method should offer new opportunities for immunological studies, especially in the context of identifying novel therapeutic targets.

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.

The Link between Autoimmunity and Lymphoma: Does NOTCH Signaling Play a Contributing Role?

An association between certain autoimmune conditions and increased risk of developing lymphoma is well documented. Recent evidence points to NOTCH signaling as a strong driver of autoimmunity. Furthermore, a role for NOTCH in various lymphomas, including classical Hodgkin lymphoma, non-Hodgkin lymphoma, and T cell lymphoma has also been described. In this mini-review, we will outline what is known about involvement of NOTCH signaling in those autoimmune conditions, such as rheumatoid arthritis and primary Sjörgren's syndrome, which show an increased risk for subsequent diagnosis of lymphoma. Furthermore, we will detail what is known about the lymphomas associated with these autoimmune conditions and how aberrant or sustained NOTCH signaling in the immune cells that mediate these diseases may contribute to lymphoma.

NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.

Therapeutic targeting of NOTCH signaling ameliorates immune-mediated bone marrow failure of aplastic anemia.

Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1(IC) and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1(IC) was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1(IC) levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.