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.

Common and Exclusive Features of Intestinal Intraepithelial γδ T Cells and Other γδ T Cell Subsets.

Murine peripheral lymph node TCR γδ T cells have been divided into type 1 and type 17 functional categories based on phenotypic and functional markers. Localized in the gut epithelial barrier, intestinal intraepithelial lymphocytes (iIEL) γδ T cells constitute a peculiar subset of T lymphocytes involved in intestinal homeostasis. However, whether iIEL γδ T cells obey the type 1/type 17 dichotomy is unclear. Using both global transcriptional signatures and expression of cell surface markers, we reveal that murine iIEL γδ T cells compose a distinct population, expressing ∼1000 specific genes, in particular genes that are responsible for cytotoxicity and regulatory functions. The expression of the transcription factor Helios is a feature of iIEL γδ T cells, distinguishing them from the other TCR γδ T subsets, including those present in the epithelia of other tissues. The marked expression of Helios is also shared by the other iIELs, TCRαßCD8αα lymphocytes present within the intestinal epithelium. Finally, we show that Helios expression depends in part on TGF-ß signaling but not on the microbiota. Thus, our study proposes iIEL γδ T cells as a distinct subset and identifies novel markers to differentiate them from their peripheral counterparts.

SMAD4 TGF-ß-independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation.

SMAD4, a mediator of TGF-ß signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-ß receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-ß signature in naive CD8+ T cells. Mechanistically, prior to TGF-ß signaling, SMAD4 binds to promoters and enhancers of several TGF-ß target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-ß signal, SMAD4 limits the expression of TGF-ß negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-ß. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-ß control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-ß-independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.

A T cell-intrinsic function for NF-κB RelB in experimental autoimmune encephalomyelitis.

NF-kappaB (NF-κB) is a family of transcription factors with pleiotropic functions in immune responses. The alternative NF-κB pathway that leads to the activation of RelB and NF-κB2, was previously associated with the activation and function of T cells, though the exact contribution of these NF-κB subunits remains unclear. Here, using mice carrying conditional ablation of RelB in T cells, we evaluated its role in the development of conventional CD4+ T (Tconv) cells and their function in autoimmune diseases. RelB was largely dispensable for Tconv cell homeostasis, activation and proliferation, and for their polarization toward different flavors of Thelper cells in vitro. Moreover, ablation of RelB had no impact on the capacity of Tconv cells to induce autoimmune colitis. Conversely, clinical severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS) was significantly reduced in mice with RelB-deficient T cells. This was associated with impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) specifically in the central nervous system. Our data reveal a discrete role for RelB in the pathogenic function of Tconv cells during EAE, and highlight this transcription factor as a putative therapeutic target in MS.