The p.Arg63Trp polymorphism controls Vav1 functions and Foxp3 regulatory T cell development

A single nucleotide polymorphism causing constitutive activation of Vav1 results in increased natural Treg generation and is responsible for the imbalance between Vav1 GEF and adaptor functions

A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BNm for “BN mutated.” In BNm rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4+ CD25bright regulatory T cells (Treg) is defective in BNm rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BNm rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BNm rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BNm×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment

Essais de production de protéines phosphatases sensibles aux microcystines et à l'acide okadaïque pour la conception de biocapteurs

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34+ and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing

BN^m rats develop inflammatory bowel disease.

<p>(A) Small intestine and colon from 12 week-old BN and BN^m rats. (B, C) Length of small intestine and colon (B) and thickness of the duodenum, jejunum, ileum and colon (C) of BN (n = 10) and BN^m rats with macroscopic signs of intestinal lesions (n = 7). (D) Hematoxylin-eosin staining of duodenum, jejunum and colon from 8 to 10 week-old BN and BN^m rats. Images are representative of microscopic lesions observed in all affected BN^m rats (original magnification: 100 X). Stars indicate granulomas; arrows point to infiltration and double head arrows indicate the thickness of the intestinal wall. (E) CD68, CD3 and B220 immunoperoxydase staining on sections of jejunum from 8 week-old BN^m rats. Positive staining results in a brown reaction product. HE: hematoxylin-eosin staining showing the presence of polymorphonuclear leucocytes (original magnification: 400 X). (F) Myeloperoxidase (MPO) activity in the duodenum, jejunum, ileum and colon tissue samples from BN (n = 30) and BN^m rats with macroscopic lesions (n = 21). (G) Cytokine and chemokine protein expression in duodenum from BN (n = 7) and BN^m rats with macroscopic lesions (n = 7). (H) Relative mRNA expression of IL-2 and IL-17A in duodenum from BN (n = 4) and BN^m rats exhibiting intestinal macroscopic lesions (n = 7). (BN: black columns, BN^m: white columns).</p

Impaired suppressive function of BN^m Treg is involved in the development of intestinal lesions.

<p>(A) Absolute numbers of Foxp3+ CD4+ T cells in thymus, spleen and mLN from BN (n = 7) and BN^m (n = 13) rats. Data are representative of three independent experiments. (B) Suppressive activity of thymic CD25^bright CD4+ SP cells (top panel) and peripheral CD25^bright CD4+ T cells (bottom panel) from BN or BN^m rats was assessed in co-culture experiments with CFSE-labeled naive LEW CD4 T cells as effector cells. Proliferation was assessed by CFSE dilution (percentages indicate the proportion of CFSE^low cells). Data are representative of three independent experiments. (C, D) Disease frequency (C) and duodenum microscopic scores (D) in 12 week-old BN^m rats injected with PBS (white column or symbols; n = 21) or with 4.10⁶ BN CD25^bright CD4+ T cells (grey columns or symbols; n = 12) at 4 weeks of age. (E) Cytokine protein expression in duodenum from control BN^m rats with microscopic intestinal scores (white columns; n = 8) and from BN^m rats transferred with CD25^bright CD4+ T cells (grey columns; n = 12).</p

BN^m rats exhibit a T cell autonomous lymphopenia restricted to CD4 T cells.

<p>(A) Percentage of TCRαβ positive cells in PBMC from BN (n = 15, black bars), BN^m (n = 9, white bars) and (BN×BN^m) F1 (n = 5, grey bars) rats. (B) CD4 and CD8 expression in BN and BN^m spleen T cells; numbers indicate the cell percentages in the outlined area. (C) Absolute numbers of BN (n = 4) and BNm (n = 9) T cells, CD4+ and CD8+ T cells, and B cells in spleen. Data in B and C are representative of five independent experiments. (D) CD4 and CD8 expression in BN and BN^m thymocytes; numbers indicate cell percentages in the outlined area. (E) Absolute numbers of BN (n = 5) and BN^m (n = 5) in thymocyte subsets (DN: double negative; DP: double positive; SP: simple positive). Data in D and E are representative of six independent experiments. (F) Absolute numbers of lymphocytes in spleen of lethally irradiated (LEW×BN) F1 rats reconstituted with BN (n = 6) or BN^m (n = 8) T cell-depleted bone marrow cells. Data are representative of three independent experiments. (G) Absolute numbers of donor CD4 SP and CD8 SP cells in thymus of sub-lethally irradiated (LEW×BN) F1 rats 15 days after intrathymic injection of DN thymocytes from BN (n = 4) and BN^m (n = 4) rats. (BN: black columns, BN^m: white columns).</p

BN^m rats carry a disrupted <i>Themis</i> gene.

<p>(A) Genome scan for loci controlling the percentage of CD4 T cells in the blood of 44 (BN^m×DA)×BN^m rats. Horizontal lines represent genome-wide significance thresholds of 5% (significant) and 0.1% (highly significant) as determined by permutation tests. (B) Percentages of CD4 T cells in 44 (BN^m×DA)×BN^m backcross rats classified according to their genotypes at the microsatellite marker of chromosome 1 nearest to the QTL. In each group, horizontal bars represent the mean values. nn: homozygous BN (n = 23); nd: heterozygous BN-DA (n = 21). (C) Fine mapping of the BN^m mutation in 28 (BN^m×DA) F2 or (BN^m×DA)×BN^m backcross rats, among which 16 showed normal proportions of CD4 T lymphocytes (Unaffected) and 12 showed CD4 T cell lymphopenia (Affected). The position of each microsatellite marker on chromosome 1 is indicated in megabases (Mb). White: homozygous BN^m; black: heterozygous BN^m/DA. Rec: number of rats characterized by a given recombination. The physical map of the critical 1.5 Mb interval containing 6 genes is shown underneath. (D) Relative mRNA expression of the 6 genes in BN^m (n = 4) and BN (n = 4) thymocytes. Data are representative of two independent experiments. (E) Electrophoregram, nucleic acid sequences and corresponding amino acids of the BN and BN^m Themis gene in the region surrounding the 4 nucleotide insertion corresponding to the BN^m mutation (boxed). (F) Immunoblot analysis of Themis and β-actin in thymocytes from BN, BN^m, LEW and DA rats, C57BL/6 mice and in human PBMC using anti-Themis antibodies specific either for the mouse C-terminal (left panel) or the human N-terminal (right panel) portion of the protein.</p