Journal: Nature immunology

Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

doi: 10.1038/ni.3808

Figure Lengend Snippet: CD4 + CD45RA + T cells from healthy individuals were stimulated in the absence or presence of 3PO (200 nM), an inhibitor of the glycolytic enzyme PFKFB3, or C75 (20 μM), an inhibitor of the fatty acid synthase (FAS). ( a, b ) Expression of the SH3PXD2A gene assessed by RT-PCR (8 control-RA pairs). ( c ) Expression of the mobility gene module (see ) assessed by RT-PCR. Percent change from the vehicle control is shown for each individual gene (8 RA-control pairs). ( d–g ) TKS5 protein expression determined by flow cytometry. Representative histograms and summary from 8 experiments. In a–c , e and g , data are mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 (paired t -test). ( h–m ) T cell invasion measured in the 3D-collagen matrix system as in . ( h , k ) Representative confocal images of DAPI-stained nuclei taken at indicated depths. Scale bars, 200 μm. ( i, l ) DAPI indices (signal at defined depth/signal at surface) quantified in 4 experiments. ( j, m ) Maximum invasion distances determined in 4 healthy and 4 RA samples. In j and m , data are mean ± s.e.m. ** P < 0.01, *** P < 0.001 (Mann-Whitney U-test).

Article Snippet: For experiments targeting anabolic lipogenesis, chimeric mice carrying the same synovial tissue were randomly assigned to three treatment arms: (A) control (vehicle); (B) treatment with FAS inhibitor C75 (5 mg/kg/mouse qad, # 191282-48-1 Cayman Chemical); or ML265 (10 mg/kg/mouse qd, #1221186-53-3 Cayman Chemical).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Nature immunology

Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

doi: 10.1038/ni.3808

Figure Lengend Snippet: CD4 + CD45RA + T cells from healthy age-matched individuals and RA patients were stimulated for 72 h. ( a ) Gene expression for 20 lipogenic genes was profiled by RT-PCR. Data from a discovery cohort of 10 healthy-RA pairs are shown as a heat map. Scaled z-score: red indicates high and blue low transcript levels. ( b ) Gene sets of de novo fatty acid synthesis, esterification, and FA β-oxidation pathways compared in a validation cohort of 6 healthy-RA pairs. Relative gene expression measured by RT-PCR. ( c ) Expression kinetics of lipid biosynthesis genes following T cell activation ( n = 6 independent samples per time point). ( d ) Immunoblot analysis of key enzymes involved in FA synthesis (ACC, FAS, SCD1) in 4 control-RA pairs. Data are representative for 8 experiments. ( e ) Expression kinetics of carnitine acyltransferases ( CPT1A and CPT1B , involved in lipid transport into mitochondria) following T cell activation ( n = 5 independent samples per time point). ( f ) Expression levels of genes involved in cholesterol synthesis (8 healthy-RA pairs). ( g, h ) Representative images showing lipid droplets deposited in the cytoplasm. Fluorescent signal quantification of neutral lipids in C75 treatment groups ( n = 5 patients/group). Scale bar, 20 μm. ( i, j ) Flow cytometry of Bodipy-stained T cells. Left: Representative histograms. Right: Mean fluorescence intensities from 5 experiments. All data are mean ± s.e.m. * P < 0.05. ** P < 0.01 (Mann-Whitney U-test). Non-significant (ns). FA, fatty acid; MUFA, mono-unsaturated fatty acid; DG, diacylglycerol; TG, triacylglycerol.

Article Snippet: For experiments targeting anabolic lipogenesis, chimeric mice carrying the same synovial tissue were randomly assigned to three treatment arms: (A) control (vehicle); (B) treatment with FAS inhibitor C75 (5 mg/kg/mouse qad, # 191282-48-1 Cayman Chemical); or ML265 (10 mg/kg/mouse qd, #1221186-53-3 Cayman Chemical).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Activation Assay, Western Blot, Control, Flow Cytometry, Staining, Fluorescence, MANN-WHITNEY

Journal: Nature immunology

Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells

doi: 10.1038/ni.3808

Figure Lengend Snippet: CD4 + CD45RO − PBMC were isolated from RA patients with active disease and adoptively transferred into human synovium-NSG chimeric mice. Mice were randomly assigned to one of three treatment arms ( n = 14 synovial grafts/treatment arm): Vehicle arm (vehicle injection); RA treated with C75 (5 mg/kg, i.p. every other day) or RA treated with ML265 (10 mg/kg, i.p. daily). Synovial tissues were harvested, tissue sections were stained with anti-human CD3 (brown) and anti-RANKL (pink) antibodies and frequencies of RANKL positive cells were quantified in randomly selected high-powered fields. ( a ) Representative tissue stains showing human CD3 + and RANKL + T cells infiltrating into synovial tissue, where they form cellular clusters. Scale bars, 20 μm. ( b ) Frequencies of CD3 + RANKL + T cells in the tissue as percent of total cells. ( c ) RT-PCR-based quantification of T cell receptor ( TRB ) transcripts. ( d ) Gene expression of IFNG and IL17 . ( e ) Gene expression of transcription factors, key cytokines and TKS5 determined by RT-PCR. ( f , g ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3. Frequencies of tissue-residing IFN-γ + CD3 + T cells in the different treatment arms. Scale bars, 20 μm. All data are mean ± s.e.m. * P < 0.05. ** P < 0.01. *** P < 0.001 (Mann-Whitney U-test). Non-significant (ns).

Article Snippet: For experiments targeting anabolic lipogenesis, chimeric mice carrying the same synovial tissue were randomly assigned to three treatment arms: (A) control (vehicle); (B) treatment with FAS inhibitor C75 (5 mg/kg/mouse qad, # 191282-48-1 Cayman Chemical); or ML265 (10 mg/kg/mouse qd, #1221186-53-3 Cayman Chemical).

Techniques: Isolation, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Immunofluorescence, MANN-WHITNEY