Journal: Nature immunology
Article Title: Metabolic control of the scaffold protein TKS5 in tissue-invasive, pro-inflammatory T cells
Figure Lengend Snippet: Reducing glycolytic flux renders CD4 + T cells tissue-invasive and pro-inflammatory CD4 + CD45RO − PBMC were isolated from healthy individuals. Glycolytic activity was inhibited by transfecting the cells with PFKFB3 siRNA ( a–f ) or treating with the PFKFB3 inhibitor 3PO (200 nM) ( g–l ). Cells were adoptively transferred into human synovium-engrafted NSG mice. Seven days later, synovial tissue grafts were harvested and processed for immuno-histochemical analysis or for RT-PCR analysis of cytokine and transcription factor gene expression. Data indicate mean ± s.e.m. from 20 different synovial tissues. ( a , g ) Immunohistochemical staining for tissue-residing CD3 + T cells. Scale bars, 20 μm. ( b , h ) Quantification of T cell receptor ( TRB ) transcripts and of tissue-invading CD3 + T cells, expressed as percent of total cells. ( c , i ) Gene expression of IFNG and IL17 . ( d , j ) Co-immunofluorescence staining of tissue sections for IFN-γ and CD3 + T cells. Scale bars, 20 μm. ( e , k ) Frequencies of IFN-γ + CD3 + T cells in the tissue. ( f , l ) Gene expression of lineage-determining transcription factors, key inflammatory markers and TKS5 were determined by RT-PCR. Scale bars, 20 μm. All data are mean ± s.e.m. * P
Article Snippet: Antibodies Primary antibodies used: APC/Cy7 anti-human CD3 (Clone SK7, BioLegend), FITC anti-human CD4 (Clone RPA-T4, BioLegend), anti-human CD8a PE-Cy5 (Clone RPA-T8, eBioscience), PE-Cy™7 Mouse Anti-Human CD45RA (Clone HI100, BD Biosciences), APC anti-human CD45RA (Clone HI100, BioLegend), APC Mouse Anti-Human CD45RO (Clone UCHL1, BD Biosciences), Brilliant Violet 711™ anti-human CD45 (HI30, BioLegend), cortactin antibody (PA5-29799) (ThermoFisher Scientific), FAS mouse monoclonal antibody (M01) (Clone 3F2-1F3, Abnova), Acetyl-CoA Carboxylase (C83B10) Rabbit mAb (Cell Signaling Technology), SCD1 (C12H5) Rabbit mAb (Cell Signaling Technology), RANKL/CD254 Antibody (12A668) (ThermoFisher Scientific), Monoclonal Mouse Anti-Human CD3 (Clone F7.2.38, Dako), Polyclonal Rabbit Anti-Human CD3 (#A045201-2, Dako), β-Actin (8H10D10) Mouse mAb (#3700, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling), WesternSure HRP Goat anti-Mouse IgG (H+L) (LI-COR), Anti-Interferon gamma antibody (ab25101, Abcam), Anti-PPAR gamma antibody (ab178860, Abcam), PPARγ (81B8) Rabbit mAb (Cell Signaling), Monoclonal anti-flag (HRP) antibody (A8592-.2MG, Sigma-Aldrich).
Techniques: Isolation, Activity Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Immunofluorescence