Oligomerization Regulates the Localization of Cdc24, the Cdc42 Activator in Saccharomyces cerevisiae

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High endothelial venules as traffic control points maintaining lymphocyte population homeostasis in lymph nodes

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Isolation and enrichment of mouse splenic T cells for ex vivo and in vivo T cell receptor stimulation assays

International audienceSpecific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation

PTX Instructs the Development of Lung-Resident Memory T Cells in <i>Bordetella pertussis</i> Infected Mice

Whooping cough is a severe, highly contagious disease of the human respiratory tract, caused by Bordetellapertussis. The pathogenicity requires several virulence factors, including pertussis toxin (PTX), a key component of current available vaccines. Current vaccines do not induce mucosal immunity. Tissue-resident memory T cells (Trm) are among the first lines of defense against invading pathogens and are involved in long-term protection. However, the factors involved in Trm establishment remain unknown. Comparing two B.pertussis strains expressing PTX (WT) or not (ΔPTX), we show that the toxin is required to generate both lung CD4+ and CD8+ Trm. Co-administering purified PTX with ΔPTX is sufficient to generate these Trm subsets. Importantly, adoptive transfer of lung CD4+ or CD8+ Trm conferred protection against B. pertussis in naïve mice. Taken together, our data demonstrate for the first time a critical role for PTX in the induction of mucosal long-term protection against B. pertussis

MYC deficiency impairs the development of effector/memory T lymphocytes

International audienceIn the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation

Novel mouse models based on intersectional genetics to identify and characterize plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are the main source of type I interferon (IFN-I) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DCs (tDCs), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. In the present report, we present a reporter mouse, pDC-Tom, designed through intersectional genetics based on unique Siglech and Pacsin1 coexpression in pDCs. The pDC-Tom mice specifically tagged pDCs and, on breeding with Zbtb46GFP mice, enabled transcriptomic profiling of all splenic DC types, unraveling diverging activation of pDC-like cells versus tDCs during a viral infection. The pDC-Tom mice also revealed initially similar but later divergent microanatomical relocation of splenic IFN+ versus IFN− pDCs during infection. The mouse models and specific gene modules we report here will be useful to delineate the physiological functions of pDCs versus other DC types

Phenotype of CD21⁻ RFP⁺ stromal cells.

<p>(A and E) Confocal images of a LN section from a CD21cre-RFP chimera stained for the indicated markers. RFP⁺ cells appear in red. The dashed line delineates B cell follicle boundary. (B, C, D) CD21⁻ RFP⁻ gp38⁺ CD31⁻ CD45⁻ FRCs and CD21⁻ RFP⁺ gp38⁺ CD31⁻ CD45⁻ cells were sorted by flow cytometry and their transcriptomic profiles were analyzed by microarrays (B and C) or RT-PCR (D). (B) Scatter plot of global comparison of gene expression between CD21⁻ RFP⁺ cells and FRCs. Each gene in the microarray is represented by a dot with coordinates consisting of average gene expression computed from three independent CD21⁻ RFP⁺ samples (<i>y</i>-axis) and from the three matched FRC samples (<i>x</i>-axis). Genes with Log₂ average expression level at least 1.5-fold higher in CD21⁻ RFP⁺ cells are shown in red, while genes with Log₂ average expression level at least 1.5-fold higher in FRCs are shown in blue. These genes are separated from the other genes by colored lines representing these fold change cutoffs. (C) Heat map for the 50 genes with the most significantly higher expression in CD21⁻ RFP⁺ cells. A subset of the genes shown in red in panel B was further selected based on a <i>p</i> value<0.05 for differential expression between CD21⁻ RFP⁺ cells and FRCs, as computed by Limma. Genes (rows) and samples (columns) were clustered by complete linkage hierarchical clustering, using Euclidean distance measure. For each gene, expression levels close to the mean value across all six samples are shown in black, high expression levels in red, and low expression levels in green. (D) Relative expression of <i>Cxcl13</i>, <i>Ccl21</i>, <i>Ccl19</i>, <i>Tgfb1</i>, and <i>Adrb2</i> mRNA levels quantified by RT-PCR. Data are representative of three different experiments (4–5 mice per sample). (E) Confocal images of a LN B cell follicle and its adjacent T cell area from a CD21cre-RFP chimera stained for CXCL13 (green), CD21/35 (blue), and B220 (white). RFP⁺ cells appear in red. The dashed line represents the delineation of the B220 staining. Data are representative of two different experiments (two mice per experiment).</p

CD21⁻ RFP⁺ stromal cells are surrounded by inflamed B cell follicles.

<p>CD21cre-RFP chimeras were untreated (A, upper panel) or injected (A, lower panel and B) with an emulsion of CFA/PBS in the ears. (A) Three weeks later, ear draining LNs were sectioned, stained for collagen IV (green), IgD (white), and FDC-M2 expression (blue) and imaged by confocal microscopy. RFP⁺ cells appear in red. (B) Confocal pictures of an inflamed B cell follicle stained for collagen IV (white), B220 (blue), and CD21/35 (green) expression. Arrows highlight the region of the inflamed B cell follicle enriched in collagen IV. Insert displays high magnification of the boundary between T and B cell areas (dashed line). Data are representative of three different experiments (two mice per experiment).</p