Mobilization of Plasmids From Bacteria Into Diatoms by Conjugation Technique.

Diatoms serve as a source for a variety of compounds with particular biotechnological interest. Therefore, redirecting the flow to a specific pathway requires the elucidation of the gene's specific function. The most commonly used method in diatoms is biolistic transformation, which is a very expensive and time-consuming method. The use of episomes that are maintained as closed circles at a copy number equivalent to native chromosomes has become a useful genetic system for protein expression that avoids multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. These episomes can be introduced from bacteria into diatoms via conjugation. Here, we describe a detailed protocol for gene expression that includes 1) the gateway cloning strategy and 2) the conjugation protocol for the mobilization of plasmids from bacteria to diatoms.

Addressing the Role of Conventional CD8αß+ T Cells and CD4+ T Cells in Intestinal Immunopathology Using a Bone Marrow-Engrafted Model.

Inflammatory bowel disease (IBD) is characterized by an aberrant immune response against microbiota. It is well established that T cells play a critical role in mediating the pathology. Assessing the contribution of each subset of T cells in mediating the pathology is crucial in order to design better therapeutic strategies. This protocol presents a method to identify the specific effector T-cell population responsible for intestinal immunopathologies in bone marrow-engrafted mouse models. Here, we used anti-CD4 and anti-CD8ß depleting antibodies in bone marrow-engrafted mouse models to identify the effector T-cell population responsible for intestinal damage in a genetic mouse model of chronic intestinal inflammation. Key features • This protocol allows addressing the role of CD4+ or CD8αß+ in an engrafted model of inflammatory bowel disease (IBD). • This protocol can easily be adapted to address the role of other immune cells or molecules that may play a role in IBD.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

Regulatory T cells promote cancer immune-escape through integrin αvß8-mediated TGF-ß activation.

Presence of TGFß in the tumor microenvironment is one of the most relevant cancer immune-escape mechanisms. TGFß is secreted in an inactive form, and its activation within the tumor may depend on different cell types and mechanisms than its production. Here we show in mouse melanoma and breast cancer models that regulatory T (Treg) cells expressing the ß8 chain of αvß8 integrin (Itgß8) are the main cell type in the tumors that activates TGFß, produced by the cancer cells and stored in the tumor micro-environment. Itgß8 ablation in Treg cells impairs TGFß signalling in intra-tumoral T lymphocytes but not in the tumor draining lymph nodes. Successively, the effector function of tumor infiltrating CD8+ T lymphocytes strengthens, leading to efficient control of tumor growth. In cancer patients, anti-Itgß8 antibody treatment elicits similar improved cytotoxic T cell activation. Thus, this study reveals that Treg cells work in concert with cancer cells to produce bioactive-TGFß and to create an immunosuppressive micro-environment.